Anti-CD93 antibody or antigen-binding fragment thereof and use thereof

Abstract

The present invention relates to the field of biotechnology. Provided are an anti-CD93 antibody or an antigen-binding fragment thereof and the use thereof. The antibody can specifically bind to CD93, and can block the binding of CD93 to MMRN2 and / or the binding of CD93 to IGFBP7, thereby preventing the normal development and maturation of tumor blood vessels, exerting an anti-tumor effect, and providing a new possibility for the treatment and / or prevention of cancer.

Description

Anti-CD93 antibodies or antigen-binding fragments thereof and their applications

[0001] Priority Declaration

[0002] This application claims priority to Chinese invention patent application number 202311760668.1, filed on December 19, 2023, entitled “Anti-CD93 antibodies or antigen-binding fragments thereof and their applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biotechnology, and in particular to an anti-CD93 antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0004] CD93 (C1qRp) is a C-type lectin-like, type I, single-pass transmembrane protein. Its extracellular C-type lectin domain can bind to MMRN2 (polyprotein-2), making it a member of the Group XIV C-type lectin family. Under normal physiological conditions, CD93 mediates vascular and endothelial cell migration, with MMRN2 and IGFBP7 (insulin-like growth factor binding protein 7) as its ligands. During tumorigenesis, CD93 is overexpressed on the surface of tumor blood vessels. IGFBP7, also highly expressed in tumors, binds to CD93, accelerating the migration of tumor endothelial cells. MMRN2 also assists this process. These effects lead to excessive tumor vascular growth. Excessive migration causes vascular leakage, further deteriorating the tumor microenvironment, enabling immune cell infiltration into the tumor site and hindering drug delivery through vascular access. If antibodies are used to block the interaction between CD93 and its ligands IGFBP7 and MMNR2, tumor blood vessels can develop and mature normally, reversing the deterioration of the tumor microenvironment. This allows other drugs and the immune system to attack the tumor, achieving the goal of treatment. Therefore, the development and optimization of anti-CD93 antibodies is of great significance for the prevention or treatment of tumor-related diseases.

[0005] In view of this, the present disclosure is proposed. Summary of the Invention

[0006] One of the objectives of the present disclosure is to provide an antibody or antigen-binding fragment thereof that can specifically bind to CD93 and its use.

[0007] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0008] In a first aspect, an anti-CD93 antibody or antigen-binding fragment thereof is provided, comprising a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region;

[0009] The complementarity determining regions of the heavy chain variable region include HCDR1, HCDR2 and HCDR3, and the complementarity determining regions of the light chain variable region include LCDR1, LCDR2 and LCDR3;

[0010] The HCDR1 comprises amino acid residue SY, amino acid residue NY or amino acid residue DY;

[0011] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 14, 36, 57 or 73;

[0012] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 19, 41, 62 or 78;

[0013] The LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 23, amino acid residues GTN, amino acid residues GTS or amino acid residues NSA;

[0014] The LCDR2 comprises amino acid residue AA, amino acid residue SA or amino acid residue YA;

[0015] The LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 29, 50, 71 or 87.

[0016] In a second aspect, the present disclosure provides an anti-CD93 antibody or an antigen-binding fragment thereof, comprising:

[0017] (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7; and,

[0018] (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8;

[0019] The above-mentioned HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition or the IMGT definition.

[0020] In a third aspect, the present disclosure provides an anti-CD93 antibody or an antigen-binding fragment thereof, which competes with the anti-CD93 antibody of the first or second aspect for binding to CD93, or the epitope thereof that binds to the CD93 antigen is the same as the epitope of the anti-CD93 antibody of the first or second aspect that binds to the CD93 antigen.

[0021] In a fourth aspect, the present disclosure provides a biomaterial, comprising any one of the following (i) to (vi):

[0022] (i) a multispecific antibody comprising the anti-CD93 antibody or antigen-binding fragment thereof as described in any one of the preceding aspects;

[0023] (ii) a chimeric antigen receptor (CAR), comprising an extracellular region comprising an antigen-binding domain; and wherein the antigen-binding domain comprises the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9;

[0024] (iii) a nucleic acid molecule encoding the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antibody according to (i), or the chimeric antigen receptor according to (ii);

[0025] (iv) a vector carrying the nucleic acid molecule described in (iii);

[0026] (v) a recombinant cell, characterized in that the recombinant cell comprises the nucleic acid molecule described in (iii), or comprises the vector described in (iv), or expresses the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or expresses the multispecific antibody described in (i);

[0027] (vi) An engineered immune cell, wherein the immune cell expresses the chimeric antigen receptor described in (ii) or contains a nucleic acid molecule encoding the chimeric antigen receptor described in (iii).

[0028] In a fifth aspect, the present disclosure provides use of the anti-CD93 antibody or antigen-binding fragment thereof of the first, second, or third aspect, or the biomaterial of the fourth aspect, in any of the following:

[0029] (I) Detection of CD93 or cells expressing CD93;

[0030] (II) preparing products for detecting CD93 or cells expressing CD93;

[0031] (III) blocking CD93 binding to MMRN2, and / or blocking CD93 binding to IGFBP7;

[0032] (IV) preparing a blocking agent for blocking CD93 binding to MMRN2; or preparing a blocking agent for blocking CD93 binding to IGFBP7;

[0033] (V) preparing a pharmaceutical composition for treating, preventing or ameliorating a disease, disorder or condition associated with CD93;

[0034] (VI) Preparing a pharmaceutical composition for promoting normalization of tumor microenvironment blood vessels.

[0035] In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned anti-CD93 antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, or the aforementioned engineered immune cell.

[0036] In a seventh aspect, the present disclosure provides a method for producing the aforementioned anti-CD93 antibody or antigen-binding fragment thereof, comprising the following steps:

[0037] (a) culturing the aforementioned recombinant cells under conditions for expressing an anti-CD93 antibody or an antigen-binding fragment thereof;

[0038] (b) isolating and purifying the anti-CD93 antibody or antigen-binding fragment thereof obtained in step (a).

[0039] In an eighth aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease, disorder, or condition associated with CD93, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-CD93 antibody or antigen-binding fragment thereof and / or the aforementioned pharmaceutical composition. The present disclosure also provides an anti-CD93 antibody or antigen-binding fragment according to any of the preceding aspects for use as a medicament.

[0040] In a ninth aspect, a method for promoting normalization of tumor microenvironment blood vessels is provided, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-CD93 antibody or antigen-binding fragment thereof and / or the aforementioned pharmaceutical composition.

[0041] In a tenth aspect, the present disclosure provides a kit for detecting CD93, comprising the aforementioned anti-CD93 antibody or antigen-binding fragment thereof.

[0042] The present disclosure includes the following benefits:

[0043] In some embodiments, the provided anti-CD93 antibodies or antigen-binding fragments thereof are capable of specifically binding to the CD93 protein and blocking the binding of CD93 to MMRN2 and / or blocking the binding of CD93 to IGFBP7. Functional methods based on HUVECs (human umbilical vein endothelial cells) are also used to simulate the mechanism by which candidate antibodies exert their efficacy in vivo, thereby demonstrating that the screened candidate antibodies have clear biological activity. Notably, the present disclosure discovered that IGFBP7 is more effective than MMRN2 in promoting the formation of HUVECs-simulated blood vessels, and the anti-CD93 antibodies provided in some embodiments are more effective than antibodies known in the prior art in blocking the binding of CD93 to IGFBP7, providing new possibilities for the treatment and / or prevention of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] FIG1 is a curve showing the binding activity of CD93 monoclonal antibodies (CG002-01-11) to CHO-hCD93 cells under 3-fold dilution conditions as determined by flow cytometry fluorescence sorting in Example 2;

[0046] FIG2 is a curve showing the binding activity of CD93 monoclonal antibodies (CG002-12-22) to CHO-hCD93 cells under 5-fold dilution conditions as determined by flow cytometry fluorescence sorting in Example 2;

[0047] FIG3 is a curve showing the binding activity of CD93 monoclonal antibodies (CG002-02, 06, 08-10, 15, 17, 20-22) to CHO-hCD93 cells under 5-fold dilution conditions as determined by flow cytometry fluorescence sorting in Example 2;

[0048] FIG4 is a blocking curve of the CD93 monoclonal antibody (CG002-01-11) in Example 3 blocking the binding between CHO-hCD93 and MMRN2 under 3-fold dilution conditions;

[0049] FIG5 is a blocking curve of the CD93 monoclonal antibody (CG002-12-22) in Example 3 blocking the binding between CHO-hCD93 and MMRN2 under 5-fold dilution conditions;

[0050] FIG6 is a blocking curve of the CD93 monoclonal antibodies (CG002-02, 06, 08-10, 15, 17, 20-22) in Example 3 blocking the binding between CHO-hCD93 and MMRN2 under 5-fold dilution conditions;

[0051] FIG7 is a blocking curve of the CD93 monoclonal antibody (CG002-01-11) in Example 4 blocking the binding between CHO-hCD93 and IGFBP7 under 3-fold dilution conditions;

[0052] FIG8 is a blocking curve of the CD93 monoclonal antibody (CG002-12-22) in Example 4 blocking the binding between CHO-hCD93 and IGFBP7 under 5-fold dilution conditions;

[0053] FIG9 is a blocking curve of the CD93 monoclonal antibodies (CG002-02, 06, 08-10, 15, 17, 20-22) in Example 4 blocking the binding between CHO-hCD93 and IGFBP7 under 3-fold dilution conditions;

[0054] FIG10 is a binding activity curve of CD93 monoclonal antibodies (CG002-02, 10, 15, 20) to CHO-hCD93 cells under 5-fold dilution conditions;

[0055] FIG11 shows the blocking curves of CD93 monoclonal antibodies (CG002-02, 10, 15, 20) blocking the binding between CHO-hCD93 and MMRN2 at 5-fold dilution;

