EP4735480A1

EP4735480A1 - Multi-specific antibodies comprising antigen-binding domains specifically recognizing c5ar1 and gm-csfr alpha

Info

Publication number: EP4735480A1
Application number: EP24830855.3A
Authority: EP
Inventors: Chong HE; Wenwu Zhai; Zhiwen Zhou; Michael George Strainic; Ping Yuan; Zuoan YI
Original assignee: Staidson Beijing Biopharmaceutical Co Ltd
Current assignee: Staidson Beijing Biopharmaceutical Co Ltd
Priority date: 2023-06-27
Filing date: 2024-06-27
Publication date: 2026-05-06
Also published as: CN121816363A; WO2025002221A1

Abstract

Provides multi-specific (e.g., bispecific) antibodies that specifically recognize C5aR1 and GM-CSFRα, and pharmaceutical compositions comprising multi-specific (e.g., bispecific) antibodies that specifically recognize C5aR1 and GM-CSFRα. Also provided are methods of making and using these antibodies.

Description

MULTI-SPECIFIC ANTIBODIES COMPRISING ANTIGEN-BINDING DOMAINS SPECIFICALLY RECOGNIZING C5aR1 AND GM-CSFR alpha

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission of sequence list is incorporated herein by reference in its entirety: C5aR1 and GM-CSFRα bispecific antibody seqlist 20230627. xml, date recorded: June 27, 2023, size: 127KB) .

FIELD

This application relates to multi-specific (e.g., bispecific) antibodies comprising a first antigen-binding domain that specifically recognizing C5aR1 and a second antigen-binding domain that specifically recognizing GM-CSFRα. The application further relates to pharmaceutical compositions comprising antibodies specifically recognizing C5aR1 and antibodies specifically recognizing GM-CSFRα; pharmaceutical compositions comprising the multi-specific (e.g., bispecific) antibody recognizing C5aR1 and GM-CSFRα; as well as methods of manufacture and uses thereof, including methods of treating and preventing inflammatory disease or condition.

BACKGROUND

Granulocyte-macrophage colony stimulating factor (GM-CSF) is also known as colony-stimulating factor 2 (CSF2) . GM-CSF is a type I proinflammatory cytokine which plays a role in exacerbating inflammatory, respiratory and autoimmune diseases. The GM-CSF receptor is a member of the hematopoietic receptor superfamily. It is heterodimeric, consisting of an alpha and a beta subunit. GM-CSF is able to bind with relatively low affinity to the αsubunit alone (Kd 1-5nM) but not at all to the β subunit alone. However, the presence of both α and β subunits results in a high affinity ligand-receptor complex (Kd≈100pM) . Neutralization of GM-CSF binding to GM-CSFRα is therefore a therapeutic approach to treating diseases and conditions mediated through GM-CSFR.
C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) is a member of the rhodopsin superfamily of G-protein-coupled receptor (GPCR) (Gerard NP et al., J Biol Chem 264: 1760-1766, 1989) . C5aR1, with its high affinity ligand C5a, is an essential part of the complement system, playing important role in eliciting the broad immune responses. The structure of C5aR1 conforms to the seven transmembrane receptor family and contains 4 extracellular domains, seven membrane-spanning helices, and 4 intracellular C domains (Dixon et al. Nature 326, 73-77, 1987; Findlay et al. Biochem. J. 238, 625-642, 1986) . C5aR1 is a classical G protein-coupled receptor that signals through Gαi and Gα16 which both are G- proteins. Activated C5aR1 has been shown to associate with two of the four mammalian β-arrestins (β-arrestin 1, 2) , which have different dependencies on the phosphorylation status of the receptor. Upon agonist biding, C5aR1 undergoes rapid phosphorylation on the six serine residues present in the C-terminal region followed by desensitization and internalization. Serine residues in the C5aR1 C terminus were identified that control the intracellular trafficking of the C5aR1-arrestin complex in response to C5a. (Braun et al. J Biol Chem. 278: 4277-4285, 2003) .
C5aR1 has been found to be expressed on various inflammatory cells and smooth muscle cells to promote an inflammatory reaction. It is also present on subsets of T cells and dendritic cells to regulate the adaptive immune response (Kemper et al. Nature Rev. Immunol. 7.9-18, 2007) . C5a-C5aR1 interaction is required for the optimal oxidative burst and phagocytosis, which form the fundamental machinery in innate immunity for bacterial clearance. The binding of C5a ligand and its receptor C5aR1 thereof induces an inflammatory reaction by inducing cytokine and chemokine expression (Monsinjon et al. FASEB J 17: 1003–1014, 2003.; Fukuoka et al. Clin Exp Immunol 131: 248–253, 2003) . C5a-C5aR1 interaction is also an essential step in neutrophil migration by causing adhesion molecule expression and inducing chemokine production (Riedemann et al. Immunity 19: 193–202, 2003. ) . It has been reported that C5aR1 and its ligand C5a play a key role in initiating and maintaining several inflammatory responses by recruiting and activating neutrophils and monocytes in the lungs (Julien Carvelli et al. Nature 588, 146-150, 2020) . In addition, C5a perturbs coagulation and fibrinolysis pathway, resulting in damaging intravascular coagulation (Muhlfelder et al. J Clin Invest 63: 147–150, 1979.; Laudes et al. Am J Pathol 160: 1867–1875, 2002. ) . In addition, it has been reported that activated C5aR1 and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR1 and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR1 mRNA levels were associated with decreased overall survival (Cho Ms et al. Cell Rep. 2014) .
Inhibition of C5a-C5aR1 interaction should reduce the acute inflammatory response mediated via C5a. The blockade of C5aR1 signal, thereby reducing of the inflammatory reaction, has also been researched. WO2003/062278A and WO2008/022390A disclose anti-C5aR1 antibodies and methods to obtain and use them. However, as an optimal therapeutic directed to a target in this pathway has yet to be commercialized, a significant unmet medical need exists.
The multi-specific (e.g., bispecific) antibody capable of targeting multiple antigens in a single agent, blocked two or more pathologies pathways at the same time, so they may have some novel activities that do not exist in mixtures of the parental or reference monoclonal antibodies. For example (i) the multi-specific (e.g., bispecific) antibody directly targets effector cells to tumor cells, enhancing cytotoxicity thereof; (ii) the multi-specific (e.g., bispecific) antibody can simultaneously identify two antigens, thereby improving the selectivity and affinity of the bispecific antibody, a more comprehensive, more efficient treatment effect can be provided; (iii) compared with the combination therapy of two monoclonal antibodies, the multi-specific (e.g., bispecific) antibody reduces the development and clinical trial costs.
Given this, for the treatment of inflammatory diseases, autoimmune diseases, or cancers and the like, monoclonal antibody-based therapy still needs to be improved, and there is a need for novel strategies, e.g., multi-specific or bispecific antibody for preventing and treating such diseases.
The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.
BRIEF SUMMARY
The present application provides multi-specific (e.g., bispecific) antibodies comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the first antigen-binding domain comprises: (i) a V_H comprising an HC-CDR1 comprising SGYSWH (SEQ ID NO: 1) , an HC-CDR2 comprising YIHSSGDTDYNPSLKR (SEQ ID NO: 2) , and an HC-CDR3 comprising SIGDY (SEQ ID NO: 3) ; and a V_L comprising an LC-CDR1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 11) ; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 12) , and an LC-CDR3 comprising SQSTHVPWT (SEQ ID NO: 13) ; or a V_H comprising an HC-CDR1 comprising SYWMN (SEQ ID NO: 4) ; an HC-CDR2 comprising RIDPX₁DSX₂TRYNQKFED (SEQ ID NO: 49) , wherein X₁ is Y, G or R, and X₂ is E or G; and an HC-CDR3 comprising FVX₁PX₂GX₃FAY (SEQ ID NO: 50) , wherein X₁ is I or W, X₂ is S or V, and X₃ is G or A; and a V_L comprising an LC-CDR1 comprising RSSX₁SPX₂HSNGNTYLH (SEQ ID NO: 51) , wherein X₁ is Q or R, and X₂ is V or L; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 17) , and an LC-CDR3 comprising SQSTX₁VPPT (SEQ ID NO: 52) , wherein X₁ is H or L.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; and a LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 18-19, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the first antigen-binding domain comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising ELSIH (SEQ ID NO: 34) ; an HC-CDR2 comprising GFDPEDGETIYAQKX₁QG (SEQ ID NO: 53) , wherein X₁ is S or F; and an HC-CDR3 comprising GRYX₁X₂LX₃X₄X₅YGFDY (SEQ ID NO: 54) , wherein X₁ is T or V, X₂ is E or S, X₃ is A, F or Y, X₄ is T, F or Q, and X₅ is T or N; and a V_L comprising an LC-CDR1 comprising RASQSVSSYLA (SEQ ID NO: 40) ; an LC-CDR2 comprising GASSRAT (SEQ ID NO: 41) , and an LC-CDR3 comprising QQYX₁NWPX₂T (SEQ ID NO: 55) , wherein X₁ is D or N, and X₂ is Y or P.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 35-36, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 37-39, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 42-43, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: (i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the first antigen-binding domain comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23 or 59; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61; or (iv) a V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30 or 66; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66; or (v) a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67; or (vi) a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68; or (vii) a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein comprising a first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the second antigen-binding domain comprises a V_H comprising an HC-CDR1 comprising ELSIH (SEQ ID NO: 34) ; an HC-CDR2 comprising GFDPEDGETIYAQKX₁QG (SEQ ID NO: 53) , wherein X₁ is S or F; and an HC-CDR3 comprising GRYX₁X₂LX₃X₄X₅YGFDY (SEQ ID NO: 54) , wherein X₁ is T or V, X₂ is E or S, X₃ is A, F or Y, X₄ is T, F or Q, and X₅ is T or N; and a V_L comprising an LC-CDR1 comprising RASQSVSSYLA (SEQ ID NO: 40) , an LC-CDR2 comprising GASSRAT (SEQ ID NO: 41) , and an LC-CDR3 comprising QQYX₁NWPX₂T (SEQ ID NO: 55) , wherein X₁ is D or N, X₂ is Y or P.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the second antigen-binding domain comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, the format of the multi-specific (e.g., bispecific) antibody is selected from the group consisting of DVD-Ig, IgG- (scFv) ₂ and Hetero H, CrossMab.
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format, comprising four polypeptide chains: wherein: the two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-L-V_H2-C_H1, wherein V_H1 is a heavy-chain variable region that specifically binds to the C5aR1; V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; L is a linker; C_H1 is a heavy chain constant region C_H1 domain; wherein the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and the other two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-L-V_L2-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1; V_L2 is a light chain variable region that specifically binds to GM-CSFRα; L is a linker; and C_L is a light chain constant region.
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format, comprising four polypeptide chains: wherein: the two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-L-V_H2-C_H1, wherein V_H1 is a heavy-chain variable region that specifically binds to the GM-CSFRα; V_H2 is a heavy chain variable region that specifically binds to C5aR1; L is a linker; C_H1 is a heavy chain constant region C_H1 domain; wherein the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and the other two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-L-V_L2-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα; V_L2 is a light chain variable region that specifically binds to C5aR1; L is a linker; and C_L is a light chain constant region.
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format, comprising the amino acid sequence of any one of SEQ ID NOs: 101-102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101-102; and/or the amino acid sequence of any one of SEQ ID NOs: 79-80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79-80.
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format, comprising the amino acid sequence of any one of SEQ ID NOs: 103-104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 103-104; and/or the amino acid sequence of any one of SEQ ID NOs: 81-82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81-82.
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format, comprising: (i) the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79; or (ii) the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80; or (iii) the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79; or (iv) the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80; or (v) the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81; or (vi) the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82; or (vii) the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81; or (viii) the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format, comprising: (i) the amino acid sequence of any one of SEQ ID NOs: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101; and the amino acid sequence of any one of SEQ ID NOs: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79; or (ii) the amino acid sequence of any one of SEQ ID NOs: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101; and the amino acid sequence of any one of SEQ ID NOs: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 80; or (iii) the amino acid sequence of any one of SEQ ID NOs: 102, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 102; and the amino acid sequence of any one of SEQ ID NOs: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79; or (iv) the amino acid sequence of any one of SEQ ID NOs: 102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 102; and the amino acid sequence of any one of SEQ ID NOs: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 80; or (v) the amino acid sequence of any one of SEQ ID NOs: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 103; and the amino acid sequence of any one of SEQ ID NOs: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81; or (vi) the amino acid sequence of any one of SEQ ID NOs: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 103; and the amino acid sequence of any one of SEQ ID NOs: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 82; or (vii) the amino acid sequence of any one of SEQ ID NOs: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 104; and the amino acid sequence of any one of SEQ ID NOs: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81; or (viii) the amino acid sequence of any one of SEQ ID NOs: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 104; and the amino acid sequence of any one of SEQ ID NOs: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 82.
In some embodiments, the multi-specific (e.g., bispecific) is IgG- (scFv) ₂ format, comprising four chains, wherein two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, wherein V_H1 is a heavy chain variable region that specifically binds to C5aR1; V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; V_L2 is a light chain variable region that specifically binds to GM-CSFRα; L1 and L2 are linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc region comprises C_H2 and C_H3 domains; and the other two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1, and C_L is a light chain constant region.
In some embodiments, the multi-specific (e.g., bispecific) is IgG- (scFv) ₂ format, comprising four chains, two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, wherein V_H1 is a heavy chain variable region that specifically binds to GM-CSFRα; V_H2 is a heavy chain variable region that specifically binds to C5aR1; V_L2 is a light chain variable region that specifically binds to C5aR1; L1 and L2 are linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc region comprising C_H2 and C_H3 domains; and the other two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα, and C_L is a light chain constant region.
In some embodiments, the multi-specific (e.g., bispecific) is IgG- (scFv) ₂ format, wherein the V_H2 and V_L2 may further comprise cysteines substitution.
In some embodiments, the multi-specific (e.g., bispecific) is IgG- (scFv) ₂ format, comprising: (i) the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 83; and/or the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87; or (ii) the amino acid sequence of SEQ ID NO: 84, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 84; and/or the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88; or (iii) the amino acid sequence of SEQ ID NO: 85, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 85; and/or the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87; or (iv) the amino acid sequence of SEQ ID NO: 86, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 86; and/or the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88.
In some embodiments, the multi-specific (e.g., bispecific) antibody is CrossMab format comprising four chains, wherein: one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1, wherein V_H1 is a heavy chain variable region specifically binding to C5aR1; C_H1 is a heavy chain constant region C_H1 domain; the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1, and C_L is a light chain constant region; one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H2-C_L, wherein V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; C_L is a light chain constant region; the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L2-C_H1, wherein V_L2 is a light-chain variable region that specifically binds to GM-CSFRα, and C_H1 is a heavy-chain constant region C_H1 domain.
In some embodiments, the multi-specific (e.g., bispecific) antibody is CrossMab format comprising four chains, wherein: one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1, wherein V_H1 is a heavy chain variable region specifically binding to GM-CSFRα; C_H1 is a heavy chain constant region C_H1 domain; the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα, and C_L is a light chain constant region; one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H2-C_L, wherein V_H2 is a heavy chain variable region that specifically binds to C5aR1; C_L is a light chain constant region; the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L2-C_H1, wherein V_L2 is a light-chain variable region that specifically binds to C5aR1, and C_H1 is a heavy-chain constant region C_H1 domain.
In some embodiments, the multi-specific (e.g., bispecific) antibody is CrossMab format comprising: (i) the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 105; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 106, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 106; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98; or (ii) the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 107; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 108; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, the multi-specific (e.g., bispecific) antibody is CrossMab format comprising: (i) the amino acid sequence of SEQ ID NO: 89, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 89; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 90, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 90; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98; or (ii) the amino acid sequence of SEQ ID NO: 91, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 91; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 92, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 92; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98; or (iii) the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 93; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 94; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100; or (iv) the amino acid sequence of SEQ ID NO: 95, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 95; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 96, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 96; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the Fc region is selected from the group consisting of an Fc region from an IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the Fc region comprises one or more amino acid mutations.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the Fc region is aglycosylated or deglycosylated.
In some embodiments, the multi-specific (e.g., bispecific) antibody provided herein, wherein the Fc region has reduced fucosylation or is afucosylated.
Also provided are pharmaceutical compositions, kits and articles of manufacture comprising any one of the multi-specific (e.g., bispecific) antibodies recognizing C5aR1 and GM-CSFRα, nucleic acids, vectors, isolated host cells described above. In addition, a method of treating or preventing a disease in an individual using thereof is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the results of exemplary anti-C5aR1 antibodies in the C5aR1-TEV/β-Arrestin-Luc reporter assay.
Fig. 2A shows the exemplary schematic structure diagram of DVD-Ig BsAb molecule, Fig. 2B shows the exemplary schematic structure diagram of IgG- (scFv) ₂ BsAb molecule, and Fig. 2C shows the exemplary schematic structural diagram of CrossMab BsAb molecules.
Fig. 3A shows the binding curves of the exemplary bispecific antibodies to human GM-CSFRα, and Fig. 3B shows the binding curves of the exemplary bispecific antibodies to human C5aR1.
Figs. 4A-4C show the results of the exemplary bispecific antibodies in the non-specific binding assay for insulin, dsDNA and BVP, respectively.
Fig. 5 shows the result of activity of the exemplary bispecific antibodies in the TF-1 proliferation assay.
Fig. 6 shows the result of activity of the exemplary bispecific antibodies in the C5aR1-TEV/β-Arrestin-Luc reporter assay.

