WO2025035451A1 - Anticorps anti-cd93, composition et utilisation associées - Google Patents

Anticorps anti-cd93, composition et utilisation associées Download PDF

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WO2025035451A1
WO2025035451A1 PCT/CN2023/113535 CN2023113535W WO2025035451A1 WO 2025035451 A1 WO2025035451 A1 WO 2025035451A1 CN 2023113535 W CN2023113535 W CN 2023113535W WO 2025035451 A1 WO2025035451 A1 WO 2025035451A1
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amino acid
acid sequence
seq
antibody
antigen
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Chinese (zh)
Inventor
周进伟
刘丹丹
彭静
何阳
周岳华
冯辉
姚盛
沈懿娟
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Shanghai Junshi Biosciences Co Ltd
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Shanghai Junshi Biosciences Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the field of medical technology, and specifically relates to anti-CD93 antibodies, compositions and uses thereof.
  • CD93 (also known as C1q R1, C1q R, C1q RP, MXRA4 or ECSM3) is a transmembrane glycoprotein that consists of an extracellular domain (amino acid sequence from 24 to 580) composed of a C-type lectin domain, an epidermal growth factor (EGF)-like domain, a highly glycosylated mucin-like domain, a transmembrane domain (amino acid sequence from 581 to 601) and a short cytoplasmic domain (amino acid sequence from 601 to 652). It is mainly expressed in endothelial cells, platelets, stem cells and bone marrow cells (granulocytes and monocytes).
  • EGF epidermal growth factor
  • IGFBP7 insulin-like growth factor binding protein 7
  • the present invention provides an anti-CD93 antibody or an antigen-binding fragment thereof, wherein the anti-CD93 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, or a HCDR1 having 1, 2 or 3 amino acid differences with the HCDR1, a HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and a HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3; and/or the anti-CD93 antibody or an antigen-binding fragment thereof comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 defined by Kabat.
  • LCDR light chain variable region CDR
  • LCDR1 or a sequence having 1, 2 or 3 amino acid differences with said LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with said LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with said LCDR3.
  • Different sequence
  • the present invention provides an isolated anti-CD93 antibody or antigen-binding fragment thereof, which has at least one of the following properties: (I) binds to the same, completely overlapping, or partially overlapping epitope of human CD93 protein as the antibody or antigen-binding fragment thereof described in any embodiment of the present invention; (II) competes with the antibody or antigen-binding fragment thereof described in any embodiment of the present invention for binding to the epitope of human CD93 protein.
  • the present invention provides a polynucleotide molecule encoding an anti-CD93 antibody or antigen-binding fragment thereof described herein.
  • the present invention provides a nucleic acid construct comprising the polynucleotide molecule according to any embodiment of the present invention.
  • the present invention provides an expression vector comprising the polynucleotide molecule or nucleic acid construct described herein; preferably, the vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising a polynucleotide molecule described herein or an expression vector described herein, and/or expressing an anti-CD93 antibody or antigen-binding fragment thereof as described in any embodiment herein.
  • the present invention provides a method for preparing the anti-CD93 antibody or antigen-binding fragment thereof described herein, the method comprising culturing the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, allowing it to express the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.
  • the present invention provides an anti-CD93 antibody or an antigen-binding fragment thereof prepared by the method for preparing an anti-CD93 antibody or an antigen-binding fragment thereof described in any embodiment herein.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CD93 antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, a nucleic acid construct or expression vector described herein, a host cell described herein, a multispecific antibody described herein, or a single-chain antibody described herein, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a drug combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, a nucleic acid construct or expression vector as described herein, a host cell as described herein, a multispecific antibody as described herein, a single-chain antibody as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.
  • the therapeutic agent is one or a combination of an anti-PD-1 antibody, a PD-L1 antibody, and an anti-CTLA4 antibody.
  • the present invention provides use of the antibodies or antigen-binding fragments thereof described herein, the polynucleotide molecules described herein, the nucleic acid constructs or expression vectors described herein, the host cells described herein, the multispecific antibodies described herein, the single-chain antibodies described herein, the pharmaceutical compositions described herein, or the pharmaceutical combinations described herein in the preparation of a medicament for treating and/or preventing a CD93-mediated disease or disorder.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, a nucleic acid construct or expression vector described herein, a method for treating and/or preventing a CD93-mediated disease or condition.
  • the present invention provides a method for treating and/or preventing a CD93-mediated disease or condition, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the antibody or antigen-binding fragment thereof described herein, the nucleic acid construct or polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the multispecific antibody described herein, the single-chain antibody described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the nucleic acid construct or polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the multispecific antibody described herein, the single-chain antibody described herein, the pharmaceutical composition described herein, or the drug combination described herein, preferably further comprising a drug delivery device.
  • the present invention provides a method for detecting the presence of CD93 in a sample using the antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof.
  • the method comprises the step of contacting the antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof with the sample.
  • FIG1 Graph showing the results of the cell-level binding experiment of anti-CD93 chimeric antibodies.
  • Figure 2 Blocking experiment results of anti-CD93 chimeric antibody blocking the binding of IGFBP7 protein to human CD93.
  • FIG3 Graph showing the results of the cell-level binding experiment of anti-CD93 humanized antibodies.
  • Figure 4 Blocking experiment results of anti-CD93 humanized antibody blocking the binding of IGFBP7 protein to human CD93.