[0056] FIG12 shows the blocking curves of CD93 monoclonal antibodies (CG002-02, 10, 15, 20) blocking the binding between CHO-hCD93 and IGFBP7 under 3-fold dilution conditions;

[0057] FIG13 shows the inhibition of HUVECs simulated angiogenesis by CD93 monoclonal antibodies (CG002-02, 10, 15, 20) in the presence of only antibodies in Example 5;

[0058] FIG14 shows the total length of simulated blood vessels formed by HUVECs after treatment with CD93 monoclonal antibodies (CG002-02, 10, 15, 20) in the presence of only antibodies in Example 5, calculated using Image J software;

[0059] FIG15 shows the inhibition of HUVECs simulated angiogenesis by CD93 monoclonal antibodies (CG002-02, 10, 15, 20) in the presence of the ligand MMRN2 in Example 5;

[0060] FIG16 shows the total length of simulated blood vessels formed by HUVECs after treatment with CD93 monoclonal antibodies (CG002-02, 10, 15, 20) in the presence of the ligand MMRN2 in Example 5, calculated using Image J software;

[0061] FIG17 shows the inhibition of HUVECs simulated angiogenesis by CD93 monoclonal antibodies (CG002-02, 10, 15, 20) in the presence of ligand IGFBP7 in Example 5;

[0062] FIG18 shows the total length of simulated blood vessels formed by HUVECs after treatment with CD93 monoclonal antibodies (CG002-02, 10, 15, 20) in the presence of ligand IGFBP7 in Example 5, as calculated using Image J software. DETAILED DESCRIPTION

[0063] The following will clearly and completely describe the technical solutions of the present disclosure in conjunction with the embodiments. Obviously, the embodiments described are only a part of the embodiments of the present disclosure, not all of them. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0064] In the present disclosure, the articles "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

[0065] In this disclosure, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of this disclosure, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0066] In the present disclosure, the term "CD93", also known as C1qRp, is a C-type lectin-like type I single-pass transmembrane protein. "CD93", "CD93 antigen" and "CD93 protein" are used interchangeably in the present disclosure. Unless otherwise indicated, the term includes human CD93, isoforms and species homologs expressed by cells naturally expressed or transfected with CD93 genes. The term also includes artificially modified CD93, including but not limited to polypeptides or proteins that have been mutated, truncated or fused with other domains, and retain the necessary antigenic epitopes for antibody binding. The anti-CD93 antibodies or antigen-binding fragments thereof provided in the present disclosure are capable of specifically binding to CD93.

[0067] In this disclosure, the term "MMRN2" refers to multimerin-2, an ECM glycoprotein tightly associated with the cell surface. MMRN2 comprises four distinct disulfide-bonded subunits, p125, p140, p110, and p200, and is an EMILIN-like protein with a signal peptide, an N-terminal EMI domain, and a C-terminal C1q domain separated by a central coiled-coil-rich region. Unless otherwise indicated, the term "MMRN2" includes human MMRN2, isoforms, and species homologs expressed by cells naturally expressed or transfected with the MMRN2 gene. The term also includes artificially modified MMRN2, including, but not limited to, polypeptides or proteins that have undergone mutations, truncations, or fusions with other domains.

[0068] In this disclosure, the term "IGFBP7" refers to insulin-like growth factor binding protein 7 (IGFBP7), a member of the IGFBP family that binds to insulin with high affinity and insulin-like growth factor with low affinity. Unless otherwise specified, the term "IGFBP7" includes human IGFBP7, isoforms, and species homologs expressed by cells naturally expressed or transfected with the IGFBP7 gene. The term also includes artificially modified IGFBP7, including, but not limited to, polypeptides or proteins that have been mutated, truncated, or fused to other domains.

[0069] In this disclosure, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The term "antibody" is used in the broadest sense to encompass various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, full-length antibodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Typically, a natural, intact antibody comprises two heavy (H) chains and two light (L) chains. Antibodies can be divided into five major classes or isotypes: IgA, IgD, IgE, IgG, and IgM, depending on whether they contain α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). Each heavy chain consists of a variable region (VH) and first, second, third, and optionally fourth constant regions (CH1, CH2, CH3, and CH4, respectively). The light chains of mammals can be divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region (CL). The variable regions of the light and heavy chains determine the binding of antigens. The variable region of each chain usually contains three hypervariable regions, called "complementarity determining regions (CDRs)", of which the light chain CDRs include LCDR1, LCDR2, LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions (VH and VL) are each composed of three complementarity determining regions connected by four framework regions (FRs). Generally, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0070] In the present disclosure, the term "antigen-binding fragment" is a substance comprising an antibody CDR that lacks some of the amino acids present in the full-length chain but is still capable of specific binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies). The above-mentioned antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0071] In the present disclosure, the term "Fab" of an antibody refers to a portion of an antibody composed of a single light chain (including a variable region and a constant region) and the variable region and the first constant region of a single heavy chain bound together by a disulfide bond. A "Fab' fragment" refers to a Fab fragment that includes part of the hinge region. "F(ab')2" refers to a dimer of Fab'. An "Fv fragment" is composed of the variable region of a single light chain and / or the variable region of a single heavy chain. A "single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by directly connecting the light chain variable region and the heavy chain variable region to each other or connected by a peptide linker sequence. The "minimum recognition unit of an antibody" refers to a structure containing only a single CDR in the variable region. Although the minimum recognition unit has a small molecular weight and low affinity, it has the ability to bind to the antigen.

[0072] The CDR boundaries of the antibodies or antigen-binding fragments thereof in the present disclosure can be defined or identified according to the IMGT, Kabat, Chothia, AbM, or Contact definitions. CDRs defined in other ways acceptable in the art also fall within the scope of protection of the present disclosure (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); RM MacCallum et al.,. Antibody–antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, ACR Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,Developmental and Comparative Immunology 27(2003)55–77).

[0073] In this disclosure, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Common naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bonded to a hydrogen, carboxyl, amino, and R groups), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids. In the present disclosure, the term "percent identity" refers to the extent to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Comparison of amino acid sequence identity percentages can be performed in various ways known in the art, such as software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, which are well known in the art. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

[0074] In the present disclosure, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, for example, a reaction between an antibody and an antigen. In some specific embodiments, for example, the binding is determined by flow cytometry fluorescence sorting technology.

[0075] In the present disclosure, the term "anti-CD93 antibody" refers to an antibody that can specifically bind to CD93. In some specific embodiments, the "anti-CD93 antibody" specifically binds to human CD93.

[0076] In the present disclosure, the term "affinity" or "avidity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen. The strength or affinity of an immunological binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. KD can be determined using any conventional method known in the art, including but not limited to Biacore assays, Octet methods, microthermophoresis, HPLC MS methods, and flow cytometry fluorescence sorting techniques.

[0077] The binding of the antibodies or antigen-binding fragments thereof provided herein to CD93 can also be measured using a "half-maximal effective concentration (EC50)." This refers to the concentration of the drug or antibody that achieves 50% of the maximal biological effect after a specified exposure time. Generally, a lower EC50 indicates better affinity, indicating that binding to the target protein occurs at lower concentrations. EC50 values ​​can be determined using binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting, and other binding assays).

[0078] In the present disclosure, the term "epitope" refers to any antigenic determinant on an antigen that is bound by the paratope of an antibody. Antigenic determinants are typically special chemical groups with a certain composition and structure. An epitope can be linear (i.e., continuous) or conformational (i.e., comprising spaced-apart amino acid residues, non-continuous). An epitope defines the minimum binding site for an antibody and is therefore the specific target of an antibody or its antigen-binding fragment. An epitope can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy).

[0079] In the present disclosure, the term "multispecific antibody" refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.

[0080] The “chimeric antigen receptor” in the present disclosure, CAR is an artificial receptor that simulates the function of TCR, which is composed of an extracellular domain, a transmembrane domain and an intracellular signaling domain connected in sequence. When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal can be transmitted to the cell through the hinge region and the transmembrane region. The intracellular signaling domain then converts the signal into an activation signal to activate effector cells. The effector cells kill tumor cells by secreting perforin or producing cytokines. At the same time, the effector cells themselves also amplify, further expanding the immune killing effect. The extracellular domain is generally composed of a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to the antigen and a hinge region (Hinge) that acts as a connection. The single-chain variable fragment is the antigen binding domain of CAR, which determines the specificity and function of CAR-immune cells. The hinge region is the extracellular domain of the CAR that connects the single-chain variable fragment and the transmembrane domain. It generally maintains the stability required for robust CAR expression and activity in effector cells. The hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8α / CD28. The type and length of the hinge region have a significant impact on the functional activity of the CAR. The transmembrane domain connects the extracellular domain of the CAR to the intracellular signaling domain. Commonly used transmembrane domains are derived from CD4, CD8, CD28, and CD3ζ or their derivatives. The choice of transmembrane domain influences the degree of activation of the CAR structure in cellular function. The intracellular domain consists of a costimulatory domain and a signaling domain. The costimulatory domain is typically derived from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). The signaling domain is typically the T cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain.

[0081] The CAR of the present disclosure (including functional parts and functional variants thereof) can be obtained by methods known in the art, for example, can be prepared by any suitable method for preparing a polypeptide or protein.

[0082] In the present disclosure, the term "engineered immune cell" refers to an immune cell expressing CAR or an immune cell modified by CAR, wherein the immune cell includes but is not limited to T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells (Natural killer T cell), dendritic cells, granulocytes, B cells, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0083] In this disclosure, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, the encoding may optionally encode a sense strand or an antisense strand. Nucleic acid molecules may be naturally occurring, synthetic, recombinant, or any combination thereof. "Nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably.