DETAILED DESCRIPTION

Definitions
As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of the disease (such as, for example, tumor volume for cancer) . The methods of the application contemplate any one or more of these aspects of treatment.
The term “prevent, ” and similar words such as “prevented” , “preventing” , “prevention” or “prophylactic” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., a pathogenic infection. It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to occurrence or recurrence of the disease or condition. As used herein, “prevention” and similar words also includes reducing the risk and susceptibility to occurrence or recurrence of the disease or condition, e.g., a pathogenic infection.
The term “antibody” herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific, multi-specific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen binding activity. In some embodiments, a full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991) . The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain) , IgG2 (γ2 heavy chain) , IgG3 (γ3 heavy chain) , IgG4 (γ4 heavy chain) , IgA1 (α1 heavy chain) , or IgA2 (α2 heavy chain) .
Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy-and light-chain variable domains and also the constant domain of the light chain and the first constant domain (C_H1) of the heavy chain. As used herein, Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a V_L domain and a constant domain of a light chain (C_L) , and a V_H domain and a first constant domain (C_H1) of a heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain C_H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH are Fab' fragments wherein the cysteine residue (s) of the constant domains bear a free thiol group. Pepsin treatment yields an F (ab') 2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.
The term “antigen-binding fragment” as used herein includes an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F (ab’) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment also includes a fusion protein comprising the antibody fragment described above. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
The term “multi-specific antibody” as used herein refers to an antibody having binding specificity to two or more different antigens or to different epitopes on the same antigen in one molecule, e. g, bispecific antibody. The term “bispecific antibody” as used herein refers to an antibody having binding specificity to two different antigen or to different epitopes on the same antigen in one molecule. In preferred embodiments, the multi-specific antibody is preferably a bispecific antibody. Bispecific antibody is produced through a process that involves design of the intact molecule, synthesis and cloning of the nucleotide sequences for each domain, expression in mammalian cells and purification of the final product. Exemplary bispecific antibody structures include structures known in the art, such as DVD-Ig format, IgG-(scFv) 2 format or Hetero H, CrossMab format etc. (see Labrijn AF, et al. Nat Rev Drug Discov. 2019 Aug; 18 (8) : 585-608) .
The term "valent" as used within the current application denotes the presence of a specified number of binding domains in an antigen binding molecule. As such, the terms "bivalent" , or "tetravalent" denote the presence of two binding domain, and four binding domains, respectively, in an antigen binding molecule. The bispecific antibodies according to the application are at least "bivalent" and may be "tetravalent " or "multivalent" .
The term “antigen-binding domain” as used herein refers to the portion of an antigen binding molecule that specifically binds to an antigen. More specifically, the term “antigen-binding domain” refers to a portion of an antibody that comprises a region that specifically binds to and is complementary to a portion or the entirety of an antigen. In the case of large antigens, the antigen binding molecule may bind only a specific part of the antigen, which part is called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also referred to as variable regions) . Preferably, the antigen-binding domain comprises an antibody light chain variable domain (V_L) and an antibody heavy chain variable domain (V_H) . In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen. In some embodiments, the antigen-binding domains that specifically recognizing C5aR1 or GM-CSFRα can be provided as being contained in antibodies and antigen-binding fragments thereof as further defined herein.
The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
As used herein, a first antibody “competes” for binding to a target with a second antibody when the first antibody inhibits the target binding of the second antibody by at least about 50% (such as at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99%) in the presence of an equimolar concentration of the first antibody, or vice versa. A high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03/48731.
As used herein, the term “specifically binds, ” “specifically recognizing, ” or “is specific for” refers to measurable and reproducible interactions, such as binding between a target and an antibody, that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically recognizes a target (which can be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity that is at least about 10 times its binding affinity for other targets.
An “isolated” antibody as used herein refers to an antibody that (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, (3) is expressed by a cell from a different species, or, (4) does not occur in nature.
The term “isolated nucleic acid” as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the “isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991) ; Chothia et al., J. Mol. Biol. 196: 901-917 (1987) ; Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997) ; MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) ; Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008) ; Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003) ; and Honegger and Plückthun, J. Mol. Biol., 309: 657-670 (2001) , where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008) ; Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010) ; and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015) . The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present application and for possible inclusion in one or more claims herein.
TABLE 1: CDR DEFINITIONS
¹Residue numbering follows the nomenclature of Kabat et al., supra
²Residue numbering follows the nomenclature of Chothia et al., supra
³Residue numbering follows the nomenclature of MacCallum et al., supra
⁴Residue numbering follows the nomenclature of Lefranc et al., supra
⁵Residue numbering follows the nomenclature of Honegger and Plückthun, supra
The term “chimeric antibodies” refer to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984) ) .
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy-and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
“Single-chain Fv, ” also abbreviated as “sFv” or “scFv, ” are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .
The term “diabodies” refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) typically with short linkers (such as about 5 to about 10 residues) between the V_H and V_L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the V_H and V_L domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404, 097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) .
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986) ; Riechmann et al., Nature 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992) .
“Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skilled in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) , or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, %amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32 (5) : 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5 (1) : 113, 2004) .
Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy-and light-chain variable domains and also the constant domain of the light chain and the first constant domain (C_H1) of the heavy chain. As used herein, Thus, the term "Fab" refers to an antibody fragment comprising a light chain fragment comprising a V_L domain and a constant domain of a light chain (C_L) , and a V_H domain and a first constant domain (C_H1) of a heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain C_H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH are Fab' fragments wherein the cysteine residue (s) of the constant domains bear a free thiol group. Pepsin treatment yields an F (ab') 2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.
The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region, and in some embodiments, includes a partial hinge region, whether a monomer form or a multimeric form. The natural Fc monomer can be linked to a dimer or multimeric form by covalent (i.e., disulfide bond) and non-covalent association. The term includes native sequence Fc regions and variant Fc regions. The antibody Fc domain may be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2) , IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) , and IgM. In one embodiment, the antibody Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. Particularly, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The amino acid sequences of the heavy chains are always presented with the C-terminal lysine, however variants without the C-terminal lysine are included in the application. An IgG Fc region comprises an IgG C_H2 and an IgG C_H3 domain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Rabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) .
In some embodiments, each of the two Fc monomers in the Fc dimer comprises one or more amino acid substitutions that facilitates two monomer heterodimerization. In some embodiments, heterodimerization of an Fc monomer may be facilitated by introducing different but compatible substitutions in two Fc monomers, such as "Knob-into-Hole " . The "Knob-into-hole " technology is also disclosed in US Patent Publication No. 8,216,805. In some embodiments, one Fc monomer includes Knob mutation T366W, and another Fc monomer includes Hole mutations T366S, L358A, and Y407V. In some embodiments, two Cys residues were introduced to form a stabilized disulfide bridge, the Fc monomer comprising the knob modification additionally comprise the amino acid substitution S354C, and the Fc monomer comprising the hole modification additionally comprise the amino acid substitution Y349C.
The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR of this application is one that binds an IgG antibody (a γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor” ) and FcγRIIB (an “inhibiting receptor” ) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. inAnnu. Rev. Immunol. 15: 203-234 (1997) ) . The term includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and/or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) ; Capel et al., Immunomethods 4: 25-34 (1994) ; and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995) . Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) .
The term “FcRn” refers to the neonatal Fc receptor (FcRn) . FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an α-chain noncovalently bound to β2-microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and the regulation of serum IgG levels. FcRn can act as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway.
The “C_H1 domain” of a human IgG Fc region usually extends from about amino acid 118 to about amino acid 215 (EU numbering system) . The C_H1 region herein may be a native sequence C_H1 domain or a variant C_H1 domain.
“Hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985) ) . Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-Sbonds in the same positions.
The “C_H2 domain” of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340. The C_H2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two C_H2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the C_H2 domain. Burton, Molec Immunol. 22: 161-206 (1985) . The C_H2 domain herein may be a native sequence C_H2 domain or variant C_H2 domain.
The “C_H3 domain” comprises the stretch of residues C-terminal to a C_H2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG) . The C_H3 region herein may be a native sequence C_H3 domain or a variant C_H3 domain.
A “functional Fc fragment” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity (CDC) ; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR) , etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art.
An antibody with a variant IgG Fc with “altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity (e.g., FcγR or FcRn) and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. The variant Fc which “exhibits increased binding” to an FcR binds at least one FcR with higher affinity (e.g., lower apparent Kd or IC₅₀ value) than the parent polypeptide or a native sequence IgG Fc. According to some embodiments, the improvement in binding compared to a parent polypeptide is about 3-fold, such as about any of 5, 10, 25, 50, 60, 100, 150, 200, or up to 500-fold, or about 25%to 1000%improvement in binding. The polypeptide variant which “exhibits decreased binding” to an FcR, binds at least one FcR with lower affinity (e.g., higher apparent Kd or higher IC₅₀ value) than a parent polypeptide. The decrease in binding compared to a parent polypeptide may be about 40%or more decrease in binding.
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) . To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998) .
The polypeptide comprising a variant Fc region which “exhibits increased ADCC” or mediates ADCC in the presence of human effector cells more effectively than a polypeptide having wild type IgG Fc or a parent polypeptide is one which in vitro or in vivo is substantially more effective at mediating ADCC, when the amounts of polypeptide with variant Fc region and the polypeptide with wild type Fc region (or the parent polypeptide) in the assay are essentially the same. Generally, such variants will be identified using any in vitro ADCC assay known in the art, such as assays or methods for determining ADCC activity, e.g., in an animal model etc. In some embodiments, the variant is from about 5-fold to about 100-fold, e.g. from about 25 to about 50-fold, more effective at mediating ADCC than the wild type Fc (or parent polypeptide) .
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in US patent No. 6,194,551B1 and WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron (s) .
The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60%homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50%homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
An “effective amount” of an antibody or composition as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and by known methods relating to the stated purpose.
The term “therapeutically effective amount” refers to an amount of an antibody or composition as disclosed herein, effective to “treat” a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount of the antibody or composition as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that extends the survival of a patient. In some embodiments, the therapeutically effective amount is an amount that improves progression free survival of a patient.
As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
It is understood that embodiments of the application described herein include “consisting of” and/or “consisting essentially of” embodiments.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X” .
As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat infection of type X means the method is used to treat infection of types other than X.
As used herein and in the appended claims, the singular forms “a, ” “or, ” and “the” include plural referents unless the context clearly dictates otherwise.
Anti-C5aR1 antigen-binding domain and Anti-GM-CSFRα antigen-binding domain
In some embodiments, the antigen-binding domain specifically recognizing C5aR1 (i.e., anti-C5aR1 antigen-binding domain) can be contained in an antibody, including but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein. In some embodiments, the antigen-binding domains are contained in isolated antibodies that bind to C5aR1. Contemplated antigen-binding domains specifically recognizing C5aR1 can be contained in the entirety or a fragment of full-length antibodies (e.g., full-length IgG1, IgG2, IgG3 or IgG4) , scFvs, multi-specific (such as bispecific) antibodies, immunoconjugates, and the like. In some embodiments, the antibody comprising antigen-binding domains specifically recognizing C5aR1 is a Fab, a Fab’, a F (ab) ’2, a Fab’-SH, a single-chain Fv (scFv) , an Fv fragment, a dAb, a Fd, or a diabody. In some embodiments, the antibody comprising antigen-binding domains specifically recognizing C5aR1 is a multi-specific antibody (e.g., bispecific antibody) . In some embodiments, reference to an antigen-binding domain that specifically binds to C5aR1 means that the antigen-binding domain binds to C5aR1 with an affinity that is at least about 10 times (including for example at least about any of 10, 10², 10³, 10⁴, 10⁵, 10⁶, or 10⁷ times) its binding affinity for non-target. In some embodiments, the non-target is an antigen that is not C5aR1.
In some embodiments, the antigen-binding domain specifically recognizing GM-CSFRα (i.e., anti-GM-CSFRα antigen-binding domain) can be contained in an antibody, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein. In some embodiments, the antigen-binding domains are contained in isolated antibodies that bind to GM-CSFRα. Contemplated antigen-binding domains specifically recognizing GM-CSFRα can be contained in the entirety or a fragment of full-length anti-GM-CSFRα antibodies (e.g., full-length IgG1, IgG2, IgG3 or IgG4) , scFvs, multi-specific (such as bispecific) antibodies, immunoconjugates, and the like. In some embodiments, the antibody comprising antigen-binding domains specifically recognizing GM-CSFRα is a Fab, a Fab’, a F (ab) ’2, a Fab’-SH, a single-chain Fv (scFv) , an Fv fragment, a dAb, a Fd, or a diabody. In some embodiments, reference to an antigen-binding domain that specifically binds to GM-CSFRα means that the antigen-binding domain binds to GM-CSFRα with an affinity that is at least about 10 times (including for example at least about any of 10, 10², 10³, 10⁴, 10⁵, 10⁶, or 10⁷ times) its binding affinity for non-target. In some embodiments, the non-target is an antigen that is not GM-CSFRα.
Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA) . Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay or biolayer interferometry (BLI) .
Although antigen-binding domains containing human sequences (e.g., human heavy and light chain variable domain sequences comprising human CDR sequences) are extensively discussed herein, non-human antigen-binding domains or are also contemplated. In some embodiments, non-human antigen-binding domains comprise human CDR sequences from an antigen-binding domain as described herein and non-human framework sequences. Non-human framework sequences include, in some embodiments, any sequence that can be used for generating synthetic heavy and/or light chain variable domains using one or more human CDR sequences as described herein, including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) , etc. In some embodiments, a non-human antigen-binding domains includes an antigen-binding domain generated by grafting one or more human CDR sequences as described herein onto a non-human framework sequence (e.g., a mouse or chicken framework sequence) .
The anti-C5aR1 antigen-binding domain of the present application can be selected from those recorded in International Application No. PCT/US2022/082260, which was incorporated herein in its entirety. For example, 1C2, 1C2.30 or 1C2.35 used in the examples of the present application is specific for C5aR1, whose CDR sequences is shown in Table 2.
The anti-GM-CSFRα antigen-binding domain of the present application can be selected from those disclosed in International Publication No. WO2020/108423 A1, which was incorporated herein in its entirety. For example, E35 used in the examples of the present application is specific for GM-CSFRα, whose CDR sequences is shown in Table 5.
Combination of an antigen-binding domain specifically recognizing C5aR1 and an antigen-binding domain specifically recognizing GM-CSFRα
In some embodiments, the combination of the present application is provided in the form of a multi-specific (e.g., bispecific) antibody, which comprises a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα.
In some embodiments, the combination of the present application is provided in the form of a combination of an antibody comprising anti-C5aR1 antigen-binding domain and an antibody comprising anti-GM-CSFRα antigen-binding domain. In some embodiments, the antibody comprising anti-C5aR1 antigen-binding domain is an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1. In some embodiments, the antibody comprising anti-GM-CSFRα antigen-binding domain is an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα.
In some embodiments, the combination of the present application is provided in the form of a composition (e.g., pharmaceutical composition) , which comprise anti-C5aR1 antigen-binding domain and anti-GM-CSFRα antigen-binding domain.
In some embodiments, the combination of the present application is provided in the form of a composition (e.g., pharmaceutical composition) , which comprise an antibody comprising anti-C5aR1 antigen-binding domain and an antibody comprising anti-GM-CSFRαantigen-binding domain. In some embodiments, the antibody comprising anti-C5aR1 antigen-binding domain is an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1. In some embodiments, the antibody comprising anti-GM-CSFRα antigen-binding domain is an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα.
In some embodiments, the anti-C5aR1 antigen-binding domain comprises a V_H comprising an HC-CDR1 comprising SGYSWH (SEQ ID NO: 1) , an HC-CDR2 comprising YIHSSGDTDYNPSLKR (SEQ ID NO: 2) , and an HC-CDR3 comprising SIGDY (SEQ ID NO: 3) ; and a V_L comprising an LC-CDR1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 11) ; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 12) , and an LC-CDR3 comprising SQSTHVPWT (SEQ ID NO: 13) .
In some embodiments, the anti-C5aR1 antigen-binding domain comprise a V_H comprising an HC-CDR1 comprising SYWMN (SEQ ID NO: 4) ; an HC-CDR2 comprising RIDPX₁DSX₂TRYNQKFED (SEQ ID NO: 49) , wherein X₁ is Y, G or R, and X₂ is E or G; and an HC-CDR3 comprising FVX₁PX₂GX₃FAY (SEQ ID NO: 50) , wherein X₁ is I or W, X₂ is S or V, and X₃ is G or A; and a V_L comprising an LC-CDR1 comprising RSSX₁SPX₂HSNGNTYLH (SEQ ID NO: 51) , wherein X₁ is Q or R, and X₂ is V or L; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 17) , and an LC-CDR3 comprising SQSTX₁VPPT (SEQ ID NO: 52) , wherein X₁ is H or L.
In some embodiments, the anti-C5aR1 antigen-binding domain comprise a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; and a LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 18-19, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the anti-C5aR1 antigen-binding domain comprise: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the anti-C5aR1 antigen-binding domain comprise: (i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or (ii) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69.
In some embodiments, the anti-C5aR1 antigen-binding domain comprise: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23 or 59; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61; or (iv) a V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30 or 66; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66; or (v) a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67; or (vi) a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68; or (vii) a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69.
In some embodiments, the anti-GM-CSFRα antigen-binding domain comprise a V_H comprising an HC-CDR1 comprising ELSIH (SEQ ID NO: 34) ; an HC-CDR2 comprising GFDPEDGETIYAQKX₁QG (SEQ ID NO: 53) , wherein X₁ is S or F; and an HC-CDR3 comprising GRYX₁X₂LX₃X₄X₅YGFDY (SEQ ID NO: 54) , wherein X₁ is T or V, X₂ is E or S, X₃ is A, F or Y, X₄ is T, F or Q, and X₅ is T or N; and a V_L comprising an LC-CDR1 comprising RASQSVSSYLA (SEQ ID NO: 40) ; an LC-CDR2 comprising GASSRAT (SEQ ID NO: 41) , and an LC-CDR3 comprising QQYX₁NWPX₂T (SEQ ID NO: 55) , wherein X₁ is D or N, and X₂ is Y or P.
In some embodiments, the anti-GM-CSFRα antigen-binding domain comprise a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 35-36, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 37-39, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 42-43, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the anti-GM-CSFRα antigen-binding domain comprise: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, the anti-GM-CSFRα antigen-binding domain comprise a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74.
