OA21300A - Anti-ccr8 monoclonal antibodies and uses thereof. - Google Patents

Anti-ccr8 monoclonal antibodies and uses thereof. Download PDF

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Publication number
OA21300A
OA21300A OA1202300162 OA21300A OA 21300 A OA21300 A OA 21300A OA 1202300162 OA1202300162 OA 1202300162 OA 21300 A OA21300 A OA 21300A
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amino acid
acid sequence
seq
antibody
cancer
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OA1202300162
Inventor
Runsheng LI
Wentao Huang
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Lanova Medecines Limited
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Publication of OA21300A publication Critical patent/OA21300A/en

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Abstract

Provided are antibodies or fragment thereof having binding specificity to the human chemokine (C-C motif) receptor 8 (CCR8) protein.These antibodies are capable of binding to CCR8 at high affinity and can mediate antibody-dependent cellular cytotoxicity (ADCC).

Description

ANTI-CCR8 MONOCLONAL ANTIBODIES AND USES THEREOF
BACKGROUND
[1] Chemokine (C-C motif) receptor 8 (CCR8) is a member of the beta chemokine receptor family, and is a seven transmembrane protein similar to G protein-coupled receptors. Chemokines and their receptors are important for the migration of varions cell types into the inflammatory sites. This receptor protein preferentially expresses in the thymus. The ligand of thé CCR8 is CCL1. CCL8 also fonctions as a CCR8 agonist.
[2] CCR8 is expressed principally on regulatory T cells (Treg) and is important for CCR8+ Treg-mediated immunosuppression. Recent studies hâve demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of cancer patients. It was also demonstrated that CCR8+ myeloid cells were expanded in patients with cancer.
[3] Antibodies targeting CCR8 hâve been shown fo significantly suppress tumor growth and improve long-term survival in animal models. This antitumor activity correlated with increased tumorspecificT cells, and enhanced infiltration of CD4+ and CD8+ T cells.
Treatment with the antibodies prevented induction and suppressivefonction of Tregs without affecting CD8+ T cells. Targeting CCR8, therefore, is a promising cancer immunotherapy approach.
SUMMARY
[4] Anti-CCR8 antibodies are discovered herein that hâve high binding affinity to the human CCR8 protein, and are efficient in mediating antibody-dependent cellular cytotoxicity (ADCC).
[5] In one embodiment, an antibody or fragment thereof is provided, having binding specificity to a human chemokine (C-C motif) receptor 8 (CCR8) protein. The antibody or fragment thereof comprises a heavy chain variable région comprising heavy chain complementarity determining régions CDRH1, CDRH2, and CDRH3 and a light chain variable région light chain comprising complementarity determining régions CDRL1, CDRL2, and CDRL3. In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 22, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 23, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 24, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27. .
[6] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 35, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 37, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27.
[7] Also provided, in some embodiments, are compositions comprising the antibody or fragment thereof and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a second antibody having specificity to a tumor antigen. In some embodiments, the second antibody is a tumor-opsonizing antibody.
[8] Methods and uses for the treatment of diseases and conditions are also provided. In one embodiment, provided is a method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or fragment thereof of the présent disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[9] FIG. 1 shows the CCR8-binding affinity of the chimeric antibodies with murine
VH/VL.
[10] FIG. 2 shows the ADCC activity of three of the chimeric antibodies.
[11] FIG. 3 showsthat humanized 88D2C6 antibodies hâve high affmities.
[12] FIG. 4 shows that humanized 137D1H10 antibodies hâve high affmities.
[13] FIG. 5 shows that, compared to the chimeric antibodies, the humanized antibodies hâve significantly higher ADCC activities.
[14] FIG. 6 shows the results of cell-based binding of LM-108 and reference antibody to human CCR8 by flow cytometry. Upper panel: FACS binding of LM-108 on human CCR8 expressing HEK293. Lower panel: FACS binding of LM-108 on HEK293 cells.
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[15] FIG. 7 shows cell-based binding of LM-108 to human CCR8 high expressing U2OS cells by flow cytometry.
[16] FIG. 8 shows cell-based binding of LM-108 to human CCR8 low expressing Jurkat cells by flow cytometry.
[17] FIG. 9 shows the results of ADCC Reporter Gene Assay of LM-108 with Jurkat/CD16a(158v)/NFAT-luc cells, targeting CCR8 high expressing U2OS cells.
[18] FIG. 10 shows the results of ADCC Reporter Gene Assay of LM-108 with Jurkat/CD16a(158v)/NFAT cells, targeting CCR8 low expressing Jurkat cells.
[19] FIG. 11 shows the results of ADCC testing of LM-108 with hPBMCs targeting CCR8-expressing cells (upper panel) or control cells (lower panel).
[20] FIG. 12 shows ADCP of LM-108 with Monocyte-derived Macrophage by FACS. Upper panel: ADCP of LM-108 LM-108 towards human CCR8 over expressing CHO-K1 cells. Lower panel: ADCP of LM-108 LM-108 towards control CHO-K1 cells.
[21] FIG. 13 présents imaging results of ADCP of LM-108 with Monocyte-derived Macrophage by Operetta.
[22] FIG. 14 shows tumor growth curves of CT26 tumor-bearing mice post administration of LM-108m and anti-mPD-1 Ab, in an in vivo effîcacy study of LM-108m on CT26 syngeneic model with BALB/c mice.
[23] FIG. 15 shows tumor growth curves of MC3 8 tumor-bearing mice post administration of LM-108m and vehicle, in an in vivo effîcacy study of LM-108 on MC38 syngeneic model with hCCR8 kimice.
[24] FIG. 16 shows the results of cytotoxicity évaluation of LM-108-vc-MMAE on hCCR8 expressing cells.
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DETAILED DESCRIPTION
Définitions
[25] It is to be noted that the terni “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the ternis “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
[26] As used herein, the terni “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molécule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a spécifie length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the définition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acétylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by Chemical synthesis.
[27] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molécules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molécules are homologous at that position. A degree of homology between sequences is a fimetion of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the présent disclosure.
[28] A polynucleotide or polynucleotide région (or a polypeptide or polypeptide région) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %,
-421300 % or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
[29] The term “an équivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucléotide sequence having a certain degree of homology, or sequence identity, with the nucléotide sequence of the nucleic acid or complément thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucléotide sequence which has a certain degree of homology with or with the complément thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complément thereof. Likewise, “an équivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the équivalent polypeptide or polynucleotide has one, two, three, four or five addition, délétion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the équivalent sequence retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence. .
[30] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molécule that comprises at least a portion of an immunoglobulin molécule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining région (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable région, a heavy chain or light chain constant région, a framework (FR) région, or any portion thereof, or at least one portion of a binding protein.
[31] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a spécifie antigen to form a complex.
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[32] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable régions of the heavy (Vh) and light chains (Vl) of immunoglobuline. In some aspects, the régions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specifîcity of the original immunoglobulin, despite removal of the constant régions and the introduction of the linker. ScFv molécules are known in the art and are described, e.g., in US patent 5,892,019.
[33] The term antibody encompasses varions broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ 1- γ4). It is the nature of this chain that détermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGi, IgGi, IgGa, IgG4, IgGs, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. Ail immunoglobulin classes are clearly within the scope of the présent disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molécules. With regard to IgG, a standard immunoglobulin molécule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable région.
. , - i ' I
[34] Antibodies, antigen-binding polypeptides, variants, or dérivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain | antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti- idiotypie (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein). Immunoglobulin or antibody molécules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA,
-621300 and Ig Y), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molécule.
[35] Light chains are classified as either kappa or lambda (K, λ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains 1 o are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
[36] Both the light and heavy chains are divided into régions of structural and functional 15 homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions détermine antigen récognition and specifîcity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CH1, CH2 or CH3) confer important biological . properties such as sécrétion, transplacental mobility, Fc receptor binding, complément binding, and the like. By convention the numbering of the constant région domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody. The N-terminal portion is a variable région and at the C-terminal portion is a constant région; the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
[37] As indicated above, the variable région allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining régions (CDRs), of an antibody combine to form the variable région that defines a three-dimensional antigen-binding site. This quatemary antibody structure forms the antigen-binding site présent at the end of each arm of the Y.