[0084] In alternative embodiments, the nucleic acid molecule is RNA or DNA. The nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid molecule is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. When it is incorporated into a vector, DNA is preferably used.

[0085] In the present disclosure, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can also contain a replication initiation site. A vector can also include components that assist its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. A vector can be an expression vector or a cloning vector. In some embodiments, the vectors provided by the present disclosure (e.g., expression vectors) contain a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof as described in the present disclosure, at least one promoter (e.g., SV40, CMV, EF 1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0086] In this disclosure, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, e.g., if it is naturally occurring, from its natural environment. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0087] In this disclosure, the term "recombinant cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced. The exogenous polynucleotide may or may not be integrated into the genome of the "recombinant cell." When the recombinant cell contains a vector, the vector can be introduced into a mammalian cell to construct a recombinant cell, which can then be used to express the antibodies or antigen-binding fragments provided herein. Culturing the recombinant cells can yield the corresponding antibodies. Examples of mammalian cells that can be used include CHO cells.

[0088] In the present disclosure, the term "pharmaceutical composition" refers to a composition that is in a form that allows for the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. In some specific embodiments, the antibody contained in the pharmaceutical composition or the expressed antibody can specifically target and bind to CD93, blocking CD93 from binding to MMRN2 and / or IGFBP7.

[0089] In the present disclosure, the term "binding to CD93" includes binding to isolated CD93 molecules and / or cells expressing CD93.

[0090] In the present disclosure, the term “blocking CD93 from binding to MMRN2” includes isolated CD93 molecules and / or cells expressing CD93, and MMRN2 includes isolated MMRN2 molecules and / or cells expressing MMRN2.

[0091] In the present disclosure, the term “blocking CD93 from binding to IGFBP7” includes isolated CD93 molecules and / or cells expressing CD93, and IGFBP7 includes isolated IGFBP7 molecules and / or cells expressing IGFBP7.

[0092] In the present disclosure, the term "a blocker that blocks CD93 binding to MMRN2" refers to any agent that reduces the level of binding between CD93 and MMRN2 compared to the level of binding between CD93 and MMRN2 in the absence of the agent.

[0093] In the present disclosure, the term "a blocker that blocks CD93 binding to IGFBP7" refers to any agent that reduces the level of binding between CD93 and IGFBP7 compared to the level of binding between CD93 and IGFBP7 in the absence of the agent.

[0094] In the present disclosure, the term "IL-17D-CD93 axis" refers to the recognition of IL-17D by its receptor CD93.

[0095] In this disclosure, "pharmaceutically acceptable carrier" can include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0096] In this disclosure, the term "subject" or "patient" refers to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0097] In this disclosure, the term "diagnosis" refers to the identification of a pathological state, disease, or condition, such as the identification of a CD93-associated disease, or the identification of a subject with a CD93-associated disease who may benefit from a particular treatment regimen. In some embodiments, diagnosis comprises the identification of abnormal levels or activity of CD93. In some embodiments, diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0098] As used herein, a "CD93-associated" disease, disorder, or condition refers to any disease or condition caused by, exacerbated by, or associated with an increase or decrease in the expression or activity of CD93. In some embodiments, a CD93-associated disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as cancer. In some embodiments, a CD93-associated disease or condition is characterized by expression or overexpression of CD93 and / or a CD93-associated gene.

[0099] In the present disclosure, the term "CD93-expressing" cancer refers to a cancer in which CD93 is expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells, and the level of CD93 expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells is significantly higher than the level expected for normal cells.

[0100] In the present disclosure, the term "effective amount" refers to a therapeutic amount, which is sufficient to reduce or improve the severity and / or duration of a condition or one or more symptoms thereof; prevent the progression of the disease; cause the disease to regress; prevent the recurrence, development or progression of one or more symptoms associated with the disease; detect the disease; or enhance or improve the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). The therapeutically effective dose of the antibody or antigen-binding fragment thereof described in the present disclosure depends on a variety of factors well known in the art, such as body weight, age, past medical history, current treatment, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the degree of tumor development. A person skilled in the art (e.g., a doctor or veterinarian) may proportionally reduce or increase the dose according to these or other conditions or requirements.

[0101] In a first aspect, an anti-CD93 antibody or antigen-binding fragment thereof is provided, comprising a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region;

[0102] The complementarity determining regions of the heavy chain variable region include HCDR1, HCDR2 and HCDR3, and the complementarity determining regions of the light chain variable region include LCDR1, LCDR2 and LCDR3;

[0103] The HCDR1 comprises amino acid residue SY, amino acid residue NY or amino acid residue DY.

[0104] In an optional embodiment, the HCDR1 includes amino acid residue SY, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 9 to 13.

[0105] In an optional embodiment, the HCDR1 includes amino acid residue NY, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 31 to 35.

[0106] In an optional embodiment, the HCDR1 includes amino acid residue DY, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 52 to 56.

[0107] The HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 14, 36, 57 or 73.

[0108] In an optional embodiment, the HCDR2 includes the amino acid sequence shown in SEQ ID NO. 14, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 14 to 18.

[0109] In an optional embodiment, the HCDR2 includes the amino acid sequence shown in SEQ ID NO. 36, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 36 to 40.

[0110] In an optional embodiment, the HCDR2 includes the amino acid sequence shown in SEQ ID NO. 57, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 57 to 61.

[0111] In an optional embodiment, the HCDR2 includes the amino acid sequence shown in SEQ ID NO. 73, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 73 to 77.

[0112] The HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 19, 41, 62 or 78.

[0113] In an optional embodiment, the HCDR3 includes the amino acid sequence shown in SEQ ID NO. 19, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 20 to 22.

[0114] In an optional embodiment, the HCDR3 includes the amino acid sequence shown in SEQ ID NO. 41, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 42 to 44.

[0115] In an optional embodiment, the HCDR3 includes the amino acid sequence shown in SEQ ID NO. 62, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 63 to 65.

[0116] In an optional embodiment, the HCDR3 includes the amino acid sequence shown in SEQ ID NO. 78, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 79 to 81.

[0117] The LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 23, amino acid residue GTN, amino acid residue GTS or amino acid residue NSA.

[0118] In an optional embodiment, the LCDR1 includes the amino acid sequence shown in SEQ ID NO. 23, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 24 to 26.

[0119] In an optional embodiment, the LCDR1 includes amino acid residues GTN, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 45 to 47.

[0120] In an optional embodiment, the LCDR1 includes amino acid residues GTS, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 66 to 68.

[0121] In an optional embodiment, the LCDR1 includes the amino acid residue NSA, for example, but not limited to, any one of the sequences shown in SEQ ID NOs. 82 to 84.

[0122] The LCDR2 comprises amino acid residue AA, amino acid residue SA or amino acid residue YA.

[0123] In an optional embodiment, the LCDR2 includes amino acid residue AA, for example, but not limited to, amino acid residue AA or any sequence shown in SEQ ID NO. 27, 28, 85 or 86.

[0124] In an optional embodiment, the LCDR2 includes amino acid residue SA, for example, but not limited to, amino acid residue SA, or any sequence shown in SEQ ID NO. 48 or 49.

[0125] In an optional embodiment, the LCDR2 includes amino acid residue YA, for example, but not limited to, amino acid residue YA, or any sequence shown in SEQ ID NO. 69 or 70.

[0126] The LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 29, 50, 71 or 87.

[0127] In an optional embodiment, the LCDR3 includes an amino acid sequence as shown in SEQ ID NO. 29, for example, but not limited to, any sequence shown in SEQ ID NO. 29 or 30.

[0128] In an optional embodiment, the LCDR3 includes an amino acid sequence as shown in SEQ ID NO. 50, for example, but not limited to, the sequence shown in either SEQ ID NO. 50 or 51.

[0129] In an optional embodiment, the LCDR3 includes an amino acid sequence as shown in SEQ ID NO. 71, for example, but not limited to, the sequence shown in either SEQ ID NO. 71 or 72.

[0130] In an optional embodiment, the LCDR3 includes an amino acid sequence as shown in SEQ ID NO. 87, for example, but not limited to, the sequence shown in either SEQ ID NO. 87 or 88.

[0131] In an optional embodiment, according to the IMGT definition, in the anti-CD93 antibody or antigen-binding fragment thereof, the HCDR1 includes the amino acid sequence shown in SEQ ID NO.13, 35 or 56; the HCDR2 includes the amino acid sequence shown in SEQ ID NO.18, 40, 61 or 77; the HCDR3 includes the amino acid sequence shown in SEQ ID NO.22, 44, 65 or 81; the LCDR1 includes the amino acid sequence shown in SEQ ID NO.26, 47, 68 or 84; the LCDR2 includes amino acid residue AA, amino acid residue SA or amino acid residue YA; the LCDR3 includes the amino acid sequence shown in SEQ ID NO.30, 51, 72 or 88.

[0132] In an optional embodiment, the complementarity determining region of the anti-CD93 antibody or antigen-binding fragment thereof is selected from any one of the following (a)-(d):

[0133] (a) the HCDR1 comprises the amino acid sequence set forth by amino acid residues SY, the HCDR2 comprises the amino acid sequence set forth by SEQ ID NO.14, the HCDR3 comprises the amino acid sequence set forth by SEQ ID NO.19, the LCDR1 comprises the amino acid sequence set forth by SEQ ID NO.23, the LCDR2 comprises the amino acid residues AA, and the LCDR3 comprises the amino acid sequence set forth by SEQ ID NO.29; or

[0134] (b) the HCDR1 comprises amino acid residues NY, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 36, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 41, the LCDR1 comprises amino acid residues GTN, the LCDR2 comprises amino acid residues SA, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 50; or

[0135] (c) the HCDR1 comprises amino acid residues DY, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 57, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 62, the LCDR1 comprises amino acid residues GTS, the LCDR2 comprises amino acid residues YA, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 71; or

[0136] (d) the above-mentioned HCDR1 comprises amino acid residue DY, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.73, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.78, LCDR1 comprises amino acid residue NSA, LCDR2 comprises amino acid residue SA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.87.

