Anti-CD93 antibody, and composition and use thereof

Abstract

The present invention provides an anti-CD93 antibody, and a composition and a use thereof. The present invention further provides a polynucleotide molecule encoding the antibody or an antigen-binding fragment thereof of the present application, an expression vector and a host cell for expressing the antibody or the antigen-binding fragment thereof of the present application, and a use of the antibody or the antigen-binding fragment thereof of the present application.

Description

Anti-CD93 antibodies, compositions and uses thereof Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to anti-CD93 antibodies, compositions and uses thereof. Background Art

[0002] CD93 (also known as C1q R1, C1q R, C1q RP, MXRA4 or ECSM3) is a transmembrane glycoprotein composed of an extracellular domain (amino acid sequence 24-580) consisting of a C-type lectin domain, an epidermal growth factor (EGF)-like domain, a highly glycosylated mucin-like domain, a transmembrane domain (amino acid sequence 581-601) and a short cytoplasmic domain (amino acid sequence 601-652). It is mainly expressed in endothelial cells, platelets, stem cells and bone marrow cells (granulocytes and monocytes).

[0003] Recent studies have found that the interaction between CD93 and insulin-like growth factor binding protein 7 (IGFBP7) is crucial for the formation of abnormal tumor blood vessels. Inhibiting this interaction with anti-CD93 monoclonal antibodies can improve the anti-cancer effects of chemotherapy drugs and increase the number of effector T cells in tumors, thereby increasing the sensitivity of tumors to immune checkpoint inhibitors.

[0004] Currently, no anti-CD93 monoclonal antibody drugs have entered the clinical stage, and new anti-CD93 antibodies are needed.

[0005] Summary of the Invention

[0006] The present invention provides anti-CD93 antibodies or antigen-binding fragments thereof that block the binding of CD93 to IGFBP7. The anti-CD93 antibodies or antigen-binding fragments provided herein can be used as standalone therapies or in combination with other therapies and / or other anticancer agents for the treatment of CD93-mediated diseases, such as cancer.

[0007] In one aspect, the present invention provides an anti-CD93 antibody or antigen-binding fragment thereof, wherein the anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 as defined by Kabat, or a HCDR1 having 1, 2 or 3 amino acid differences therefrom, a HCDR2 or a sequence having 1, 2 or 3 amino acid differences therefrom, and a HCDR3 or a sequence having 1, 2 or 3 amino acid differences therefrom; and / or the anti-CD93 antibody or antigen-binding fragment thereof comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 as defined by Kabat. NO: 5, 13, 21, 28, 31, 32 or 36 LCDR1 or a sequence having 1, 2 or 3 amino acid differences therefrom, LCDR2 or a sequence having 1, 2 or 3 amino acid differences therefrom, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences therefrom.

[0008] In another aspect, the present invention provides an isolated anti-CD93 antibody or antigen-binding fragment thereof having at least one of the following properties: (I) binding to the same, completely overlapping, or partially overlapping epitope of human CD93 protein as the antibody or antigen-binding fragment thereof according to any embodiment of the present invention; (II) competing with the antibody or antigen-binding fragment thereof according to any embodiment of the present invention for binding to an epitope of human CD93 protein.

[0009] In yet another aspect, the present invention provides polynucleotide molecules encoding the anti-CD93 antibodies or antigen-binding fragments thereof described herein.

[0010] In yet another aspect, the present invention provides a nucleic acid construct comprising the polynucleotide molecule according to any embodiment of the present invention.

[0011] In another aspect, the present invention provides an expression vector comprising the polynucleotide molecule or nucleic acid construct described herein; preferably, the vector is a eukaryotic expression vector.

[0012] In yet another aspect, the present invention provides a host cell comprising a polynucleotide molecule described herein or an expression vector described herein, and / or expressing an anti-CD93 antibody or antigen-binding fragment thereof as described in any embodiment herein.

[0013] In another aspect, the present invention provides a method for preparing the anti-CD93 antibody or antigen-binding fragment thereof described herein, comprising culturing the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, allowing it to express the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0014] In another aspect, the present invention provides an anti-CD93 antibody or an antigen-binding fragment thereof prepared by the method for preparing an anti-CD93 antibody or an antigen-binding fragment thereof described in any embodiment herein.

[0015] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD93 antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, a nucleic acid construct or expression vector described herein, a host cell described herein, a multispecific antibody described herein, or a single-chain antibody described herein, and a pharmaceutically acceptable carrier or excipient.

[0016] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, a nucleic acid construct or expression vector as described herein, a host cell as described herein, a multispecific antibody as described herein, a single-chain antibody as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents. Preferably, the therapeutic agent is one or a combination of an anti-PD-1 antibody, a PD-L1 antibody, and an anti-CTLA4 antibody.

[0017] In another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, a polynucleotide molecule, a nucleic acid construct or expression vector, a host cell, a multispecific antibody, a single-chain antibody, a pharmaceutical composition, or a pharmaceutical combination as described herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by CD93.

[0018] In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, a nucleic acid construct or expression vector described herein, a host cell described herein, a multispecific antibody described herein, a single-chain antibody described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein for use in treating and / or preventing a CD93-mediated disease or condition.

[0019] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by CD93, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof described herein, a nucleic acid construct or polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a multispecific antibody described herein, a single-chain antibody described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein.

[0020] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the nucleic acid construct or polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the multispecific antibody described herein, the single-chain antibody described herein, the pharmaceutical composition described herein, or the drug combination described herein, preferably further comprising a drug delivery device.

[0021] In another aspect, the present invention provides a method for detecting the presence of CD93 in a sample using an antibody or antigen-binding fragment thereof as described herein, or a detection composition comprising the antibody or antigen-binding fragment thereof. In some embodiments, the method comprises contacting the sample with an antibody or antigen-binding fragment thereof as described herein, or a detection composition comprising the antibody or antigen-binding fragment thereof.

[0022] A more detailed description of various aspects of this disclosure is provided below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: Graph showing the results of cell-level binding experiments of anti-CD93 chimeric antibodies.

[0024] Figure 2: Blocking experiment results of anti-CD93 chimeric antibody blocking the binding of IGFBP7 protein to human CD93.