More specifically, the antigen-binding site is defîned by three CDRs on each of the VH and VK chains (z.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molécules derived from camelid species or engineered based on camelid immunoglobulins, a complété immunoglobulin molécule may consist of heavy chains only, with no light chains. See, e.g., Hamers-CastermaneZ al., Nature 35 363:446-448 (1993).
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[38] In naturally occurring antibodies, the six “complementarity determining régions” or “CDRs” présent in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” régions, show less inter-molecular variability. The framework régions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β -sheet structure. Thus, framework régions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promûtes the noncovalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework régions, respectively, can be readily identified for any given heavy or light chain variable région by one of ordinary skill in the art, since they hâve been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917(1987)).
[39] In the case where there are two or more définitions of a terni which is used and/or accepted within the art, the définition of the term as used herein is intended to include ail such meanings unless explicitly stated to the contrary. A spécifie example is the use of the term “complementarity determining région” (“CDR”) to deseribe the non-contiguous antigen combining sites found within the variable région of both heavy and light chain polypeptides. This particular région has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR définitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either définition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can
-821300 routinely détermine which residues comprise a particular CDR given the variable région amino acid sequence of the antibody.
Kabat Chothia
CDR-H1 31-35 26-32
CDR-H2 50-65 52-58
CDR-H3 95-102 95-102
CDR-L1 24-34 26-32
CDR-L2 50-56 50-52
CDR-L3 89-97 91-96
[40] Kabat et al. also defined a numbering System for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this o System of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering System set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).
[41] In addition to table above, the Kabat number System describes the CDR régions as 15 follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR20 H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the 25 end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.
[42] Antibodies disclosed herein may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable région may 30 be condricthoid in origin (e.g., from sharks).
[43] As used herein, the term “heavy chain constant région” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy
-921300 chain constant région comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge région) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., ail or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant région may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molécule.
[44] The heavy chain constant région of an antibody disclosed herein may be derived from different immunoglobulin molécules. For example, a heavy chain constant région of a polypeptide may comprise a CH1 domain derived from an IgGi molécule and a hinge région derived from an IgG3 molécule. In another example, a heavy chain constant région can comprise a hinge région derived, in part, from an IgGj molécule and, in part, from an IgG3 molécule. In another example, a heavy chain portion can comprise a chiimeric hinge derived, in part, from an IgGj molécule and, in part, from an IgÛ4 molécule.
[45] As used herein, the terni “light chain constant région” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant région comprises at least one of a constant kappa domain or constant lambda domain.
[46] A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
[47] As previously indicated, the subunit structures and three-dimensional configuration of the constant régions of the various immunoglobulin classes are well known. As used herein, the tenu “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant
-1021300 région domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge région of an immunoglobulin heavy chain molécule.
[48] As used herein the term “CH2 domain” includes the portion of a heavy chain molécule that extends, e.g, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering System; and residues 231-340, EU numbering System; see Kabat et al., U.S. Dept. of Health and Hurrian Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 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 CH2 domains of an intact native IgG molécule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molécule and comprises approximately 108 residues.
[49] As used herein, the term “hinge région” includes the portion of a heavy chain molécule that joins the CH1 domain to the CH2 domain. This hinge région comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding régions to move independently. Hinge régions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).
[50] As used herein the term “disulfîde bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfîde bond or bridge with a second thiol group. In most naturally occurring IgG molécules, the CH1 and CK régions are linked by a disulfîde bond and the two heavy chains are linked by two disulfîde bonds at positions corresponding to 239 and 242 using the Kabat numbering System (position 226 or 229, EU numbering System).
[51] As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive région or site is obtained or derived from a first species and the constant région (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In certain embodiments the target binding région or site will be from a non-human source (e.g. mouse or primate) and the constant région is human.
[52] As used herein, “percent humanization” is calculated by determining the number of framework amino acid différences (Le., non-CDR différence) between the humanized domain
-il21300 and the germline domain, subtracting that number from the total number of amino acids, and then dividing that by the total number of amino acids and multiplying by 100.
[53] By “specifically binds” or “has specifïcity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this définition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The terni “specifïcity” is used herein to qualify the relative affînity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to hâve a higher specifïcity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specifïcity than it has for related epitope “D.” '
[54] As used herein, the ternis “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Bénéficiai or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) State of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether détectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to hâve the condition or disorder or those in which the condition or disorder is to be prevented.
[55] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animais, farm animais, and zoo, sport, or pet animais such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. '
[56] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefît from
-1221300 administration of an antibody or composition of the présent disclosure used, e.g., for détection, for a diagnostic procedure and/or for treatment.
Anti-CCR8 Antibodies
[57] The présent disclosure provides anti-CCR8 antibodies and fragments that hâve high affmity to the human CCR8 protein, are potent in mediating ADCC and ADCP, and showed potent in vivo tumor inhibitor effîcacy. Interesting, when using an anti-CCR8 antibody (from WO2020138489) as a benchmark, the instantly disclosed antibodies exhibited no binding to non-CCR8-expressing cells, while the benchmark antibody bound to those control cells as well. The présent antibodies, therefore, can be expected to hâve less undesired adverse effects in clinical uses. These antibodies, therefore, are suitable agents for treating various diseases featuring overexpressed CCR8, such as cancer.
[58] In accordance with one embodiment of the présent disclosure, therefore, provided are antibodies and antigen-binding fragments thereof that are able to bind to CCR8. Example antibodies include those murine ones listed in Table l(e.g., 137D1H10, 88D2C6, 89B6F8, 132H8E10,10F11B2,40H10F3, 53D6A9, 352H11C11,362G7D3, 362H10A3, 367D10E7, 370D2C10), as well as humanized ones of Tables 2-3. Also included are those that include the same CDRs as illustrated herein. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as those illustrated here, and those that compete with the instantly disclosed in binding to CCR8.
[59] In accordance with one embodiment of the présent disclosure, provided is an antibody or fragment thereof that includes the heavy chain and light chain variable domains with the CDR régions disclosed herein, as well as their biological équivalents.
[60] In one embodiment, the CDRs are those of 88D2C6 or its humanized counterparts, as exemplified in Tables 2B and 2D. In one embodiment, the CDRH1 comprises the amino acid sequence of SEQID NO: 22 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL1 comprises the amino acid
-1321300 sequence of SEQ ID NO: 25 or 28 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof.
[61] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof.
[62] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 22, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 23, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 24, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27.
[63] Also provided, in some embodiments, are those that include the same CDRs as 88D2C6 or its humanized counterparts. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as 88D2C6 or its humanized counterparts, and those that compete with any of them in binding to CCR8.
[64] In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 (mouse or chimeric) and 29-31 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence
-1421300 selected from the group consisting of SEQID NO:3(mouse or chimeric) and 29-31 (humanized).
[65] In some embodiments, the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 (mouse or chimeric), 32-34 and 41-43 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO (mouse or chimeric):2, 32-34 and 41-43 (humanized).
[66] In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:29 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO:32-34 and 41-43. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:30 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO:32-34 and 4143.In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:31 and the light chain variable région comprises the amino acid sequence of any one Of SEQ ID NO:32-34 and 41-43.In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:29 and the light chain variable région comprises the amino acid sequence of SEQ ID NO:33.
[67] In one embodiment, the CDRs are those of 137D1H10 or its humanized counterparts,. as exemplified in Tables 3B and 3D. In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof. .
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[68] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof.
[69] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 35, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 37, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27.
[70] Also provided, in some embodiments, are those that include the same CDRs as 137D1H10 or its humanized counterparts. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as 137D1H10 or its humanized counterparts, and those that compete with any of them in binding to CCR8.
[71] In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 (mouse or chimeric) and 38-40 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3 (mouse or chimeric) and 29-31 (humanized).
[72] In some embodiments, the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 (mouse or chimeric), 32-34 and 41-43 (humanized), or a peptide having at least 90% sequence identity to an amino acid
-1621300 sequence selected from the group consisting of SEQ ID NO (mouse or chimeric):2, 32-34 and 41-43 (humanized).