In some embodiments, the anti-GM-CSFRα antigen-binding domain comprise: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a combination comprising (i) an antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: (a) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (b) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (c) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (d) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a combination comprising (i) an antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an antibody or antigen- binding fragment specifically recognizing GM-CSFRα, wherein: the antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a combination comprising (i) an antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a combination comprising (i) an antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an antibody or antigen- binding fragment specifically recognizing GM-CSFRα, wherein: the antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a combination comprising (i) an antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a combination comprising (i) an antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an antibody or antigen- binding fragment specifically recognizing GM-CSFRα, wherein: the antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
Multi-specific (e.g., bispecific) antibodies recognizing C5aR1 and GM-CSFRα
In one aspect, the present application provides a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: (i) a V_H comprising an HC-CDR1 comprising SGYSWH (SEQ ID NO: 1) , an HC-CDR2 comprising YIHSSGDTDYNPSLKR (SEQ ID NO: 2) , and an HC-CDR3 comprising SIGDY (SEQ ID NO: 3) ; and a V_L comprising an LC-CDR1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 11) ; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 12) , and an LC-CDR3 comprising SQSTHVPWT (SEQ ID NO: 13) ; or (ii) a V_H comprising an HC-CDR1 comprising SYWMN (SEQ ID NO: 4) ; an HC-CDR2 comprising RIDPX₁DSX₂TRYNQKFED (SEQ ID NO: 49) , wherein X₁ is Y, G or R, and X₂ is E or G; and an HC-CDR3 comprising FVX₁PX₂GX₃FAY (SEQ ID NO: 50) , wherein X₁ is I or W, X₂ is S or V, and X₃ is G or A; and a V_L comprising an LC-CDR1 comprising RSSX₁SPX₂HSNGNTYLH (SEQ ID NO: 51) , wherein X₁ is Q or R, and X₂ is V or L; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 17) , and an LC-CDR3 comprising SQSTX₁VPPT (SEQ ID NO: 52) , wherein X₁ is H or L.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising ELSIH (SEQ ID NO: 34) ; an HC-CDR2 comprising GFDPEDGETIYAQKX₁QG (SEQ ID NO: 53) , wherein X₁ is S or F; and an HC-CDR3 comprising GRYX₁X₂LX₃X₄X₅YGFDY (SEQ ID NO: 54) , wherein X₁ is T or V, X₂ is E or S, X₃ is A, F or Y, X₄ is T, F or Q, and X₅ is T or N; and a V_L comprising an LC-CDR1 comprising RASQSVSSYLA (SEQ ID NO: 40) ; an LC-CDR2 comprising GASSRAT (SEQ ID NO: 41) , and an LC-CDR3 comprising QQYX₁NWPX₂T (SEQ ID NO: 55) , wherein X₁ is D or N, and X₂ is Y or P.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; and a LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 18-19, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the first antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 35-36, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 37-39, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 42-43, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23 or 59. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20; and a V_L comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23; or a V_H comprising the amino acid sequence of SEQ ID NO: 56, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 56; and a V_L comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 59.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21; and a V_L comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24; or a V_H comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 60.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22; and a V_L comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25; or a V_H comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 61.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30 or 66. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26; and a V_L comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30; or a V_H comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62; and a V_L comprising the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27; and a V_L comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31; or a V_H comprising the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 67.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28; and a V_L comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32; or a V_H comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 68.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69. In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69.
In some embodiments, the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and a V_L comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33; or a V_H comprising the amino acid sequence of SEQ ID NO: 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 69.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73. In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44; and a V_L comprising the amino acid sequence of SEQ ID NO: 47, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47; or a V_H comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 73.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73. In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45; and a V_L comprising the amino acid sequence of SEQ ID NO: 47, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47; or a V_H comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 73.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74. In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46; and a V_L comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48; or a V_H comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 74.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC- CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a multi-specific (e.g., bispecific) antibody comprising a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα, wherein the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, the multi-specific (e.g., bispecific) antibody comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises an IgG1 heavy chain constant region. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises an IgG2 heavy chain constant region. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises an IgG3 heavy chain constant region. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises an IgG4 heavy chain constant region. In some embodiments, the IgG is a human IgG. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 109. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 110. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises a lambda light chain constant region. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 111. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 112. In some embodiments, the multi-specific (e.g., bispecific) antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.
Linkers in Multi-specific (e.g., bispecific) Antibody
In some embodiments, the linkers (L, L1, L2, L3…) may play important roles (such as regulating binding affinity, expression yields, physiochemical properties, activity, pharmacokinetics, etc) in a multi-specific (e.g., bispecific) antibody construct. Linkers may be used to join domains and/or regions of the chimeric heavy chain of the multi-specific (e.g., bispecific) antibody into a contiguous molecule. In some embodiments, the multi-specific (e.g., bispecific) antibody includes at least two linker polypeptides. In some embodiments, the multi-specific (e.g., bispecific) antibody may include such as a flexible linker interconnecting the variable heavy and light chains of an scFv. In some embodiments, the multi-specific (e.g., bispecific) antibody may include additional linkers that connect other binding units to the core structure of the multi-specific (e.g., bispecific) antibody.
An exemplary, non-limiting example of a linker is a polypeptide chain comprising at least 4 amino acid residues. Portions of such linkers may be flexible, hydrophilic and have little or no secondary structure of their own (linker portions or flexible linker portions) . Linkers of at least 4 amino acids may be used to join domains and/or regions that are positioned near to one another after the molecule has assembled. Longer linkers may also be used. In some embodiments, linkers may be about any one of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues. When multiple linkers are used to interconnect portions of the molecule, the linkers may be the same or different (e.g., the same or different length and/or amino acid sequence) .
In some aspects, the polypeptide linker comprises or consists of a Gly-Ser linker. As used herein, the term "Gly-Ser linker" refers to a peptide that consists of glycine and serine residues. An exemplary Gly-Ser linker comprises an amino acid sequence of the formula (Gly₄Ser) _n, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) . A preferred Gly-Ser linker is (Gly₄Ser) ₂ or (Gly₄Ser) ₄. Another exemplary Gly-Ser linker is (Gly₄Ser) ₃. In yet other aspects, two or more Gly-Ser linker are incorporated in series in a polypeptide linker. In some aspects, the polypeptide linker comprises at least a portion of a hinge region (e.g., derived from an IgGl, IgG2, IgG3, or IgG4 molecule) and a series of Gly-Ser amino acid residues (e.g., a Gly-Ser linker such as (Gly₄Ser) _n) .
In some embodiments, the linker comprises both a hinge portion and a linker portion, such as a linker portion comprising a Gly-Ser linker. In other aspects, the linker comprises only a hinge portion or only a linker portion, such as a Gly-Ser linker. In some embodiments, the linkers comprise a Gly-Ser linker portion. In certain aspects, the Gly-Ser linker portion of different linker is the same length, whereas in other aspects, the Gly-Ser linker portion of different linker is different length. When a bispecific antibody comprises an scFv, the heavy and light chains of the scFv may be connected by a flexible linker. In some embodiments, this flexible linker generally does not comprise a hinge portion, but rather, is a Gly-Ser linker or other flexible linker. The length and amino acid sequence of a flexible linker interconnecting domains of an scFv may be selected and optimized.
DVD-Ig format (dual variable domain immunoglobulin) BsAb:
In some embodiments, the multi-specific (e.g., bispecific) antibody is DVD-Ig format bispecific antibody. The DVD-Ig bispecific antibody (DVD-Ig BsAb) is a bispecific, tetravalent IgG-like molecule similar to a conventional IgG, and the DVD-Ig BsAb is unique in that each arm of the molecule contains two variable domains (VDs) (i.e., two antigen-binding domains) for different antigens or to different epitopes on the same antigen. The variable domains in each arm are connected in tandem by separate heavy and light chain linkers, and together they form an inner VD and an outer VD capable of binding to different antigens or to different epitopes on the same antigen. The schematic structure diagram of DVD-Ig BsAb molecule was shown in Fig. 2A. The DVD-Ig bispecific antibody is a homodimer composed of two identical monomers. Each monomer of the DVD-Ig bispecific antibody comprises two polypeptide chains, which are a chimeric heavy chain and a chimeric light chain, respectively. The DVD-Ig technology was described in “Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules. MAbs. 2009 Jul-Aug; 1 (4) : 339-47; DiGiammarino E, et al., Design and generation of DVD-Ig^TMmolecules for dual-specific targeting. Methods Mol Biol. 2012; 899: 145-56” .
In one embodiment of the present application, one antigen-binding domain of DVD-Ig BsAb specifically recognizing C5aR1, and the other antigen-binding domain of DVD-Ig BsAb specifically recognizing GM-CSFRα. In some embodiments, the bispecific antibody recognizes C5aR1 and GM-CSFRα at the same time.
In some embodiments, the chimeric heavy chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_H1-L-V_H2-C_H1, the chimeric light chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_L1-L-V_L2-C_L, wherein V_H1 and V_L1 are a heavy chain variable region and a light chain variable region that specifically bind to one antigen or one epitope in one antigen, respectively; V_H2 and V_L2 are a heavy chain variable region and a light chain variable region that specifically bind to another antigen or another epitope on the same antigen, respectively; L is a linker peptide; C_H1 is a heavy chain constant region C_H1 domain; and C_L is a light chain constant region, wherein V_H1 and V_L1 form an antigen-binding domain (i.e., outer VD) , and V_H2 and V_L2 form another antigen-binding domain (i.e., inner VD) of the DVD-Ig BsAb. In some embodiments, the heavy chain may further comprise Fc region comprising C_H2 and C_H3 domains.
In some embodiments, the two heavy chains of the DVD-Ig BsAb comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_H1-L-V_H2-C_H1, wherein V_H1 is a heavy-chain variable region that specifically binds to the C5aR1; V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; L is a linker; C_H1 is a heavy chain constant region C_H1 domain; wherein the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and the two light chains of the DVD-Ig BsAb comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_L1-L-V_L2-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1; V_L2 is a light chain variable region that specifically binds to GM-CSFRα; L is a linker; and C_L is a light chain constant region. The V_H1 and V_L1 form an antigen-binding domain specifically recognizing C5aR1, the V_H2 and V_L2 form an antigen-binding domain specifically recognizing GM-CSFRα.
In some embodiments, the two heavy chains of the DVD-Ig BsAb comprise polypeptide sequences in the orientation from N-terminus to C-terminus: a V_H1-L-V_H2-C_H1, wherein V_H1 is a heavy-chain variable region that specifically binds to the GM-CSFRα; V_H2 is a heavy chain variable region that specifically binds to C5aR1; L is a linker; C_H1 is a heavy chain constant region C_H1 domain; the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and the two light chains of the DVD-Ig BsAb comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_L1-L-V_L2-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα; V_L2 is a light chain variable region that specifically binds to C5aR1; L is a linker; and C_L is a light chain constant region. The V_H1 and V_L1 form an antigen-binding domain specifically recognizing GM-CSFRα, the V_H2 and V_L2 form an antigen-binding domain specifically recognizing C5aR1.
The linkers, which span from the C-terminus of the outer VD’s heavy and light chain to the N-terminus of the inner VD’s heavy and light chain respectively, connected the two variable domains (VDs) within an arm of the DVD-Ig BsAb. For any given DVD-Ig BsAb, linker design may affect expression yields, physiochemical properties, activity, pharmacokinetics, and efficacy in ways that may be difficult to predict and therefore choice of linker sequence may need to be empirically determined. Three categories of example linkers and their corresponding sequences have been found to work well in DVD-Ig BsAb. Elbow linkers are naturally occurring sequences derived from the linkage between the constant domain 1 and the variable domain of human IgG1. Long and short versions of these linkers, as well as kappa and lambda-specific light chain versions are described. Alternative naturally occurring linker sequences are the hinge-based linkers which are derived from sequences found between the CH1 and CH2 of human IgG1. Glycine/serine linkers are another notable category, which are thought to impart flexibility, pose low immunogenicity risks and have been widely used in the construction of antibody-like molecules (Robinson CR, Sauer RT (1998) Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc Natl Acad Sci U S A 95: 5929–5934) .
In some embodiments, the linker in DVD-Ig BsAb heavy chain comprises the amino acid sequence “ASTKGP” (SEQ ID NO: 114) or the amino acid sequence “ASTKGPSVFPLAP” (SEQ ID NO: 113) .
In some embodiments, the linker in DVD-Ig BsAb light chain comprises the amino acid sequence “TVAAP” (SEQ ID NO: 116) or the amino acid sequence “TVAAPSVFIFPP” (SEQ ID NO: 115) .
In some embodiments, the linkers in DVD-Ig BsAb comprises the amino acid sequence of any of SEQ ID NOs: 113-116, or a or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 113-116.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 75.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 76.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 77, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 77.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 78.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 102.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 103.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 104.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 80.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81.
In some embodiments, the DVD-Ig BsAb comprises the amino acid sequence of any one of SEQ ID NOs: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 82.
In some embodiments, the bispecific antibody comprises the amino acid sequence of any one of SEQ ID NOs: 75-76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 75-76; and the amino acid sequence of any one of SEQ ID NOs: 79-80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79-80.
In some embodiments, the bispecific antibody comprises the amino acid sequence of any one of SEQ ID NOs: 77-78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 77-78; and the amino acid sequence of any one of SEQ ID NOs: 81-82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81-82.
In some embodiments, the bispecific antibody comprises the amino acid sequence of any one of SEQ ID NOs: 101-102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101-102; and the amino acid sequence of any one of SEQ ID NOs: 79-80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79-80.
In some embodiments, the bispecific antibody comprises the amino acid sequence of any one of SEQ ID NOs: 103-104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 103-104; and the amino acid sequence of any one of SEQ ID NOs: 81-82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81-82.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 101; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 101; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 102; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 102; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 103; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 103; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 104; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 104; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
IgG- (scFv) 2 format BsAb:
In some embodiments, the multi-specific (e.g., bispecific) antibody is IgG- (scFv) ₂ format bispecific antibody. IgG- (scFv) ₂ BsAb is a tetravalent IgG-like molecule which contains a full-length IgG-scFv fusion, in which, a single-chain Fv (scFv) antibody fragment of specificity for one antigen is genetically fused to the C-terminus of a conventional IgG of specificity for a different antigen. Wherein scFv fragment and Fv domain in conventional IgG form antigen-binding domains recognizing different antigens or different epitopes on the same antigen, respectively. In some embodiments, the scFv is fused to the C-terminus of a conventional IgG C_H3 domain via a peptide linker (L1) . In some embodiments, the schematic structure diagram of IgG- (scFv) ₂ BsAb molecule was shown in Fig. 2B. The technology of IgG- (scFv) ₂ format was described in “Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15 (2) : 159-63” .
In one embodiment of the present application, one antigen-binding domain of IgG- (scFv) ₂ BsAb specifically recognizing C5aR1, and the other antigen-binding domain of IgG- (scFv) ₂ BsAb specifically recognizing GM-CSFRα. In some embodiments, the bispecific antibody recognizes C5aR1 and GM-CSFRα at the same time.
In some embodiments, the IgG- (scFv) ₂ bispecific antibody is a homodimer composed of two identical monomers, wherein each monomer contains two antigen-binding domains recognizing different antigens or different epitopes on the same antigen and comprises two polypeptide chains, which are a chimeric heavy chain and a light chain pair.
In some embodiments, the chimeric heavy chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_H1-C_H1-Fc-L1-V_H2- L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, the light chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_L1-C_L, wherein V_H1 and V_L1 are a heavy chain variable region and a light chain variable region that specifically bind to one antigen, respectively; V_H2 and V_L2 are a heavy chain variable region and a light chain variable region that specifically bind to different antigen or different epitope on the same antigen; L1 and L2 are peptide linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc comprises C_H2 and C_H3 domains; and C_L is a light chain constant region, wherein V_H1 and V_L1 form an antigen-binding domain, and V_H2 and V_L2 form another antigen-binding domain of the IgG- (scFv) ₂.
In some embodiments, the chimeric heavy chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, wherein V_H1 is a heavy chain variable region that specifically binds to C5aR1; V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; V_L2 is a light chain variable region that specifically binds to GM-CSFRα; L1 and L2 are linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc region comprises C_H2 and C_H3 domains; and the light chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1, and C_L is a light chain constant region, wherein V_H1 and V_L1 form an antigen-binding domain specifically recognizing C5aR1, and V_H2 and V_L2 form another antigen-binding domain specifically recognizing GM-CSFRα of the IgG- (scFv) ₂.
In some embodiments, the chimeric heavy chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, wherein V_H1 is a heavy chain variable region that specifically binds to GM-CSFRα; V_H2 is a heavy chain variable region that specifically binds to C5aR1; V_L2 is a light chain variable region that specifically binds to C5aR1; L1 and L2 are linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc region comprises C_H2 and C_H3 domains; and the light chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα, and C_L is a light chain constant region, wherein V_H1 and V_L1 form an antigen-binding domain specifically recognizing GM-CSFRα, and V_H2 and V_L2 form another antigen-binding domain specifically recognizing C5aR1 of the IgG- (scFv) ₂.
In some embodiments according to any one of the IgG- (scFv) ₂ bispecific antibodies described herein, the single-chain variable fragment (scFv) specifically recognizing C5aR1 or GM-CSFRα comprises engineered cysteine mutations. In some embodiments, a disulfide-stabilized bispecific antibody was obtained by introducing two cysteine mutations at the V_H and V_L interface.
In some embodiments, the linker (L2) between V_H and V_L also affect the stability and production of the scFv and the final BsAb. The most commonly used linker is (Gly₄Ser) _n (n=3-4) .
In some embodiments, the linker (L1) between the scFv and IgG C_H3 domain also affects significantly the stability and solution behavior of the IgG-scFv constructs (Schaeter G, et al (2011) A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies. Cancer Cell 20: 472–486) . A flexible peptide, usually in the form of (Gly₄Ser) _n (n=0-4) , is often used as the linker between the scFv and its IgG fusion partner, when constructing the IgG- (scFv) ₂ BsAb molecule. The optimal linker length may vary between 0 and 20 amino acids, among different IgG-scFv fusion partners, thus needs to be experimentally tested. In some embodiments, an example of the linker (L1) between Fc region and scFv comprises the amino acid sequence of GGGGSGGGGTGGGGS (SEQ ID NO: 117) .
In some embodiments, the linker L1 in IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 117, or a or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 117.
In some embodiments, the linker L2 in IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 118, or a or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 118.