[0025] FIG3 shows the results of cell-based binding experiments of humanized anti-CD93 antibodies.

[0026] Figure 4: Blocking experiment results of anti-CD93 humanized antibody blocking the binding of IGFBP7 protein to human CD93.

[0027] FIG5 : Experimental results of binding of anti-CD93 chimeric antibody to human CD93.

[0028] Figure 6: Blocking experiment results of anti-CD93 chimeric antibody blocking the binding of IGFBP7 protein to human CD93.

[0029] Figure 7: Binding assay results of anti-CD93 monoclonal antibodies to CHO hCD93 cells.

[0030] Figure 8: Anti-CD93 monoclonal antibody blocking the binding of IGFBP7 to CHO hCD93 cells. DETAILED DESCRIPTION

[0031] definition

[0032] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0033] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined as follows. Unless otherwise clearly defined elsewhere in this article, the scientific and technological terms used herein all have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form unless otherwise clearly provided in the text.

[0034] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0035] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0036] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in a claim, "consisting of..." will refer to only one of the listed numbers or one of the elements of the list.

[0037] Unless expressly stated otherwise, as used herein, the words "a" and "an" should be understood to mean "at least one."

[0038] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0039] The terms "activity" or "biological activity," or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CD93 activity in vivo or in vitro, IC50, in vivo stability of the antibody, and immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0040] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0041] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0042] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0043] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0044] The precise amino acid sequence boundaries of the variable region CDRs of an antibody can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al., 1989, Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948, 1997), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT)) (1999 Nucleic Acids Research, 27, 209-212), and affinity propagation clustering based on large numbers of crystal structures. propagation clustering) North.

[0045] Unless otherwise indicated, the CDR of an antibody of the present invention can be determined by those skilled in the art according to any scheme (e.g., different assignment systems or combinations) of this area. In some embodiments, the CDR of an antibody of the present invention is defined by Kabat, Chothia, AbM, Contact, North, or IMGT. In some embodiments, the CDR of an antibody of the present invention is defined by Kabat.

[0046] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0047] Antibodies with different specificities (that is, different binding sites for different antigens) have different CDRs. However, although CDR is different between antibodies, there are only a limited number of amino acid positions in the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the CDR sequence remainder can be determined. Therefore, the present invention also contemplates the variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0048] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least a fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When the binding activity for the antigen is expressed on a molar basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0049] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0050] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or a light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0051] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0052] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0053] The term "murine antibody" or "hybridoma antibody" as used herein refers to a monoclonal antibody against human CD93 prepared according to the knowledge and skills in the art. A subject is injected with CD93 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0054] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0055] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0056] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0057] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0058] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0059] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0060] As used herein, the term "cross-reactivity" refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Thus, "cross-reactivity" should be understood as interspecies reactivity with the same molecule X expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human CD93, monkey, and / or murine CD93 (mouse or rat) can be determined by FACS analysis.

[0061] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is a function of the dissociation rate constant and the association rate constant (kD, respectively). dis and k on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0062] The term "not binding" to a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the K of the protein or cell is less than 1%. D 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0063] The term "high affinity" for IgG antibodies refers to the K D 1.0×10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype refers to a K D is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0064] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited otherwise, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0065] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner. Recombinant constructs will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0066] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0067] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0068] "Activation," "stimulation," and "treatment" as applied to a cell or receptor may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response," e.g., a response of a cell, tissue, organ, or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, rate of gene expression, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0069] As used herein, the terms "treatment" or "treating" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or preventing or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, "treatment" or "treating" refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless expressly described herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0070] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.

[0071] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in either order.

[0072] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a CD93 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, e.g., to treat, cure, prevent, or ameliorate the relevant medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients, whether administered in combination, sequentially, or simultaneously, that results in a therapeutic effect. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0073] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0074] The term "cancer" is used herein to refer to a group of cells that exhibit an abnormally high level of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. The term "tumor" as used herein refers to one or more cells comprising a cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.

[0075] Anti-CD93 antibodies and antigen-binding fragments thereof

[0076] The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CD93. The terms "anti-CD93 antibody," "anti-CD93," "CD93 antibody," or "antibody that binds to CD93" refer to antibodies that are capable of binding to a CD93 protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CD93.

[0077] In some embodiments, the present invention provides antibodies that bind to CD93 protein.

[0078] In some embodiments, the antibodies of the invention bind to human CD93 protein.

[0079] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDRs (HCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1, 9, 17, or 39 defined by Kabat, and / or comprise light chain variable region CDRs (LCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5, 13, 21, 28, 31, 32, or 36 defined by Kabat.

[0080] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDRs (HCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1 defined by Kabat, and / or comprise light chain variable region CDRs (LCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5 defined by Kabat.

[0081] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDRs (HCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9 defined by Kabat, and / or comprise light chain variable region CDRs (LCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 13 defined by Kabat.

[0082] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDRs (HCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 17 defined by Kabat, and / or comprise light chain variable region CDRs (LCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 21 defined by Kabat.

[0083] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDRs (HCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 40 defined by Kabat, and / or comprise light chain variable region CDRs (LCDRs) defined by Kabat, Chothia, AbM, Contact, North, or IMGT, preferably LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 32 defined by Kabat.

[0084] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable region CDRs (HCDRs) and light chain variable region CDRs (LCDRs) that are the HCDR and LCDR sequences defined by Kabat, Chothia, AbM, Contact, North, or IMGT in the heavy chain variable region and light chain variable region, respectively, as shown in the following groups of sequences, preferably the HCDR and LCDR sequences defined by Kabat:

[0085] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof comprise heavy chain variable region CDRs (HCDRs) of HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1, 9, 17, or 39 as defined by Kabat, and / or comprise light chain variable region CDRs (LCDRs) of LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5, 13, 21, 28, 31, 32, or 36 as defined by Kabat.

[0086] In some embodiments, in the anti-CD93 antibody or antigen-binding fragment thereof of the present invention, HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, 10 or 18, HCDR2 comprises the amino acid sequence of SEQ ID NO: 3, 11 or 19 or 57, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, 12 or 20, and / or LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, 14, 22, 53, 54 or 55, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, 15 or 23, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8, 16, 24 or 56.