  • FIG5 Experimental results of binding of anti-CD93 chimeric antibody to human CD93.
  • Figure 6 Blocking experiment results of anti-CD93 chimeric antibody blocking the binding of IGFBP7 protein to human CD93.
  • Figure 7 Binding experiment results of anti-CD93 monoclonal antibody and CHO hCD93 cells.
  • Figure 8 Experimental results of anti-CD93 monoclonal antibody blocking the binding of IGFBP7 to CHO hCD93 cells.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above.
  • “or” or “and/or” should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as “only one” or “exactly one” or when “consisting of" is used in the claims, it will refer to only one number listed or one element of the list.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps introduced where necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity.
  • Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software.
  • One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.
  • activity or “biological activity”, or the terms “biological property” or “biological characteristic” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize CD93 activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells.
  • antibody refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of its.
  • isolated does not imply the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule that contains at least four peptide chains when naturally present: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs).
  • CDRs complementary determining regions
  • FRs conservative framework regions
  • Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • variable region CDRs of an antibody can be determined using any of a number of well-known schemes, including Chothia (Chothia et al., 1989, Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948, 1997), Kabat (Kabat et al., Sequences of Proteins of Immunolobulins), and the like, based on the variability of antibody sequences. gical Interest, 4th ed., U.S.
  • the CDR of antibodies of the present invention can be determined by those skilled in the art according to any scheme (e.g., different assignment systems or combinations) of this area.
  • the CDR of antibodies of the present invention is defined by Kabat, Chothia, AbM, Contact, North or IMGT. In some embodiments, the CDR of antibodies of the present invention is defined by Kabat.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of the antibody also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose declared CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of a different scheme (such as a different assignment system or combination).
  • Antibodies with different specificities have different CDRs.
  • CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of a CDR.
  • the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.
  • antigen-binding fragment of an antibody includes fragments or derivatives of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
  • the binding fragment or derivative When the binding activity of the antigen is expressed on a molar basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody.
  • the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as “conservative variants” or “functional conservative variants” of antibodies).
  • binding compound refers to both antibodies and their binding fragments.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide generally also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.
  • diabody refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen binding sites.
  • hybridomas refers to an anti-human CD93 antibody prepared according to the knowledge and skills in the art. Monoclonal antibodies are prepared by injecting a test subject with the CD93 antigen and then isolating hybridomas that express antibodies with the desired sequence or functional properties.
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from antibodies such as rodents ("parent antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects than the parent rodent antibodies.
  • humanized antibody refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • a humanized antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat sugar chains. Similarly, a “mouse antibody” refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat sugar chains. Similarly, a "rat antibody” refers to an antibody that contains only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, such as to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, such as Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • epitope refers to a protein determinant that is capable of specific binding to an antibody.
  • Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that in the presence of denaturing solvents, the binding to the former but not to the latter is lost.
  • cross-reactivity refers to the binding to antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Therefore, “cross-reactivity” should be understood as an interspecies reaction with the same molecule X expressed in different species.
  • the cross-reactivity specificity of monoclonal antibodies recognizing human CD93, monkey, and/or murine CD93 (mouse or rat) can be determined by FACS analysis.
  • Affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the dissociation rate constant.
  • KD equilibrium dissociation constant
  • Affinity is the ratio of the binding number and the association rate constant ( kdis and kon , respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.
  • the term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD for binding to the protein or cell is 1.0 ⁇ 10-6 M or higher, more preferably 1.0 ⁇ 10-5 M or higher, more preferably 1.0 ⁇ 10-4 M or higher, 1.0 ⁇ 10-3 M or higher, more preferably 1.0 ⁇ 10-2 M or higher.
  • high affinity for IgG antibodies refers to a KD for an antigen of 1.0 ⁇ 10-6 M or less, preferably 5.0 ⁇ 10-8 M or less, more preferably 1.0 ⁇ 10-8 M or less, 5.0 ⁇ 10-9 M or less, and more preferably 1.0 ⁇ 10-9 M or less.
  • "high affinity” binding may vary.
  • “high affinity” binding for the IgM subtype refers to a KD of 10-6 M or less, preferably 10-7 M or less, and more preferably 10-8 M or less.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.
  • RNA ribonucleic acid
  • nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.
  • a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construction refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner.
  • a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more
  • Recombinant constructs will typically include a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell.
  • a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell.
  • Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the invention.
  • vectors After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a vector that is capable of replicating and expressing a target cell when transformed, transfected or transduced into a host cell.
  • the expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host when necessary.
  • Activation may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies.
  • Activation may refer to cell activation regulated by internal mechanisms as well as external or environmental factors.
  • Response/reaction such as the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.
  • biochemical or physiological behavior e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state
  • treatment refers to improving the disease or condition (i.e., slowing down or preventing or reducing the progression of the disease or at least one of its clinical symptoms) in one embodiment.
  • treatment or “treating” refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient.
  • treatment or “treating” refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or in both aspects.
  • methods for assessing the treatment and/or prevention of a disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
  • cyno or “cynomolgus monkey” refers to cynomolgus monkeys.
  • Administration "in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) administration and consecutive administration in either order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to an amount of the CD93 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the development of the disease or condition.
  • a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount to treat, cure, prevent or ameliorate a related medical condition or to increase the rate of treatment, cure, prevention or amelioration of such a condition.