[73] In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:38 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO:32-34 and 41-43. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:39 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO:32-34 and 4143.In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:40 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO:32-34 and 41-43.In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:40 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO:42.
[74] The antibodies that contained these CDR régions, whether mouse, humanized or chimeric, had potent CCR8 binding and inhibitory activities. As shown in Example 4, certain residues within the CDR can be modifîed to retain or improve the property or reduce their potential to hâve post-translational modifications (PTMs). Such modifîed CDR can be referred to as affînity matured or de-risked CDRs (e.g., SEQ ID NO:25).
[75] Modifîed CDRs can include those having one, two or three amino acid addition, délétion and/or substitutions. In some embodiments, the substitutions can be conservative substitutions.
[76] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains hâve been defined in the art, including basic side chains {e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, méthionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another
-1721300 embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
[77] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.
o Table A. Amino Acid Similarity Matrix
C G P S A T D E N Q H K R V M 1 L F Y w
w -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6
1 -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 . 0 2 6
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6
K -5 -2 -1 0 -1 0 0 0 1 1 0 5
H -3 -2 0 -1 -1 -1 1 1 2 3 6
Q -5 -1 0 -1 0 -1 2 2 1 4
N -4 0 -1 1 0 0 2 1 2
E -5 0 -1 0 0 0 3 4
D -5 1 -1 0 0 0 4
T -2 0 0 1 1 3
A -2 1 1 1 2
S 0 1 1 1
P -3 -1 6
G -3 5
C 12
Table B. Conservative Amino Acid Substitutions
For Amino Acid Substitution With
Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys
Arginine D-Arg, Lys, D-Lys, Orn D-Orn
Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gin, D-GIn
Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-GIn
Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser
Glutamine D-GIn, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-GIn
Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala
Isoleucine D-lle, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine Val, D-Val, Met, D-Met, D-lle, D-Leu, Ile
Lysine D-Lys, Arg, D-Arg, Orn, D-Orn
Méthionine D-Met, S-Me-Cys, Ile, D-lle, Leu, D-Leu,Val, D-Val
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Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp
Proline D-Pro
Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys
Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,Val, D-Val
Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp
Valine D-Val, Leu, D-Leu, Ile, D-lle, Met, D-Met
[78] It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g, hâve a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.
[79] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a fimctional moiety (e.g., PEG, a drug, a toxin, or a label).
[80] Antibodies, variants, or dérivatives thereof of the disclosure include dérivatives that are modified, i.e., by the covalent attachment of any type of molécule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acétylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous Chemical modifications may be carried out by known techniques, including, but not limited to spécifie Chemical cleavage, acétylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.
[81] In some embodiments, the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
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[82] The antibodies may be conjugated or fused to a therapeutic agent, which may include détectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
[83] The antibodies can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium sait and oxalate ester.
[84] The antibodies can also be detectably labeled using fluorescence emitting metals such as Eu, or others of the lanthanide sériés. These metals can be attached to the antibody using such métal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to an antibody are well known, see, e.g., Arnone/ al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeldétf al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623- 53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pincheraei al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Détection And Therapy, Baldwin et al. (eds.), Academie Press pp. 303-16 (1985), and Thorpe et al., “The Préparation And Cytotoxic Properties Of Antibody-Toxin Conjugales ”, Immunol. Rev. (52:119-58 (1982)).
Bi-functional Molécules and Combination Thérapies
[85] CCR8 is a chemokine and a tumor antigen. As a tumor antigen targeting molécule, an antibody or antigen-binding fragment spécifie to CCR8 can be combined with a second antigen-binding fragment spécifie to an immune cell, or an antigen-binding fragment spécifie to an immune checkpoint to generate a combination therapy or a bispecific antibody.
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I
[86] In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell. Molécules on the immune cell which can be targeted include, for example, CCL1, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1,PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD 122, 4-
IBB (also known as CD 137), TIM3, OX-40 or OX40L, CD40orCD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer-cell immunoglobulin-like receptors (KIRs), and CD47.
[87] As an immune checkpoint inhibitor, an antibody or antigen-binding fragment spécifie to CCR8 can be combined with a second antigen-binding fragment spécifie to a tumor •15 antigen to generate a bispecific antibody. A “tumor antigen” is an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for use in cancer therapy. Normal proteins in the body are not antigenic. Certain proteins, however, are produced or overexpressed during tumorigenesis and thus appear “foreign” to the body. This may include
2ù normal proteins that are well sequestered from the immune System, proteins that are normally produced in extremely small quantifies, proteins that are normally produced only in certain stages of development, or proteins whose structure is modifîed due to mutation.
[88] An abundance of tumor antigens are known in the art and new tumor antigens can be readily identified by screening. Non-limiting examples of tumor antigens include EGFR, 25 Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, aVp3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.
[89] In some aspects, the monovalent unit has specifîcity to a protein that is overexpressed on a tumor cell as compared to a corresponding non-tumor cell. A “corresponding non-tumor cell” as used here, refers to a non-tumor cell that is of the same cell type as the origin of the tumor cell. It is noted that such proteins are not necessarily different from tumor antigens. Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectum, breast, lung, pancréas and gastrointestinal tract carcinomas; heregulin receptors (HÈR-2, neu or c-erbB-2), which is frequently overexpressed in breast, ovarian, colon, lung, prostate and cervical cancers; epidermal growth factor receptor (EGFR), which is
-2121300 highly expressed in a range of solid tumors including those of the breast, head and neck, nonsmall cell lung and prostate; asialoglycoprotein receptor; transferrin receptor; serpin enzyme complex receptor, which is expressed on hépatocytes; fîbroblast growth factor receptor (FGFR), which is overexpressed on pancreatic ductal adenocarcinoma cells; vascular endothélial growth factor receptor (VEGFR), for anti-angiogenesis gene therapy; folate receptor, which is selectively overexpressed in 90% of nonmucinous ovarian carcinomas; cell surface glycocalyx; carbohydrate receptors; and polymeric immunoglobulin receptor, which is useful for gene delivery to respiratory épithélial cells and attractive for treatment of lung diseases such as Cystic Fibrosis.
[90] Different format of bispecifîc antibodies are also provided. In some embodiments, each of the anti-PD-Ll fragment and the second fragment each is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecifîc antibody further includes a Fc fragment.
[91] Bifunctional molécules that include not just antibody or antigen binding fragment are also provided. As a tumor antigen targeting molécule, an antibody or antigen-binding fragment spécifie to CCR8, such as those described here, can be combined with an immune cytokine or ligand optionally through a peptide linker. The linked immune cytokines or ligands include, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bi-functional molécules can combine the immune checkpoint blocking effect with tumor site local immune modulation.
Antibody-Drug Conjugales
[92] In some embodiments, the antibodies or fragments may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifîers, pharmaceutical agents, or PEG.
[93] In one embodiment, the antibodies or fragments of the disclosure are covalently attached to a drug moiety. The drug moiety may be, or be modified to include, a group reactive with a conjugation point on the antibody. For example, a drug moiety can be attached by alkylation (e.g., at the epsilon-amino group lysines or the N-terminns of antibodies), reductive amination of oxidized carbohydrate, transestérification between
-2221300 hydroxyl and carboxyl groups, amidation at amino groups or carboxyl groups, and conjugation to thiols.
[94] In some embodiments, the number of drug moieties, p, conjugated per antibody molécule ranges from an average of 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p ranges from an average of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4 or about 2 to about 3.
[95] For example, when Chemical activation of the protein results in formation of free thiol groups, the protein may be conjugated with a sulfhydryl reactive agent. In one aspect, the agent is one which is substantially spécifie for free thiol groups. Such agents include, for example, malemide, haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio.
[96] The drug can be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l-carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, —SH), while its Sulfo-NHS ester is reactive toward primary amines (as found in Lysine and the protein or peptide N-terminus). Yet another linker is maleimidocaproyl (me). Other suitable linkers include linkers hydrolyzable at a spécifie pH or a pH range, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers The linker may be covalently bound to the antibody to such an extent that the antibody must be degraded intracellularly in order for the drug to be released e.g. the me linker and the like.