[0087] In some embodiments, the heavy chain variable region of the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises: (I) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 2, SEQ ID NO: 3 or 57, and SEQ ID NO: 4, respectively, or HCDR1, HCDR2, and HCDR3 that have 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3 or 57, and SEQ ID NO: 4, respectively; or (II) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, or HCDR1, HCDR2, and HCDR3 that have 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; or (III) HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, or HCDR1, HCDR2, and HCDR3 that have 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 1 : SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; and / or its light chain variable region comprises: (I) LCDR1, LCDR2 and LCDR3 as represented by amino acid sequences of SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or LCDR1, LCDR2 and LCDR3 as represented by amino acid sequences of SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or LCDR1, LCDR2 and LCDR3 as represented by amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or LCDR1, LCDR2 and LCDR3 as represented by amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or LCDR1, LCDR2 and LCDR3 as represented by amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or (III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 or 56, or LCDR1, LCDR2 and LCDR3 whose amino acid sequences have 1, 2 or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 or 56, respectively.

[0088] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.

[0089] In some embodiments, an anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom.

[0090] In some embodiments, an anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom.

[0091] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO:3 or 57, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.

[0092] In some embodiments, an anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 54 or 55, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom.

[0093] In some embodiments, an anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom.

[0094] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention comprise heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.

[0095] In some embodiments, the anti-CD93 antibodies or antigen-binding fragments thereof comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3 and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO:55, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO:15, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:16, or an amino acid sequence having 1, 2, or 3 amino acid differences therefrom.

[0096] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and / or a light chain variable region:

[0097] The heavy chain variable region comprises:

[0098] (I) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, or SEQ ID NO: 4, respectively; or

[0099] (II) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; or

[0100] (III) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively;

[0101] The light chain variable region comprises:

[0102] (I) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 or SEQ ID NO: 8, respectively; or

[0103] (II) LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or

[0104] (III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or 56, respectively.

[0105] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8.

[0106] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0107] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0108] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3 or 57, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 53, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8.

[0109] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 54 or 55, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0110] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56.

[0111] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 57, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8.

[0112] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 55, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0113] In some embodiments, the heavy chain variable region of an anti-CD93 antibody or antigen-binding fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 37-48, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1, 9, 17, 37-48, and / or the light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, 25-36, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 13, 21, 25-36.

[0114] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:

[0115] (I) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; or

[0116] (II) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:13, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:13; or

[0117] (III) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0118] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof described herein comprises: a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48; and a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

[0119] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

[0120] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD93 antibody or antigen-binding fragment thereof of the present invention are selected from the amino acid sequence of the heavy chain variable region or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the amino acid sequence of the light chain variable region or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively, as shown in any one of the following sequence numbers:

[0121] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof of the present invention comprises:

[0122] (I) a heavy chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 49 or 51, and a light chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 50 or 52; or

[0123] (II) a heavy chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 49 or 51, and a light chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 50 or 52; or

[0124] (III) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 49 and a light chain having an amino acid sequence as shown in SEQ ID NO: 50, or

[0125] (IV) A heavy chain having an amino acid sequence as shown in SEQ ID NO: 51 and a light chain having an amino acid sequence as shown in SEQ ID NO: 52.

[0126] In another aspect, the present invention provides an isolated anti-CD93 antibody or antigen-binding fragment thereof, which has one or more of the following properties: (1) the epitope of human CD93 protein bound by it is identical to, completely overlaps with, or partially overlaps with the anti-CD93 antibody or antigen-binding fragment thereof described herein; (2) it competes with the anti-CD93 antibody or antigen-binding fragment thereof described herein for binding to the epitope of human CD93 protein.

[0127] In some embodiments, the anti-CD93 antibody or antigen-binding fragment thereof described in the present invention is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.

[0128] In some embodiments, the antibody of the present invention is a murine antibody or a chimeric antibody. In some embodiments, the antibody of the present invention further comprises a heavy chain framework region and / or a light chain framework region derived from a murine antibody.

[0129] In some embodiments, the antibodies of the present invention are humanized antibodies. In some embodiments, the antibodies of the present invention comprise a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene), and / or a light chain framework region (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene). The heavy chain framework region and / or light chain framework region optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from human residues to mouse residues.

[0130] In some embodiments, the antibodies described herein are monoclonal antibodies.

[0131] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab; Fab′; F(ab′)2; Fv fragment; diabody; linear antibody; single-chain antibody molecule, such as sc-Fv; nanobody formed from antibody fragments and multispecific antibody.

[0132] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of CD93 can be used as an immunogen (antigen) for producing antibodies. By way of example and not limitation, any CD93 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-human CD93 antibodies can be produced by methods known in the art.

[0133] Antibodies derived from rodents (e.g., mice) can cause unwanted antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can lead to immune responses against the therapeutic antibodies in the human body. Such immune responses can lead to loss of therapeutic efficacy at the very least and, in severe cases, potentially lethal allergic reactions. One approach to reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the retention of intact rodent variable regions in chimeric antibodies can still cause unwanted immunogenicity in patients. Grafting the complementarity determining region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences (Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0134] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal hybridoma antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0135] In some embodiments, the chimeric CD93 antibodies of the present invention can be prepared by operatively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant region comprised by the chimeric antibodies of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG4.

[0136] In some embodiments, chimeric CD93 antibodies of the present invention can be obtained by "mixing and matching" chimeric light chain and chimeric heavy chain expression plasmids into expressing cells, and the CD93 binding of such "mixed and matched" antibodies can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).

[0137] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0138] In some embodiments, one or more of the three HCDR sequences and three LCDR sequences of the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention have amino acid differences compared to the corresponding three HCDR sequences and three LCDR sequences defined above. Herein, the amino acid differences include amino acid deletions, insertions, or substitutions. In some embodiments, one or more of the three HCDR sequences and three LCDR sequences of the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention have 1, 2, or 3 amino acid differences selected from amino acid deletions, amino acid insertions, and amino acid substitutions compared to the corresponding three HCDR sequences and three LCDR sequences defined above.