  • a therapeutically effective dose refers only to that ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter by at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.
  • “Pharmaceutically acceptable carrier” refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth.
  • Cancer may be benign (also known as a benign tumor), pre-malignant or malignant.
  • Cancer cells may be solid cancer cells or leukemia cancer cells.
  • tumor refers to one or more cells comprising a cancer.
  • tumor growth is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.
  • the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CD93.
  • the term "anti-CD93 antibody”, “anti-CD93”, “CD93 antibody” or “antibody that binds to CD93” refers to an antibody that is capable of binding to a CD93 protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD93.
  • the present invention provides antibodies that bind to CD93 protein.
  • the antibodies of the invention bind to human CD93 protein.
  • the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, and/or comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 5, 13, 21, 28, 31, 32 or 36 defined by Kabat.
  • HCDR heavy chain variable region CDR
  • LCDR3 light chain variable region CDR
  • the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1 defined by Kabat, and/or comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 5 defined by Kabat.
  • HCDR heavy chain variable region CDR
  • LCDR3 light chain variable region CDR
  • the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 9 defined by Kabat, and/or the light chain variable region CDR (LCDR) comprised thereof comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 13 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 13 defined by Kabat.
  • HCDR heavy chain variable region CDR
  • LCDR3 light chain variable region CDR
  • the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 17 defined by Kabat, and/or, the light chain variable region CDR (LCDR) comprised thereof is LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 21 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably defined by Kabat.
  • HCDR heavy chain variable region CDR
  • the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 40 defined by Kabat, and/or the light chain variable region CDR (LCDR) comprised thereof comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 32 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 32 defined by Kabat.
  • HCDR heavy chain variable region CDR
  • LCDR3 light chain variable region CDR
  • the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region CDR
  • the heavy chain variable region (HCDR) and light chain variable region (LCDR) are the HCDR and LCDR sequences defined by Kabat, Chothia, AbM, Contact, North or IMGT in the heavy chain variable region and light chain variable region shown in the sequences of the following groups, respectively, preferably the HCDR and LCDR sequences defined by Kabat:
  • the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) of HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, and/or comprises a light chain variable region CDR (LCDR) of LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 5, 13, 21, 28, 31, 32 or 36 defined by Kabat.
  • HCDR heavy chain variable region CDR
  • LCDR3 light chain variable region CDR
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, 10 or 18, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, 11 or 19 or 57, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4, 12 or 20, and/or LCDR1 comprises The LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, 15 or 23, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8, 16, 24 or 56.
  • its heavy chain variable region comprises: (I) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, and SEQ ID NO: 4, respectively, or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, and SEQ ID NO: 4, respectively; or (II) HDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • CDR1, HCDR2 and HCDR3, or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or (III) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
  • CDR3; and/or its light chain variable region comprises: (I) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (II) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:
  • the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with this
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises The invention further comprises an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20
  • the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having 1, 2 or 3 amino acid differences with
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or 57 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 or 58.
  • the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence
  • the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence
  • the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 54 or 55 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20
  • the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having 1, 2 or 3 amino acid differences with
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having 6 amino acid differences with the amino acid sequence,
  • the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and/or a light chain variable region:
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, SEQ ID NO: 4, respectively; or
  • the light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7, or SEQ ID NO: 8, respectively; or
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 24.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3 or 57, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 53, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 54 or 55, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 56.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 57, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 55, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the heavy chain variable region of the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 17, 37-48, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 17, 37-48, and/or, its light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 13, 21, 25-36, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 5, 13, 21, 25-36.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:1, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:1; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:5, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:5; or
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:9, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:13, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:13; or
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 17; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 21, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 21.
  • the anti-CD93 antibody or its antigen-binding fragment described in the present invention comprises: a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.
  • the anti-CD93 antibody or its antigen-binding fragment described in the present invention comprises: a heavy chain variable region, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.
  • amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD93 antibody or antigen-binding fragment thereof of the present invention are selected from the amino acid sequence of the heavy chain variable region shown in any of the following sequence numbers or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto and the amino acid sequence of the light chain variable region or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively.
  • the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises:
  • the present invention provides an isolated anti-CD93 antibody or antigen-binding fragment thereof, which has one or more of the following characteristics: (1) the epitope of human CD93 protein bound by it is the same as, completely overlaps with, or partially overlaps with the anti-CD93 antibody or antigen-binding fragment thereof described herein; (2) the epitope of human CD93 protein bound by it is the same as, completely overlaps with, or partially overlaps with the anti-CD93 antibody or antigen-binding fragment thereof described herein; The fragments compete for binding to an epitope of the human CD93 protein.
  • the anti-CD93 antibody or antigen-binding fragment thereof described in the present invention is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.
  • the antibody of the present invention is a murine antibody or a chimeric antibody. In some embodiments, the antibody of the present invention further comprises a heavy chain framework region and/or a light chain framework region derived from a murine antibody.
  • the antibody of the present invention is a humanized antibody.
  • the antibody of the present invention comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene), and/or a light chain framework region (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene).
  • the heavy chain framework region and/or the light chain framework region optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to mouse residues.
  • the antibodies described herein are monoclonal antibodies.
  • the antigen-binding fragment is selected from: Fab; Fab'; F(ab')2; Fv fragment; diabody; linear antibody; single-chain antibody molecule, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • Any suitable method for producing antibodies can be used to produce antibodies of the present invention.