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[97] A linker can include a group for linkage to the antibody. For example, linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio).
[98] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the like. The conjugate can be used for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The conjugate can be used accordingly in a variety of settings for the treatment of animal cancers. The conjugate can be used to deliver a drug to a tumor cell or cancer cell. Without being bound by theory, in some embodiments, the conjugate binds to or associâtes with a cancer cell expressing claudin 18.2, and the conjugate and/or drug can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.
[99] Once inside the cell, one or more spécifie peptide sequences within the conjugate (e.g., in a linker) are hydrolytically cleaved by one or more tumor-cell or cancer-cellassociated proteases, resulting in release of the drug. The released drug is then free to migrate within the cell and induce cytotoxic or cytostatic or other activities. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug subsequently pénétrâtes the cell, or acts at the cell surface.
[100] Examples of drug moieties or payloads are selected from the group consisting of DM1 (maytansine, N2’-deacetyl-N2’-(3-mercapto-l-oxopropyl)- or N2’-deacetyl-N2’-(3mercapto-l-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproylmonomethylauristatin-D or N-methyl-L-valyl-N-[(lS,2R)-2-methoxy-4-[(2S)-2-[(lR,2R)-lmethoxy-2-methyl-3-oxo-3-[[(lS)-2-phenyl-l-(2-thiazolyl)ethyl]amino]propyl]-l-pyr rolidinyl]-1 -[(1 S)-1 -methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)l-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4(methylamino)heptanoyl-(aR, PR,2S)-P-methoxy-a-methyl-2-pyrrolidinepropanoyl-Lphenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(paminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxolH-pyrrol-l-yl)-l-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-
-2421300 omithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(lS,2R)-4-[(2S)-2[( 1 R,2R)-3 -[[( 1 R,2S)-2-hydroxy-1 -methyl-2-phenylethyl] amino]-1 -methoxy-2-methyl-3oxopropyl]-l-pyrrolidinyl] -2-methoxy-l-[(lS)-l-methylpropyl]-4-oxobutyl]-N-methyl-Lvalinamide). DM1 is a dérivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF are auristatin dérivatives. In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE. In some embodiments, the drug moiety is a maytansinoid or an auristatin.
[101] The antibodies or fragments may be conjugated or fused to a therapeutic agent, which may include détectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
[102] The antibodies can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a Chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium sait and oxalate ester.
[103] The antibodies can also be detectably labeled using fluorescence emitting metals such as Eu, or others of the lanthanide sériés. These metals can be attached to the antibody using such métal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to an antibody are well known, see, e.g., Arnonet al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeldei al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623- 53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pincheraef al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Détection And Therapy, Baldwin et al. (eds.),
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Academie Press pp. 303-16 (1985), and Thorpe et al., “The Préparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunot Rev. (52:119-58 (1982)).
Polynucleotides Encoding the Antibodies andMethods of Preparing the Antibodies
[104] The présent disclosure also provides isolated polynucleotides or nucleic acid molécules encoding the antibodies, variants or dérivatives thereof of the disclosure. The polynucleotides of the présent disclosure may encode the entire heavy and light chain variable régions of the antigen-binding polypeptides, variants or dérivatives thereof on the same polynucleotide molécule or on separate polynucleotide molécules. Additionally, the polynucleotides of the présent disclosure may encode portions of the heavy and light chain variable régions of the antigen-binding polypeptides, variants or dérivatives thereof on the same polynucleotide molécule or on separate polynucleotide molécules.
[105] Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant régions of the antigen-binding polypeptides of the présent disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a spécifie antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci hâve been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties.
Treatment Methods
[106] As described herein, the antibodies, variants or dérivatives of the présent disclosure may be used in certain treatment and diagnostic methods. .
[107] The présent disclosure is further directed to antibody-based thérapies which involve administering the antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and dérivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and dérivatives thereof as described herein).
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[108] The antibodies of the disclosure can also be used to treat or inhibit cancer. In some embodiments, the cancer cells in the patient express or overexpress CCR8. As provided above, CCR8 can be overexpressed in tumor cells, in particular gastric, pancreatic, esophageal, ovarian, and lung tumors. Inhibition of CCR8 has been shown to be useful for treating the tumors.
[109] Accordingly, in some embodiments, provided are methods for treating a cancer in a patient in need thereof. The method, in one embodiment, entails administering to the patient an effective amount of an antibody of the présent disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient over-express CCR8.
[110] Cellular thérapies, such as chimeric antigen receptor (CAR) T-cell thérapies, are also provided in the présent disclosure. A suitable cell can be used, that is put in contact with an anti-CCR8 antibody of the présent disclosure (or altematively engineered to express an antiCCR8antibody of the présent disclosure). In some embodiments, the antibody is presented in a chimeric antigen receptor (CAR). Upon such contact or engineering, the cell can then be introduced to a cancer patient in need of a treatment. The cancer patient may hâve a cancer of any of the types as disclosed herein. The cell (e.g., T cell) can be, for instance, a tumorinfiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation.
[111] In some embodiments, the cell was isolated from the cancer patient him- or her-self. In some embodiments, the cell was provided by a donor or from a cell bank. When the cell is isolated from the cancer patient, undesired immune reactions can be minimized
[112] Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endométrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric, pancreatic, esophageal, ovarian, and lung cancers.
[113] Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the antibodies or variants, or dérivatives thereof of the disclosure include, but are not limited to, progression, and/or métastasés of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic,
-2721300 myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, ostéogénie sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, rénal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, épithélial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.
[114] A spécifie dosage and treatment regimen for any particular patient will dépend upon a variety of factors, including the particular antibodies, variant or dérivative thereof used, the patient's âge, body weight, general health, sex, and diet, and the time of administration, rate of excrétion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The âmount will also dépend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
[115] Methods of administration of the antibodies, variants or include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subeutaneous, intranasal, épidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through épithélial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Thus, pharmaceutical
-2821300 compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
[116] The term “patenterai” as used herein refers to modes of administration which include 10 intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intra-articular injection and infusion.
[117] Administration can be systemic or local. In addition, it may be désirable to introduce the antibodies of the disclosure into the central nervous System by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be 15 facilitated by an intraventricular cathéter, for example, attached to a réservoir, such as an
Ommaya réservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[118] It may be désirable to administer the antigen-binding polypeptides or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, 20 and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a cathéter, by means of a suppository, or by means of an implant, said implant being of a porous, nonporous, or gelâtinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the disclosure, caré must 25 be taken to use materials to which the protein does not absorb.
[119] The amount of the antibodies of the disclosure which will be effective in the treatment, inhibition and prévention of an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The précisé dose 30 to be employed in the formulation will also dépend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test Systems.
[120] As a general proposition, the dosage administered to a patient of the antigen-binding 35 polypeptides of the présent disclosure is typically 0.1 mg/kg to 100 mg/kg of the patient's
-2921300 body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies hâve a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the disclosure may be reduced by enhancing uptake and tissue pénétration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
[121] In an additional embodiment, the compositions of the disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, 15 IL-13, IL-15, anti-CD40, CD40L, and TNF-a.
[122] In additional embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
Diagnostic Methods
[123] Over-expression of CCR8 is observed in certain tumor samples, and patients having
CCR8-over-expressing cells are likely responsive to treatments with the anti-CCR8 antibodies of the présent disclosure. Accordingly, the antibodies of the présent disclosure can also be used for diagnostic and prognostic purposes.
[124] A sample that preferably includes a cell can be obtained from a patient, which can be 25 a cancer patient or a patient desiring diagnosis. The cell be a cell of a tumor tissue or a tumor block, a blood sample, a urine sample or any sample from the patient. Upon optional pretreatment of the sample, the sample can be incubated with an antibody of the présent disclosure under conditions allowing the antibody to internet with a CCR8 protein potentially présent in the sample. Methods such as ELISA can be used, taking advantage of the anti-
CCR8 antibody, to detect the presence of the CCR8 protein in the sample.