[0139] In some embodiments, the framework regions of the anti-CD93 antibodies or antigen-binding fragments thereof of the present invention have 1, 2, 3, 4, or 5 amino acid differences selected from amino acid deletions, amino acid insertions, and amino acid substitutions compared to the corresponding framework regions in the specific sequences disclosed herein.

[0140] In some embodiments, the present invention provides CDR sequences, heavy chain variable regions, light chain variable regions, heavy chains and light chains that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding CDR sequences, heavy chain variable regions, light chain variable regions, heavy chains and light chains specifically disclosed herein after one or more (e.g., 1-10, 1-5 or 1-3) amino acid deletions, insertions and / or substitutions.

[0141] It should be understood that the introduction of these amino acid differences will not affect the biological activity of the resulting anti-CD93 antibody or antigen-binding fragment thereof, including the ability to bind to the CD93 protein or fragment thereof with sufficient affinity to enable the antibody to be used as a diagnostic and / or therapeutic agent targeting CD93. In other words, the antibody or antigen-binding fragment thereof obtained by mutation retains at least the binding affinity of the antibody or antigen-binding fragment thereof to the CD93 protein or fragment thereof as compared to the antibody or antigen-binding fragment thereof before mutation.

[0142] In some embodiments, the anti-CD93 antibodies described herein are IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4 antibodies, or modified forms thereof, as described in the following sections.

[0143] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an anti-CD93 antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0144] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0145] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0146] In some embodiments, a multispecific antibody is provided herein. The multispecific antibody comprises a second functional module (e.g., a second antibody) having a different binding specificity than the anti-CD03 antibody provided herein, thereby being able to bind to at least two different binding sites and / or target molecules. Therefore, the present invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region of an antibody or antigen-binding fragment thereof described herein.

[0147] In some embodiments, provided herein is a single-chain antibody comprising the light chain variable region and the heavy chain variable region of an anti-CD93 antibody or antigen-binding fragment thereof described herein.

[0148] Polynucleotide molecules and antibody expression

[0149] In yet another aspect, the present invention provides a polynucleotide molecule encoding an anti-CD93 antibody or antigen-binding fragment thereof as described herein. The present invention also includes the full-length complementary sequence of the polynucleotide molecule. The polynucleotide molecule may comprise a polynucleotide molecule encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide molecule comprising the amino acid sequence of the light chain and / or heavy chain of the antibody. In some embodiments, the polynucleotide molecule encoding the antibody of the present invention includes a polynucleotide molecule that has been mutated by nucleotide deletion, insertion or substitution, but still has at least about 60%, 70%, 80%, 90% or 95% identity with the CDR corresponding coding region depicted in the sequence described above.

[0150] In yet another aspect, the present invention provides a nucleic acid construct comprising a polynucleotide molecule as described herein. The nucleic acid construct can be, for example, an expression cassette comprising a promoter and a transcription terminator operably linked to the polynucleotide molecule. In some embodiments, the nucleic acid construct is an expression vector.

[0151] In yet another aspect, the present invention provides an expression vector comprising a polynucleotide molecule or nucleic acid construct as described herein. Preferably, the vector is a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0152] In another aspect, the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein and / or expressing an anti-CD93 antibody or antigen-binding fragment thereof as described in any embodiment herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0153] In yet another aspect, the present invention provides a method for preparing an anti-CD93 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell as described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0154] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

[0155] In one embodiment, the present invention provides a method for producing an anti-CD93 antibody, wherein the method comprises introducing an expression vector into a mammalian host cell and culturing the host cell for a sufficient period of time to allow expression of the antibody in the host cell, or more preferably, to allow secretion of the antibody into the culture medium in which the host cell is grown, thereby producing the antibody. The antibody can be recovered from the culture medium using standard protein purification methods.

[0156] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Likewise, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are less likely to be immunogenic because their carbohydrate structures are normal components of natural human serum IgG.

[0157] Pharmaceutical compositions and kits

[0158] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD93 antibody or antigen-binding fragment thereof as described herein, a nucleic acid construct or polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or a multispecific antibody or single-chain antibody as described herein, and a pharmaceutically acceptable carrier or excipient. It should be understood that the anti-CD93 antibody or pharmaceutical composition provided herein can be incorporated into a suitable carrier, excipient, and other agents in the formulation for co-administration, thereby providing improved transfer, delivery, tolerance, etc.

[0159] The term "pharmaceutical composition" refers to a preparation that permits the active ingredient contained therein to exist in biologically effective form, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0160] Pharmaceutical formulations comprising the anti-CD93 antibodies described herein can be prepared by mixing an anti-CD93 antibody of the invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0161] The pharmaceutical composition or formulation of the present invention may also include one or more other active ingredients, which are required for the specific indication being treated, preferably those active ingredients that do not adversely affect each other's complementary activities. In some embodiments, the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics or various known anti-tumor or anti-cancer agents, and the active ingredients are suitably combined in an amount effective for the intended use. In some embodiments, the pharmaceutical composition of the present invention further comprises a composition of a polynucleotide molecule encoding an anti-CD93 antibody.

[0162] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule or nucleic acid construct described herein, an expression vector described herein, a host cell described herein, a multispecific antibody or single-chain antibody described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0163] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule or nucleic acid construct described herein, the expression vector described herein, the host cell described herein, or the multispecific antibody or single-chain antibody described herein, or the pharmaceutical composition or drug combination described herein.

[0164] In some embodiments, the kit is a detection kit containing the antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof. Optionally, the antibody or antigen-binding fragment thereof can be labeled with a marker well known in the art.

[0165] Medical uses and treatments

[0166] Any of the anti-CD93 antibodies or antigen-binding fragments thereof provided herein can be used in therapeutic methods. It should also be understood that when discussing "antibodies," compositions comprising antibodies or antigen-binding fragments thereof are also included. The anti-CD93 antibodies or antigen-binding fragments thereof of the present invention can be used in therapeutic or prophylactic methods described in any embodiment of the present invention in a therapeutically effective amount or a prophylactically effective amount.