  • Any suitable form of CD93 can be used as an immunogen (antigen) for producing antibodies.
  • any CD93 variant or fragment thereof can be used as an immunogen.
  • hybridoma cells producing monoclonal anti-human CD93 antibodies of murine origin can be produced by methods known in the art.
  • Antibodies derived from rodents may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use leads to the body's immune response to the therapeutic antibodies, which at least results in loss of therapeutic efficacy and, in severe cases, potentially lethal allergic reactions.
  • One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of intact rodent variable regions in chimeric antibodies may still cause harmful immunogenicity in patients.
  • CDR complementarity determining region
  • chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the prepared mouse monoclonal hybridoma antibody.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric CD93 antibody of the present invention can be prepared by effectively linking the heavy and light chain variable regions of the immunoglobulin derived from hybridomas to the human IgG constant region (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy chains and chimeric light chains.
  • the constant region contained in the chimeric antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric CD93 antibodies of the present invention can be obtained by "mixing and matching" chimeric light chain and chimeric heavy chain expression plasmids to transfect expression cells, and the CD93 binding of such "mixed and matched" antibodies can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).
  • the humanized antibodies of the present invention can be prepared by inserting mouse CDR regions into human germline framework regions using methods known in the art, such as Winter et al. U.S. Pat. No. 5,225,539 and Queen et al. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
  • one or more of the three HCDR sequences and three LCDR sequences in the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention have amino acid differences compared to the corresponding three HCDR sequences and three LCDR sequences defined above.
  • the amino acid differences include amino acid deletions, insertions or substitutions.
  • one or more of the three HCDR sequences and three LCDR sequences of the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention have amino acid differences selected from 1, 2 or 3 amino acids selected from amino acid deletions, amino acid insertions and amino acid substitutions compared to the corresponding three HCDR sequences and three LCDR sequences defined above.
  • the framework regions of the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention have 1, 2, 3, 4 or 5 amino acid differences selected from amino acid deletions, amino acid insertions and amino acid substitutions compared to the corresponding framework regions in the specific sequences disclosed herein.
  • the present invention provides CDR sequences, heavy chain variable regions, light chain variable regions, heavy chains and light chains that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding CDR sequences, heavy chain variable regions, light chain variable regions, heavy chains and light chains specifically disclosed herein after one or more (such as 1-10, 1-5 or 1-3) amino acid deletions, insertions and/or substitutions.
  • the introduction of these amino acid differences will not affect the biological activity of the resulting anti-CD93 antibody or antigen-binding fragment thereof, including the activity of binding to the CD93 protein or fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic agent and/or therapeutic agent targeting CD93.
  • the antibody or antigen-binding fragment obtained by mutation compared with the antibody or antigen-binding fragment before mutation, at least retains the binding affinity of the antibody or antigen-binding fragment before mutation to the CD93 protein or fragment thereof.
  • the anti-CD93 antibodies described herein are IgG antibodies, such as IgG1, IgG2, IgG3 or IgG4 antibodies or modified forms thereof, as described in the following sections.
  • one or more amino acid modifications can be introduced into the Fc region of the anti-CD93 antibodies provided herein to generate Fc region variants.
  • the Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.
  • cysteine engineered antibodies such as "thioMAbs,” in which one or more residues of an antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of polysaccharides include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol
  • a multispecific antibody is provided herein.
  • the multispecific antibody comprises a second functional module (e.g., a second antibody) having a binding specificity different from that of the anti-CD03 antibody provided herein, thereby being able to bind to at least two different binding sites and/or target molecules. Therefore, the present invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region of an antibody or an antigen-binding fragment thereof described herein.
  • a single-chain antibody comprising a light chain variable region and a heavy chain variable region of an anti-CD93 antibody or antigen-binding fragment thereof described herein.
  • the present invention provides a polynucleotide molecule encoding an anti-CD93 antibody or antigen-binding fragment thereof as described herein.
  • the present invention also includes the full-length complementary sequence of the polynucleotide molecule.
  • the polynucleotide molecule may comprise a polynucleotide molecule encoding the amino acid sequence of the light chain variable region and/or the heavy chain variable region of the antibody, or a polynucleotide molecule comprising the amino acid sequence of the light chain and/or the heavy chain of the antibody.
  • the polynucleotide molecule encoding the antibody of the present invention includes a polynucleotide molecule that has been mutated by nucleotide deletion, insertion or substitution, but still has at least about 60%, 70%, 80%, 90% or 95% identity with the CDR corresponding coding region depicted in the sequence described above.
  • the present invention provides a nucleic acid construct comprising a polynucleotide molecule as described herein.
  • the nucleic acid construct can be, for example, an expression cassette comprising a promoter and a transcription terminator operably linked to the polynucleotide molecule.
  • the nucleic acid construct is an expression vector.
  • the present invention provides an expression vector comprising a polynucleotide molecule or a nucleic acid construct as described herein.
  • the vector is a eukaryotic expression vector.
  • the polynucleotide molecule as described herein is contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein and/or expressing an anti-CD93 antibody or antigen-binding fragment thereof as described in any embodiment herein.
  • the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method for preparing an anti-CD93 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.
  • the present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including those obtainable from American Type Culture Collection (ATCC)
  • ATCC American Culture Collection
  • Many immortalized cell lines of the American Culture Collection (ATCC) are available. These especially include Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines.
  • Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.
  • the present invention provides a method for preparing an anti-CD93 antibody, wherein the method comprises, when an expression vector is introduced into a mammalian host cell, culturing the host cell for a sufficient time to allow the antibody to be expressed in the host cell, or more preferably allowing the antibody to be secreted into the culture medium in which the host cell grows, thereby preparing the antibody.
  • the antibody can be recovered from the culture medium using standard protein purification methods.
  • antibodies expressed by different cell lines or expressed in transgenic animals have different glycosylation from one another.
  • all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies.
  • non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structures are normal components of native human serum IgG.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CD93 antibody or antigen-binding fragment thereof as described herein, a nucleic acid construct or polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or a multispecific antibody or single-chain antibody as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the anti-CD93 antibody or pharmaceutical composition thereof provided by the present invention can be integrated with suitable carriers, excipients and other agents in the formulation for combined administration, thereby providing improved transfer, delivery, tolerance, etc.
  • composition refers to a preparation which permits the active ingredient contained therein to exist in biologically effective form, and which contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.
  • compositions containing the anti-CD93 antibodies described herein can be prepared by mixing an anti-CD93 antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized preparation.
  • pharmaceutical excipients Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)
  • the pharmaceutical composition or preparation of the present invention may also include one or more other active ingredients, which are required for the specific indications to be treated, preferably those active ingredients that do not adversely affect the complementary activities of each other.
  • other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics or various known anti-tumor or anti-cancer agents, and the active ingredients are appropriately combined in an amount effective for the intended use.
  • the pharmaceutical composition of the present invention also includes a composition of a polynucleotide molecule encoding an anti-CD93 antibody.
  • the present invention provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule or nucleic acid construct described herein, the expression vector described herein, the host cell described herein, the multispecific antibody or single-chain antibody described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents. dose.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule or nucleic acid construct described herein, the expression vector described herein, the host cell described herein, or the multispecific antibody or single-chain antibody described herein, or the pharmaceutical composition or drug combination described herein.
  • the kit is a detection kit, which contains the antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof can be labeled with a marker well known in the art.
  • anti-CD93 antibody or antigen-binding fragment thereof can be used in a method of treatment. It should also be understood that when discussing "antibodies", compositions comprising antibodies or antigen-binding fragments thereof are also included.
  • the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention can be used in a therapeutic or preventive method described in any embodiment of the present invention in a therapeutically effective amount or a preventively effective amount.
  • the present invention provides use of the antibodies or antigen-binding fragments thereof described herein, the polynucleotide molecules or nucleic acid constructs described herein, the expression vectors described herein, the host cells described herein, or the multispecific antibodies or single-chain antibodies described herein, or the pharmaceutical compositions or pharmaceutical combinations described herein in the preparation of drugs for treating and/or preventing CD93-mediated diseases or disorders.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule or nucleic acid construct described herein, an expression vector described herein, a host cell described herein, or a multispecific antibody or single-chain antibody described herein, or a pharmaceutical composition or pharmaceutical combination described herein for use in treating and/or preventing a CD93-mediated disease or condition.
  • the present invention provides a method for treating and/or preventing a CD93-mediated disease or condition, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule or nucleic acid construct described herein, an expression vector described herein, a host cell described herein, or a multispecific antibody or single-chain antibody described herein, or a pharmaceutical composition or pharmaceutical combination described herein.
  • the CD93-mediated disease or condition includes a disease or condition caused by the interaction between CD93 and insulin-like growth factor binding protein 7 (IGFBP7), such as abnormal tumor vasculature.
  • IGFBP7 insulin-like growth factor binding protein 7
  • the CD93-mediated disease or disorder is cancer; more preferably, the characteristics of the cancer include abnormal tumor vasculature; further preferably, the cancer is selected from colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, brain tumor, bone cancer, melanoma.
  • the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration, etc.
  • the present invention provides a method for detecting the presence of CD93 in a sample using the antibodies or antigen-binding fragments thereof described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other liquid samples of biological origin.
  • the biological sample comprises cells or tissues.
  • the method comprises the steps of contacting the sample with the antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal generated by the binding of the antibody or antigen-binding fragment thereof to CD93.
  • the antibodies or antigen-binding fragments thereof described herein may be labeled to indicate whether the conjugate is formed.
  • the method may be used for diagnostic purposes, or for non-diagnostic purposes.
  • the present invention provides use of the antibody or antigen-binding fragment thereof described herein, or a detection composition containing the antibody or antigen-binding fragment thereof, in the preparation of a detection reagent for detecting the presence of CD93 in a sample.
  • the antibodies of the present invention can be prepared by a variety of methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.
  • Example 1 Preparation of recombinant protein for anti-CD93 antibody activity detection
  • the cDNA sequence encoding the CD93 extracellular domain was amplified by conventional PCR technology, and the amplified fragment was cloned into a self-constructed eukaryotic expression plasmid system (MX2-mFc, containing the mouse IgG2a Fc domain) using conventional cloning technology, which contains a puromycin selection system, thereby generating a recombinant fusion protein expression plasmid hCD93 ECDmFC.
  • MX2-mFc eukaryotic expression plasmid system
  • hCD93 ECD mFC recombinant protein was expressed and purified using expression cells 293E.