In some embodiments, the chimeric heavy chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, the light chains of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_L1-C_L.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 83.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 84, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 84.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 85, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 85.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 86, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 86.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 83; and the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 84, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 84; and the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 85, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 85; and the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87.
In some embodiments, the IgG- (scFv) ₂ BsAb comprises the amino acid sequence of SEQ ID NO: 86, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 86; and the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88.
Hetero, H CrossMab format BsAb:
In some embodiments, the multi-specific (e.g., bispecific) antibody is Hetero, H CrossMab format bispecific antibody. The major challenge in the generation of bispecific IgG antibody is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobin domain crossover, known as CrossMab technology, which is based on the crossover of the antibody domain within one Fab-arm of a bispecific antibody, and can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology. Hetero, H CrossMab BsAb is a heterodimer consists of two monomers recognizing different antigens or different epitopes on the same antigen, respectively. The complete Fab domain can be exchanged in the CrossMab^Fab, or either only the variable domains is exchanged in the CrossMab ^VH-VL, or the constant domain of the Fab arm is exchanged in the CrossMab^CH1-CL. The Hetero H, CrossMab format bispecific antibody was also described in Klein C, et al. The use of CrossMAb technology for the generation of bi-and multi-specific antibodies. MAbs. 2016 Aug-Sep; 8 (6) : 1010-20. The application is specially described by using a CrossMab^CH1-CLstructure (Hetero, H CrossMab) . The schematic structural diagram of the CrossMab BsAb molecules was shown in Fig. 2C.
In some embodiments, The KiH technology that is simultaneously introducing two Cys residue mutations forming the stabilized disulfide bridge (e.g., S354 C on the "Knob " side and Y349 C on the "Hole " side) in the Fc domain. In some embodiments, through the KiH method, the 366 threonine (T) residue in an antibody C_H3 domain is replaced with tryptophan (W) to form a "knob " structure, and the 366 threonine (T) residue in the other matched antibody C_H3 domain is replaced with serine (S) , the 368 leucine (L) residue is replaced with alanine (A) , the 407 tyrosine (Y) residue is replaced with valine (V) to form a "hole" structure, and heterologous heavy chain dimerization is promoted by means of the reduction of the spatial steric hindrance effect after mutation and the covalent disulfide bond formed in the hinge region, wherein the numbering is in accordance with the EU numbering.
In some embodiments, the Hetero, H CrossMab BsAb is a bivalent IgG like bispecific antibody with C_H1 and C_L crossover and KiH mutation in the Fc region. In some embodiments, the Fc is derived from a wild-type human IgG1.
In one embodiment of the present application, one antigen-binding domain of Hetero, H CrossMab BsAb specifically recognizing C5aR1, and the other antigen-binding domain of Hetero, H CrossMab BsAb specifically recognizing GM-CSFRα. In some embodiments, the bispecific antibody recognizes C5aR1 and GM-CSFRα at the same time.
In some embodiments, the Hetero, H CrossMab bispecific antibody is a heterodimer composed of two different monomers, each monomer comprises two polypeptide chains, wherein the first monomer contains an antigen-binding domain that specifically binds to one antigen and comprises a first heavy chain and a first light chain, and the second monomer contains an antigen-binding domain that specifically binds to another antigen or different epitope on the same antigen and comprises a second heavy chain and a second light chain. In some embodiments, the first heavy chain of the bispecific antibody comprises polypeptide sequences in the orientation from N-terminus to C-terminus: V_H1-C_H1; the first light chain of the bispecific antibody comprise polypeptide sequences in the orientation from N-terminus to C-terminus: V_L1-C_L; the second heavy chain of the bispecific antibody comprises polypeptide sequences in the orientation from N-terminus to C-terminus: V_H2-C_L; the second light chain of the bispecific antibody comprises polypeptide sequences in the orientation from N-terminus to C-terminus: V_L2-C_H1; wherein V_H1 and V_L1 are a heavy chain variable region and a light chain variable region that specifically bind to one antigen, respectively; V_H2 and V_L2 are a heavy chain variable region and a light chain variable region that specifically bind to different antigen or different epitope on the same antigen; C_H1 is a heavy chain constant region C_H1 domain; and C_L is a light chain constant region, wherein V_H1 and V_L1 form an antigen-binding domain, and V_H2 and V_L2 form another antigen-binding domain of the Hetero, H CrossMab BsAb. In some embodiments, the first heavy chain and the second heavy chain may further comprise Fc region comprising C_H2 and C_H3 domains.
In some embodiments, the first heavy chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1, wherein V_H1 is a heavy chain variable region specifically binding to C5aR1; C_H1 is a heavy chain constant region C_H1 domain; the first heavy chain may further comprise Fc region comprising C_H2 and C_H3 domains; the first light chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1, and C_L is a light chain constant region; the second heavy chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H2-C_L, wherein V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; C_L is a light chain constant region; the second heavy chain may further comprise Fc region comprising C_H2 and C_H3 domains; and the second light chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L2-C_H1, wherein V_L2 is a light-chain variable region that specifically binds to GM-CSFRα, and C_H1 is a heavy-chain constant region C_H1 domain.
In some embodiments, the first heavy chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1, wherein V_H1 is a heavy chain variable region specifically binding to GM-CSFRα; C_H1 is a heavy chain constant region C_H1 domain; the first heavy chain may further comprise Fc region comprising C_H2 and C_H3 domains; the first light chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα, and C_L is a light chain constant region; the second heavy chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H2-C_L, wherein V_H2 is a heavy chain variable region that specifically binds to C5aR1; C_L is a light chain constant region; the second heavy chain may further comprise Fc region comprising C_H2 and C_H3 domains; and the second light chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L2-C_H1, wherein V_L2 is a light-chain variable region that specifically binds to C5aR1, and C_H1 is a heavy-chain constant region C_H1 domain.
In some embodiments, one or more amino acid residues in the C_H3 domain of the first heavy chain are substituted with one or more amino acid residues with a larger side chain to form "knob " , and one or more amino acid residues in the C_H3 domain of the second heavy chain is substituted with one or more amino acid residues with a smaller side chain to form a "hole " . In another embodiments, one or more amino acid residues in the C_H3 domain of the second heavy chain is substituted with one or more amino acid residues with a larger side chain to form "knob " , and one or more amino acid residues in the C_H3 domain of the first heavy chain is substituted with one or more amino acid residues with a smaller side chain to form a " hole" . In some embodiments, the Fc knob comprise the amnio acid substitution T366W and the Fc hole comprise the amino acid substitution T366S, L368A and Y407V. In some embodiments, the Fc knob further comprise the amino acid substitution S354C, the Fc hole further comprise the amino acid substitution Y349C, wherein the numbering is in accordance with the EU numbering.
In some embodiments, the Hetero, H CrossMab BsAb comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 105; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 106, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 106; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98.
In some embodiments, the Hetero, H CrossMab BsAb comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 107; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 108; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, the Hetero, H CrossMab BsAb comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 89, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 89; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 90, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 90; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98.
In some embodiments, the Hetero, H CrossMab BsAb comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 91, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 91; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 92, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 92; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98.
In some embodiments, the Hetero, H CrossMab BsAb comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 93; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 94; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, the Hetero, H CrossMab BsAb comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 95, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 95; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 96, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 96; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
Exemplary antibody sequences are shown in Tables 2-15, wherein the CDR numbering is according to the Kabat numbering. Those skilled in the art will recognize that many algorithms are known for prediction of CDR positions and for delimitation of antibody heavy chain and light chain variable regions. Anti-C5aR1, anti-GM-CSFRα antibodies or bispecific antibodies that specifically recognizing C5aR1 and GM-CSFRα comprising CDRs, V_H and/or V_L sequences from antibodies described herein, but based on prediction algorithms other than those exemplified in the tables below, are within the scope of this application. The anti-C5aR1 antibody sequences can be selected from those disclosed in International application No. PCT/US2022/082260, which was incorporated herein in its entirety. The anti-GM-CSFRα antibody sequences can be selected from those disclosed in International Publication No. WO2020/108423 A1, which was incorporated herein in its entirety.
Table 2 Exemplary anti-C5aR1 antibody CDR sequences
Table 3 Exemplary anti-C5aR1 antibody V_H and V_L sequences
Table 4 Exemplary anti-C5aR1 antibody V_H and V_L Cys mutation sequences
Table 5 Exemplary anti-GM-CSFRα antibody CDR sequences
Table 6 Exemplary anti-GM-CSFRα antibody V_H and V_L sequences
Table 7 Exemplary anti-GM-CSFRα antibody V_H and V_L Cys mutation sequences
Table 8 Exemplary bispecific antibodies sequences of DVD-Ig format
Table 9 Exemplary bispecific antibodies sequences of DVD-Ig format
Table 10 Exemplary bispecific antibodies sequences of IgG- (scFv) 2 format
Table 11 Exemplary bispecific antibodies sequences of Hetero, H CrossMab format
Table 12 Exemplary bispecific antibodies sequences of Hetero, H CrossMab format
Table 13 Exemplary sequence
Table 14 Exemplary linker in DVD-Ig format BsAb
Table 15 Exemplary linker in IgG- (scFv) 2 format BsAb
Binding affinity
Binding affinity can be indicated by Kd, Koff, Kon, or Ka. The term “Koff” , as used herein, is intended to refer to the off-rate constant for dissociation of an antigen-binding domain from the antigen-binding domain/antigen complex, as determined from a kinetic selection set up. The term “Kon” , as used herein, is intended to refer to the on-rate constant for association of an antibody to the antigen to form the antigen-binding domain/antigen complex. The term dissociation constant “Kd” , as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antigen-binding domains present in a solution of antibody molecules at equilibrium, and is equal to Koff/Kon. The measurement of Kd presupposes that all binding agents are in solution. In the case where the antigen-binding domain is tethered to a cell wall, e.g., in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives a good approximation of Kd. The affinity constant, Ka, is the inverse of the dissociation constant, Kd. In some embodiments, the antigen-binding domain is comprised within an antigen-binding protein.
The dissociation constant (Kd) is used as an indicator showing affinity of antigen-binding domain moieties to antigens. For example, easy analysis is possible by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using Biacore (made by Amersham Biosciences) , analysis of biomolecular interactions by surface plasmon resonance, according to the user's manual and attached kit. The Kd value that can be derived using these methods is expressed in units of M. An antibody that specifically binds to a target may have a Kd of, for example, ≤ 10^-7 M, ≤ 10^-8 M, ≤ 10^-9 M, ≤ 10^-10 M, ≤ 10^-11 M, ≤ 10^-12 M, or ≤ 10^-13 M.
Binding specificity of the antibody can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans.
In some embodiments, the antigen-binding domain specifically recognizing C5aR1 specifically binds to a target C5aR1 with a Kd of about 10^-7M to about 10^-13M (such as about 10^-7M to about 10^-13M, about 10^-8M to about 10^-13M, about 10^-9M to about 10^-13M, or about 10^- ¹⁰ M to about 10^-12 M) . Thus in some embodiments, the Kd of the binding between the antigen-binding domain specifically recognizing C5aR1 and C5aR1, is about 10^-7M to about 10^-13M, about 1×10^-7M to about 5×10^-13M, about 10^-7M to about 10^-12M, about 10^-7M to about 10^-11M, about 10^-7M to about 10^-10M, about 10^-7M to about 10^-9M, about 10^-8M to about 10^-13M, about 1×10^-8M to about 5×10^-13M, about 10^-8M to about 10^-12M, about 10^-8M to about 10^-11M, about 10^-8M to about 10^-10M, about 10^-8M to about 10^-9M, about 5×10^-9M to about 1×10^-13M, about 5×10^-9M to about 1×10^-12M, about 5×10^-9M to about 1×10^-11M, about 5×10^-9M to about 1×10^-10M, about 10^-9M to about 10^-13M, about 10^-9M to about 10^-12M, about 10^-9M to about 10^-11M, about 10^-9M to about 10^-10M, about 5×10^-10M to about 1×10^-13M, about 5×10^-10M to about 1×10^-12M, about 5×10^-10M to about 1×10^-11M, about 10^-10M to about10^-13M, about 1×10^-10M to about 5×10^-13M, about 1×10^-10M to about 1×10^-12M, about 1×10^-10M to about 5×10^-12M, about 1×10^-10M to about 1×10^-11M, about 10^-11M to about 10^-13M, about 1×10^-11M to about 5×10^-13M, about 10^-11M to about 10^-12M, or about 10^-12M to about 10^-13M. In some embodiments, the Kd of the binding between the antigen-binding domain specifically recognizing C5aR1 and a C5aR1 is about 10^-7M to about 10^-13M.
In some embodiments, the Kd of the binding between the antigen-binding domain specifically recognizing C5aR1 and a non-target is higher than the Kd of the binding between the antigen-binding domain specifically recognizing C5aR1 and the target, and is herein referred to in some embodiments as the binding affinity of the antigen-binding domain specifically recognizing C5aR1 to the target (e.g., C5aR1) is higher than that to a non-target. In some embodiments, the non-target is an antigen that is not C5aR1. In some embodiments, the Kd of the binding between antigen-binding domain specifically recognizing C5aR1 and a non-C5aR1 target can be at least about 10 times, such as about 10-100 times, about 100-1000 times, about 10³-10⁴ times, about 10⁴-10⁵ times, about 10⁵-10⁶ times, about 10⁶-10⁷ times, about 10⁷-10⁸ times, about 10⁸-10⁹ times, about 10⁹-10¹⁰ times, about 10¹⁰-10¹¹ times, or about 10¹¹-10¹² times of the Kd of the binding between the antigen-binding domain specifically recognizing C5aR1 and a target C5aR1.
In some embodiments, the antigen-binding domain specifically recognizing GM-CSFRα specifically binds to a target GM-CSFRα with a Kd of about 10^-7M to about 10^-13M (such as about 10^-7M to about 10^-13M, about 10^-8M to about 10^-13M, about 10^-9M to about 10^- ¹³M, or about 10^-10M to about 10^-12M) . Thus in some embodiments, the Kd of the binding between the antigen-binding domain specifically recognizing GM-CSFRα and GM-CSFRα, is about 10^-7M to about 10^-13M, about 1×10^-7M to about 5×10^-13M, about 10^-7M to about 10^-12M, about 10^-7 M to about 10^-11M, about 10^-7M to about 10^-10M, about 10^-7M to about 10^-9M, about 10^-8 M to about 10^-13M, about 1×10^-8M to about 5×10^-13M, about 10^-8M to about 10^-12M, about 10^-8M to about 10^-11M, about 10^-8M to about 10^-10M, about 10^-8M to about 10^-9M, about 5×10^-9M to about 1×10^-13M, about 5×10^-9M to about 1×10^-12M, about 5×10^-9M to about 1×10^-11M, about 5×10^-9M to about 1×10^-10M, about 10^-9M to about 10^-13M, about 10^-9M to about 10^-12M, about 10^-9M to about 10^-11M, about 10^-9M to about 10^-10M, about 5×10^-10M to about 1×10^-13M, about 5×10^-10M to about 1×10^-12M, about 5×10^-10M to about 1×10^-11M, about 10^-10M to about10^-13M, about 1×10^-10M to about 5×10^-13M, about 1×10^-10M to about 1×10^-12M, about 1×10^-10M to about 5×10^-12M, about 1×10^-10M to about 1×10^-11M, about 10^-11M to about 10^-13M, about 1×10^-11M to about 5×10^-13M, about 10^-11M to about 10^-12M, or about 10^-12M to about 10^-13M. In some embodiments, the Kd of the binding between the antigen-binding domain specifically recognizing GM-CSFRα and a GM-CSFRα is about 10^- ⁷M to about 10^-13M.
In some embodiments, the Kd of the binding between the antigen-binding domain specifically recognizing GM-CSFRα and a non-target is higher than the Kd of the binding between the antigen-binding domain specifically recognizing GM-CSFRα and the target, and is herein referred to in some embodiments as the binding affinity of the antigen-binding domain specifically recognizing GM-CSFRα to the target (e.g., GM-CSFRα) is higher than that to a non-target. In some embodiments, the non-target is an antigen that is not GM-CSFRα. In some embodiments, the Kd of the binding between antigen-binding domain specifically recognizing GM-CSFRα and a non-GM-CSFRα target can be at least about 10 times, such as about 10-100 times, about 100-1000 times, about 10³-10⁴ times, about 10⁴-10⁵ times, about 10⁵-10⁶ times, about 10⁶-10⁷ times, about 10⁷-10⁸ times, about 10⁸-10⁹ times, about 10⁹-10¹⁰ times, about 10¹⁰- 10¹¹ times, or about 10¹¹-10¹² times of the Kd of the binding between the antigen-binding domain specifically recognizing GM-CSFRα and a target GM-CSFRα.
Nucleic Acids, Vectors and Host cells
Nucleic acid molecules encoding the anti-C5aR1 antibodies or anti-GM-CSFRαantibodies, and the multi-specific (e.g., bispecific) antibodies specifically recognizing C5aR1 and GM-CSFRα are also contemplated. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding a full-length anti-C5aR1 antibody or anti-GM-CSFRαantibody, including any of the full-length anti-C5aR1 or anti-GM-CSFRα antibodies described herein. In some embodiments, the nucleic acid (or a set of nucleic acids) encoding the antibody, antigen-binding fragment or multi-specific (e.g., bispecific) antibody described herein may further comprises a nucleic acid sequence encoding a peptide tag (such as protein purification tag, e.g., His-tag, HA tag) .
Also contemplated here are isolated host cells comprising an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody that specifically recognizing C5aR1 and GM-CSFRα, an isolated nucleic acid encoding the polypeptide components of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody that specifically recognizing C5aR1 and GM-CSFRα, or a vector comprising a nucleic acid encoding the polypeptide components of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody that specifically recognizing C5aR1 and GM-CSFRα described herein.
The present application also includes variants to these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibodies, antigen-binding fragments or multi-specific (e.g., bispecific) antibodies of the present application under at least moderately stringent hybridization conditions.
The present application also provides vectors in which a nucleic acid of the present application is inserted.
In brief summary, the expression of an antibody, antigen-binding fragment or multi-specific (e.g., bispecific) antibody by a natural or synthetic nucleic acid encoding the antibody, antigen-binding fragment or multi-specific (e.g., bispecific) antibody can be achieved by inserting the nucleic acid into an appropriate expression vector, such that the nucleic acid is operably linked to 5’ and 3’ regulatory elements, including for example a promoter (e.g., a lymphocyte-specific promoter) and a 3’ untranslated region (UTR) . The vectors can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
The nucleic acids of the present application may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346; 5,580,859; 5,589,466, incorporated by reference herein in their entireties. In some embodiments, the application provides a gene therapy vector.
The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals. Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) .
A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Factor-1α (EF-1α) . However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the application should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the application. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
In some embodiments, the expression of the anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRαis inducible. In some embodiments, a nucleic acid sequence encoding the anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is operably linked to an inducible promoter, including any inducible promoter described herein.
Inducible promoters
The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Exemplary inducible promoter systems for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90: 5603-5607) , synthetic ligand-regulated elements (see, e.g., Spencer, D. M. et al 1993) Science 262: 1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et al. (1993) Biochemistry 32: 10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1014-10153) . Further exemplary inducible promoter systems for use in in vitro or in vivo mammalian systems are reviewed in Gingrich et al. (1998) Annual Rev. Neurosci 21: 377-405. In some embodiments, the inducible promoter system for use to express the anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRαantibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is the Tet system. In some embodiments, the inducible promoter system for use to express the anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is the lac repressor system from E. coli.
An exemplary inducible promoter system for use in the present application is the Tet system. Such systems are based on the Tet system described by Gossen et al. (1993) . In an exemplary embodiment, a polynucleotide of interest is under the control of a promoter that comprises one or more Tet operator (TetO) sites. In the inactive state, Tet repressor (TetR) will bind to the TetO sites and repress transcription from the promoter. In the active state, e.g., in the presence of an inducing agent such as tetracycline (Tc) , anhydrotetracycline, doxycycline (Dox) , or an active analog thereof, the inducing agent causes release of TetR from TetO, thereby allowing transcription to take place. Doxycycline is a member of the tetracycline family of antibiotics having the chemical name of 1-dimethylamino-2, 4a, 5, 7, 12-pentahydroxy-11-methyl-4, 6-dioxo-1, 4a, 11, 11a, 12, 12a-hexahydrotetracene-3-carboxamide.
In one embodiment, a TetR is codon-optimized for expression in mammalian cells, e.g., murine or human cells. Most amino acids are encoded by more than one codon due to the degeneracy of the genetic code, allowing for substantial variations in the nucleotide sequence of a given nucleic acid without any alteration in the amino acid sequence encoded by the nucleic acid. However, many organisms display differences in codon usage, also known as “codon bias” (i.e., bias for use of a particular codon (s) for a given amino acid) . Codon bias often correlates with the presence of a predominant species of tRNA for a particular codon, which in turn increases efficiency of mRNA translation. Accordingly, a coding sequence derived from a particular organism (e.g., a prokaryote) may be tailored for improved expression in a different organism (e.g., a eukaryote) through codon optimization.