[0167] In another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule or nucleic acid construct described herein, an expression vector described herein, a host cell described herein, or a multispecific antibody or single-chain antibody described herein, or a pharmaceutical composition or pharmaceutical combination described herein in the preparation of a medicament for treating and / or preventing a CD93-mediated disease or condition.

[0168] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule or nucleic acid construct described herein, an expression vector described herein, a host cell described herein, or a multispecific antibody or single-chain antibody described herein, or a pharmaceutical composition or pharmaceutical combination described herein for treating and / or preventing a CD93-mediated disease or condition.

[0169] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by CD93, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule or nucleic acid construct described herein, an expression vector described herein, a host cell described herein, or a multispecific antibody or single-chain antibody described herein, or a pharmaceutical composition or pharmaceutical combination described herein.

[0170] In some embodiments, the CD93-mediated disease or condition includes a disease or condition caused by the interaction between CD93 and insulin-like growth factor binding protein 7 (IGFBP7), such as abnormal tumor vasculature.

[0171] Preferably, the CD93-mediated disease or disorder is cancer; more preferably, the characteristics of the cancer include abnormal tumor vasculature; further preferably, the cancer is selected from colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, brain tumor, bone cancer, melanoma.

[0172] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration.

[0173] Methods for diagnosis and detection

[0174] In another aspect, the present invention provides a method for detecting the presence of CD93 in a sample using the antibodies or antigen-binding fragments thereof described herein. The term "detection" includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. The method comprises the steps of contacting the sample with the antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal generated by the binding of the antibody or antigen-binding fragment thereof to CD93. When used for detection purposes, the antibodies or antigen-binding fragments thereof described herein may be labeled to indicate whether the conjugate is formed. In certain embodiments, the method can be used for diagnostic purposes or non-diagnostic purposes.

[0175] In yet another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described herein, or a detection composition containing the antibody or antigen-binding fragment thereof, in the preparation of a detection reagent for detecting the presence of CD93 in a sample.

[0176] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0177] The antibodies of the present invention can be prepared by a variety of methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0178] Example

[0179] The present invention is illustrated by the following examples, but is not intended to be limiting thereof. The present invention has been described in detail herein, and specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0180] Example 1. Preparation of recombinant protein for anti-CD93 antibody activity detection

[0181] The cDNA sequence encoding the CD93 extracellular domain was amplified using conventional PCR. Using conventional cloning techniques, the amplified fragment was cloned into a self-constructed eukaryotic expression plasmid system (MX2-mFc, containing the murine IgG2a Fc domain) with puromycin selection, generating the recombinant fusion protein expression plasmid hCD93 ECDmFC. The hCD93 ECD mFC recombinant protein was expressed and purified using 293E expression cells.

[0182] The amino acid sequence of the human CD93 extracellular domain (CD93 ECD) is amino acids Ala24-Lys580 (UniProtKB / Swiss-Prot: Q9NPY3.3), which is used for the activity detection of the antibody of the present invention.

[0183] Example 2. Preparation of mouse hybridoma cells

[0184] 2.1. Immunization of Animals

[0185] Five BALB / c mice (8-week-old female, purchased from Simonsen Laboratories of Gilroy) were immunized with CD93-Fc recombinant protein. Following the initial immunization (50 μg / mouse), booster immunizations (25 μg / mouse) were performed every one or two weeks for a total of six immunizations.

[0186] 2.2 Cell Fusion

[0187] Four days after the final booster immunization, inguinal and popliteal lymph nodes and spleens were harvested from the mice, ground in physiological saline, and the lymphocyte-rich suspension was fused with mouse myeloma Sp2 / 0 cells by conventional electroporation. The fusion product was cultured for 5 days in DMEM complete medium supplemented with 1:50 HAT (hypoxanthine, methotrexate, and thymidine) to screen for successfully fused cells (hybridomas). The medium was then switched to DMEM complete medium supplemented with 1:50 HT (hypoxanthine and thymidine) until the end of the screening process.

[0188] The DMEM complete culture medium ratio is: 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U / mL penicillin-streptomycin + 1:100 OPI (oxaloacetate, pyruvate and insulin), and the incubator conditions are 8% CO2, 37°C.

[0189] Example 3. Screening of mouse hybridoma cells and performance testing of the resulting anti-CD93 mouse antibodies

[0190] From a pool of 9,600 different polyclonal hybridoma cell lines, 181 hybridoma cell lines were screened by ELISA for antibodies that specifically bind to hCD93. Of these 181 binding-positive hybridoma cell lines, 15 expressed antibodies that blocked the binding of IGFBP7 to hCD93 expressed on the cell surface using FACS. Subsequent ELISA blocking experiments confirmed that seven of these polyclonal cell lines were able to block the binding of hCD93 to IGFBP7.

[0191] The ELISA procedure involves coating a 96-well MaxiSorp plate (Thermo Fisher Scientific, Nunc) with 2 μg / mL hCD93 (mouse Fc tag, Junmeng) and blocking with 1% BSA. Igfbp7 at a final concentration of 0.5 μg / mL is incubated with hybridoma culture supernatant on the microplate for 30 minutes. The mixture is then transferred to the MaxiSorp plate and incubated for 30 minutes. Detection is performed using a horseradish peroxidase-conjugated goat anti-human secondary antibody.

[0192] The FACS assay involves incubating CHO CD93 cells overexpressing human CD93 with a 0.5 μg / ml hIGFBP7mFc protein solution in staining buffer (PBS + 1% FBS) and various concentrations of anti-CD93 hybridoma antibodies at 4°C for 30 minutes. Unbound antibody is then washed away with staining buffer. The cells are then incubated with a 1% (v / v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch) in staining buffer at 4°C for 30 minutes in the dark. Finally, the cells are collected using flow cytometry, and cell surface-bound fluorescent antibody is detected. Raw data are analyzed using FlowJo to obtain MFI values, and GraphPad Prism software is used to perform a four-parameter curve fit to determine IC50 values.