[125] Presence of the CCR8 protein in the sample (optionally with the amount or concentration) can be used for diagnosis of cancer, as an indication that the patient is suitable for a treatment with the antibody, or as an indication that the patient has (or has not) responded to a cancer treatment. For a prognostic method, the détection can be done at once,
-3021300 twice or more, at certain stages, upon initiation of a cancer treatment to indicate the progress of the treatment.
Compositions
[126] The présent disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody, and an acceptable carrier. In some embodiments, the composition further includes a second anticancer agent (e.g., an immune checkpoint inhibitor).
[127] In a spécifie embodiment, the terni “pharmaceutically acceptable” means approved by a regulatory agency of the Fédéral or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animais, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
[128] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be stérile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, minerai oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stéarate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, éthanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acétates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, émulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycérides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnésium stéarate, sodium saccharine, cellulose, magnésium carbonate, etc. Examples of
-311 suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The w parental préparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[129] In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in stérile isotonie aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingrédients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing stérile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of stérile water for injection or saline can be provided so that the ingrédients may be mixed prior to administration.
[130] The compounds of the disclosure can be formulated as neutral or sait forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferrie hydroxides, isopropylamine, triethylamine, 2-ethylamino éthanol, histidine, procaine, etc.
EXAMPLES
3Ό Example 1: Génération of murine monoclonal antibodies against human CCR8
[131] The humanCCR8 protein was used to immunize different strains of mice, and hybridomaswere generated accordingly. CCR8 positive binders wereselected and subcloned. Subsequently, in vitro binding and functional screening were carried out and lead antibodies with highest binding affînity and strongest functional potency were identified.
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[132] The VH/VL sequences of the lead murine antibodies are provided in the table below.
Table 1. VH/VL sequence of the lead murine antibodies
Name Sequence (CDRs are underlined) SEQ ID NO:
137D1H10 VH EVQLVESGGGLVQPKGSLKLSCTASGFTFNTYAMNWVRQAPGKGL EWVARIRSKANNYATYYADSVKDRFTISRDDSQRILYLQMNNLKA EDTAMYYCVRDRSRGEDYAMDYWGQGTSVTVSS 1
137D1H10 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYP FTFGAGTKLELK 2
88D2C6 VH ΞVQLVETGGGLVQPKGS LKLS CAAS GFTFNPNAMNWVRQAPGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKT EDTAMYYCVRGKDDGYRHYAMDYWGQGTSVTVS S . 3
88D2C6 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYP FTFGAGTKLELK 2
89B6F8 VH DVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRL EWVATISSGDSYTYYPDSVKGRFTISRDNAKNTLYLRMSSLKSED TAMYYCTRDHYRYDVYAMDYWGQGTSVTVSS 4
89B6F8 VL DIVMTQS PS SLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQK PGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA VYYCQNDYSYPLTFGAGTKLELK 5
132H8E10 VH QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYAVNWVRQPTGKGL EWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLOTDDT ARYYCARDSLYGNYPAYWGQGTLVTVSA 6
132H8E10 VL DIQMTQSPSSLSASLGGKVTITCKASQDTNKYMAWYQHKPGKGPR LLIHSTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQ YDNLLTFGGGTKLEIK 7
10F11B2 VH QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYSVHWVRQHSGKTL VWMGRLWSDGDTSYNSAFTSRLSISRDTSKSQVFLKMNSLQTEDT GTYYCARKAPNGGAFDYWGQGVMVTVS S 8
10F11B2 VL QWLTQPKS VSTS LE ST VKLS CKLNSGNIGSYYVHWYQQHEGRS P TNMIYRDDKRPDGVPDRFSGSIDS S SNSAFLTINNVQTEDEAIYF CHSSDSSIKCIFGGGTKLTVL 9
40H10F3 VH QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGL EWMGRMRYYGDTSFSSALK5RLSISRDTSKNQVFLKMSSLQTDDT GTYYCTRGPP SYYRGDFFDYWGQGVMVAVSS 10
40H10F3 VL DIQMTQSPSLLSASVGDRVTINCKASLNINNYLNWYQQKLGEAPK LLIDNTNNLQTGIPSRFSGSGSGTDYTLTISNLQPEDFGTYFCFQ HNGWPLTFGSGTKLEIK 11
53D6A9 VH EVQLVESGGSLVQPGRSLKLSCAASGFTYNNYVMAWVRRAPTKGL EWVASISTDGVSTQYRDSVKGRFTISRDNAKTSLFLHMDSLRSED TATYYCAKDAARGLYGQGGYFDFWGQGVMVTVSS 12
53D6A9 VL DIQMTQSPASLSASLGETVSIECLASEGISNDLAWYQQKSGKSPQ LLIYAATRLEGGVPSRFSGSGSGTRFSLKISGMQFEDEADYFCQQ SYKYPWTFGGGTKLELK 13
352H11C11 VH EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMJWVRQAPGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKT EDTAMYYCVRGGPIYHMDCWGQGTSVTVSS 14
352H11C11 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGSGTKLEIK 15
362G7D3 VH EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKT 16
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EDTAMYYCVRDPGLRQGMDYWGQGTSVTVSS
362G7D3 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGSGTKLEIK 15
362H10A3 VH EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSOSMLYLOMNNLKT EDTAMYYCVRGGGNYRGDYFDYWGQGTTLTVSS 17
362H10A3 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGSGTKLEIK 15
367D10E7 VH EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSOSMLYLOMNNLKT EDTAMYY CVRYDRSYAMDYWGQGTSVTVSS 18
367D10E7 VL vl QIVLTQSPAIMSAS PGEKVTITCSAS S SVSYMHWFQQKPGTSPKL WIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQR SSYPLTFGAGTKLELK 19
367D10E7 VL v2 DIVMTQAAPSVPVTPGE SVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGAGTKLELK 2
370D2C10 VH EVQLIETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKDL EWVSRIRTKSNNYATYYADSVKDRFTISRDDSQSMLYLOMNNLKT EDTAMYYCVTGTTWAKEFAYWGQGTLVTVSA 20
370D2C10 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPHLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGSGTKLEIK 21
Example 2.Binding to CCR8 on U2OS Cells
[133] This example tested the antibodies’ activity in binding to human CCR8 protein expressed on U2OS cells.
[134] Chimeric antibodies (fused to human IgGl constant régions) of a few of the above o identified murine antibodies were expressed from cDNA. The antibodies were incubated with U2OS cells expressing the human CCR8.Among the antibodies tested, 88D2C6 and 137D1H10 exhibited considerably higher affmity than the rest, in a dose-dependent manner (FIG. 1).
Example 3. ADCC Measurement
[135] The potency of 88D2C6 and 137D1H10, along with 370D2C10 (ail fused to human
IgGl constant régions, with ADCC-activating mutations in Fc), in mediating ADCC was measured with CCR8-overexpressing CHO Kl and U2OS cells. Ail three antibodies exhibited high ADCC activities (FIG. 2).
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[136] Ail three antibodies exhibited potent ADCC activities. Among them, however, 88D2C6 had considerably higher potency (EC50: 0.01743 nM with CHO cells and 0.1108 for U2OS cells).
Example 4. Humanization of the mouse mAbs
[137] The murine antibody variable région genes of 88D2C6 and 352H1 ICI 1 were employed to create humanized mAbs. The amino acid sequences of the VH and VL of mAb were compared against the available database of human Ig gene sequences to find the overall best-matching human germline Ig gene sequences. The CDRs of the murine antibodies were then grafted into the matched human sequences. The cDNA was synthesized and used to produce the humanized antibodies. Certain back mutations from the murine antibodies were then introduced back to the humanized antibodies. Certain amino acids were mutated to reduce the chance of post-translational modification.
[138] The amino acid sequences of the humanized antibodies are provided below.