Other specific variations of the Tet system include the following “Tet-Off” and “Tet-On” systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In that system, a tetracycline-controlled transactivator protein (tTA) , which is composed of TetR fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target nucleic acid that is under transcriptional control of a tetracycline-responsive promoter element (TRE) . The TRE is made up of TetO sequence concatamers fused to a promoter (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter) . In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.
Conversely, in the Tet-On system, transcription is active in the presence of Tc or Dox. The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain. However, a four amino acid change in the TetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of Dox. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.
Another inducible promoter system is the lac repressor system from E. coli (See Brown et al., Cell 49: 603-612 (1987) ) . The lac repressor system functions by regulating transcription of a polynucleotide of interest operably linked to a promoter comprising the lac operator (lacO) . The lac repressor (lacR) binds to LacO, thus preventing transcription of the polynucleotide of interest. Expression of the polynucleotide of interest is induced by a suitable inducing agent, e.g., isopropyl-β-D-thiogalactopyranoside (IPTG) .
In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tel et al., 2000 FEBS Letters 479: 79-82) . Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
In some embodiments, there is provided nucleic acid encoding an anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα according to any of the antigen-binding domain, antibody or multi-specific (e.g., bispecific) antibody described herein. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the antibody, antigen-binding domain, antigen-binding fragment or multi-specific (e.g., bispecific) antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in separate vectors. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all of the nucleic acid sequences are contained in the same vector. Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses) .
Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) . In some embodiments, the introduction of a polynucleotide into a host cell is carried out by calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) .
In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo) . In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present application, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the application.
Preparation of antibody or antigen-binding fragment
In some embodiments, the antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment that specifically recognizes C5aR1 or GM-CSFRα) is a monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment or the multi-specific (e.g., bispecific) antibody are derived from a monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment or the multi-specific (e.g., bispecific) antibody comprises V_H and V_L domains, or variants thereof, from the monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment or the multi-specific (e.g., bispecific) antibody further comprises C_H1 and C_L domains, or variants thereof, from the monoclonal antibody. Monoclonal antibodies can be prepared, e.g., using known methods in the art, including hybridoma methods, yeast display, phage display methods, or using recombinant DNA methods. Additionally, exemplary yeast display and phage display methods are described herein and in the Examples below.
In a hybridoma method, a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent can include a polypeptide or a fusion protein of the protein of interest. Generally, peripheral blood lymphocytes ( “PBLs” ) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) , the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ( “HAT medium” ) , which prevents the growth of HGPRT-deficient cells.
In some embodiments, the immortalized cell lines fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies.
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. The binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) . Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980) .
After the desired hybridoma cells are identified, the clones can be sub cloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the sub clones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
In some embodiments, according to any of the antigen-binding proteins or bispecific antibodies described herein, the antigen-binding protein or bispecific antibody comprises sequences from a clone selected from an antibody library (such as a phage library presenting scFv or Fab fragments) . The clone may be identified by screening combinatorial libraries for antibody fragments with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991) ; Marks et al., J. Mol. Biol. 222: 581-597 (1992) ; Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, N.J., 2003) ; Sidhu et al., J. Mol. Biol. 338 (2) : 299-310 (2004) ; Lee et al., J. Mol. Biol. 340 (5) : 1073-1093 (2004) ; Fellouse, Proc. Natl. Acad. Sci. USA 101 (34) : 12467-12472 (2004) ; and Lee et al., J. Immunol. Methods 284 (1-2) : 119-132 (2004) .
In certain phage display methods, repertoires of V_H and V_L genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994) . Phages typically display antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993) . Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992) . Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
The anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα can be prepared using phage display to screen libraries for antigen-binding moieties specific to the target antigen (such as C5aR1 and GM-CSFRα) . The library can be a human scFv phage display library having a diversity of at least 1 × 10⁹ (such as at least about any of 1 × 10⁹, 2.5 × 10⁹, 5 × 10⁹, 7.5 × 10⁹, 1 × 10¹⁰, 2.5 × 10¹⁰, 5 × 10¹⁰, 7.5 × 10¹⁰, or 1 × 10¹¹) unique human antibody fragments. In some embodiments, the library is ahuman library constructed from DNA extracted from human PMBCs and spleens from healthy donors, encompassing all human heavy and light chain subfamilies. In some embodiments, the library is ahuman library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library, wherein heavy chain CDR3 is completely randomized, with all amino acids (with the exception of cysteine) equally likely to be present at any given position (see, e.g., Hoet, R.M. et al., Nat. Biotechnol. 23 (3) : 344-348, 2005) . In some embodiments, the heavy chain CDR3 of the semi-synthetic human library has a length from about 5 to about 24 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some embodiments, the library is a fully-synthetic phage display library. In some embodiments, the library is a non-human phage display library.
Phage clones that bind to the target antigen (such as C5aR1 or GM-CSFRα) with high affinity can be selected by iterative binding of phage to the target antigen, which is bound to a solid support (such as, for example, beads for solution panning or mammalian cells for cell panning) , followed by removal of non-bound phage and by elution of specifically bound phage. The bound phage clones are then eluted and used to infect an appropriate host cell, such as E. coli XL1-Blue, for expression and purification. The panning can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds with solution panning, cell panning, or a combination of both, to enrich for phage clones binding specifically to the target antigen. Enriched phage clones can be tested for specific binding to the target antigen by any methods known in the art, including for example ELISA and FACS.
Monoclonal antibodies and multi-specific (e.g., bispecific) antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the application can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) . Hybridoma cells as described above or antigen-specific phage clones of the application can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy-and light-chain constant domains and/or framework regions in place of the homologous non-human sequences (U.S. Patent No. 4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the application, or can be substituted for the variable domains of one antigen-combining site of an antibody of the application to create a chimeric bivalent antibody. In some embodiments, additional variable domains targeting a different epitope or antigen can be included to generate a chimeric bispecific antibody.
The antibody or antigen-binding fragment can be monovalent antibodies. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using any method known in the art.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, C_H2, and C_H3 regions. In some embodiments, the first heavy-chain constant region (C_H1) containing the site necessary for light-chain binding is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. In some embodiments, antibody variable domains targeting different epitopes or different antigens can be fused to immunoglobulin constant-domain sequences to generate a chimeric multi-specific (e.g., bispecific) antibody.
Human and Humanized Antibodies
The antibody, antigen-binding fragment or multi-specific (e.g., bispecific) antibody can comprise humanized antibody moieties or human antibody moieties. Humanized forms of non-human (e.g., murine) antibody moieties are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F (ab’) ₂, scFv, or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from non-human immunoglobulin. Humanized antibody moieties include human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibody moieties can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody can comprise substantially at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
Generally, a humanized antibody or humanized antibody moiety has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. According to some embodiments, humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ) , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibody moieties are antibody moieties (U.S. Patent No. 4,816,567) , wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibody moieties are typically human antibody moieties in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
As an alternative to humanization, human antibody moieties can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., PNAS USA, 90: 2551 (1993) ; Jakobovits et al., Nature, 362: 255-258 (1993) ; Bruggemann et al., Year in Immunol., 7: 33 (1993) ; U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97/17852. Alternatively, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio/Technology, 10: 779-783 (1992) ; Lonberg et al., Nature, 368: 856-859 (1994) ; Morrison, Nature, 368: 812-813 (1994) ; Fishwild et al., Nature Biotechnology, 14: 845-851 (1996) ; Neuberger, Nature Biotechnology, 14: 826 (1996) ; Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995) .
Human antibodies or human antibody moieties may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) . The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 (1) : 86-95 (1991) .
Antibody Variants of Anti-C5aR1, anti-GM-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα
In some embodiments, amino acid sequences of the antibody variants of anti-C5aR1 antigen-binding domain, anti-GMR-CSFRα antigen-binding domain, anti-C5aR1 antibody, anti-GMR-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antigen-binding domain or antigen-binding fragment. Amino acid sequences of an antigen-binding entity variants may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding entity, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antigen-binding entity. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In some embodiments, variants of the antibody, antigen-binding fragment, or multi-specific (e.g., bispecific) antibody having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., improved bioactivity, retained/improved antigen binding, decreased immunogenicity. In some embodiments, the amino acid substitutions described herein are limited to “exemplary substitutions” shown in Table A of this application. In some embodiments, the amino acid substitutions are limited to “preferred substitutions” shown in Table A of this application.
Conservative substitutions are shown in Table A below
Amino acids may be grouped into different classes according to common side-chain properties:
a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
c. acidic: Asp, Glu;
d. basic: His, Lys, Arg;
e. residues that influence chain orientation: Gly, Pro;
f. aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g., bioactivity based on RBC lysis inhibition assay or binding affinity) . Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve bioactivity based on TF-1 cell proliferation assay or antibody affinity. Such alterations may be made in HVR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) , and/or specificity determining residues (SDRs) , with the resulting variant V_H and V_L being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001) . )
In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) . A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In some embodiments of the variant V_H and V_L sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., Ala or Glu) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations to demonstrate functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antigen-binding moiety with an N-terminal methionyl residue. Other insertional variants of the antigen-binding moiety include the fusion to the N-or C-terminus of the antigen-binding moiety to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antigen-binding moiety.
Fc Region Variants
In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody (e.g., a full-length anti-C5aR1, anti-GM-CSFRα antibody, multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα, anti-C5aR1, anti-GM-CSFRα, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and Fc fusion protein) provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs) . In some embodiments, the Fc region variant has decreased ADCC effector function. There are many examples of changes or mutations to Fc sequences that can alter effector function. For example, WO 00/42072 and Shields et al. J Biol. Chem. 9 (2) : 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell (e.g., an infected cell) , whose membrane-surface antigens have been bound by specific antigen-binding moieties (e.g., an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) . The typical ADCC involves activation of NK cells by antibodies. An NK cell expresses CD16 which is an Fc receptor. This receptor recognizes, and binds to, the Fc portion of an antibody bound to the surface of a target cell. The most common Fc receptor on the surface of an NK cell is called CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of an antibody results in NK cell activation, release of cytolytic granules and consequent target cell apoptosis.
In some embodiments, the application contemplates an monoclonal antibody or multi-specific (e.g., bispecific) antibody variant (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα variant) comprising an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity) , but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) . Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985) ; U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987) ) . Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI^TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96^TM non-radioactive cytotoxicity assay (Promega, Madison, Wis. ) . Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998) . C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) ; Cragg, M.S. et al., Blood 101: 1045-1052 (2003) ; and Cragg, M.S. and M. J. Glennie, Blood 103: 2738-2743 (2004) ) . FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18 (12) : 1759-1769 (2006) ) .
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056) . Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581) .
Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) . )
In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) opsonization, e.g. such as described in Moore et al., MAbs. 2 (2) : 181–189 (2010) .
In some embodiments, there is provided an anti-C5aR1 antibody, anti-GM-CSFRαantibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variant comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn) . Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et al. ) . Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826) .
See also Duncan &Winter, Nature 322: 738-40 (1988) ; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
Anti-C5aR1 antibodies, anti-GM-CSFRα antibodies or multi-specific (e.g., bispecific) antibodies specifically recognizing C5aR1 and GM-CSFRα (such as full-length anti-C5aR1 antibodies, anti-GM-CSFRα antibodies, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) comprising any of the Fc variants described herein, or combinations thereof, are contemplated.
Glycosylation Variants
In some embodiments, an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) provided herein is altered to increase or decrease the extent to which the anti-C5aR1 antibody, anti-GM-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is glycosylated. Addition or deletion of glycosylation sites to an anti-C5aR1 antibody, anti-GM-CSFRα antibody or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα may be conveniently accomplished by altering the amino acid sequence of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα or polypeptide portion thereof such that one or more glycosylation sites is created or removed.
Where the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the C_H2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997) . The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα of the application may be made in order to create the antibody or multi-specific (e.g., bispecific) antibody variants with certain improved properties.
The N-glycans attached to the C_H2 domain of Fc is heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 2006, 140: 777-83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum. N-glycosylation of the Fc is important for binding to FcγR; and afucosylation of the N-glycan increases Fc's binding capacity to FcγRIIIa. Increased FcγRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications in which cytotoxicity is desirable.
In some embodiments, an enhanced effector function can be detrimental when Fc-mediated cytotoxicity is undesirable. In some embodiments, the Fc fragment or C_H2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the C_H2 domain is mutated to prevent from glycosylation.
In some embodiments, anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variants are provided comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, anti-C5aR1 antibodies, anti-GM-CSFRα antibodies or multi-specific (e.g., bispecific) antibodies specifically recognizing C5aR1 and GM-CSFRα are contemplated herein that have reduced fucose relative to the amount of fucose on the same anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα produced in a wild-type CHO cell. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells (e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene) . In some embodiments, the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5%of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. In some embodiments, the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is completely without fucose, or has no fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L. ) ; US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd) . Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) . Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ; US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11) , and knockout cell lines, such asα-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ; Kanda, Y. et al., Biotechnol. Bioeng., 94 (4) : 680-688 (2006) ; and WO2003/085107) .
Anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is bisected by GlcNAc. Such anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al. ) ; U.S. Pat. No. 6,602,684 (Umana et al. ) ; US 2005/0123546 (Umana et al. ) , and Ferrara et al., Biotechnology and Bioengineering, 93 (5) : 851-861 (2006) . Anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al. ) ; WO 1998/58964 (Raju, S. ) ; and WO 1999/22764 (Raju, S. ) .
In some embodiments, the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variants comprising an Fc region are capable of binding to an FcγRIII. In some embodiments, the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) variants comprising an Fc region have ADCC activity in the presence of human effector cells (e.g., T cell) or have increased ADCC activity in the presence of human effector cells compared to the otherwise same anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) comprising a human wild-type Fc region.
Cysteine Engineered Variants
In some embodiments, it may be desirable to create cysteine engineered anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) in which one or more amino acid residues are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and may be used to conjugate the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα to other moieties, such as drug moieties or linker-drug moieties, to create an anti-C5aR1, anti-GM-CSFRα or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα immunoconjugate, as described further herein. Cysteine engineered anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
Derivatives
In some embodiments, a multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the multi-specific (e.g., bispecific) antibodies specifically recognizing C5aR1 and GM-CSFRα include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers) , and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol) , polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the multi-specific (e.g., bispecific) antibodies specifically recognizing C5aR1 and GM-CSFRα may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα to be improved, whether the multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα derivative will be used in a therapy under defined conditions, etc.
Pharmaceutical Compositions
Also provided herein are compositions (such as pharmaceutical compositions, also referred to herein as formulations) comprising any of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) , nucleic acids encoding the antibodies, vectors comprising the nucleic acids encoding the antibodies, or host cells comprising the nucleic acids or vectors described herein. In some embodiments, there is provided a pharmaceutical composition comprising any one of the anti- C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα described herein and a pharmaceutically acceptable carrier.
Suitable formulations of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα are obtained by mixing an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) , in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG) . Exemplary formulations are described in WO98/56418, expressly incorporated herein by reference. Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the individual to be treated herein. Lipofectins or liposomes can be used to deliver the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα of this application into cells.
The formulation herein may also contain one or more active compounds in addition to the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent in addition to the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or about from 1 to 99%of the heretofore employed dosages.
The anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (e.g., full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Sustained-release preparations may be prepared.
Sustained-release preparations of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα(e.g., full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody (or fragment thereof) , which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate ) , or poly (vinylalcohol) ) , polylactides (U.S. Pat. No. 3,773,919) , copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT^TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) , and poly-D (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydro gels release proteins for shorter time periods. When encapsulated antibody remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 ℃, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization of anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-Sbond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