  • CD93 ECD The amino acid sequence of the human CD93 extracellular domain (CD93 ECD) is the amino acid at position Ala24-Lys580 (UniProtKB/Swiss-Prot: Q9NPY3.3), which is used for the activity detection of the antibody of the present invention.
  • CD93-Fc recombinant protein was used as an antigen to immunize 5 BALB/c mice (purchased from Simonsen Laboratories of Gilroy, female BALB/c, 8 weeks old). After the initial immunization (50ug/mouse), booster immunization (25ug/mouse) was performed every 1 or 2 weeks for a total of 6 immunizations.
  • the inguinal lymph nodes, popliteal lymph nodes and spleen of the mice were taken, and the lymphocyte-rich suspension was obtained after grinding in physiological saline, and it was fused with mouse myeloma cells Sp2/0 according to the conventional electroporation method.
  • the fusion product was cultured in DMEM complete medium containing 1:50 HAT (hypoxanthine, methotrexate and thymidine) for 5 days to screen the successfully fused cells (hybridoma cells). Then it was changed to DMEM complete medium containing 1:50 HT (hypoxanthine and thymidine) until the end of the screening.
  • the DMEM complete medium had a ratio of: 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U/mL penicillin-streptomycin + 1:100 OPI (oxaloacetate, pyruvate and insulin), and the incubator conditions were 8% CO 2 , 37°C.
  • FBS fetal bovine serum
  • L-glutamine 100 U/mL penicillin-streptomycin + 1:100
  • OPI oxaloacetate, pyruvate and insulin
  • 181 hybridoma cells were screened by ELISA, and the antibodies secreted by them could specifically bind to hCD93.
  • 181 hybridoma cells with positive binding antibodies expressed by 15 hybridoma cells could block the binding of IGFBP7 to hCD93 expressed on the cell surface in FACS.
  • 7 of the polyclonal cell lines were determined to be able to block the binding of hCD93 to IGFBP7.
  • the above ELISA includes: 96-well MaxiSorp plates (Thermo Fisher Nunc) were coated with 2ug/mL hCD93 (mouse Fc tag, Junmeng) and then blocked with 1% BSA. Igfbp7 with a final concentration of 0.5ug/ml was incubated with hybridoma culture supernatant on the microplate for 30 minutes. The mixture was then transferred to the MaxiSorp plate and incubated for 30 minutes. Detection was performed with horseradish peroxidase-labeled goat anti-human secondary antibody.
  • the above FACS includes: incubating the self-constructed human CD93-overexpressing CHO CD93 cells with hIGFBP7mFc protein solution (0.5 ⁇ g/ml) prepared with staining buffer (PBS+1% FBS) and anti-CD93 hybridoma antibodies of different concentrations at 4°C for 30 minutes. Then wash away the unbound antibodies with staining buffer, and then incubate the cells with 1% (v/v) sheep anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch) prepared with staining buffer at 4°C in the dark for 30 minutes. Finally, collect the cells with flow cytometry to detect the fluorescent antibodies bound to the cell surface. FlowJo was used to analyze the raw data to obtain the MFI value, and GraphPad Prism software was used to perform a four-parameter curve fitting to obtain the IC50 value.
  • hIGFBP7mFc protein solution 0.5 ⁇ g/ml
  • staining buffer PBS+1% FBS
  • Seven blocking monoclonal cell lines were obtained through subclone screening.
  • the antibodies secreted by them were purified and analyzed.
  • mRNA was extracted from monoclonal hybridoma cells, and the variable regions of the antibodies were sequenced. The seven sequences were determined to be 1H2.3, 2A6.5, 2D9.4, 3D9.7, 3E2.7, 4B5.3 and 4C5.4.
  • the DNA coding sequence corresponding to the variable region of the anti-CD93 mouse antibody was determined using a degenerate primer PCR method.
  • Candidate hybridoma cells were cultured, the cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol.
  • the first-strand cDNA was synthesized using this as a template, and then the first-strand cDNA was used as a subsequent template to PCR amplify the corresponding variable region DNA coding sequence.
  • the primer sequences used in the amplification reaction are complementary to the first framework region and constant region of the variable region of the antibody (Larrick, J.W. et al., 1990, Scand. J. Immunol., 32, 121-128 and Coloma, J.J.
  • Consensus sequences were searched in germline and rearranged Ig variable region sequence databases using NCBI Ig-Blast (http://www.ncbi.nlm.nih.gov/projects/igblast/).
  • Complementarity determining region (CDR) amino acid sequences were identified by sequence annotation based on the Kabat (Wu, T.T and Kabat, E.A. 1970 J. Exp. Med., 132: 211-250) and IMGT systems (Lefranc M.-P.
  • the light chain and The heavy chain variable region was used to construct an anti-CD93 chimeric antibody.
  • the coding sequences of the heavy chain constant region IgG4 and the light chain constant region ⁇ were cloned from human B lymphocytes (from Beijing Institute of Hematology) and introduced into the pCDNA3.1 plasmid to form vectors HXT1S-KDL and HXT2, respectively.
  • the heavy chain and light chain variable region coding sequences of the aforementioned anti-CD93 mouse antibody were synthesized by tsingke, digested by BSPQI, and subcloned into HXT4S and HXT2, respectively.