[0193] Seven blocking monoclonal cell lines were obtained through subcloning and screening. The antibodies secreted by these cells were purified and analyzed. mRNA was extracted from the monoclonal hybridoma cells, and the antibody variable regions were sequenced. The seven sequences were identified as 1H2.3, 2A6.5, 2D9.4, 3D9.7, 3E2.7, 4B5.3, and 4C5.4.

[0194] Example 4. Determination of variable region sequences of anti-CD93 murine antibodies (expressed according to Kabat or IMGT)

[0195] The DNA coding sequence corresponding to the variable region of the anti-CD93 murine antibody was determined using a degenerate primer-based PCR method. Candidate hybridoma cells were cultured, harvested by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. This was used as a template to synthesize first-strand cDNA. The first-strand cDNA was then used as a template for subsequent PCR amplification of the corresponding variable region DNA coding sequence. The primer sequences used in the amplification reaction were complementary to the first framework region and constant region of the antibody variable region (Larrick, JW et al., 1990, Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). To a 50 μl reaction system, 1 μl of cDNA, 5 μl of 10× PCR buffer, 1 μl of each upstream and downstream primer (25 pmol), 1 μl of dNTPs, 1 μl of 25 mmol / L MgCl₂, and 39 μl of H₂O were added. The reaction was pre-denatured at 95°C for 10 min, and 1 μl of Taq enzyme was added. PCR amplification was performed using a temperature cycler. The reaction conditions were denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 15 s for 32 cycles, followed by a 10-min incubation at 72°C. The PCR products were recovered and purified. The amplified products were sequenced to obtain the amino acid sequences of the heavy and light chain variable regions of the anti-human CD93 mouse antibody.

[0196] Consensus sequences were searched against the germline and rearranged Ig variable region sequence database using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ). Complementarity-determining region (CDR) amino acid sequences were identified by sequence annotation based on the Kabat (Wu, TT and Kabat, EA 1970 J. Exp. Med., 132: 211-250) and IMGT systems (Lefranc M.-P. et al. 1999 Nucleic Acids Research, 27, 209-212) and by internet-based sequence analysis (http: / / www.imgt.org / IMGT_vquest / vquest and http: / / www.ncbi.nlm.nih.gov / igblast / ).

[0197] The corresponding chimeric antibodies of the selected anti-CD93 murine antibodies are shown in Table 1 below.

[0198] Table 1: Correspondence between mouse antibodies and chimeric antibodies

[0199] Example 5. Construction of anti-CD93 chimeric antibody

[0200] Considering the expression amount, activity, and type of antibodies expressed by hybridoma cells, the light chain and heavy chain variable regions of the anti-human CD93 mouse antibodies TAB1913-1, TAB1913-2, TAB1913-3, TAB1913-4, TAB1913-5, TAB1913-6, and TAB1913-7 were selected to construct anti-CD93 chimeric antibodies.

[0201] The coding sequences for the IgG4 heavy chain constant region and the κ light chain constant region were cloned from human B lymphocytes (from the Beijing Institute of Hematology) and introduced into the pCDNA3.1 plasmid to form the vectors HXT1S-KDL and HXT2, respectively. The heavy and light chain variable region coding sequences of the aforementioned anti-CD93 murine antibody were synthesized by tsingke, digested with BSPQI, and subcloned into HXT4S and HXT2, respectively. Various chimeric heavy and light chain expression plasmids were mixed and paired and transfected into expression cells (CHOK118, Suzhou Junmeng), resulting in 49 anti-human CD93 chimeric antibodies. Their numbers and pairings are shown in Table 2. Subsequent experimental materials were extracted from cells transfected with these plasmids.

[0202] Table 2: Chimeric antibody numbers and sources of their heavy and light chain variable regions

[0203] Example 6. Screening of chimeric antibodies

[0204] 6.1 Binding of anti-CD93 chimeric antibodies to human CHO-CD93-expressing cells

[0205] CHO-CD93 cells overexpressing human CD93 (CHO-K1 cells were purchased from the ATCC cell bank, number CCL-61. A stable CD93-overexpressing cell line was constructed based on these cells; the amino acid sequence of CD93 is from NCBI accession number NP_036204.2) were incubated with different concentrations of anti-CD93 chimeric antibody (starting at 50 μg / ml, diluted 3-fold, with a total of 10 concentration gradients) at 4°C for 30 min. Unbound antibody was then washed with staining buffer (PBS + 1% FBS) and incubated with 1% (v / v) fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), prepared in staining buffer at 4°C for 30 min in the dark. Cells were harvested using a flow cytometer (BD Biosciences, Canto II model) and cell surface-bound fluorescent antibody was detected. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curves were fitted using GraphPad to calculate the EC50.

[0206] As shown in Figure 1 and Table 3, most of the anti-CD93 chimeric antibodies of the present invention can bind to human CD93 highly expressed on the surface of CHO-CD93 cells with high affinity, among which C17 and C33 have the highest binding top values, while C21 has no binding activity.

[0207] Table 3: Cell-level affinity of anti-CD93 chimeric antibodies (EC 50 )

[0208] 6.2 Anti-CD93 chimeric antibody blocks IGFBP7 protein binding to human CD93-expressing cells

[0209] CHO-CD93 cells overexpressing human CD93 were incubated with hIGFBP7mFc protein solution (Junshi Biotechnology, Lot #20200318, working concentration 10 μg / ml) prepared in staining buffer (PBS + 1% FBS) and various concentrations of anti-CD93 chimeric antibody (starting at 50 μg / ml, diluted 3-fold, 10 concentration steps) at 4°C for 30 min. Unbound antibody was then washed away with staining buffer, and the cells were incubated with 1% (v / v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch, Cat #115-175-071) in staining buffer at 4°C for 30 min in the dark. Cells were harvested using a flow cytometer (BD Biosciences, Canto II model) and cell surface-bound fluorescent antibody was detected. The raw data were analyzed with FlowJo to obtain the MFI value, and the IC50 value was obtained by four-parameter curve fitting using GraphPad Prism software.

[0210] As shown in Figure 2 and Table 4, among the following 12 chimeric antibodies, C17, C21, C36, C40, and C41 could not effectively block the binding of hIGFBP7 protein to CHO-CD93 cells, while the other antibodies could block the binding of hIGFBP7 protein to CHO-CD93 cells.