Humanized sequences
Table 2A. Humanization of 88D2C6 — VH
A. 88D2C6
Naine Sequence SEQ ID NO:
88D2C6 VH EVQLVETGGGLVQPKGSLKLSCAASGFTFNPNAMNWVRQAPGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKT EDTAMYYCVRGKDDGYRHYAMDYWGQGTSVTVSS 3
VI (CDR grafting) EVQLVESGGGLVQPGGSLKLSCAASGFTFNPNAMNWVRQASGKGL EWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKT EDTAVYYCTRGKDDGYRHYAMDYWGQGTTVTVSS 29
V2 (with back mutations) EVQLVESGGGLVQPGGSLKLSCAASGFTFNPNAMNWVRQASGKGL EWVGRIRSKSNNYATYYADSVKDRFTISRDDS KNTAYLQMNNLKT EDTAVYYCVRGKDDGYRHYAMDYWGQGTTVTVS S 30
V3 (with back mutations) EVQLVESGGGLVQPGGSLKLSCAASGFTFNPNAMNWVRQASGKGL EWVARIRSKSNNYATYYADSVKDRFTISRDDSKNTLYLQMNNLKT EDTAVYYCVRGKDDGYRHYAMDYWGQGTTVTVSS 31
Table 2B. CDR Sequences
CDR Sequence SEQ ID NO:
CDR-H1 PNAMN 22
CDR-H2 RIRSKSNNYATYYADSVKD 23
CDR-H3 GKDDGYRHYAMDY 24
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Table 2C. Humanisation of 88D2C6 - VL
Naine Sequence SEQ ID NO:
88D2C6 VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGAGTKLELK 2
VI (CDR grafting) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNANTYLYWYLQKP GQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPFTFGGGTKVEIK 32
V2 (with back mutations) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNANTYLYWFLQKP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGV YYCMQHLEYPFTFGGGTKVEIK 33
V3 (with back mutations) DIVMTQAPLSLPVTPGEPVSISCRSSKSLLHSNANTYLYWFLQKP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGV YYCMQHLEYPFTFGGGTKLEIK 34
Table 2D. CDR Sequences
CDR Sequence SEQ ID NO:
CDR-L1 RSSKSLLHSNGNTYLY 28
CDR-L1 (PM site mutated) RSSKSLLHSNANTYLY 25
CDR-L2 RMSNLAS 26
CDR-L3 MQHLEYPFT 27
Table 2E. Humanized antibodies
VL VLvl VLv2 VLv3
VH 88-xi
VHvl 88-H1L2 88-H1L3
VHv2 88-H2L2 88-H2L3
VH v3 88-H3L2 88-H2L3
B. 137D1H10
Table 3A. Humanisation of 137D1H10 - VH
Name Sequence SEQ ID NO:
137D1H10 VH EVQLVESGGGLVQPKGSLKLSCTASGFTFNTYAMNWVRQAPGKGL EWVARIRSKANNYATYYADSVKDRFTISRDDSQRILYLOMNNLKA EDTAMYYCVRDRSRGEDYAMDYWGQGTSVTVSS 1
VI (CDR grafting) EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGL EWVGRIRSKANNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKT EDTAVYYCTSDRSRGEDYAMDYWGQGTLVTVSS 38
V2 (with back mutations) EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGL EWVGRIRS KANNYATYYAD SVKDRFTISRDDSKNTAYLOMNNLKT EDTAVYYCVRDRSRGEDYAMDYWGQGTLVTVSS 39
V3 (with back mutations) EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGL EWVARIRSKANNYATYYADSVKDRFTISRDDSKNTLYLOMNNLKT EDTAVYYCVRDRSRGEDYAMDYWGQGTLVTVSS ~ ~ 40
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Table 3B. CDR Sequences
CDR Sequence SEQID NO:
CDR-H1 TYAMN 35
CDR-H2 RIRSKANNYATYYADSVKD 36
CDR-H3 DRSRGEDYAMDY 37
Table 3C. Humanization of 137D1H10 - VL
Naine Sequence SEQ ID NO:
137D1H10VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGV YYCMQHLEYPFTFGAGTKLELK 2
VI (CDR grafting) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNANTYLYWYLQKP GQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPFTFGQGTKLEIK 41
V2 (with back mutations) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNANTYLYWFLQKP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGV YYCMQHLEYPFTFGQGTKLEIK 42
V3 (with back mutations) DIVMTQSPLSLPVTPGEPVSISCRSSKSLLHSNANTYLYWFLQKP GQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLKISRVEAEDVGV YYCMQHLEYPFTFGQGTKLEIK · 43
Table 3D. CDR Sequences
CDR Sequence SEQ ID NO:
CDR-L1 RS S KSLLHSNGNTYLY 28
CDR-L1 (PM site mutated) RSSKSLLHSNANTYLY 25
CDR-L2 RMSNLAS 26
CDR-L3 MQHLEYPFT 27
Table 3E. Humanized antibodies
VL VLvl VLv2 VLv3
VH 137-xi
VHvl
VHv2 137-H2L2 137-H2L3
VHv3 137-H3L2 137-H3L3
£xample 5. Testing of Humanized Antibodies
[139] This example tested some of the humanized antibodiesfor their binding affmity to
CCR8 and their ability to induce ADCC.
[140] The experimental procedures are similar to Examples 2-3. FIG. 3 shows the affinities of humanized antibodies of 88D2C6. Apparently, ail of them had high affmity (see also Table 4)·
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Table 4A. Binding to CCR8 expressed in CHO-K1 cells
88-H1L2 88-H1L3 88-H2L2 88-H2L3 88-H3L2 88-H3L3 88-xi
Bottom 2669 2143 2494 2276 3937 3474 3631
Top 32782 31947 32830 31610 32495 32214 33947
LogEC50 -0.7515 -0.5189 -0.6391 -0.5461 -0.6861 -0.5826 -0.6548
HillSIope 1.502 1.377 1.404 1.408 1.382 1.345 1.480
EC50 0.1772 0.3028 0.2296 0.2844 0.2060 0.2615 0.2214
Span 30113 29804 30336 29334 28557 28740 30316
Table 4B. Binding to CCR8 expressed in U2OS cells
88-H1L2 88-H1L3 88-H2L2 88-H2L3 88-H3L2 88-H3L3 88-xi
Bottom 2106 1542 2756 1530 2826 1623 1027
Top 28891 27635 28329 25780 27435 27421 29659
LogEC50 -0.6779 -0.5797 -0.7241 -0.5119 -0.6841 -0.6289 -0.6921
HillSIope 1.409 1.387 1.524 1.450 1.521 1.378 1.097
EC50 0.2099 0.2632 0.1888 0.3077 0.2070 0.2350 0.2032
Span 26785 26092 25574 24250 24609 25798 28632
[141] FIG. 4 shows the affinities of humanized antibodies of 137D1H10 (data in Table 5).
Table SA. Binding to CCR8 expressed in CH0-K1 cells
137-H2L2 137-H2L3 137-H3L2 137-H3L3 137-xi
Bottom 2548 2244 4264 3820 3032
Top 29347 30182 33198 33389 34947
LogEC50 -0.7893 -0.5055 -0.6843 -0.7538 -0.7792
HillSIope 1.141 1.272 1.592 1.449 1.581
EC50 0.1625 0.3123 0.2069 0.1763 0.1663
Span 26799 27937 28934 29569 31915
Table SB. Binding to CCR8 expressed in U20S cells
137-H2L2 137-H2L3 137-H3L2 137-H3L3 137-xi
Bottom 915.4 1322 2065 2098 2172 ·
Top 26251 27254 32257 31927 33430
LogECSO -0.8422 -0.4841 -0.6897 -0.7169 -0.6052
HillSIope 1.171 1.122 1.351 1.492 1.422
EC50 0.1438 0.3280 0.2043 0.1919 0.2482
Span 25336 25932 30192 29829 31258
[142] In tenus of ADCC (FIG. 5), surprisingly, ail humanized antibodies exhibited greatly 15 higher (about 5 to 10-fold) ADCC inducing activity than the chimeric counterparts.
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Example 6. Cell-Based Antibody Binding
[143] This example compared 137-H3L2 (LM-108) with reference antibodies with respect to cell-based binding
[144] Cell based binding of LM-108 and reference Ab (synthesized according to
WO2020138489) to human CCR8 overexpressing cells (HEK293/H_CCR8) were assessed using flow cytometry. The human CCR8 overexpressing HEK293 (HEK293/H_CCR8) and parental HEK293 cells were incubated with titrated LM-108, reference Ab and its isotype control (from 200nM, 4 folds dilutions, 12 points) at 4°C for 60 minutes. The cells were washed with FACS buffer twice, then stained with fluorescent conjugated secondaryantibody (Alexa Fluor® 647 Goat Anti-Human IgG, Jackson, 109-605-098)at 4°C for 60 minutes. After the cells were washed twice and they were analyzed by flow cytometry.