[0137] In an optional embodiment, according to the IMGT definition: the complementarity determining region of the anti-CD93 antibody or antigen-binding fragment thereof is selected from any one of the following (a')-(d'):

[0138] (a') HCDR1 comprises the amino acid sequence set forth in SEQ ID NO.13, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO.18, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO.22, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO.26, LCDR2 comprises amino acid residue AA, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO.30; or

[0139] (b') HCDR1 comprises the amino acid sequence set forth in SEQ ID NO.35, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO.40, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO.44, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO.47, LCDR2 comprises amino acid residue SA, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO.51; or

[0140] (c') HCDR1 comprises the amino acid sequence set forth in SEQ ID NO.56, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO.61, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO.65, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO.68, LCDR2 comprises amino acid residues YA, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO.72; or

[0141] (d') HCDR1 comprises the amino acid sequence shown in SEQ ID NO.56, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.77, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.81, LCDR1 comprises the amino acid sequence shown in SEQ ID NO.84, LCDR2 comprises amino acid residue SA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.88.

[0142] In a second aspect, the present disclosure provides an anti-CD93 antibody or an antigen-binding fragment thereof, comprising:

[0143] (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7; and,

[0144] (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8;

[0145] The above-mentioned HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition or the IMGT definition.

[0146] In an optional embodiment, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-CD93 antibodies or antigen-binding fragments thereof provided by the present disclosure can be independently selected from Tables 1 to 4.

[0147] Tables 1 to 4 respectively show the CDR amino acid sequences defined according to different definition methods for the exemplary antibody CG002-10 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2), the exemplary antibody CG002-15 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4), the exemplary antibody CG002-02 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.6), and the exemplary antibody CG002-20 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8).

[0148] Table 1

[0149] Table 2

[0150] Table 3

[0151] Table 4

[0152] In an optional embodiment, the above-mentioned anti-CD93 antibody or its antigen-binding fragment contains a heavy chain framework region and / or a light chain framework region, and the above-mentioned heavy chain framework region and / or light chain framework region are derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0153] In an optional embodiment, the three CDRs of the above-mentioned anti-CD93 antibody or its antigen-binding fragment are separated by flanking portions called framework regions (FR, light chain FR includes LFR1, LFR2, LFR3 and LFR4, heavy chain FR includes HFR1, HFR2, HFR3 and HFR4).

[0154] In an optional embodiment, the HFR1 comprises an amino acid sequence as shown in any one of SEQ ID NO.89, 103 and 116, or comprises a sequence having at least 89% (e.g., 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.89, 103 and 116.

[0155] In an optional embodiment, the HFR2 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 90, 104, 117 and 128, or comprises a sequence having at least 84% (e.g., 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NOs. 90, 104, 117 and 128.

[0156] In an optional embodiment, the HFR3 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 92, 106, 119 and 130, or comprises a sequence having at least 92% (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NOs. 92, 106, 119 and 130.

[0157] In an optional embodiment, the HFR4 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 94, 108 and 132, or comprises a sequence having at least 87% (e.g., 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NOs. 94, 108 and 132.

[0158] In an optional embodiment, the LFR1 includes an amino acid sequence as shown in any one of SEQ ID NO.95, 109, 121 and 133, or includes a sequence having at least 73% (e.g., 73%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.95, 109, 121 and 133.

[0159] In an optional embodiment, the LFR2 includes an amino acid sequence as shown in any one of SEQ ID NO.97, 111 and 123, or includes a sequence having at least 77% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.97, 111 and 123.

[0160] In an optional embodiment, the LFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO.99, 113, 125 and 136, or comprises a sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.99, 113, 125 and 136.

[0161] In an optional embodiment, the LFR4 includes an amino acid sequence as shown in any one of SEQ ID NO.101, 115, 127 and 138, or includes a sequence having at least 84% (e.g., 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.101, 115, 127 and 138.

[0162] In an optional embodiment, the HFR1 comprises the HFR1 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7.

[0163] In an optional embodiment, the HFR2 comprises the HFR2 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7.

[0164] In an optional embodiment, the HFR3 comprises the HFR3 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7.

[0165] In an optional embodiment, the HFR4 comprises the HFR4 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7.

[0166] In an optional embodiment, the LFR1 comprises the LFR1 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8.

[0167] In an optional embodiment, the LFR2 comprises the LFR2 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8.

[0168] In an optional embodiment, the LFR3 comprises the LFR3 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8.

[0169] In an optional embodiment, the LFR4 comprises the LFR4 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8.

[0170] In an optional embodiment, the HFR1, HFR2, HFR3, and HFR4 comprise the HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region set forth in SEQ ID NO. 1. The CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 1, and the FR regions of the corresponding definition are obtained based on the structure of the heavy chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3, and HFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 89, 91, 93, and 94, respectively.

[0171] In an optional embodiment, the HFR1, HFR2, HFR3, and HFR4 comprise the HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region set forth in SEQ ID NO. 3. The CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 2, and the FR regions of the corresponding definition are obtained based on the structure of the heavy chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3, and HFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 103, 105, 107, and 108, respectively.

[0172] In an optional embodiment, the HFR1, HFR2, HFR3, and HFR4 comprise the HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region set forth in SEQ ID NO. 5. The CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 3, and the FR regions of the corresponding definition are obtained based on the structure of the heavy chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3, and HFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 116, 118, 120, and 108, respectively.

[0173] In an optional embodiment, the HFR1, HFR2, HFR3, and HFR4 comprise the HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region set forth in SEQ ID NO. 7. The CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 4, and the FR regions of the corresponding definition are obtained based on the structure of the heavy chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3, and HFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 116, 129, 131, and 132, respectively.

[0174] In an optional embodiment, the LFR1, LFR2, LFR3, and LFR4 comprise LFR1, LFR2, LFR3, and LFR4 of the light chain variable region set forth in SEQ ID NO. 2. These CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 1, and the FR regions of the corresponding definition are obtained based on the structure of the light chain variable region. Taking the IMGT definition as an example, LFR1, LFR2, LFR3, and LFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 96, 98, 100, and 102, respectively.

[0175] In an optional embodiment, the LFR1, LFR2, LFR3, and LFR4 comprise LFR1, LFR2, LFR3, and LFR4 of the light chain variable region set forth in SEQ ID NO. 4. These CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 2, and the FR regions of the corresponding definition are obtained based on the structure of the light chain variable region. Taking the IMGT definition as an example, LFR1, LFR2, LFR3, and LFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 110, 112, 114, and 115, respectively.

[0176] In an optional embodiment, the LFR1, LFR2, LFR3, and LFR4 comprise the LFR1, LFR2, LFR3, and LFR4 of the light chain variable region set forth in SEQ ID NO. 6. These CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 3, and the FR regions of the corresponding definition are obtained based on the structure of the light chain variable region. Taking the IMGT definition as an example, LFR1, LFR2, LFR3, and LFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 122, 124, 126, and 127, respectively.

[0177] In an optional embodiment, the LFR1, LFR2, LFR3, and LFR4 comprise LFR1, LFR2, LFR3, and LFR4 of the light chain variable region set forth in SEQ ID NO. 8. These CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 4, and the FR regions of the corresponding definition are obtained based on the structure of the light chain variable region. Taking the IMGT definition as an example, LFR1, LFR2, LFR3, and LFR4 comprise the amino acid sequences set forth in SEQ ID NOs. 134, 135, 137, and 138, respectively.

[0178] In an alternative embodiment, the anti-CD93 antibody or antigen-binding fragment thereof comprises an appropriate framework region (FR) sequence, as long as the antibody can specifically bind to CD93. For example, the CDR sequences shown in Tables 1 to 4 above are obtained from mouse antibodies, but can be grafted onto any suitable FR sequence of any suitable species (e.g., mouse, human, rat, rabbit, etc.) using suitable methods known in the art (e.g., recombinant technology).

[0179] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NO. 1, 3, 5 or 7, or a heavy chain variable region having at least 77% (e.g., 77%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in any one of SEQ ID NO. 1, 3, 5 or 7;

[0180] And / or, the anti-CD93 antibody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NO. 2, 4, 6 or 8, or a light chain variable region having at least 72% (e.g., 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in any one of SEQ ID NO. 2, 4, 6 or 8.

[0181] In an optional embodiment, the above-mentioned anti-CD93 antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.2.

[0182] In an optional embodiment, the above-mentioned anti-CD93 antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.3; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.4.

[0183] In an optional embodiment, the above-mentioned anti-CD93 antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.5; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.6.

[0184] In an optional embodiment, the above-mentioned anti-CD93 antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.7; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.8.

[0185] In an optional embodiment, the antigen-binding fragment includes one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv and the minimum recognition unit of an antibody.

[0186] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof comprises a partial or complete sequence of a constant region, wherein the constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof;

[0187] In an optional embodiment, the heavy chain constant region contains a sequence of a partial or complete constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0188] In an alternative embodiment, the light chain constant region is a kappa or lambda chain.

[0189] In an optional embodiment, the constant region is derived from species comprising one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans.

[0190] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof has one or more of the following properties (i)-(v):

[0191] (i) blocking the binding of CD93 to IGFBP7, and / or blocking the binding of CD93 to MMRN2;

[0192] (ii) does not block the binding of CD93 to IGFBP7, or does not block the binding of CD93 to MMRN2;

[0193] (iii) binding to CD93 with an EC50 of no more than 0.3 μg / mL (e.g., no more than 0.1 μg / mL, 0.15 μg / mL, 0.2 μg / mL, or 0.25 μg / mL), wherein the binding is determined by flow cytometry fluorescence sorting, and the EC50 is determined by flow cytometry fluorescence sorting, which can be determined with reference to Example 2.