[0211] Table 4: Ability of anti-CD93 chimeric antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC50)

[0212] The heavy and light chains of the chimeric antibodies C1, C33, and C49 were selected for humanization design and modification. The chimeric antibody sequences are as follows:

[0213] C1-VH:TAB1913-1HC

[0214] CDR1:DYFMY(SEQ ID NO:2);

[0215] CDR2:YITNGGGGTYYPDTVKG(SEQ ID NO:3);

[0216] CDR3:DYYGSGAWFAY(SEQ ID NO:4);

[0217] C1-VL:TAB1913-1LC

[0218] CDR1:RSSQSLIHRNGDTYLH(SEQ ID NO:6);

[0219] CDR2:KVSNRFS(SEQ ID NO:7);

[0220] CDR3:SQSTHVPPWT(SEQ ID NO:8);

[0221] C33-VH:TAB1913-5HC

[0222] CDR1:DYALH(SEQ ID NO:10);

[0223] CDR2:FINTDYGNTHFNQKFKG(SEQ ID NO:11);

[0224] CDR3:NYYGNSWGYAMDY(SEQ ID NO:12);

[0225] C33-VL:TAB1913-5LC

[0226] CDR1:RSNKSLLHSNGLTYLY(SEQ ID NO:14);

[0227] CDR2:QMSNLAS(SEQ ID NO:15);

[0228] CDR3:AQNLEVPWT(SEQ ID NO:16);

[0229] C49-VH:TAB1913-7HC

[0230] CDR1:SYWMQ(SEQ ID NO:18);

[0231] CDR2:AIYPGDGDTRYTQKFKG(SEQ ID NO:19);

[0232] CDR3: HYYGYDMDS (SEQ ID NO: 20);

[0233] C49-VL:TAB1913-7LC

[0234] CDR1: RASQSISNNLH (SEQ ID NO: 22);

[0235] CDR2: FTSQSIS (SEQ ID NO: 23);

[0236] CDR3: QQSNGWPYT (SEQ ID NO: 24).

[0237] Example 7: Humanization of Antibody Variable Regions

[0238] To humanize the antibody variable region, the human immunoglobulin gene database at NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) is first searched for human germline IgG genes homologous to the murine antibody cDNA sequence. The amino acid sequence and precise boundaries of the variable region CDRs are then defined using the Kabat or IMGT numbering systems. In principle, a human IGHV with high homology to the murine antibody is selected as the humanization template, and humanization of the antibody variable region is performed via CDR grafting.

[0239] Based on the sequence of the murine antibody obtained above, humanization was performed. Briefly, the humanization process involves the following steps: A. Aligning the murine antibody gene sequence with the human embryonic antibody gene sequence to identify sequences with high homology; B. Analyzing and examining HLA-DR affinity to select human embryonic framework sequences with low affinity; C. Using computer simulation techniques and molecular docking, the variable region and surrounding framework amino acid sequences were analyzed to examine their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, the key amino acids in the murine antibody gene sequence that interact with human CD93 and maintain the spatial structure were identified. These amino acids were then grafted back into the selected human embryonic gene framework. Based on this, the framework amino acid positions that must be retained were standardized to synthesize the humanized antibody. This process resulted in the generation of a variety of humanized antibody variable regions. The designed humanized anti-CD93 antibody variable regions were then self-paired and combined to generate 48 humanized anti-CD93 antibodies. Their numbering and the amino acid sequences of the light and heavy chain variable regions are detailed below.

[0240] TAB1913-1LC-1

[0241] TAB1913-1LC-2

[0242] TAB1913-1LC-3

[0243] TAB1913-1LC-4

[0244] TAB1913-5LC-1

[0245] TAB1913-5LC-2

[0246] TAB1913-5LC-3

[0247] TAB1913-5LC-4

[0248] TAB1913-7LC-1

[0249] TAB1913-7LC-2

[0250] TAB1913-7LC-3

[0251] TAB1913-7LC-4

[0252] TAB1913-1HC-1

[0253] TAB1913-1HC-2

[0254] TAB1913-1HC-3

[0255] TAB1913-1HC-4

[0256] TAB1913-5HC-1

[0257] TAB1913-5HC-2

[0258] TAB1913-5HC-3

[0259] TAB1913-5HC-4

[0260] TAB1913-7HC-1

[0261] TAB1913-7HC-2

[0262] TAB1913-7HC-3

[0263] TAB1913-7HC-4

[0264] Example 8. Construction of anti-CD93 humanized antibody

[0265] Various chimeric heavy and light chain expression plasmids were mixed and paired and transfected into expression cells to obtain 48 anti-human CD93 humanized antibodies. Their numbering and pairing are shown in Table 5. Subsequent experimental materials were extracted from cells transfected with this series of plasmids.

[0266] Table 5: Numbering of humanized anti-CD93 antibodies and sources of their heavy and light chain variable regions

[0267] Exemplary light chain and heavy chain amino acid sequences of Hu37 and Hu92 are shown below.

[0268] Hu37-HC:

[0269] Hu37-LC:

[0270] hu92-HC:

[0271] hu92-LC:

[0272] Example 9. Screening of humanized anti-CD93 antibodies

[0273] 9.1 Binding of Humanized Anti-CD93 Antibodies to Human CHO-CD93-Expressing Cells

[0274] CHO-CD93 cells overexpressing human CD93 (CHO-K1 cells were purchased from the ATCC cell bank, number CCL-61. A stable CD93-overexpressing cell line was constructed based on these cells; the amino acid sequence of CD93 is from NCBI accession number NP_036204.2) were incubated with various concentrations of anti-CD93 humanized antibody (starting at 10 μg / ml, diluted 3-fold, with a total of 10 concentration gradients) at 4°C for 30 min. Unbound antibody was then washed with staining buffer (PBS + 1% FBS) and incubated with 1% (v / v) fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), in staining buffer at 4°C for 30 min in the dark. Cells were harvested using a flow cytometer (BD Biosciences, Canto II model) and cell surface-bound fluorescent antibody was detected. The raw data were analyzed using FlowJo to obtain MFI values, and the antibody dose-dependent binding curves were fitted using GraphPad to calculate EC50, with C1 serving as the positive control.