In some embodiments, anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) is formulated in a buffer comprising a citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80) , or any combination of the foregoing.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.
Methods of treatment or prevention a disease
In certain aspects, there is provided a method of treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual by using a combination of an antigen-binding domain specifically recognizing C5aR1 and an antigen-binding domain specifically recognizing GM-CSFRα of the present application.
In some embodiments, the method of treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual described herein, where the combination of the present application is provided in the form of a multi-specific (e.g., bispecific) antibody, which comprises a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα.
In some embodiments, the method of treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual described herein, where the combination of the present application is provided in the form of a combination of an antibody comprising anti-C5aR1 antigen-binding domain and an antibody comprising anti-GM-CSFRα antigen-binding domain. In some embodiments, the antibody comprising anti-C5aR1 antigen-binding domain is an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1. In some embodiments, the antibody comprising anti-GM-CSFRα antigen-binding domain is an anti-GM-CSFRαantibody or antigen-binding fragment specifically recognizing GM-CSFRα.
In some embodiments, the combination of the present application is provided in the form of a composition (e.g., pharmaceutical composition) , which comprise anti-C5aR1 antigen-binding domain and anti-GM-CSFRα antigen-binding domain.
In some embodiments, the combination of the present application is provided in the form of a composition (e.g., pharmaceutical composition) , which comprise an antibody comprising anti-C5aR1 antigen-binding domain and an antibody comprising anti-GM-CSFRαantigen-binding domain. In some embodiments, the antibody comprising anti-C5aR1 antigen-binding domain is an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1. In some embodiments, the antibody comprising anti-GM-CSFRα antigen-binding domain is an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα.
Methods of treatment or prevention a disease using a multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα
In certain aspects, there is provided a method of treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of the multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα described herein or a composition comprising thereof.
In some embodiments, there is provided the use of any one of the multi-specific (e.g., bispecific) antibodies or pharmaceutical compositions comprising thereof in the manufacture of a medicament for treating a disease and/or disorder associated with C5aR1 or GM-CSFRαsignaling (e.g., inflammatory diseases, autoimmune disease, or cancers) .
Diseases and/or conditions associated with C5aR1 or GM-CSFRα signaling include, but are not limited to allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and IgE-mediated allergic reactions, shock, rheumatoid arthritis, atherosclerosis, multiple sclerosis, allograft rejection, fibrotic disease, asthma, inflammatory glomerulopathies, chronic obstructive pulmonary disease, multiple sclerosis, myeloid leukemia, atherosclerosis and acute respiratory distress syndrome (ARDS) . In some embodiments, the individual is human.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: (i) a V_H comprising an HC-CDR1 comprising SGYSWH (SEQ ID NO: 1) , an HC-CDR2 comprising YIHSSGDTDYNPSLKR (SEQ ID NO: 2) , and an HC-CDR3 comprising SIGDY (SEQ ID NO: 3) ; and a V_L comprising an LC-CDR1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 11) ; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 12) , and an LC-CDR3 comprising SQSTHVPWT (SEQ ID NO: 13) ; or (ii) a V_H comprising an HC-CDR1 comprising SYWMN (SEQ ID NO: 4) ; an HC-CDR2 comprising RIDPX₁DSX₂TRYNQKFED (SEQ ID NO: 49) , wherein X₁ is Y, G or R, and X₂ is E or G; and an HC-CDR3 comprising FVX₁PX₂GX₃FAY (SEQ ID NO: 50) , wherein X₁ is I or W, X₂ is S or V, and X₃ is G or A; and a V_L comprising an LC-CDR1 comprising RSSX₁SPX₂HSNGNTYLH (SEQ ID NO: 51) , wherein X₁ is Q or R, and X₂ is V or L; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 17) , and an LC-CDR3 comprising SQSTX₁VPPT (SEQ ID NO: 52) , wherein X₁ is H or L.
In some embodiments, the method provided herein, wherein the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; and a LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 18-19, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the method provided herein, wherein the first antigen-binding domain specifically recognizing C5aR1 comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising ELSIH (SEQ ID NO: 34) ; an HC-CDR2 comprising GFDPEDGETIYAQKX₁QG (SEQ ID NO: 53) , wherein X₁ is S or F; and an HC-CDR3 comprising GRYX₁X₂LX₃X₄X₅YGFDY (SEQ ID NO: 54) , wherein X₁ is T or V, X₂ is E or S, X₃ is A, F or Y, X₄ is T, F or Q, and X₅ is T or N; and a V_L comprising an LC-CDR1 comprising RASQSVSSYLA (SEQ ID NO: 40) ; an LC-CDR2 comprising GASSRAT (SEQ ID NO: 41) , and an LC-CDR3 comprising QQYX₁NWPX₂T (SEQ ID NO: 55) , wherein X₁ is D or N, and X₂ is Y or P.
In some embodiments, the method provided herein, wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 35-36, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 37-39, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 42-43, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the method provided herein, wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: (i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56- 58; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or (ii) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69.
In some embodiments, the method provided herein, wherein the first antigen-binding domain specifically recognizing C5aR1 comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23 or 59; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59; (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61; or (iv) a V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30 or 66; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66; or (v) a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67; or (vi) a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68; or (vii) a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74.
In some embodiments, the method provided herein, wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising an first antigen-binding domain specifically recognizing C5aR1 and a second antigen-binding domain specifically recognizing GM-CSFRα, or a composition comprising thereof, wherein: the first antigen-binding domain specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the second antigen-binding domain specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 101; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 101, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 101; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 102; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 102, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 102; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 103; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 103, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 103; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 104; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 81.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 104; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 82.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 83; and the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 84, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 84; and the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 85; and the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of SEQ ID NO: 86, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 86; and the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 105; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 106, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 106; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 107; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 108; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 89, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 89; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 90, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 90; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 91, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 91; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 92, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 92; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 98.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 93; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 94; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) antibody comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 95, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 95; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 96, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 96; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.
In some embodiments, the method of treating or preventing a disease associated with C5aR1 and/or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering an effective amount of a multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα is more efficacious than administering a same valency amount of an antibody specifically recognizing C5aR1 or a same valency amount of an antibody specifically recognizing GM-CSFRα. In some embodiments, the method comprising administering an effective amount of a multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα inhibits the activity of TF-1 cell proliferation stimulated with GM-CSF by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more compared to administering a same valency amount of an antibody specifically recognizing GM-CSFRα. In some embodiments, the method comprising administering an effective amount of a multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα inhibits the activity of C5aR1 function by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more compared to administering a same valency amount of an antibody specifically recognizing C5aR1 or a same valency amount of an antibody specifically recognizing GM-CSFRα. In some embodiments, the method comprising administering an effective amount of a multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα improves patient survival by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more compared to administering a same valency amount of an antibody specifically recognizing C5aR1 or a same valency amount of an antibody specifically recognizing GM-CSFRα.
Methods of treatment or prevention a disease using a combination of antibodies recognizing C5aR1 and GM-CSFRα or a pharmaceutical composition comprising antibodies recognizing C5aR1 and GM-CSFRα
In some embodiments, there is provided a method of treating or preventing treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα. In some embodiments, there is provided a method of treating or preventing treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a combination of an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα.
In some embodiments, there is provided the use of any one of the pharmaceutical compositions comprising (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα in the manufacture of a medicament for treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) . In some embodiments, there is provided the use of any one of the combinations of an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα in the manufacture of a medicament for treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) .
Diseases and/or conditions associated with C5aR1 or GM-CSFRα signaling include, but are not limited to allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and IgE-mediated allergic reactions, shock, rheumatoid arthritis, atherosclerosis, multiple sclerosis, allograft rejection, fibrotic disease, asthma, inflammatory glomerulopathies, chronic obstructive pulmonary disease, multiple sclerosis, myeloid leukemia, atherosclerosis and acute respiratory distress syndrome (ARDS) . In some embodiments, the individual is human.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: (i) a V_H comprising an HC-CDR1 comprising SGYSWH (SEQ ID NO: 1) , an HC-CDR2 comprising YIHSSGDTDYNPSLKR (SEQ ID NO: 2) , and an HC-CDR3 comprising SIGDY (SEQ ID NO: 3) ; and a V_L comprising an LC-CDR1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 11) ; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 12) , and an LC-CDR3 comprising SQSTHVPWT (SEQ ID NO: 13) ; or (ii) a V_H comprising an HC-CDR1 comprising SYWMN (SEQ ID NO: 4) ; an HC-CDR2 comprising RIDPX₁DSX₂TRYNQKFED (SEQ ID NO: 49) , wherein X₁ is Y, G or R, and X₂ is E or G; and an HC-CDR3 comprising FVX₁PX₂GX₃FAY (SEQ ID NO: 50) , wherein X₁ is I or W, X₂ is S or V, and X₃ is G or A; and a V_L comprising an LC-CDR1 comprising RSSX₁SPX₂HSNGNTYLH (SEQ ID NO: 51) , wherein X₁ is Q or R, and X₂ is V or L; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 17) , and an LC-CDR3 comprising SQSTX₁VPPT (SEQ ID NO: 52) , wherein X₁ is H or L.
In some embodiments, the pharmaceutical composition or the combination described herein, wherein the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; and a LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 18-19, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the pharmaceutical composition or the combination described herein, wherein the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising ELSIH (SEQ ID NO: 34) ; an HC-CDR2 comprising GFDPEDGETIYAQKX₁QG (SEQ ID NO: 53) , wherein X₁ is S or F; and an HC-CDR3 comprising GRYX₁X₂LX₃X₄X₅YGFDY (SEQ ID NO: 54) , wherein X₁ is T or V, X₂ is E or S, X₃ is A, F or Y, X₄ is T, F or Q, and X₅ is T or N; and a V_L comprising an LC-CDR1 comprising RASQSVSSYLA (SEQ ID NO: 40) ; an LC-CDR2 comprising GASSRAT (SEQ ID NO: 41) , and an LC-CDR3 comprising QQYX₁NWPX₂T (SEQ ID NO: 55) , wherein X₁ is D or N, and X₂ is Y or P.
In some embodiments, the pharmaceutical composition or the combination described herein, wherein the anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 35-36, or a variant thereof comprising up to about 3 amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 37-39, or a variant thereof comprising up to about 3 amino acid substitutions; and a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 amino acid substitutions; an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 amino acid substitutions; and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 42-43, or a variant thereof comprising up to about 3 amino acid substitutions.
In some embodiments, the pharmaceutical composition or the combination described herein, wherein the anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: (i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing a C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: (i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 20-22 and 56-58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 23-25 and 59-61; or (ii) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 26-29 and 62-65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 30-33 and 66-69.
In some embodiments, the pharmaceutical composition or the combination described herein, wherein the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23 or 59; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 20 or 56; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 23 or 59; (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61; or (iv) a V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30 or 66; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 26 or 62; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 30 or 66; or (v) a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67; or (vi) a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68; or (vii) a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74, or a variant thereof having at least about 90%(for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74; or a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 44-46 and 70-72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 47-48 and 73-74.
In some embodiments, the pharmaceutical composition or the combination described herein, wherein the anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: (i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73; or (iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74; or a V_H comprising an HC-CDR1, an HC-CDR2 and an HC-CDR3 of the V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 of the V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an GM-anti-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 27 or 63, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27 or 63; and a V_L comprising the amino acid sequence of SEQ ID NO: 31 or 67, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31 or 67; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM- CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, there is provided a method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising or a combination of (i) an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and (ii) an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα, wherein: the anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the antibody or antigen-binding fragment specifically recognizing GM-CSFRα comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
In some embodiments, the method of treating or preventing a disease associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual comprising administering an effective amount of a pharmaceutical composition comprising or a combination of anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα is more efficacious than administering a same valency amount of an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 or a same valency amount of an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα. In some embodiments, the method comprising administering an effective amount of a pharmaceutical composition comprising or a combination of anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and anti-GM-CSFRαantibody or antigen-binding fragment specifically recognizing GM-CSFRα inhibits the activity of TF-1 cell proliferation stimulated with GM-CSF by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) , 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more compared to administering a same valency amount of an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα. In some embodiments, the method comprising administering an effective amount of a pharmaceutical composition comprising or a combination of anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα inhibits the activity of C5aR1 function by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) , 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more compared to administering a same valency amount of an antibody specifically recognizing C5aR1. In some embodiments, the method comprising administering an effective amount of a pharmaceutical composition comprising or a combination of anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 and anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα improves patient survival by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% (for example at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) , 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more compared to administering a same valency amount of an anti-C5aR1 antibody or antigen-binding fragment specifically recognizing C5aR1 or a same valency amount of an anti-GM-CSFRα antibody or antigen-binding fragment specifically recognizing GM-CSFRα.
Articles of Manufacture and Kits
In some embodiments of the application, there is provided an article of manufacture containing materials useful for treating or preventing a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual, or for delivering a multi-specific (e.g., bispecific) antibody recognizing C5aR1 and GM-CSFRα or a pharmaceutical composition comprising anti-C5aR1 antibody, anti-GM-CSFRα antibody to a cell expressing C5aR1 and GM-CSFRα. The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . In some embodiments, at least one active agent in the composition is a multi-specific (e.g., bispecific) antibody or an antigen-binding domain or an antibody of the application. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for administering the multi-specific (e.g., bispecific) antibody or the pharmaceutical composition to the patient. Articles of manufacture and kits comprising combinatorial therapies described herein are also contemplated.
Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is used for treating bacterial infections. In some embodiments, the package insert indicates that the composition is used for treating a disease and/or disorder associated with C5aR1 or GM-CSFRαsignaling (e.g., inflammatory diseases, autoimmune disease, or cancers) .
Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
Kits are also provided that are useful for various purposes, e.g., useful for treating or preventing a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) , or for delivering a multi-specific (e.g., bispecific) antibody recognizing C5aR1 and GM-CSFRα or a pharmaceutical composition comprising anti-C5aR1 antibody, anti-GM-CSFRα antibody to a cell expressing C5aR1 and GM-CSFRα to a cell expressing C5aR1 or GM-CSFRα, optionally in combination with the articles of manufacture. Kits of the application include one or more containers comprising anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα composition (or unit dosage form and/or article of manufacture) , and in some embodiments, further comprise another agent (such as the agents described herein) and/or instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of selection of individuals suitable for treatment. Instructions supplied in the kits of the application are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit) , but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
For example, in some embodiments, the kit comprises a composition comprising an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) . In some embodiments, the kit comprises a) a composition comprising any one of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα described herein, and b) an effective amount of at least one other agent, wherein the other agent enhances the effect (e.g., treatment effect, detecting effect) of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα. In some embodiments, the kit comprises a) a composition comprising any one of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα described herein, and b) instructions for administering the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα composition to an individual for treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual. In some embodiments, the kit comprises a) a composition comprising any one of the anti-C5aR1 antibody, anti-GM-CSFRαantibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα described herein, b) an effective amount of at least one other agent, wherein the other agent enhances the effect (e.g., treatment effect, detecting effect) of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα, and c) instructions for administering the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα composition and the other agent (s) to an individual for useful for treating a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual. The anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and the other agent (s) can be present in separate containers or in a single container. For example, the kit may comprise one distinct composition or two or more compositions wherein one composition comprises an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and another composition comprises another agent.