[0194] (iv) blocking MMRN2 binding to CD93 with an EC50 of no more than 0.2 μg / mL (e.g., no more than 0.05 μg / mL, 0.1 μg / mL, or 0.15 μg / mL), as determined by flow cytometry fluorescence sorting technology. The determination method can be referred to Example 3.

[0195] (v) blocking IGFBP7 binding to CD93 with an EC50 of no more than 3 μg / mL (e.g., no more than 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL or 2.5 μg / mL); the binding is determined by flow cytometry fluorescence sorting technology, and the determination method can be referred to Example 4.

[0196] In an optional embodiment, the CD93 in (i) to (v) above comprises human CD93.

[0197] In an alternative embodiment, the CD93 in (i) to (v) above is CD93 expressed by mammalian cells.

[0198] In an alternative embodiment, the above (iv) blocks MMRN2 binding to CHO cells expressing human CD93 with an EC50 of no more than 0.2 μg / mL.

[0199] In an alternative embodiment, the above (v) blocks the binding of IGFBP7 to CHO cells expressing human CD93 with an EC50 of no more than 3 μg / mL.

[0200] In a third aspect, the present disclosure provides an anti-CD93 antibody or an antigen-binding fragment thereof, which competes with the anti-CD93 antibody of the first or second aspect for binding to CD93, or the epitope thereof that binds to the CD93 antigen is the same as the epitope of the anti-CD93 antibody of the first or second aspect that binds to the CD93 antigen.

[0201] In a fourth aspect, the present disclosure provides a multispecific antibody comprising any of the aforementioned anti-CD93 antibodies or antigen-binding fragments thereof.

[0202] In an optional embodiment, the multispecific antibody further comprises a second portion that specifically recognizes other antigens, such as an antibody or antigen-binding fragment thereof that recognizes other antigens.

[0203] In an optional embodiment, other antigens include immune checkpoint molecules, exemplary immune checkpoint molecules include but are not limited to PD-1 or PD-L1.

[0204] In an alternative embodiment, the other antigens include tumor antigens, such as VEGF (vascular endothelial growth factor).

[0205] In a fifth aspect, the present disclosure provides a chimeric antigen receptor (CAR), which includes an extracellular region, wherein the extracellular region includes an antigen binding domain; and the antigen binding domain contains the anti-CD93 antibody or antigen binding fragment thereof shown in any one of the aforementioned items.

[0206] In a sixth aspect, the present disclosure provides a nucleic acid molecule encoding the aforementioned anti-CD93 antibody or antigen-binding fragment thereof, or the multispecific antibody described in the fourth aspect, or the chimeric antigen receptor described in the fifth aspect.

[0207] In an alternative embodiment, the nucleic acid molecule is an isolated nucleic acid molecule.

[0208] In a seventh aspect, the present disclosure provides a vector carrying the nucleic acid molecule of the sixth aspect.

[0209] In an eighth aspect, the present disclosure provides a recombinant cell comprising the aforementioned nucleic acid molecule, or the aforementioned vector, or expressing the aforementioned anti-CD93 antibody or antigen-binding fragment thereof, or expressing the multispecific antibody described in the fourth aspect.

[0210] In a ninth aspect, the present disclosure provides an engineered immune cell that expresses the aforementioned chimeric antigen receptor or contains a nucleic acid molecule encoding the aforementioned chimeric antigen receptor.

[0211] In a tenth aspect, the present disclosure provides use of the anti-CD93 antibody or antigen-binding fragment thereof of the first, second, or third aspect, or the multispecific antibody of the fourth aspect, or the chimeric antigen receptor of the fifth aspect, or the nucleic acid molecule of the sixth aspect, or the vector of the seventh aspect, or the recombinant cell of the eighth aspect, or the engineered immune cell of the ninth aspect in any of the following:

[0212] (I) Detection of CD93 or cells expressing CD93;

[0213] (II) preparing products for detecting CD93 or cells expressing CD93;

[0214] (III) blocking CD93 binding to MMRN2, and / or blocking CD93 binding to IGFBP7;

[0215] (IV) preparing a blocking agent for blocking CD93 binding to MMRN2; or preparing a blocking agent for blocking CD93 binding to IGFBP7;

[0216] (V) preparing a pharmaceutical composition for treating, preventing or ameliorating a disease, disorder or condition associated with CD93;

[0217] (VI) Preparing a pharmaceutical composition for promoting normalization of tumor microenvironment blood vessels.

[0218] In an optional embodiment, the application of aspect (I) above can utilize anti-CD93 antibodies or antigen-binding fragments thereof to specifically target and bind to CD93, and detect CD93 or cells expressing CD93 based on immunoassay technology. For example, when the antibody or antigen-binding fragment chain is an immunoconjugate, for example, with a fluorescent group attached, a fluorescence detection device can be used to achieve localization or real-time detection of CD93. For example, it can be used in immunoblotting, immunoprecipitation, or flow cytometry techniques that involve the use of the specific binding properties of the CD93 antigen and antibody to detect CD93 or cells expressing CD93. Accordingly, in aspect (II) above, those skilled in the art can select the reagent components of the kit according to the actual detection method, including but not limited to antagonists, anti-CD93 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. The kit can also be used to detect diseases, disorders, or conditions related to CD93.

[0219] In an optional embodiment, the application of the above-mentioned (III) can enable the cells to be blocked from binding CD93 to MMRN2 and / or IGFBP7 to be fully contacted with the anti-CD93 antibody or its antigen-binding fragment, for example but not limited to, placing the cells to be blocked from binding to MMRN2 and / or IGFBP7 in a culture medium containing the anti-CD93 antibody or its antigen-binding fragment and incubating them.

[0220] In an optional embodiment, the above-mentioned blocking agent (IV) further contains excipients acceptable in the art, including but not limited to culture medium, preservatives and buffer components.

[0221] In an optional embodiment, the above aspect (I) is for non-diagnostic and non-therapeutic purposes.

[0222] In an optional embodiment, the above aspect (III) is for non-diagnostic and non-therapeutic purposes.

[0223] In an eleventh aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned anti-CD93 antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, or the aforementioned engineered immune cell.

[0224] In an optional embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipient may be any conventional carrier and / or excipient known in the art. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and the like; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, solubilizers, and emulsifiers.

[0225] In an optional embodiment, the pharmaceutical composition further comprises one or more active pharmaceutical ingredients having other therapeutic effects, including but not limited to one or more combinations of chemotherapeutic agents, anticancer drugs, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0226] In an optional embodiment, the pharmaceutical composition is used as a drug for treating, preventing or alleviating a disease, disorder or condition associated with CD93.

[0227] In a twelfth aspect, the present disclosure provides a method for producing the aforementioned anti-CD93 antibody or antigen-binding fragment thereof, comprising the following steps:

[0228] (a) culturing the aforementioned recombinant cells under conditions for expressing an anti-CD93 antibody or an antigen-binding fragment thereof;

[0229] (b) isolating and purifying the anti-CD93 antibody or antigen-binding fragment thereof obtained in step (a).

[0230] In a thirteenth aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease, disorder, or condition associated with CD93, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-CD93 antibody or antigen-binding fragment thereof and / or the aforementioned pharmaceutical composition. The present disclosure also provides an anti-CD93 antibody or antigen-binding fragment according to any of the preceding aspects for use as a medicament.

[0231] In an alternative embodiment, the "CD93-related" disease, disorder or condition in any of the above aspects includes: solid tumors, hematological tumors, inflammation or immune diseases.

[0232] In alternative embodiments, a "CD93-associated" disease, disorder or condition is characterized by high expression of CD93.

[0233] In alternative embodiments, a "CD93-associated" disease, disorder or condition is characterized by high expression of IGFBP7.

[0234] In alternative embodiments, a "CD93-associated" disease, disorder or condition is characterized by high expression of MMRN2.

[0235] In alternative embodiments, the solid tumor is highly vascularized.

[0236] In a fourteenth aspect, a method for promoting normalization of tumor microenvironment blood vessels is also provided, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-CD93 antibody or antigen-binding fragment thereof and / or the aforementioned pharmaceutical composition.

[0237] In an optional embodiment, promoting normalization of tumor microenvironment blood vessels in any of the above aspects includes maintaining the vascular density of blood vessels in the tissue, and the vascular density can be measured as the number of blood vessels per field of view, or as the total length of blood vessels per field of view, as described in Example 5.

[0238] In alternative embodiments, promoting normalization of tumor microenvironment vasculature is for non-diagnostic and non-therapeutic purposes.

[0239] In alternative embodiments, promoting normalization of tumor microenvironment vasculature is used to treat, prevent, or ameliorate a disease, disorder, or condition.

[0240] In an alternative embodiment, promoting normalization of tumor microenvironment blood vessels is used to treat, prevent or alleviate solid tumors, hematological tumors, inflammatory or immune diseases.

[0241] In an optional embodiment, the solid tumor involved in any of the above-mentioned diseases, disorders or conditions is selected from the group consisting of one or more solid tumors such as hepatocellular carcinoma and renal cell carcinoma, renal cancer, liver cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, renal cell carcinoma, breast cancer, hepatocellular carcinoma, ovarian cancer, cervical cancer, glioblastoma, pancreatic cancer, head and neck cancer, Lewis lung cancer and bladder cancer.

[0242] In an optional embodiment, the disease, disorder or condition in any of the above aspects involves a blood tumor selected from the group consisting of chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), multiple myeloma and one or more of T or B cell lymphoma.

[0243] In an alternative embodiment, the disease, disorder or condition in any of the above aspects involves an inflammatory or immune disease including an immune disease associated with the IL-17D-CD93 axis.

[0244] In an optional embodiment, the chimeric antigen receptor and / or engineered immune cell in any of the above embodiments is used to treat, prevent or alleviate hematological tumors.