  • CHO-CD93 cells overexpressing human CD93 (CHO-K1 cells were purchased from ATCC cell bank, number CCL-61. Based on this cell line, a stable cell line overexpressing CD93 was independently constructed, and the amino acid sequence of CD93 is from NCBI accession number NP_036204.2) were incubated with different concentrations of anti-CD93 chimeric antibodies (starting concentration of 50 ⁇ g/ml, 3-fold dilution, a total of 10 concentration gradients) at 4°C for 30 min, and then unbound cells were washed with staining buffer (PBS + 1% FBS).
  • staining buffer PBS + 1% FBS
  • the cells were incubated with the fluorescent secondary antibody goat anti-human IgG PE (Southern Biotech, Cat#2040-09) prepared in the staining buffer at 4°C for 30 minutes in the dark. Finally, the cells were collected by flow cytometry (BD, Canto II model) to detect the fluorescent antibody bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad and the EC50 was calculated.
  • the fluorescent secondary antibody goat anti-human IgG PE Pacificn Biotech, Cat#2040-09
  • most of the anti-CD93 chimeric antibodies of the present invention can bind to human CD93 highly expressed on the surface of CHO-CD93 cells with high affinity, among which C17 and C33 have the highest binding top values, while C21 has no binding activity.
  • Anti-CD93 chimeric antibody blocks the binding of IGFBP7 protein to human CD93-expressing cells
  • the cells overexpressing human CD93, CHO-CD93 were incubated with hIGFBP7mFc protein solution (Junshi's own expression, Lot#20200318, working concentration 10 ⁇ g/ml) prepared with staining buffer (PBS+1% FBS), and different concentrations of anti-CD93 chimeric antibodies (starting concentration of 50 ⁇ g/ml, 3-fold dilution, 10 concentration gradients) at 4°C for 30 minutes. Unbound antibodies were then washed away with staining buffer, and the cells were incubated with 1% (v/v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch, Cat#115-175-071) prepared with staining buffer at 4°C in the dark for 30 minutes.
  • hIGFBP7mFc protein solution Jobshi's own expression, Lot#20200318, working concentration 10 ⁇ g/ml
  • staining buffer PBS+1% FBS
  • different concentrations of anti-CD93 chimeric antibodies starting
  • the cells were collected using a flow cytometer (BD, CantoII model) to detect fluorescent antibodies bound to the cell surface.
  • the raw data were analyzed with FlowJo to obtain the MFI value, and the IC50 value was obtained by performing a four-parameter curve fitting with GraphPad Prism software.
  • C17, C21, C36, C40, and C41 could not effectively block the binding of hIGFBP7 protein to CHO-CD93 cells, while the other antibodies could block the binding of hIGFBP7 protein to CHO-CD93 cells.
  • Table 4 Ability of anti-CD93 chimeric antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC50)
  • the heavy and light chains of the chimeric antibodies C1, C33, and C49 were selected for humanization design and transformation.
  • the sequences of the chimeric antibodies are as follows:
  • CDR2 YITNGGGGTYYPDTVKG (SEQ ID NO: 3);
  • CDR1 RSSQSLIHRNGDTYLH (SEQ ID NO: 6);
  • CDR3 SQSTHVPPWT (SEQ ID NO: 8);
  • CDR2 FINTDYGNTHFNQKFKG (SEQ ID NO: 11);
  • CDR2 QMSNLAS(SEQ ID NO: 15);
  • CDR3 AQNLEVPWT (SEQ ID NO: 16);
  • CDR1 SYWMQ (SEQ ID NO: 18);
  • CDR2 AIYPGDGDTRYTQKFKG (SEQ ID NO: 19);
  • CDR3 HYYGYDMDS (SEQ ID NO: 20);
  • CDR1 RASQSISNNLH (SEQ ID NO: 22);
  • CDR2 FTSQSIS(SEQ ID NO: 23);
  • CDR3 QQSNGWPYT (SEQ ID NO: 24).
  • the human immunoglobulin gene database in the .NET library is used to search for human germline IgG genes that are homologous to the cDNA sequences of mouse antibodies, and then the amino acid sequences and precise boundaries of the variable region CDRs are defined using the Kabat numbering system or the IMGT numbering system.
  • human IGHVs with high homology to mouse antibodies are selected as humanization templates, and humanization of the antibody variable regions is performed by CDR grafting.
  • humanization transformation involves the following steps: A. Compare the gene sequence of the mouse antibody with the gene sequence of the human embryonic system antibody to find out the sequence with high homology; B. Analyze and examine the HLA-DR affinity and select the human embryonic system framework sequence with low affinity; C. Use computer simulation technology to apply molecular docking to analyze the variable region and its surrounding framework amino acid sequence to examine its spatial three-dimensional binding mode.
  • Table 5 Numbering of humanized anti-CD93 antibodies and sources of their heavy and light chain variable regions
  • CHO-CD93 cells overexpressing human CD93 (CHO-K1 cells were purchased from ATCC cell bank, numbered CCL-61. Based on this cell line, a stable cell line overexpressing CD93 was independently constructed, and the amino acid sequence of CD93 is from NCBI accession number NP_036204.2) were incubated with different concentrations of anti-CD93 humanized antibody (starting concentration was 10 ⁇ g/ml, 3-fold dilution, a total of 10 concentration gradients) at 4°C for 30 min, and then the unbound antibody was washed away with staining buffer (PBS + 1% FBS), and then incubated with 1% (v/v) fluorescent secondary antibody goat anti-human IgG PE prepared in staining buffer (Southern Biotech, Cat#2040-09) was incubated at 4°C in the dark for 30 minutes.