In some embodiments, the kit comprises a nucleic acid (or set of nucleic acids) encoding an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) . In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα, and b) a host cell for expressing the nucleic acid (or set of nucleic acids) . In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and b) instructions for i) expressing the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα in a host cell, ii) preparing a composition comprising the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα, and iii) administering the composition comprising the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα to an individual for treating or preventing a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual. In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα, b) a host cell for expressing the nucleic acid (or set of nucleic acids) , and c) instructions for i) expressing the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα in the host cell, ii) preparing a composition comprising the anti-C5aR1 antibody, anti-GM-CSFRαantibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and iii) administering the composition comprising the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα to an individual for treating or preventing a disease and/or disorder associated with C5aR1 or GM-CSFRα signaling (e.g., inflammatory diseases, autoimmune disease, or cancers) in an individual.
The kits of the application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.
The instructions relating to the use of the anti-C5aR1 antibody, anti-GM-CSFRαantibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα compositions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. For example, kits may be provided that contain sufficient dosages of an anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα (such as a full-length anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα) as disclosed herein to provide effective treatment of an individual for an extended period, such as any of a week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of the anti-C5aR1 antibody, anti-GM-CSFRα antibody, or multi-specific (e.g., bispecific) antibody specifically recognizing C5aR1 and GM-CSFRα and pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this application. The application will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the application but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.
Example 1: Generation of anti-C5aR1 antibodies
This example illustrates the methods of using mouse hybridoma technology to generate anti-C5aR1 antibodies, and methods to screen and select antibodies for further characterization. The content of the patent with International Application No. PCT/US2022/082260 was incorporated here in this invention.
Immunizations and fusions: Balb/c and NZB mice were immunized with human C5aR1 antigen. Endpoint titers were determined by ELISA, and spleens and lymph nodes were harvested and processed according to standard protocols. Mouse B cells were isolated by EasySep Mouse B cell isolation Kit (StemCell, cat#19854A) and fused with myeloma cells SP2/0-Ag14 cells (ATCC, CRL 1581) using PEG. Following standard protocols, the fused cells were cultured in semi-solid ClonalCell-HY Cloning-Medium D (StemCell, cat#03804) . Monoclonal hybridoma clones were picked into 96 well/plate and cultured in HT medium.
Hybridoma Screening: Hybridoma clones were confirmed by primary screen antigen binding ELISA, the positive clones were purified using Protein A resin (Thermo Fisher Scientific, cat#A26458) . The binding and blocking function of the purified hybridoma antibodies were further validated by the following experiments.
C5aR receptor binding assay: Briefly, Expi293 cells overexpressing huC5aR1 were plated in FACS buffer. 50μL of the individual purified antibodies at the indicated concentration was added to the individual well followed by incubation for 30 min at room temperature (RT) . Plates were washed, then 50μL/well of AF488-labeled anti-mouse IgG was added and incubated at RT for 30 min. Cells were washed and analyzed by flow cytometry. Mean fluorescent values were recorded at each dilution and plotted as the log [Ab] and EC₅₀ values determined in GraphPad Prism 6.0.
C5aR1-TEV/β-Arrestin-Luc reporter assay: Dual transgenic U2OS cells bearing huC5aR1 coupled to tobacco etch virus (TEV) at the C terminus and β-Arrestin coupled to luciferase with a TEV cleavage site (hereinafter referred to as C5aR1-TEV/β-Arrestin-Luc cells) were used to determine functional inhibition of C5aR1 signal transduction. When C5a is added to these cells, β-Arrestin is recruited to the C5aR1. The C5aR1-coupled TEV enzyme then cleaves the TEV site in the luciferase protein allowing it to fold correctly and resulting in a luciferase signal when a substrate is added to the cells. Briefly, C5aR1-TEV/β-Arrestin-Luc cells (BonOpus, Cat#BC050088) were plated in the plates overnight at 37℃ in McCoy’s media containing 10%FBS. Cells were washed 2 times with serum-free McCoy’s , and 50μL of the individual purified hybridoma antibodies at the indicated concentration (typically titrations starting at 100nM decreasing in 3-fold increments) in serum-free McCoy’s media was added to individual well followed by incubation for 30 min at RT. 50μl of McCoy’s media containing 100nM C5a was added to each well and cells were incubated at 37℃ for 3 hr. Following incubation, the cells were equilibrated at RT for 10 min, then Bright-Glo luciferase substrate (Promega) was added to each well, and luminescence was recorded at 5 min increments for 1 hr. Luminescence values were recorded at each dilution and plotted as the log[Ab] and IC₅₀ values were determined in GraphPad Prism 6.0.
Neutrophil C5a-induced CD11b upregulation inhibition assay: Primary human whole blood was used to assay CD11b upregulation resulting from C5a stimulation. 80μL of whole blood was added to 96-well plates and incubated with 50μL of the individual purified hybridoma antibodies at the indicated concentration (typically titrations starting at 100nM decreasing in 5-fold increments) in Hank’s balanced salt solution (HBSS) at 4℃ for 30 min. C5a was added in 20μL HBSS at 20nM concentrations and the plates were incubated for 25 min at 37℃. After incubation, the plates were cooled on ice for 5 min and a cocktail containing anti-CD66b, anti-CD14, anti-GR-1, and anti-CD11b was added to each well in 50μL HBSS and incubated for 30 min on ice. Red blood cells (RBC) were then lysed by adding 1.8 ml RBC lysis buffer (BD Biosciences, Cat#349202) to each well for 10 min at RT. Following RBC lysis, plates were washed 2 times with FACS buffer, then fixed with 300μL FACS buffer containing 2%paraformaldehyde. CD11b expression on neutrophils and monocytes was assayed by flow cytometry. Mean fluorescent values were recorded at each dilution and plotted as the log [Ab] and IC₅₀ values determined in GraphPad Prism 6.0.
Through the above experimental screening, the exemplary anti-C5aR1 hybridoma 10G1 and 1C2 showed good efficiency in C5a receptor binding and inhibition activity of C5aR1 function. The hybridoma antibody clones were amplification and sequencing. The CDRs sequences of the hybridoma 10G1 and 1C2 are summarized in Table 2. The recombinant anti-C5aR1 10G1 or 1C2 chimeric antibody construct with heavy and light chain variable domain of hybridoma antibody 10G1 or 1C2 and human IgG1 constant regions (referred to as 10G1 chimeric or 1C2 chimeric) was made by using methods well known in the field.
Preparation of humanized versions of 1C2 and 10G1: The light chain variable region (V_L) and heavy chain variable region (V_H) sequences of murine antibody from hybridoma 1C2 and 10G1 were aligned to human germline antibody sequences respectively. The human germline kappa light chain (Gene ID–V gene: IGKV2_24*01 and IGKV2-30*02) and heavy chain (Gene ID–V gene: IGHV1_69_2*01 and IGHV4-38-2*01) were used as the human frameworks.
The complementarity-determining regions (CDRs) of murine anti-C5aR1 antibody 1C2 and 10G1 light chain and heavy chain were grafted into the identified closest human frameworks respectively to generate humanized antibody clone. In this process, antibodies 1C2 and 10G1 were humanized by grafting the CDRs from the murine antibody V-regions onto human germline antibody V-region frameworks, the CDRs grafted from the donor to the acceptor sequence are as defined by Kabat (Kabat et al., 1987) . To recover the activity of the antibody, some framework residues from the murine V-regions that were found to be parts of V_H-V_L interacting interface or the framework residues acting as “Vernier” zone, which may adjust CDR structure and fine-tune to fit antigen (Foote et al., 1992) were also retained in the humanized sequence. The full-length IgG versions of humanized antibodies were generated as described above. Two of the full-length IgG versions of humanized antibodies derived from 1C2 (referred to as 1C2.30 and 1C2.35) and one of the full-length IgG versions of humanized antibodies derived from 10G1 (referred to as 10G1.44) , were selected for further experimentation.
The sequences of the antibodies were summarized in Tables 2-3.
Example 2: Characterization of the anti-C5aR1 antibodies
C5a Receptor Binding assay: the binding of anti-C5aR1 antibodies to huC5aR1-overexpressing cells was examined as described in Example 1, EC₅₀ values of the anti-C5aR1 antibodies derived from 1C2 were summarized respectively in Table 16.
Table 16
However, the exemplary humanized antibody 10G1.44 exhibited reduced binding activity as compared to its corresponding chimeric antibody 10G1, 10G1.44 antibody was optimized by affinity maturation.
Affinity maturation ofclone 10G1.44: As the humanization of 10G1 reduced its affinity, humanized clone 10G1.44 was selected for affinity maturation via phage display. The affinity maturation process was according to the manufacturing protocols known in the field. Generation of IgG1 versions of affinity-matured anti-C5aR1 antibodies derived from 10G1.44 and two of the IgG1 versions of optimized anti-C5aR1 antibodies derived from 10G1.44 (referred to as 10G1.44.1 and 10G1.44.6) were selected for further experimentation.
The binding of anti-C5aR1 antibodies derived from 10G1 to huC5aR1-overexpressing cells was examined as described above in Example 1. EC₅₀ values of the anti-C5aR1 antibodies derived from 10G1.44 were summarized in Table 17.
Table 17
As shown in Table 17, the optimized anti-C5aR1 antibodies exhibited highly better binding activity to C5aR1 as compared to their parental antibody 10G1.44, demonstrating that the affinity maturation of C5aR1 antibody markedly improved the binding function.
C5aR1-TEV/β-Arrestin-Luc reporter assay: Assays using the C5aR1-TEV/β-Arrestin-Luc reporter cells were performed as described above in Example 1. The known anti-C5aR1 antibody clone 7F3 developed by Novo Nordisk, which was described in US patent NO.8071096 and used as control in the experiment, hereinafter referred to as Reference antibody. As shown in Fig. 1, the exemplary anti-C5aR1 antibody 1C2.35 was even more potent than the Reference antibody in this assay. IC₅₀ values of the anti-C5aR1 antibodies derived from 10G1 were summarized respectively in Table 18.
Table 18
In vitro human primary neutrophil CD11b upregulation assay: Assays measuring inhibition of C5a-induced CD11b upregulation on primary neutrophils were performed with anti-C5aR1 antibodies as described above. The IC₅₀ values of anti-C5aR1 antibodies derived from 1C2 and 10G1.44 were summarized in Table 19 and Table 20. The Reference antibody was used as control in the experiment. As shown in Table 19, the exemplary anti-C5aR1 antibody 1C2.30 and 1C2.35 were even more potent than the Reference antibody in this assay.
Table 19
Table 20
As demonstrated by the above experiments, the exemplary anti-C5aR1 antibodies exhibited good binding activity to human C5aR1, good inhibitory activity of C5aR1 function in the C5aR1-TEV/β-Arrestin-Luc reporter assay and could inhibit the ability of C5a-induced CD11b upregulation on primary neutrophils in vitro human primary neutrophil CD11b upregulation assay.
Example 3: Generation and characterization of bispecific antibodies recognizing C5aR1 and GM-CSFRα
In this example, the following three kinds of bispecific antibodies were prepared. Bispecific antibodies were produced through a process that involves the design of the intact molecule, synthesis, and cloning of the nucleotide sequence for each domain, expression in mammalian cells, and purification of the final product. In the following sequence design, V_H and V_L respectively represent a heavy chain variable region and a light chain variable region of the antibody; C_H represents an antibody heavy chain constant region comprising C_H1, C_H2 and C_H3 domains; Fc represents an Fc region of an antibody, comprising C_H2 and C_H3 domain; the scFv is an antibody formed by connecting V_H and V_L of the antibody through a linker peptide; C_L represents a light chain constant region; and L, L1, L2…represent linker.
3.1 DVD-Ig format (dual variable domain immunoglobulin) bispecific antibody
DVD-Ig BsAb used in the examples of the application contains an Fc region and constant regions in a configuration similar to a conventional IgG. The DVD-Ig BsAb is unique in that each arm of the molecule contains two variable domains for different antigens. The variable domains within an arm are linked in tandem and can process different binding specificities.
The DVD-Ig technology was described in “Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules. MAbs. 2009 Jul-Aug; 1 (4) : 339-47” , the DVD-Ig protein is a bispecific, tetravalent IgG-like molecule. The DVD-Ig bispecific antibody used in the examples comprises two heavy chains and two light chains, the schematic structure diagram of which was shown in Fig. 2A.
The composition design of the DVD-Ig bispecific antibody was shown in Table 21, and the amino acid sequence of the heavy chain and light chain was shown in Table 8 and Table 9.
Expression and purification of the bispecific antibody: The various parts of the chimeric heavy chain and light chain were joined together using overlap PCR extension techniques. Plasmids expressing the light and heavy chains were extracted and used to transform 293F cells. After the cells were cultured at 37℃, 8%CO₂, and 120rpm for five days, the culture media was purified using Protein A affinity chromatography. Briefly, Protein A column was first equilibrated with a PBS buffer containing 50mM PBS and 0.15M NaCl (pH7.2) , at a flow rate of 150cm/h and with a volume that is six times the volume of the column. The supernatant of the culture media (pH was adjusted to 7.2) was passed through the column at 150cm/h. Upon further equilibration, the column was washed off using 50mM sodium citrate (pH3.5) and the elution was collected.
Table 21: the composition of DVD-Ig BsAb molecule recognizing human C5aR1 and GM-CSFRα
3.2 IgG- (scFv) ₂ BsAb
IgG- (scFv) ₂ BsAb is a tetravalent IgG-like BsAb which contains a full-length IgG-scFv fusion, in which, a single-chain Fv (scFv) antibody fragment of one antigen specificity is genetically fused to the c-terminal of a conventional IgG of a different antigen specificity. In the examples of the present application, two cysteines were introduced into the scF_V sequence by substitution (V_H-Cys44 and V_L-Cys100, positions were relative to the V_H or V_L sequence, the numbering is according to the Kabat numbering) to facilitate the formation of disulfide bond to stabilize the scFv structure.
The technology of IgG- (scFv) ₂ format was described in “Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15 (2) : 159-63” . The schematic structure diagram of the IgG- (scFv) ₂ BsAb used in the examples of the present application was shown in Fig. 2B.
The composition design of the IgG- (scFv) ₂ bispecific antibody was shown in Table 22, and the amino acid sequence of the heavy chain and light chain was shown in Table 10.
The expression and purification steps were as described in Example 3.1.
Table 22: the composition of IgG- (scFv) ₂ BsAb molecule recognizing human C5aR1 and GM-CSFRα
3.3 Hetero H, CrossMab format
Hetero, H CrossMab BsAb is a heterodimer consisting of two monomers recognizing different antigens, respectively. The complete Fab domain can be exchanged in the CrossMab^Fab, or either only the variable domains in the CrossMab ^VH-VL, or the constant domain of the Fab arm in the CrossMab^CH1-CL. The Hetero H, CrossMab format bispecific was also described in Klein C, et al. The use of CrossMAb technology for the generation of bi-and multi-specific antibodies. MAbs. 2016 Aug-Sep; 8 (6) : 1010-20. The format used in the examples of the present application is CrossMab^CH1-CL, in which only the constant domain of the Fab arm is exchanged. The schematic structural diagram of the CrossMab BsAb molecules of the examples was shown in Fig. 2C.
Moreover, KIH technology was introduced into the CrossMab^CH1-CL format of the examples. In the examples of the present application, the 366 threonine (T) residue in an antibody C_H3 domain is replaced with tryptophan (W) to form a "knob " structure, and the 366 threonine (T) residue in the other matched antibody C_H3 domain is replaced with serine (S) , the 368 leucine (L) residue is replaced with alanine (A) , the 407 tyrosine (Y) residue is replaced with valine (V) to form a "hole" structure, and heterologous heavy chain dimerization is promoted using the reduction of the spatial steric hindrance effect after mutation and the covalent disulfide bond formed in the hinge region, wherein the numbering is in accordance with the EU numbering. In addition, two Cys residue mutations were also introduced forming the stabilized disulfide bridge (S354 C on the "Knob " side and Y349 C on the "Hole " side) in the Fc domain.
The composition design of the Hetero H, CrossMab bispecific antibody was shown in Table 23, and the amino acid sequence of the heavy chain and light chain was shown in Table 11 and Table 12.
The expression and purification steps were described in Example 3.1.
Table 23: the composition of Hetero H, CrossMab BsAb molecule recognizing human C5aR1 and GM-CSFRα
Example 4: Characterization of in vitro activity of bispecific antibodies recognizing C5aR1 and GM-CSFRα
Binding ELISA Assay
The binding ability of the bispecific antibodies recognizing C5aR1 and GM-CSFRαto human GM-CSFRα (hGM-CSFRα) and human C5aR1 (hC5aR1) was determined by the binding ELISA assay. Briefly, a 96-well plate was coated with human GM-CSFRα or human C5aR1 in PBS at 0.2μg/well and left overnight at 4℃. After removing the coating solution, the plates were blocked by adding a blocking buffer and incubated at room temperature for 1 hour. Plates were then washed 3 times with wash buffer. Serial dilution of purified bispecific antibodies in PBS was added to individual wells followed by incubation at room temperature for 1 hour. Plates were washed 3 times with wash buffer. Then 50μL/well of goat anti-human IgG Fc-AP at 1: 3000 dilution in ELISA diluent was added, incubated at room temperature for 1 hour, washed 5 times with wash buffer, and developed for 30 minutes with 50μL/well of pNPP substrate. Plates were analyzed with a Bio-TEK at 405 nm. Anti-GM-CSFRα monoclonal antibody E35-hIgG1 and anti-C5aR1 monoclonal antibody 1C2.30-hIgG1 were used as a control in this assay.
EC50 values of exemplary bispecific antibodies were determined in GraphPad Prism and summarized in Table 24 (for hGM-CSFRα) and Table 25 (for hC5aR1) . The binding curves were shown in Fig. 3A (for hGM-CSFRα) and Fig. 3B (for hC5aR1) . The bispecific antibodies tested positive for binding to hGM-CSFRα and hC5aR1, the binding activity of which was even better than the corresponding monoclonal antibody of E35-hIgG1 and 1C2.30-hIgG1, respectively.
Table 24
Table 25
Binding affinity (Kd) determination:
Binding affinities of bispecific antibodies recognizing C5aR1 and GM-CSFR to hGM-CSFRα were determined using Biolayer interferometry on the Octet RED96 instrument (ForteBio) at 30 ℃ and with 1200 rpm agitation. The following kinetic assay was performed using anti-human IgG Fc capture (AHC) biosensors (ForteBio) in kinetics buffer (PBS, 0.1%Tween-20 and 1%bovine serum albumin) : (a) antibody (2 μg/mL) for 300 sec, (b) baseline for 120 sec, (c) association with His-tag-hGM-CSFRα (2.5, 0.5 and 0 μg/mL) for 420 sec and (d) dissociation for 1200 sec. Data fitting and analysis were performed with Octet data analysis software 8.0 (ForteBio) using a 1: 1 binding model after Savitzky–Golay filtering. The equilibrium dissociation constant (Kd) was calculated as the ratio of Koff/Kon and summarized in Table 26 (below) . The results showed that the exemplary bispecific antibodies would bind to GM-CSFR with relatively high binding affinity.
Table 26: Binding affinity of BsAbs binding to human GM-CSFRα
Non-specific Binding Assay
Non-specific binding of bispecific antibodies recognizing C5aR1 and GM-CSFR was assessed using insulin, double-stranded DNA (dsDNA) , and baculovirus particle (BVP) ELISA (see e.g., Hotzel et al., MAbs 2012; 4 (6) : 753-60. ) . Briefly, 96-well Maxisorp plates (BlueSky Biotech) were coated with 1%suspension of insulin, dsDNA, or baculovirus particles at 4℃ overnight. The plates were then blocked in PBS with 1%BSA and 0.05%Tween-20 at room temperature for 1 hour. Serial dilutions of bispecific antibodies in PBS containing 0.5%BSA were added to the plates, incubated for 1 hour, and then the plate was washed with PBS. Boco (Bococizumab, Pfizer) which has high non-specific binding, and Evo (Evolocumab, Amgen) which has lower non-specific binding were used as controls. Bound antibodies were detected with goat anti-human IgG Fc-AP in ELISA buffer. The plate was incubated at room temperature for 1 hour with agitation, washed 6 times with wash buffer, and developed for 3-10 minutes by the addition of 50μL/well of pNPP substrate. Plates were analyzed with a Biotek Gen5 plate reader (BioTek) at 405 nm and compared to reference antibodies.
As shown in Fig. 4A (for insulin) , Fig. 4B (for dsDNA) , and Fig. 4C (for BVP) , none of the exemplary bispecific antibodies showed detectable binding with insulin, dsDNA, and BVP.
TF-1 Proliferation Assay
TF-1 cell is a human erythroleukemic cell line that is factor-dependent and can proliferate in the presence of cytokines such as GM-CSF. The bispecific antibodies recognizing C5aR1 and GM-CSFRα able to inhibit binding between GM-CSF and GM-CSFRα were assessed for biological activity in a TF-1 proliferation assay, which analyzed the ability of the antibodies to inhibit the proliferation of TF-1 cells stimulated with GM-CSF.
The TF-1 cell line (ATCC) was maintained in growth media containing RPMI 1640 + 10%FBS + 1 %L Glutamine + 0.1 %Pen/Strep, with the addition of GM-CSF at 2 ng/mL (R&D system) . Before the assay, the GM-CSF was removed by 3 cycles of spinning down at 300 g for 5 minutes, and the cells were re-suspended in the assay media (the same growth media as above but without GM-CSF) . After this process, the TF-1 cells were re-resuspended in assay media at a final concentration of 4x10⁵ /mL and incubated in 37℃, 5%CO2 for 1 hour. The bispecific antibodies were diluted serially at different concentrations in the assay media and were added to the cells, after incubating at 37℃ for an hour, GM-CSF (Peprotech) was added at a final concentration of 200pg/mL. Assay plates were incubated for 48 hrs at 37℃ in 5%CO₂ in a humidified chamber. The results of TF1 cell proliferation under the bispecific antibodies were determined by using the ATPlite 1step Luminescence Assay kit (PerkinElmer) . According to its manual, 100μl of the substrate solution was added to each well of the 96-well plates. After mixing for 2 minutes by shaking at 700rpm, the plates were read for luminescence in the Biotek Synergy Neo2 reader. IC₅₀ was calculated by PRISM. Anti-GM-CSFRαmonoclonal antibody E35-hIgG1 was used as a positive control in this assay. Human MOPC21 IgG1 isotype antibody (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing. Nucleic Acids Res. 9 (18) : 4485-4494) was made in house, used as a negative control. Luminescence values were recorded at each dilution and plotted as the log [Ab] and IC₅₀ values were determined in GraphPad Prism and summarized in Table 27.
The result was shown in Fig. 5, where “hGM-CSF” indicated the well without adding bispecific antibody, and “none” indicated the well without adding GM-CSF and bispecific antibody. As shown in Table 27 and Fig. 5, the exemplary bispecific antibody SB1006-9 and SB1006-15 exhibited good efficacy in inhibiting GM-CSF-induced proliferation of the TF-1 cell line.
Table 27
C5aR1-TEV/β-Arrestin-Luc reporter assay
As described in Example 1, the ability of exemplary bispecific antibodies specifically recognizing C5aR1 and GM-CSFRα was tested in C5aR1-TEV/β-Arrestin-Luc reporter assay. Anti-C5aR1 monoclonal antibodies 1C2.30-hIgG1 and MOPC21 were used as a positive and negative control in this assay, respectively. Luminescence values were recorded at each dilution and plotted as the log [Ab] and IC50 values were determined in GraphPad Prism and summarized in Table 28.
The result was shown in Fig. 6, where “hC5a” indicated the well without adding bispecific antibody, and “none” indicated the well without adding C5a and bispecific antibody. As shown in Table 28 and Fig. 6, the exemplary bispecific antibody SB1006-9 and SB1006-15 exhibited good inhibition activity on C5aR1 function.
Table 28