[0245] In an optional embodiment, the chimeric antigen receptor and / or engineered immune cells in any of the above embodiments are used to prepare a pharmaceutical composition for treating, preventing or alleviating hematological tumors.

[0246] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof in any of the above embodiments is used to treat, prevent or alleviate solid tumors, inflammation or immune diseases.

[0247] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof in any of the above embodiments is used to treat, prevent or alleviate pancreatic cancer, renal cancer, head and neck cancer and colorectal cancer.

[0248] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof in any of the above embodiments is used to treat, prevent or alleviate immune diseases associated with the IL-17D-CD93 axis.

[0249] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof in any of the above embodiments is used to prepare a pharmaceutical composition for treating, preventing or alleviating solid tumors, inflammation or immune diseases.

[0250] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof in any of the above embodiments is used to prepare a pharmaceutical composition for treating, preventing or alleviating pancreatic cancer, renal cancer, head and neck cancer and colorectal cancer.

[0251] In an optional embodiment, the anti-CD93 antibody or antigen-binding fragment thereof in any of the above embodiments is used to prepare a pharmaceutical composition for treating, preventing or alleviating immune diseases associated with the IL-17D-CD93 axis.

[0252] In alternative embodiments, the antibodies or antigen-binding fragments described herein can be administered at a therapeutically effective dose of between about 0.001 mg / kg and about 1000 mg / kg. In some embodiments, the dosage can vary over the course of treatment. For example, in some embodiments, the initial dosage can be higher than subsequent dosages. In some embodiments, the dosage is adjusted over the course of treatment based on the subject's response.

[0253] In an optional embodiment, the antibodies or antigen-binding fragments thereof disclosed in the present disclosure can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed in the present disclosure can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer agent, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine).

[0254] In an optional embodiment, the antibodies or antigen-binding fragments thereof disclosed in the present disclosure, when used in combination with one or more additional therapeutic agents, can be administered simultaneously with the one or more additional therapeutic agents. In some such embodiments, the antibodies or antigen-binding fragments thereof and the additional therapeutic agents can be administered simultaneously as part of the same pharmaceutical composition. However, the antibodies or antigen-binding fragments thereof that are "used in combination" with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agents. The meaning of "used in combination" in the present disclosure also includes that antibodies or antigen-binding fragments thereof that are administered before or after another therapeutic agent are also considered to be "used in combination" with the therapeutic agent, that is, the antibody or antigen-binding fragment thereof and the second substance are administered by different administration methods.

[0255] In a fifteenth aspect, the present disclosure provides a kit for detecting CD93, comprising the aforementioned anti-CD93 antibody or antigen-binding fragment thereof.

[0256] In an optional embodiment, the product of any of the above aspects, such as but not limited to the anti-CD93 antibody or antigen-binding fragment thereof in the reagent, kit, blocking agent and pharmaceutical composition, is conjugated with at least one diagnostic agent and / or therapeutic agent to form an immunoconjugate.

[0257] The diagnostic agent is selected from one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent, and a photosensitizer;

[0258] The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photosensitizing therapeutic agent, an immunoconjugate, and an oligonucleotide.

[0259] Radionuclides include but are not limited to 110 In, 111 In, 177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I. 123 I. 124 I. 125 I. 131 I. 154-158 Gd, 32 P. 11 C. 13 N. 15 O. 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br, 76 Br, 82 mRb and 83 One or more of Sr.

[0260] Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0261] Fluorescent labels include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, europium trisbipyridyldiamine, europium cryptates or chelates, diamines, bis-anthocyanin, La Jolla blue dye, allophycocyanin, allococyanin B, one or more of phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red.

[0262] Oligonucleotides include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

[0263] Drugs include, but are not limited to, methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxel alkaloids, and L-asparaginase.

[0264] Immunomodulators include, but are not limited to, one or more of cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSFs), interferons, erythropoietins, thrombopoietins, tumor necrosis factors (TNFs), interleukins (ILs), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and stem cell growth factors.

[0265] Radionuclides include but are not limited to 111 In, 111 At 177 Lu, 211 Bi, 212 Bi, 213 Bi, 211 At 62 Cu, 67 Cu, 90 Y. 125 I. 131 I. 133 I. 32 P. 33 P. 47 Sc, 111 Ag, 67 Ga, 153 Sm, 161 Tb, 152 Dy, 166 Dy, 161 Ho, 166 Ho, 186 Re、 188 Re、 189 Re、 211 Pb, 212 Pb, 223 Ra, 225 Ac, 77 As、 89 Sr. 99 Mo, 105 Rh, 149 Pm, 169 Second, 194 Ir, 58 Co、 80 mBr, 99 mTc, 103 mRh, 109 Pt, 119 Sb, 189 mOs, 192 Ir, 219 Rn, 215 Po, 221 Fr, 255 Fm, 11 C. 13 N. 15 O. 75 Br, 198 Au, 199 Au, 224 Ac, 77 Br, 113 mIn、 95 Such as 97 Such as103 Such as 105 Such as 107 Hg, 203 Hg, 121 mTe, 122 mTe, 125 mTe, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 142 Pr, 143 Pr, 161 Tb, 57 Co、 58 Co、 51 Cr, 59 Fe, 75 Se, 201 Tl, 76 Br and 169 One or more of Yb.

[0266] The present disclosure is further illustrated below through specific examples. However, it should be understood that these examples are only used for more detailed description and should not be understood as limiting the present disclosure in any form.

[0267] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0268] Experimental Materials:

[0269] Expi293 host cells were used to transiently express the immunogens, related ligands, and reference positive antibodies required for the following examples. In addition, a stable CHO-hCD93 cell clone, designated R1923, was constructed using the PCDHF vector and lentiviral transfection. The amino acid sequences of the proteins are shown in the table below:

[0270] Table 5

[0271] Example 1

[0272] Antibody preparation:

[0273] Balb / c mice were immunized once a week using hCD93-his (R1916) as an immunogen. Orbital blood was collected weekly for titer detection starting from the fourth week of immunization. ELISA and FACS binding assays were also performed.

[0274] Mice with high titers were selected and, after a single booster immunization, sacrificed. Splenocytes were harvested and fused with SP20 cells. The culture medium was then changed for 6 days. On day 9, ELISA screening for binding to CD93 protein was performed. On day 10, FACS screening for binding to CHO-hCD93 was performed. 301 samples were selected and the corresponding wells were identified for rehydration. After rehydration on day 13, FACS screening for binding to CHO-hCD93 was performed again, retaining 129 samples. Subsequently, FACS screening for blocking CHO-hCD93 / MMRN2 was performed in two batches, retaining a pool of 101 cells.

[0275] Prepare the feeder cells for subcloning in advance. Select 27 cells from the above cell pool, resuscitate them for 3 days, and plate 2-3 96-well plates per pool for 4 days. Select from the monoclonal clones and finally obtain 818 wells. After 3 days of culture, use FACS screening based on binding to CHO-hCD93 to screen out 304 cells, and replenish 100 μL of solution per well. After 1 day of culture, perform FACS screening based on binding to CHO-hCD93 again, screen out 132 wells of cells, and replenish 100 μL of solution per well. After 1 day of culture, perform FACS screening based on blocking CHO-hCD93 / MMRN2, retain 22 cell lines for freezing, expand culture and send for purification to obtain 22 mouse monoclonal antibodies, numbered CG002-01 to CG002-22.

[0276] Example 2

[0277] Binding activity screening of mouse monoclonal antibodies:

[0278] Count CHO-hCD93 and adjust the density to 2×10 6 / ml, add 100μl / well of cell suspension to a 96-well plate, centrifuge at 400g for 5 minutes, and discard the supernatant. Hybridoma purified antibody, positive control antibody R1926, and negative control antibody Isotype (Biolegend, cat: 401411) were prepared at a concentration of 30μg / mL and diluted 3 or 5 times. Resuspend the cells with 100μl of diluted hybridoma purified antibody and incubate at 4°C for 30 minutes. After washing twice with PBS, add 100μl of PE-labeled goat anti-mouse Fc antibody diluted 1:500 and resuspend the cells. Incubate at 4°C for 30 minutes. After washing once with PBS, resuspend the cells in 100μl / well of PBS and analyze the PE fluorescence intensity by flow cytometry. The screening process was carried out in two batches. The results of the binding test of hybridoma purified antibodies to CHO-hCD93 are shown in Figures 1 and 2.

[0279] The cell binding results of the first batch showed (Figure 1 and Table 6): Compared with the positive antibody, the top values ​​of molecules 07 and 09 were significantly higher than the positive control; the EC50 and TOP values ​​of molecules 02, 06, and 08 were comparable to the positive control; the cell binding results of the second batch showed (Figure 2 and Table 6): Most of the TOP values ​​were better than the positive control molecules, and the EC50 values ​​were comparable.

[0280] The molecules selected after comprehensive blocking were validated in the same cell binding experiment with the candidate antibodies from the first two batches of screening. The combined cell binding test results showed ( Figure 3 and Table 7 ): Most molecules outperformed the positive control in top values ​​and had comparable EC50 values.

[0281] Table 6 EC50 of CD93 monoclonal antibodies binding to CHO-hCD93 cells

[0282] Table 7 EC50 of the screened CD93 monoclonal antibodies binding to CHO-hCD93 cells

[0283] Example 3

[0284] Screening of mouse monoclonal antibodies based on blocking ligand MMRN2:

[0285] Count CHO-hCD93 and adjust the density to 2.5×10 6 / ml, add 50 μl / well cell suspension to 96-well plate.