[145] Both LM-108 and the reference Ab effectively bound to HEK293/H_CCR8 cells in a dose dépendent manner, at EC50 of 1.03 nM and 0.99nM, respectively (FIG. 6aand Table 6A). There was no non-specific binding observed for LM-108 but reference Ab showed slight nonspecific binding on parental HEK293 cells (FIG. 6band Table 6B).
Table 6A. Binding to CCR8-expressing HEK293 cells
Antibody EC50 (nM) Max (MFI)
LM-108 1.028 76795
Reference Ab 0.9938 86817
Isotype N.A. 192
Table 6B. Binding to Non-CCR8-expressingHEK293 cells
Antibody EC50 (nM) Max (MFI)
LM-108 N.A. 108
Reference Ab N.A. 472
Isotype N.A. 145
[146] Cell-based binding of LM-108 on cells with high levels of CCR8 expressionwas assessed using flow cytometry. The cells used were U2OS (U2OS/H_CCR8). As shown in FIG. 7 and Table 7, LM-108 could effectively bind to U2OS/H_CCR8 cells in a dose dépendent manner with an EC50 of 0.25nM.
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Table 7. Binding to High-CCR8-expressingU2OS cells
Antibody EC5O (nM) Max(MFI)
LM-108 0.2503 43087
Isotype N.A. 215
[147] Cell-based binding of LM-108 on cells withlowlevels of CCR8 expressionwasalso assessed using flow cytometry. The cells used were Jurkat engineering cells (Jurkat/H_CCR8). As shown in FIG. 8 and Table 8, LM-108 could effectively bind to Jurkat/H_CCR8 cells in a dose dépendent manner with an EC50 of 0.21nM.
Table 8. Binding to Low-CCR8-expressingJurkat cells
Antibody EC50 (nM) Max(MFI)
LM-108 0.2124 1423
Isotype N.A. 134
Example 7. ADCC Report Gene Assay
[148] This example shows the results of an ADCC reporter gene assay targetingdifferent CCR8-expressing cells.
ADCC reporter gene assay targeting U2OS/H_CCR8
[149] The ADCC activity of LM-108 was assessed with a reporter gene assay with CD16a(158v) expressing Jurkat/NFAT-luc reporter cells as effector cells. Human CCR8 overexpressing U2OS cells were used as target cells. U2OS/H_CCR8 cells were planted at the density of 1E4 cells per well in 96-well plate ovemight, then co-cultured with 1.5x105 effecter cells each well and the titrated LM-108 and the isotype control to final concentration from 10 pg/ml, 5 times dilution, and 10 points. After incubation under 37°C for 6 hours, OneGlo(Promega) was added for luminescence détection.
[150] The results are shown in FIG. 9. LM-108 exhibited potent ADCC effect (EC5o=O.OO6O5nM) with Jurkat/CD16a/NFAT-luc reporter cells targeting U2OS/H_CCR8 cells.
ADCC reporter gene assay targeting Jurkat/H_CCR8
[151] ADCC activity of LM-108 as assessed by reporter gene assay with CD16a(158v) expressing Jurkat/NFAT-luc reporter cells as effector cells. Human CCR8 low expressing
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Jurkat cells were generated as target cells to mimic the expression level on tumor associated Tregs.
[152] Effector cells (Jurkat reporter cell) and target cells (Jurkat/H_CCR8) at ratio of 3:1 were mixed with the titrated LM-108 and the isotype control (final concentration from 200nM, 5 times dilution, 11 points). After incubation under 37°C for 6 hours, OneGlo(Promega) was added for luminescence détection. As shown in FIG. 10, LM-108 had dose-dependent ADCC effect with an EC50=0.14nM.
ADCC effect of LM-108 towards human CCR8 over-expressing HEK293 (HEK293/H_CCR8) and HEK293 cells with primary human PBMCs
[153] Effector cells (hPBMCs) and target cells (HEK293/H_CCR8 or HEK293) at a ratio of 50:1 were mixed with the titrated LM-108 and the isotype control (final concentration from lOnM, 7 folds dilution, 12 points). After incubation under 37°C for 6 hours, 50 μΐ supematant from each well was analyzed by Cytotoxicity Détection Kit (Roche, 04744934001)
[154] As shown in FIG. 11 A, LM-108 exhibited spécifie and strong ADCC effect towards CCR8-expressing HEK293 cells in a dose-dependent manner with an EC50 of 0.002nM(Fig.6a). No nonspecific killing observed on parental HEK293 cells (FIG. 11B).
Example 8. Testing of Antibody-Dependent Cellular Phagocytosis (ADCP)
[155] In this example, the antibody-dependent cellular phagocytosis (ADCP) effect of LM108 towards human CCR8-overexpressed CHO-K1 cells was evaluated with monocytederived macrophages (MDMs) by FACS.
[156] The effector cells, MDMs, were induced from monocytes isolated from human PBMC with the presence of 100 ng/mL of human M-CSF for 7 days. The target cells were CHO-K1 (négative control) and CHO-K1 cells overexpressing human CCR8 which labeled CFSE. The effector cells, CFSE-labeled target cells, and titrated test antibody LM-108 or its isotype control were incubated in a 37°C, 5% CO2 incubator for 2 hours. Cells were washed with FACS buffer (1 x DPBS + 2% FBS), then stained with APC-conjugated human CD14 antibody to identify the macrophages. After wash twice with FACS buffer, the samples were analyzed by flow cytometry. Phagocytosis index was determined as the percentage of CD14APC+/CFSE+ double positive cells in total CD 14+ positive cells.
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[157] As shown in FIG. 12A-B, LM-108 had spécifie and dose dépendent phagocytosis toward human CCR8 overexpressing CHO-K1 cells, but not control cells.
[158] ADCP of LM-108 with Monocyte-derived Macrophage (MDM) was also measured by Operetta. The effector cells, MDMs, were induced from monocytes isolated from human PBMC with the presence of 100 ng/mL of human M-CSF for 6 days. Then the effector cells were seeded into a black 96-well plate with clear bottom at 2x104 count/well and cultured for another 2 days for adhérence. The target cells were Jurkat (négative control) and human CCR8 over expressing Jurkat cells (Jurkat/H_CCR8).The CFSE-labeled target cells and test antibody LM-108 or its isotype control were added into the plate with macrophages, and incubated in a 37 °C, 5% CO2 incubator for 2 hours. The effector cell: target cell ratio is 1:2. Cells were washed and stained with APC-conjugated human CD14 antibody to identify the macrophages. After washed twice, cells were fixed using 4% paraformaldéhyde (PFA) dilution and stored in a 4 °C freezer at dark before taking photos by Operetta.
[159] As shown in FIG. 13, LM-108 induced ADCP towards human CCR8-overexpressed Jurkat cells, while its isotype control did not. Neither LM-108 nor the isotype control induced obvious phagocytosis towards Jurkat cells.
Example 9. In vivo Effîcacy
[160] In this example, in vivo effîcacy studies of LM-108 or its mouse surrogate were performed.
[161] The first experiment was performed on a CT26 syngeneic model with LM-108m, a surrogate antibody for LM-108. In this study, each mouse was inoculated subcutaneously at the right axillary (latéral) with CT26 tumor cell (5x 105) in 0.1 ml of PBS for tumor , development. The animais were randomly grouped into 4 groups (n=7) on the third day after cell inoculation, then treatment started. PBS group as vehicle, LM-108m and mPD-1 antibody at dose of lOmg/kg was administrated via introperitoneal (i.p.) as single agent or combo at dayO, day3, day7, day 10, day 14 and day 17. The experiment was terminated at dayl8 due to the average tumor volume of control group reached more than 2000 mm3.