Claims

A multi-specific antibody, which comprises a first antigen-binding domain specifically recognizing C5aR1, and a second antigen-binding domain specifically recognizing GM-CSFRα.
The multi-specific antibody of claim 1, wherein the first antigen-binding domain comprises:

(i) a V_H comprising an HC-CDR1 comprising SGYSWH (SEQ ID NO: 1) , an HC-CDR2 comprising YIHSSGDTDYNPSLKR (SEQ ID NO: 2) , and an HC-CDR3 comprising SIGDY (SEQ ID NO: 3) ; and a V_L comprising an LC-CDR1 comprising RSSQSLVHSNGNTYLH (SEQ ID NO: 11) ; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 12) , and an LC-CDR3 comprising SQSTHVPWT (SEQ ID NO: 13) ; or

(ii) a V_H comprising an HC-CDR1 comprising SYWMN (SEQ ID NO: 4) ; an HC-CDR2 comprising RIDPX₁DSX₂TRYNQKFED (SEQ ID NO: 49) , wherein X₁ is Y, G or R, and X₂ is E or G; and an HC-CDR3 comprising FVX₁PX₂GX₃FAY (SEQ ID NO: 50) , wherein X₁ is I or W, X₂ is S or V, and X₃ is G or A; and a V_L comprising an LC-CDR1 comprising RSSX₁SPX₂HSNGNTYLH (SEQ ID NO: 51) , wherein X₁ is Q or R, and X₂ is V or L; an LC-CDR2 comprising KVSNRFS (SEQ ID NO: 17) , and an LC-CDR3 comprising SQSTX₁VPPT (SEQ ID NO: 52) , wherein X₁ is H or L.
The multi-specific antibody of claim 1 or 2, wherein the first antigen-binding domain comprises:

(i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; or

(ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19.
The multi-specific antibody of any one of claims 1-3, wherein the second antigen-binding domain comprises:

(i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42;

(ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or

(iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
The multi-specific antibody of any one of claims 1-4, wherein:

(i) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42;

(ii) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or

(iii) the first antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and wherein the second antigen-binding domain comprises: a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42.
The multi-specific antibody of any one of claims 1-5, wherein the first antigen-binding domain comprises:

(i) a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60;

(ii) a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61;

(iii) a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; or

(iv) a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69.
The multi-specific antibody of any one of claims 1-6, wherein the second antigen-binding domain comprises:

(i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73;

(ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or

(iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
The multi-specific antibody of any one of claims 1-7, wherein:

(i) the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 21 or 57, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 21 or 57; and a V_L comprising the amino acid sequence of SEQ ID NO: 24 or 60, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 24 or 60; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73;

(ii) the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 22 or 58, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 22 or 58; and a V_L comprising the amino acid sequence of SEQ ID NO: 25 or 61, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 25 or 61; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73;

(iii) the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 28 or 64, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 28 or 64; and a V_L comprising the amino acid sequence of SEQ ID NO: 32 or 68, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 32 or 68; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or

(iv) the first antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 29 or 65, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 29 or 65; and a V_L comprising the amino acid sequence of SEQ ID NO: 33 or 69, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 33 or 69; and wherein the second antigen-binding domain comprises: a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73.
The multi-specific antibody of claim 1, wherein the second antigen-binding domain comprises:

(i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42;

(ii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; or

(iii) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a V_L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.
The multi-specific antibody of claim 9, wherein the second antigen-binding domain comprises:

(i) a V_H comprising the amino acid sequence of SEQ ID NO: 44 or 70, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 44 or 70; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73;

(ii) a V_H comprising the amino acid sequence of SEQ ID NO: 45 or 71, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 45 or 71; and a V_L comprising the amino acid sequence of SEQ ID NO: 47 or 73, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 47 or 73; or

(iii) a V_H comprising the amino acid sequence of SEQ ID NO: 46 or 72, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 46 or 72; and a V_L comprising the amino acid sequence of SEQ ID NO: 48 or 74, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 48 or 74.
The multi-specific antibody of any one of claims 1-10, wherein the format of which is selected form the group consisting of DVD-Ig, IgG- (scFv) ₂ and Hetero H, CrossMab.
The multi-specific antibody of any one of claims 1-11, comprising four polypeptide chains: wherein:

the two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-L-V_H2-C_H1, wherein V_H1 is a heavy-chain variable region that specifically binds to the C5aR1; V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; L is a linker; C_H1 is a heavy chain constant region C_H1 domain; optionally, wherein the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and

the other two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-L-V_L2-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1; V_L2 is a light chain variable region that specifically binds to GM-CSFRα; L is a linker; and C_L is a light chain constant region;

wherein V_H1 and V_L1 constitute the first antigen-binding domain specifically recognizing C5aR1 and V_H2 and V_L2 constitute the second antigen-binding domain specifically recognizing GM-CSFRα.
The multi-specific antibody of any one of claims 1-11, comprising four polypeptide chains: wherein:

the two polypeptide chains each comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-L-V_H2-C_H1, wherein V_H1 is a heavy-chain variable region that specifically binds to the GM-CSFRα; V_H2 is a heavy chain variable region that specifically binds to C5aR1; L is a linker; C_H1 is a heavy chain constant region C_H1 domain; optionally, wherein the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and

the other two polypeptide chains each comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-L-V_L2-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα; V_L2 is a light chain variable region that specifically binds to C5aR1; L is a linker; and C_L is a light chain constant region;

wherein V_H1 and V_L1 constitute the first antigen-binding domain specifically recognizing GM-CSFRα and V_H2 and V_L2 constitute the second antigen-binding domain specifically recognizing C5aR1.
The multi-specific antibody of claim 12 or 13, which comprises:

the amino acid sequence of any one of SEQ ID NOs: 101-102, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101-102; and/or

the amino acid sequence of any one of SEQ ID NOs: 79-80, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79-80.
The multi-specific antibody of claim 12 or 13, which comprises:

the amino acid sequence of any one of SEQ ID NOs: 103-104, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of any one of SEQ ID NOs: 103-104; and/or

the amino acid sequence of any one of SEQ ID NOs: 81-82, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of any one of SEQ ID NOs: 81-82.
The multi-specific antibody of claim 12 or 13, which comprises:

(i) the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 79;

(ii) the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 75; and the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 80;

(iii) the amino acid sequence of SEQ ID NOs: 76, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NO: 79, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 79;

(iv) the amino acid sequence of SEQ ID NO: 76, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 76; and the amino acid sequence of SEQ ID NOs: 80, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 80;

(v) the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 81;

(vi) the amino acid sequence of SEQ ID NO: 77, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 77; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 82;

(vii) the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 81, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 81; or

(viii) the amino acid sequence of SEQ ID NO: 78, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 78; and the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 82.
The multi-specific antibody of any one of claims 1-11, comprising four polypeptide chains, wherein:

two polypeptide chains each comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, wherein V_H1 is a heavy chain variable region that specifically binds to C5aR1; V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; V_L2 is a light chain variable region that specifically binds to GM-CSFRα; L1 and L2 are linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc region comprises C_H2 and C_H3 domains; and

the other two polypeptide chains each comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1, and C_L is a light chain constant region;

wherein V_H1 and V_L1 constitute the first antigen-binding domain specifically recognizing C5aR1 and V_H2 and V_L2 constitute the second antigen-binding domain specifically recognizing GM-CSFRα.
The multi-specific antibody of any one of claims 1-11, comprising four polypeptide chains, wherein:

two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1-Fc-L1-V_H2-L2-V_L2, or V_H1-C_H1-Fc-L1-V_L2-L2-V_H2, wherein V_H1 is a heavy chain variable region that specifically binds to GM-CSFRα; V_H2 is a heavy chain variable region that specifically binds to C5aR1; V_L2 is a light chain variable region that specifically binds to C5aR1; L1 and L2 are linkers; C_H1 is a heavy chain constant region C_H1 domain; Fc region comprising C_H2 and C_H3 domains; and

the other two polypeptide chains comprise polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα, and C_L is a light chain constant region;

wherein V_H1 and V_L1 constitute the first antigen-binding domain specifically recognizing GM-CSFRα and V_H2 and V_L2 constitute the second antigen-binding domain specifically recognizing C5aR1.
The multi-specific antibody of claim 17 or 18, wherein the V_H2 and V_L2 further comprise cysteines substitution.
The multi-specific antibody of any one of claims 17-19, which comprises:

(i) the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 83; and/or the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 87;

(ii) the amino acid sequence of SEQ ID NO: 84, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 84; and/or the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 88;

(iii) the amino acid sequence of SEQ ID NO: 85, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 85; and/or the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 87; or

(iv) the amino acid sequence of SEQ ID NO: 86, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 86; and/or the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 88.
The multi-specific antibody of any one of claims 1-11, comprising four polypeptide chains, wherein:

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1, wherein V_H1 is a heavy chain variable region specifically binding to C5aR1; C_H1 is a heavy chain constant region C_H1 domain; optionally, the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains;

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to C5aR1, and C_L is a light chain constant region;

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H2-C_L, wherein V_H2 is a heavy chain variable region that specifically binds to GM-CSFRα; C_L is a light chain constant region; optionally, the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L2-C_H1, wherein V_L2 is a light-chain variable region that specifically binds to GM-CSFRα, and C_H1 is a heavy-chain constant region C_H1 domain;

wherein V_H1 and V_L1 constitute the first antigen-binding domain specifically recognizing C5aR1 and V_H2 and V_L2 constitute the second antigen-binding domain specifically recognizing GM-CSFRα.
The multi-specific antibody of any one of claims 1-11, comprising four polypeptide chains, wherein:

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H1-C_H1, wherein V_H1 is a heavy chain variable region specifically binding to GM-CSFRα; C_H1 is a heavy chain constant region C_H1 domain; optionally, the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains;

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L1-C_L, wherein V_L1 is a light chain variable region that specifically binds to GM-CSFRα, and C_L is a light chain constant region;

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_H2-C_L, wherein V_H2 is a heavy chain variable region that specifically binds to C5aR1; C_L is a light chain constant region; optionally, the polypeptide chain may further comprise Fc region comprising C_H2 and C_H3 domains; and

one polypeptide chain comprises polypeptide sequences in the orientation from N-terminus to the C-terminus: V_L2-C_H1, wherein V_L2 is a light-chain variable region that specifically binds to C5aR1, and C_H1 is a heavy-chain constant region C_H1 domain;

wherein V_H1 and V_L1 constitute the first antigen-binding domain specifically recognizing GM-CSFRα and V_H2 and V_L2 constitute the second antigen-binding domain specifically recognizing C5aR1.
The multi-specific antibody of claim 21 or 22, which comprises:

(i) the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 105; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 106, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 106; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 98; or

(ii) the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 107; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 108; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 100.
The multi-specific antibody of claim 21 or 22, which comprises:

(i) the amino acid sequence of SEQ ID NO: 89, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 89; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 90, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 90; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 98; or

(ii) the amino acid sequence of SEQ ID NO: 91, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 91; and/or the amino acid sequence of SEQ ID NO: 97, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 97; and/or the amino acid sequence of SEQ ID NO: 92, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 92; and/or the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 98;

(iii) the amino acid sequence of SEQ ID NO: 93, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 93; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 94, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 94; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 100; or

(iv) the amino acid sequence of SEQ ID NO: 95, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 95; and/or the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 99; and/or the amino acid sequence of SEQ ID NO: 96, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 96; and/or the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 100.
The multi-specific antibody of any one of claims 1-24, wherein:

(i) the Fc region is selected from the group consisting of an Fc region from an IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; and/or

(ii) the Fc region comprises one or more amino acid mutations; and/or

(iii) the Fc region is aglycosylated or deglycosylated; and/or

(iv) the Fc region has reduced fucosylation or is afucosylated.
The multi-specific antibody of claim 25, wherein the Fc is a Fc knob comprising the amino acid substitutions T366W, optionally further comprises S354C, wherein the numbering is according to the EU numbering.
The multi-specific antibody of claim 25, wherein the Fc is a Fc hole comprising the amino acid substitutions T366S, L368A, and Y407V, optionally further comprises Y349C, wherein the numbering is according to the EU numbering.
The multi-specific antibody of any one of claims 1-27 is bispecific antibody.
An isolated nucleic acid molecule that encodes the multi-specific antibody of any one of claims 1-28.
A vector comprising the isolated nucleic acid molecule of claim 29.
An isolated host cell comprising the vector of claim 30.
A pharmaceutical composition comprising the multi-specific antibody of any one of claims 1-28, the isolated nucleic acid molecule of claim 29, the vector of claim 30, or the isolated host cell of claim 31.
A method of treating and/or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of the multi-specific antibody of any one of claims 1-28, the isolated nucleic acid molecule of claim 29, the vector of claim 30, or the isolated host cell of claim 31, or the pharmaceutical composition of claim 32.
The method of claim 33, wherein the disease or condition is selected from the group consisting of an inflammatory disease, respiratory disease, autoimmune disease, and cancers.
The method of claim 34, wherein the disease or condition is selected from the group consisting of allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and IgE-mediated allergic reactions, shock, rheumatoid arthritis, atherosclerosis, multiple sclerosis, allograft rejection, fibrotic disease, asthma, inflammatory glomerulopathies, chronic obstructive pulmonary disease, multiple sclerosis, myeloid leukemia, atherosclerosis, and acute respiratory distress syndrome (ARDS) .