[0286] Hybridoma-purified antibody, positive control antibody R1926, and negative control antibody Isotype (Biolegend, cat: 401411) were prepared at a concentration of 30 μg / mL and serially diluted 5-fold or 3-fold. Antibody was added to a 96-well plate at 50 μL / well and incubated at 4°C for 30 minutes. MMRN2 (R1924) ligand was prepared at a concentration of 12 μg / mL and added to a 96-well plate at 50 μL / well. The plate was incubated again for 1 hour. After washing the excess antibody and ligand twice with PBS, a 1:400 dilution of fluorescent secondary antibody (APC anti-his tag) was added and the plate was incubated for another 30 minutes. After washing twice more with PBS, the plate was resuspended in 100 μL of PBS and analyzed by flow cytometry. The assay profiles are shown in Figures 3 and 4, and the EC50 values ​​are shown in Table 8.

[0287] The experimental results of the first batch of blocking MMRN2 showed (Figure 4 and Table 8): Compared with the positive control, the antibodies with blocking activity were 02, 07, 09, and 01, and the blocking activity decreased in sequence; the second batch of experiments showed (Figure 5 and Table 8): Compared with the positive control, the antibodies with blocking activity were 21, 20, 22, 17, 18, 15, 16, and 19, and the blocking activity decreased in sequence; No. 12, 13, and 14 had no blocking activity.

[0288] Based on the combined binding experiment results, the optimal molecules for blocking MMRN2 activity after combination are as follows ( Figure 6 and Table 9 ): the blocking activity is ranked from high to low as 02, 21, 22, 20, 17, 08, 15, and 09.

[0289] Table 8 EC50 of CD93 monoclonal antibodies blocking CHO-hCD93 cell binding to MMRN2

[0290] Table 9 EC50 of the screened CD93 monoclonal antibodies blocking CHO-hCD93 cells from binding to MMRN2

[0291] Example 4

[0292] Screening of mouse monoclonal antibodies based on blocking ligand IGFBP7:

[0293] Count CHO-hCD93 and adjust the density to 2.5×10 6 / ml, add 50 μl / well cell suspension to 96-well plate.

[0294] Hybridoma-purified antibody, positive control antibody R1926, and negative control antibody Isotype (Biolegend, cat: 401411) were prepared at a concentration of 30 μg / mL and serially diluted 5-fold or 3-fold. Antibody was added to a 96-well plate at 50 μL / well and incubated at 4°C for 30 minutes. IGFBP7 (R1926) ligand was prepared at a concentration of 60 μg / mL and added to the 96-well plate at 50 μL / well, followed by another incubation for 1 hour. After washing the excess antibody and ligand twice with PBS, a 1:400 dilution of fluorescent secondary antibody (APC anti-his tag) was added and the plate was incubated for another 30 minutes. After washing twice more with PBS, the plate was resuspended in 100 μL of PBS and analyzed by flow cytometry. The detection profiles are shown in Figures 7 to 9.

[0295] The results of the first batch of IGFBP7 blocking experiments (Figure 7 and Table 10) show that compared to the positive control, molecule 10 exhibited excellent blocking activity, followed by molecule 02 and 06. The remaining antibodies, including the positive control, showed no blocking activity. The results of the second batch of blocking activity experiments (Figure 8 and Table 10) show that compared to the positive control, the antibodies 20, 21, 18, 15, 17, 20, and 22 showed blocking activity, in that order. The combined IGFBP7 results of the antibodies screened using the combined results and MMRN2 are shown in Figure 9 and Table 11.

[0296] Table 10 EC50 of CD93 monoclonal antibodies blocking CHO-hCD93 cells from binding to IGFBP7

[0297] Table 11 EC50 of the screened CD93 monoclonal antibodies blocking CHO-hCD93 cells from binding to IGFBP7

[0298] Based on the experimental results of Examples 2 to 4, four molecules, CG002-02, CG002-10, CG002-15 and CG002-20, were selected as the preferred compounds.

[0299] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the CG002-02, CG002-10, CG002-15, and CG002-20 antibodies are as follows:

[0300] To clearly demonstrate the activity of the screened CD93 monoclonal antibodies in blocking the binding of CHO-hCD93 cells to IGFBP7, the results are shown in Figures 10, 11, 12 and Table 12.

[0301] Figure 10 shows that in the CHO-hCD93 binding experiment, it can be seen that the binding activity of molecule CG002-02 / 15 is similar to that of the positive control R1926; the top value (the upper limit platform value of the binding signal) of molecule 20 is significantly better than that of the positive control.

[0302] FIG11 shows that in the experiment of CHO-hCD93 blocking MMRN2 (R1924), it can be seen that the blocking activity of molecule 02 / 20 is similar to that of the positive control R1926.

[0303] Figure 12 shows that in the experiment of CHO-hCD93 blocking IGFBP7 (R1926), we can see that molecules 15 / 10 have very strong blocking activity, much stronger than the positive control; molecules 02 and 20 are also slightly stronger than the positive control; the positive control has very weak ability to block IGFBP7.

[0304] Table 12 EC50 of CG002-02 / 10 / 15 / 20 binding to CHO-hCD93 cells, and EC50 of blocking CHO-hCD93 cell binding to MMRN2 / IGFBP7

[0305] Example 5

[0306] Functional activity validation of simulated vascular maturation:

[0307] The antibodies in CG002-02 / 10 / 15 / 20 were selected for the final functional test. 50 μL of Matrigel was first added to a 96-well plate and incubated at 37°C for 30 minutes to allow it to solidify. HUVECs (human umbilical vein endothelial cells) that had been starved and cultured for 12 hours without serum were plated at 2×10 4 50 μL of the suspension was added to a 96-well plate, followed by 100 μL of a 20 μg / mL antibody suspension. Three groups were divided: Group 1 added 50 μL of blank culture medium, Group 2 added 250 μL of 200 μM MMRN, and Group 3 added 50 μL of 200 μM IGFBP7. Cultures were continued for 4-8 hours before observation under a microscope and photographic analysis.

[0308] In the absence of ligands, but with only antibodies, the inhibition of angiogenesis is shown in Figures 13 and 14. In the presence of the ligand MMRN2, the inhibition of angiogenesis by the candidate antibodies is shown in Figures 15 and 16. In the presence of the ligand IGFBP7, the inhibition of angiogenesis by the candidate antibodies is shown in Figures 17 and 18.

[0309] It can be seen from the experiment that in the absence of ligand, molecule No. 10 can significantly inhibit the formation of HUVECs simulated blood vessels; in the presence of the ligand MMRN2, the positive antibody can inhibit the formation of blood vessels, which is consistent with the previous MMRN2 blocking activity; in the presence of the ligand IGFBP7, molecules No. 10 and No. 15 have the strongest inhibitory effect, and the positive antibody has almost no effect, which is consistent with the previous blocking activity of IGFBP7. It can also be seen that the addition of IGFB7 has a certain promoting effect on blood vessel formation.

[0310] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial Applicability

[0311] The anti-CD93 antibodies or antigen-binding fragments thereof provided herein can specifically bind to CD93 and block the binding of CD93 to MMRN2 and / or to IGFBP7, thereby preventing the normal development and maturation of tumor blood vessels, exerting anti-tumor effects, and providing new possibilities for the treatment and / or prevention of cancer. Therefore, the anti-CD93 antibodies or antigen-binding fragments thereof provided herein have excellent practical performance and broad market application prospects.

Claims

1. An anti-CD93 antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof contains a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region; The complementary determining regions of the heavy chain variable region include HCDR1, HCDR2 and HCDR3, and the complementary determining regions of the light chain variable region include LCDR1, LCDR2 and LCDR3; The HCDR1 comprises amino acid residue SY, amino acid residue NY or amino acid residue DY; The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO. 14, 36, 57 or 73; The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 19, 41, 62 or 78; The LCDR1 comprises the amino acid sequence shown in SEQ ID NO.23, amino acid residue GTN, amino acid residue GTS or amino acid residue NSA; The LCDR2 comprises amino acid residue AA, amino acid residue SA or amino acid residue YA; The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 29, 50, 71 or 87.

2. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: According to IMGT definition: The HCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 13, 35 or 56; The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO. 18, 40, 61 or 77; The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 22, 44, 65 or 81; The LCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 26, 47, 68 or 84; The LCDR2 comprises amino acid residue AA, amino acid residue SA or amino acid residue YA; The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 30, 51, 72 or 88; Optionally, the complementarity determining region of the anti-CD93 antibody or antigen-binding fragment thereof is selected from any one of the following (a)-(d): (a) HCDR1 comprises the amino acid sequence set forth by amino acid residues SY, HCDR2 comprises the amino acid sequence set forth by SEQ ID NO.14, HCDR3 comprises the amino acid sequence set forth by SEQ ID NO.19, LCDR1 comprises the amino acid sequence set forth by SEQ ID NO.23, LCDR2 comprises the amino acid residues AA, and LCDR3 comprises the amino acid sequence set forth by SEQ ID NO.29; or (b) HCDR1 comprises amino acid residues NY, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.36, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.41, LCDR1 comprises amino acid residues GTN, LCDR2 comprises amino acid residues SA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.50; or (c) HCDR1 comprises amino acid residues DY, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.57, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.62, LCDR1 comprises amino acid residues GTS, LCDR2 comprises amino acid residues YA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.71; or (d) HCDR1 includes amino acid residues DY, HCDR2 includes the amino acid sequence shown in SEQ ID NO.73, HCDR3 includes the amino acid sequence shown in SEQ ID NO.78, LCDR1 includes amino acid residues NSA, LCDR2 includes amino acid residues SA, and LCDR3 includes the amino acid sequence shown in SEQ ID NO.87; Optionally, according to the IMGT definition: the complementarity determining region of the anti-CD93 antibody or antigen-binding fragment thereof is selected from any one of the following (a')-(d'): (a') HCDR1 comprises the amino acid sequence shown in SEQ ID NO.13, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.18, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.22, LCDR1 comprises the amino acid sequence shown in SEQ ID NO.26, LCDR2 comprises amino acid residue AA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.30; or (b') HCDR1 comprises the amino acid sequence shown in SEQ ID NO.35, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.40, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.44, LCDR1 comprises the amino acid sequence shown in SEQ ID NO.47, LCDR2 comprises amino acid residue SA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.51; or (c') HCDR1 comprises the amino acid sequence shown in SEQ ID NO.56, HCDR2 comprises the amino acid sequence shown in SEQ ID NO.61, HCDR3 comprises the amino acid sequence shown in SEQ ID NO.65, LCDR1 comprises the amino acid sequence shown in SEQ ID NO.68, LCDR2 comprises the amino acid residue YA, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO.72; or (d') HCDR1 includes the amino acid sequence shown in SEQ ID NO.56, HCDR2 includes the amino acid sequence shown in SEQ ID NO.77, HCDR3 includes the amino acid sequence shown in SEQ ID NO.81, LCDR1 includes the amino acid sequence shown in SEQ ID NO.84, LCDR2 includes amino acid residue SA, and LCDR3 includes the amino acid sequence shown in SEQ ID NO.