  • staining buffer PBS + 1% FBS
  • the cells were collected by flow cytometry (BD, Canto II model) to detect the fluorescent antibodies bound to the cell surface.
  • the raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad and the EC50 was calculated.
  • C1 was used as the positive control of the experiment.
  • the anti-CD93 humanized antibodies of the present invention are all able to bind with high affinity to human CD93 highly expressed on the surface of CHO-CD93 cells.
  • Table 6-1 Cell-level affinity (EC 50 ) of anti-CD93 humanized antibodies
  • Table 6-2 Cell-level affinity (EC 50 ) of anti-CD93 humanized antibodies
  • Table 6-3 Cell-level affinity (EC 50 ) of anti-CD93 humanized antibodies
  • Table 6-4 Cell-level affinity (EC 50 ) of anti-CD93 humanized antibodies
  • Anti-CD93 humanized antibody blocks the binding of IGFBP7 protein to human CD93 expressing cells
  • CHO-CD93 cells overexpressing human CD93 were incubated with hIGFBP7mFc protein solution (Junshi's own expression, Lot#20200318, working concentration 10 ⁇ g/ml) prepared with staining buffer (PBS+1% FBS) and different concentrations of anti-CD93 humanized antibody (starting concentration of 50 ⁇ g/ml, 3-fold dilution, 10 concentration gradients) at 4°C for 30 minutes. Unbound antibodies were then washed away with staining buffer, and the cells were incubated with 1% (v/v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch, Cat#115-175-071) prepared with staining buffer at 4°C in the dark for 30 minutes.
  • hIGFBP7mFc protein solution Jobshi's own expression, Lot#20200318, working concentration 10 ⁇ g/ml
  • staining buffer PBS+1% FBS
  • different concentrations of anti-CD93 humanized antibody starting concentration of 50 ⁇ g/m
  • the anti-CD93 humanized antibodies of the present invention can effectively block the binding of hIGFBP7 protein to CHO-CD93 cells.
  • Table 7-1 Ability of anti-CD93 humanized antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC 50 )
  • Table 7-2 Ability of anti-CD93 humanized antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC 50 )
  • Table 7-3 Ability of anti-CD93 humanized antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC 50 )
  • hu37, Ref1 (17B10) and Ref2 (H3L3) antibodies can all bind to hCD93 with high affinity, and the binding activities of hu37, Ref1 (17B10) and Ref2 (H3L3) are comparable.
  • Example 12 Binding of anti-hCD93 monoclonal antibodies to CHO hCD93 cells
  • CHOhCD93 cells (CHO-K1 cells stably expressing hCD93, hCD93 positive) were incubated with different concentrations of hCD93 monoclonal antibody hu37 (starting concentration of 3.3 ⁇ g/ml, 3-fold dilution, a total of 10 concentration gradients) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, cells were collected by flow cytometry (BD Fortessa) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad ( Figure 1) and the EC 50 was calculated.
  • the positive controls were Ref1 (17B10) and Ref2 (H3L3) antibodies; the negative control was anti-KLH hu-IgG1 antibody.
  • hu37, Ref1 (17B10) and Ref2 (H3L3) antibodies can all bind to hCD93 on the surface of CHO hCD93 cells with high affinity, with EC50 of 144.2 ng/mL, 150.3 ng/mL and 128.6 ng/mL, respectively.
  • the binding activities of hu37, Ref1 (17B10) and Ref2 (H3L3) are comparable.
  • Example 13 Anti-hCD93 monoclonal antibody blocks the binding of IGFBP7 to CHO hCD93 cells
  • CHOhCD93 cells (CHO-K1 cells stably expressing hCD93, hCD93 positive) were incubated with different concentrations of hCD93 monoclonal antibody hu37 (starting concentration of 30 ⁇ g/ml, 3-fold dilution, a total of 10 concentration gradients) and 15 ⁇ g/ml hIGFBP7 (CD93 ligand) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, the cells were collected by flow cytometry (BD Fortessa) to detect fluorescent antibodies bound to the cell surface.
  • BD Fortessa flow cytometry
  • the raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent inhibition curve was fitted by GraphPad ( Figure 2) and the IC 50 was calculated.
  • the positive controls were Ref1 (17B10) and Ref2 (H3L3) antibodies; the negative control was anti- KLH hu-IgG1 antibody.
  • both Ref1 (17B10) and Ref2 (H3L3) antibodies could not block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells, while hu37 could effectively block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells with an IC50 of 559.9 ng/ml.

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Abstract

La présente invention concerne un anticorps anti-CD93, une composition et une utilisation associées. La présente invention concerne en outre une molécule polynucléotidique codant pour l'anticorps ou un fragment de liaison à l'antigène de celui-ci selon la présente demande, un vecteur d'expression et une cellule hôte pour exprimer l'anticorps ou le fragment de liaison à l'antigène de celui-ci selon la présente demande, et une utilisation de l'anticorps ou du fragment de liaison à l'antigène de celui-ci selon la présente demande.
PCT/CN2023/113535 2023-08-17 2023-08-17 Anticorps anti-cd93, composition et utilisation associées Ceased WO2025035451A1 (fr)

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CN120518769A (zh) * 2025-07-25 2025-08-22 杭州市第一人民医院(西湖大学附属杭州市第一人民医院) 一种cd93单克隆抗体及其制备方法和应用

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