[162] Tumor sizes were measured three times weekly in two dimensions using a caliper and the volume was expressed in mm3 using the formula: V = 0.5 a x b2 where a and b were the long and short diameters of the tumor, respectively. The tumor sizes were then used for the
-4221300 calculations of T/C (%) values. T/C (%) was calculated using the formula: T/C % = (Ti-T0)/ (Vj-Vo) x 100 , T, was the average tumor volume of a treatment group on a given day, To was the average tumor volume of the treatment group on the first day of treatment, Vi was the average tumor volume of the vehicle control group on the same day with Ti, and Vo was the . average tumor volume of the vehicle group on the first day of treatment. TGI was calculated for each group using the formula: TGI (%) = [100-T/C].
[163] FIG. 14 shows the tumor growth curve of CT26 tumor-bearing Balb/c mice post administration of LM-108m and anti-mPD-1 Ab.LM-108m as single agent or in combination of mPD-1 antibody showed strong anti-tumor activity (Table 9).Data points represent group (n=7) mean, error bars represent standard error of the mean (SEM). p value was calculated based on tumor size by t-test, compared with vehicle control using PBS.
Table 9. In vivo Efficacy (CT26 syngeneic model)
Days after treatment T/C(%) 0 3 6 9 12 15 18
G2: LM-108ml0mpk - 72.161 86.504 54.453 47.024 35.639 28.219
G3: Anti-mPD-1 Ab lOmpk - 59.926 70.322 44.463 38.057 35.773 38.326
G4: LM-108m+mPD-l Ab - 70.506 60.774 35.973 27.413 20.386 15.292
Days after treatment TGI(%) 0 3 6 9 12 15 18
G2: LM-108ml0mpk - 27.839 13.496 45.547 52.976 64.361 71.781
G3: Anti- mPD-1 Ab lOmpk - 40.074 29.678 55.537 61.943 64.227 61.674
G4: LM-108m+mPD-l Ab - 29.494 39.226 64.027 72.587 79.614 84.708
Days after treatment P value 0 3 6 9 12 15 18
G2: LM-108ml0mpk - 0.053 0.385 0.024 0.026 0.025 0.023
G3: Anti-mPD-1 Ab lOmpk - 0.024 0.073 0.007 0.008 0.026 0.044
G4: LM-108m+mPD-l Ab - 0.037 0.017 0.002 0.006 0.01 0.012
[164] The second experiment used human CCR8 ki mice which were purchased from BIOCYTOGEN. Each hCCR8ki mouse was inoculated subcutaneously at the right axillary (latéral) withMC38 tumor cell (lxlO6) in 0.1 ml of PBS for tumor development. The animais were randomly grouped on the third day after cell inoculation, then treatment started for the efficacy study. LM-108 at dose of lOmg/kg was administrated via introperitoneal (i.p.) at dayO, day3, day7, day 10. The experiment was terminated when average tumor volume of control group reached more than 2000 mm3’
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[165] Tumor sizes were measured three times weekly in two dimensions using a caliper and the volume was expressed in mm3 using the formula: V = 0.5 a x b2 where a and b were the long and short diameters of the tumor, respectively. The tumor sizes were then used for the calculations of T/C (%) values. T/C (%) was calculated using the formula: T/C % = (Tj-T0)/ (Vj-Vo) xlOO , T, was the average tumor volume of a treatment group on a given day, To was the average tumor volume of the treatment group on the first day of treatment, Vi was the average tumor volume of the vehicle control group on the same day with Ti5 and Vo was the average tumor volume of the vehicle group on the first day of treatment. TGI was calculated for each group using the formula: TGI (%) = [100-T/C]. Data points represent group (n=8) mean, error bars represent standard error of the mean (SEM). p value was calculated based on tumor size by t-test, compared with Vehicle Control.
[166] FIG. 15 shows the tumor growth curve of MC38 tumor-bearing mice post administration of LM-108, and the data are summarized in Table 10. LM-108 showed potent tumor inhibitory effects.
Table 10. In vivo Efficacy (MC38 syngeneic mode!)
P Value Days after treatment
0 2 4 7 9 11 14
LM-108 vs. Vehicle - 0.985 0.503 0.042 0.002 0 0.001
T/C(%) Days after treatment
0 3 5 7 10 12 14
LM-108 vs. Vehicle - 99.68 95.326 73.673 47.349 34.464 31.23
TGI(%) Days after treatment
0 3 5 7 10 12 14
LM-108 vs. Vehicle - 0.32 4.674 26.327 52.651 65.536 68.77
Example 10. Cytotoxicity Assessment
[167] This example evaluated the cytotoxicity of an antibody-drug conjugate, LM-108-vcMMAE (Monomethyl auristatin E), on human CCR8 expressing HEK293 cells.
[168] To evaluate if LM-108 can be used for ADC development, cytotoxicity of MMAE conjugated LM-108 was assessed by co-culture the human CCR8 expressing HEK293 cells and the titrated ADC of LM-108-vc-MMAE.
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[169] HEK293/H_CCR8 cells were planted at the density of 1.5 x 104 cells per well in 96well plate ovemight, then co-cultured with titrated LM-108-vc-MMAE to final concentration from lOOnm, 3 times dilution,9 points. After incubation under 37°C for 96 hours, Cell-TiterGlo (Promega) was added for cell viability évaluation.
[170] As shown in FIG. 16, MMAE conjugated LM-108 had potent cytotoxicity(IC50=0.5285nM) on human CCR8-expressing HEK293 cells.
* * *
[171] The présent disclosure is not to be limited in scope by the spécifie embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally équivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that varions modifications and variations can be made in the methods and compositions of the présent disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the présent disclosure cover the modifications and variations of this disclosure provided they corne within the scope of the appended daims and their équivalents.
[172] Ail publications and patent applications mentioned in this spécification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims (17)

1. An antibody or fragment thereof having binding specificity to a human chemokine (C-
C motif) receptor 8 (CCR8) protein, wherein the antibody or fragment thereof comprises a heavy chain variable région comprising heavy chain complementarity determining régions CDRH1, CDRH2, and CDRH3 and a light chain variable région light chain comprising complementarity determining régions CDRL1, CDRL2, and CDRL3, and wherein:
(a) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 35, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 37, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27, or (b) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 22, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 23, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 24, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25 or 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 27.
2. The antibody or fragment thereof of claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 35, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 36, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 37, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence ofSEQIDNO: 27.
3. The antibody or fragment thereof of claim 2, wherein the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38-40, and the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32-34 and 41-43.
4. The antibody or fragment thereof of claim 2, wherein the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:40, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:42.
5. The antibody or fragment thereof of claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 22, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 23, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 24, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 25, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 26, and the CDRL3 comprises the amino acid sequence ofSEQ ID NO: 27.
6. The antibody or fragment thereof of claim 5, wherein the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29-31, and the light chain variable région comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 32-34 and 41-43.
7. The antibody or fragment thereof of claim 5, wherein the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:29, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:33.
8. The antibody or fragment thereof of any one of claims 1-7, which is humanized.
9. An antibody or fragment thereof, which has binding specificity to a human chemokine (C-C motif) receptor 8 (CCR8) protein and binds to the same epitope on the CCR8 protein as the antibody or fragment thereof of claim 1, or competes with the antibody or fragment thereof of claim 1 in binding to the CCR8 protein.
10. The antibody or fragment of any one of claims 1-9, which is capable of mediating antibody-dependent cellular cytotoxicity (ADCC).
11. The antibody or fragment of claim 10, which is not afucosylated.
12. The antibody or fragment thereof of any one of claims 1-11, which further has a binding specificity to a second target protein.
13. A composition comprising the antibody or fragment thereof of any one of claims 1-12 and a pharmaceutically acceptable carrier.
14. The composition of claim 13, further comprising a second antibody having specificity to a tumor antigen.
15. The antibody or fragment thereof of any one of claims 1-12 for use in the treatment of cancer.
16. Use of the antibody or fragment thereof of any one of claims 1-12 for the préparation of a médicament for treating cancer.
17. The antibody or fragment thereof of claim 15 or the use of claim 16, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endométrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, uréthral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, oesophageal cancer, ovarian cancer, rénal cancer, melanoma, prostate cancer and thyroid cancer.
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