88.

3. An anti-CD93 antibody or an antigen-binding fragment thereof, characterized in that: Include: (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO. 1, 3, 5 or 7; and, (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NO. 2, 4, 6 or 8; Optionally, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are determined according to the IMGT definition, the Kabat definition, the Chothia definition, the AbM definition or the Contact definition.

4. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody contains a heavy chain framework region and / or a light chain framework region, and the heavy chain framework region and / or the light chain framework region are derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof; optionally, the framework region of the heavy chain variable region includes HFR1, HFR2, HFR3 and HFR4, and the framework region of the light chain variable region includes LFR1, LFR2, LFR3 and LFR4, wherein: The HFR1 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 89, 103 and 116, or comprises a sequence having at least 89% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 89, 103 and 116; and / or, the HFR2 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 90, 104, 117 and 128, or comprises a sequence having at least 84% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 90, 104, 117 and 128; and / or, the HFR3 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 92, 106, 119 and 130, or comprises a sequence having at least 92% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 92, 106, 119 and 130; and / or, the HFR4 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 94, 108 and 132, or comprises a sequence having at least 87% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 94, 108 and 132; and / or, the LFR1 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 95, 109, 121 and 133, or comprises a sequence having at least 73% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 95, 109, 121 and 133; and / or, the LFR2 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 97, 111 and 123, or comprises a sequence having at least 77% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 97, 111 and 123; and / or, the LFR3 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 99, 113, 125 and 136, or comprises a sequence having at least 80% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 99, 113, 125 and 136; and / or, the LFR4 comprises an amino acid sequence as shown in any one of SEQ ID NOs. 101, 115, 127 and 138, or comprises a sequence having at least 84% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 101, 115, 127 and 138; Optionally, according to the IMGT definition, the HFR1 comprises the amino acid sequence shown in SEQ ID NO.89, the HFR2 comprises the amino acid sequence shown in SEQ ID NO.91, the HFR3 comprises the amino acid sequence shown in SEQ ID NO.93, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.94; Or, according to the IMGT definition, the HFR1 comprises the amino acid sequence shown in SEQ ID NO.103, the HFR2 comprises the amino acid sequence shown in SEQ ID NO.105, the HFR3 comprises the amino acid sequence shown in SEQ ID NO.107, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.108; Or, according to the IMGT definition, the HFR1 comprises the amino acid sequence shown in SEQ ID NO.116, the HFR2 comprises the amino acid sequence shown in SEQ ID NO.118, the HFR3 comprises the amino acid sequence shown in SEQ ID NO.120, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.108; Or, according to the IMGT definition, the HFR1 comprises the amino acid sequence shown in SEQ ID NO.116, the HFR2 comprises the amino acid sequence shown in SEQ ID NO.129, the HFR3 comprises the amino acid sequence shown in SEQ ID NO.131, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.

132. Alternatively, according to the IMGT definition, the LFR1 comprises the amino acid sequence shown in SEQ ID NO.96, the LHFR2 comprises the amino acid sequence shown in SEQ ID NO.98, the LHFR3 comprises the amino acid sequence shown in SEQ ID NO.100, and the LHFR4 comprises the amino acid sequence shown in SEQ ID NO.102; Or, according to the IMGT definition, the LFR1 comprises the amino acid sequence shown in SEQ ID NO.110, the LHFR2 comprises the amino acid sequence shown in SEQ ID NO.112, the LHFR3 comprises the amino acid sequence shown in SEQ ID NO.114, and the LHFR4 comprises the amino acid sequence shown in SEQ ID NO.115; Or, according to the IMGT definition, the LFR1 comprises the amino acid sequence shown in SEQ ID NO.122, the LHFR2 comprises the amino acid sequence shown in SEQ ID NO.124, the LHFR3 comprises the amino acid sequence shown in SEQ ID NO.126, and the LHFR4 comprises the amino acid sequence shown in SEQ ID NO.127; Or, according to the IMGT definition, the LFR1 comprises the amino acid sequence shown in SEQ ID NO.134, the LHFR2 comprises the amino acid sequence shown in SEQ ID NO.135, the LHFR3 comprises the amino acid sequence shown in SEQ ID NO.137, and the LHFR4 comprises the amino acid sequence shown in SEQ ID NO.

138.

5. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The anti-CD93 antibody or antigen-binding fragment thereof has an amino acid sequence as shown in any one of SEQ ID NO. 1, 3, 5 or 7, or a heavy chain variable region having at least 77% sequence identity with the amino acid sequence as shown in any one of SEQ ID NO. 1, 3, 5 or 7; and / or, the anti-CD93 antibody or antigen-binding fragment thereof has a light chain variable region as shown in the amino acid sequence of any one of SEQ ID NO. 2, 4, 6 or 8, or having at least 72% sequence identity with the amino acid sequence shown in any one of SEQ ID NO. 2, 4, 6 or 8; Optionally, the anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region of any one of the following: (a) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.1; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.2; or (b) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.3; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.4; or (c) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.5; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.6; or (d) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.

8.

6. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The anti-CD93 antibody or antigen-binding fragment thereof comprises a partial or complete sequence of a constant region, wherein the constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof; Optionally, the heavy chain constant region of the antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region of the antibody is a κ or λ chain.

7. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The antigen binding fragment includes one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv and the minimum recognition unit of an antibody.

8. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The anti-CD93 antibody or antigen-binding fragment thereof has one or more of the following properties (i)-(v): (i) blocking the binding of CD93 to IGFBP7, and / or blocking the binding of CD93 to MMRN2; (ii) does not block the binding of CD93 to IGFBP7, or does not block the binding of CD93 to MMRN2; (iii) binds to CD93 with an EC50 of no more than 0.3 μg / mL, as measured by flow cytometric fluorescence sorting; (iv) blocking MMRN2 binding to CD93 with an EC50 of no more than 0.2 μg / mL, as measured by flow cytometric fluorescence sorting; (v) blocking IGFBP7 binding to CD93 with an EC50 of no more than 3 μg / mL; said binding is determined by flow cytometry fluorescence sorting technology.

9. An anti-CD93 antibody or an antigen-binding fragment thereof, characterized in that: It competitively binds to CD93 with the anti-CD93 antibody described in any one of claims 1-8, or the epitope to which it binds to the CD93 antigen is the same as the epitope to which the anti-CD93 antibody described in any one of claims 1-8 binds to the CD93 antigen.

10. A biomaterial, characterized in that: The biological material comprises any one of the following (i) to (vi): (i) a multispecific antibody, comprising the anti-CD93 antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 9; (ii) a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular region, wherein the extracellular region comprises an antigen binding domain; and wherein the antigen binding domain comprises the anti-CD93 antibody or antigen binding fragment thereof according to any one of claims 1 to 9; (iii) a nucleic acid molecule encoding the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antibody according to (i), or the chimeric antigen receptor according to (ii); (iv) a vector carrying the nucleic acid molecule described in (iii); (v) a recombinant cell, characterized in that the recombinant cell comprises the nucleic acid molecule described in (iii), or comprises the vector described in (iv), or expresses the anti-CD93 antibody or antigen-binding fragment thereof described in any one of claims 1 to 9, or expresses the multispecific antibody described in (i); (vi) An engineered immune cell, wherein the immune cell expresses the chimeric antigen receptor described in (ii) or contains a nucleic acid molecule encoding the chimeric antigen receptor described in (iii).

11. Use of the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the biomaterial according to claim 10 in any one of the following: (Ⅰ) Detection of CD93 or cells expressing CD93; (II) preparing products for detecting CD93 or cells expressing CD93; (III) blocking CD93 binding to MMRN2, and / or blocking CD93 binding to IGFBP7; (IV) preparing a blocker for blocking CD93 binding to MMRN2; or preparing a blocker for blocking CD93 binding to IGFBP7; (V) preparing a pharmaceutical composition for treating, preventing or ameliorating a disease, disorder or condition associated with CD93; (VI) Preparing a pharmaceutical composition for promoting normalization of blood vessels in the tumor microenvironment.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the biomaterial according to claim 10; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

13. A method for producing an anti-CD93 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: (a) culturing the recombinant cell of claim 10 under conditions of expressing an anti-CD93 antibody or an antigen-binding fragment thereof; (b) isolating and purifying the anti-CD93 antibody or antigen-binding fragment thereof obtained in step (a).

14. A method for treating, preventing or alleviating a disease, disorder or condition associated with CD93, characterized in that: The method comprises administering to a subject a therapeutically effective amount of the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 12.

15. A method for promoting normalization of blood vessels in the tumor microenvironment, characterized in that: The method comprises administering to a subject a therapeutically effective amount of the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 12.

16. A kit for detecting CD93, characterized in that: It comprises the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antibody according to claim 10.

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