[0275] As shown in FIG. 3 (A to D) and Tables 6-1, 6-2, 6-3, and 6-4, the anti-CD93 humanized antibodies of the present invention can bind with high affinity to human CD93 highly expressed on the surface of CHO-CD93 cells.

[0276] Table 6-1: Cell-level affinity of humanized anti-CD93 antibodies (EC 50 )

[0277] Table 6-2: Cell-level affinity of humanized anti-CD93 antibodies (EC 50 )

[0278] Table 6-3: Cell-level affinity of humanized anti-CD93 antibodies (EC 50 )

[0279] Table 6-4: Cell-level affinity of humanized anti-CD93 antibodies (EC 50 )

[0280] 6.2 Anti-CD93 humanized antibody blocks IGFBP7 protein binding to human CD93-expressing cells

[0281] CHO-CD93 cells overexpressing human CD93 were incubated with hIGFBP7mFc protein solution (Junshi Biotechnology, Lot #20200318, working concentration 10 μg / ml) prepared in staining buffer (PBS + 1% FBS) and various concentrations of humanized anti-CD93 antibody (starting at 50 μg / ml, diluted 3-fold, 10 concentration steps) at 4°C for 30 min. Unbound antibody was then washed away with staining buffer, and the cells were incubated with 1% (v / v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch, Cat #115-175-071) in staining buffer at 4°C for 30 min in the dark. Cells were harvested using a flow cytometer (BD Biosciences, Canto II model) and cell surface-bound fluorescent antibody was detected. FlowJo was used to analyze the raw data to obtain the MFI value, and GraphPad Prism software was used to perform a four-parameter curve fit to obtain the IC50 value. C1 was used as a positive control in the experiment.

[0282] As shown in FIG. 4 (A to C) and Tables 7-1, 7-2 and 7-3, the anti-CD93 humanized antibodies of the present invention can effectively block the binding of hIGFBP7 protein to CHO-CD93 cells.

[0283] Table 7-1: Ability of anti-CD93 humanized antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC 50 )

[0284] Table 7-2: Ability of anti-CD93 humanized antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC 50 )

[0285] Table 7-3: Ability of anti-CD93 humanized antibodies to block the binding of hIGFBP7 protein to CHO-CD93 cells (IC 50 )

[0286] Example 10: Binding of anti-hCD93 monoclonal antibodies to CD93 protein

[0287] Human CD93 (His Tag) (Manufacturer: Suzhou Junmeng) was diluted to 2.0 μg / mL using PBS (Manufacturer: Hyclone, Catalog Number: SH30256.01), added to the ELISA plate at 100 μl / well, and allowed to coat in a 37°C constant temperature incubator for 90 minutes; the plate was washed; 200 μl / well 2% BSA was added to the plate and incubated in a 37°C constant temperature incubator for 90 minutes; the plate was washed; the antibodies (hu37, 7B10, and H3L3; the 7B10 and H3L3 used in this article are 7B10 and H3L3 disclosed in WO2022067262A1, respectively) were diluted with 2% BSA to 10,000 ng / ml, and diluted in a 3-fold gradient to 0.056 ng / ml. All samples were added to the ELISA plate at 100 μl / well and incubated in a 37°C constant temperature incubator for 60 min; the plate was washed; horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (manufacturer: Sigma, catalog number: A0170) (Fc specific) was diluted 5000 times with 2% BSA and added to the ELISA plate at 100 μl / well, and incubated in a 37°C constant temperature incubator for 60 min; the plate was washed; the color development solution TMB (manufacturer: Sigma, catalog number: T2885) 0.1 mg / ml, 100 μl / well, was added to avoid bubbles, and the color was developed at 37°C in the dark for 15 min; finally, 2 M hydrochloric acid solution was added to terminate the reaction, 100 μl / well, and bubbles were avoided. The microplate reader reading was completed within 10 min (detection wavelength 450 nm; reference wavelength 620 nm), and the EC was fitted using a four-parameter logarithmic regression (4PL) model. 50 .

[0288] As shown in Figure 5 , hu37, Ref1 (17B10), and Ref2 (H3L3) antibodies were all able to bind to hCD93 with high affinity, and the binding activities of hu37, Ref1 (17B10), and Ref2 (H3L3) were comparable.

[0289] Example 11: Anti-hCD93 monoclonal antibody blocks the binding of IGFBP7 to CD93 protein

[0290] Human CD93 (His Tag) (Manufacturer: Suzhou Junmeng) was diluted to 2 μg / mL using PBS (Manufacturer: Hyclone, Catalog Number: SH30256.01), and 100 μl / well of the plate was added to the ELISA plate, and the plate was incubated in a 37°C constant temperature incubator for 90 minutes; the plate was washed; 200 μl / well of 2% BSA was added to the plate, and the plate was incubated in a 37°C constant temperature incubator for 90 minutes; the plate was washed; human IGFBP7 (mFc Tag) (Manufacturer: Suzhou Junmeng) was diluted to 0.3 μg / mL with 2% BSA, and the sample was diluted to 100,000 ng / ml with 0.3 μg / mL of human IGFBP7 (mFc Tag), and then diluted to 4.19 ng / ml in a 2.5-fold gradient. All samples were added to the ELISA plate at 100 μl / well and incubated in a 37°C constant temperature incubator for 60 min; the plate was washed; horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody IgG (manufacturer: Sigma, catalog number: A2554) (Fc specific) was diluted 5000 times with 2% BSA and added to the ELISA plate at 100 μl / well, and incubated in a 37°C constant temperature incubator for 60 min; the plate was washed; the color developing solution TMB (manufacturer: Sigma, catalog number: T2885) 0.1 mg / ml, 100 μl / well, was added to avoid bubbles, and the color was developed at 37°C in the dark for 15 min; finally, 2M hydrochloric acid solution was added to terminate the reaction, 100 μl / well, to avoid bubbles, and the microplate reader was read within 10 min (detection wavelength 450 nm; reference wavelength 620 nm), and the IC50 was fitted using a four-parameter logarithmic regression (4PL) model.

[0291] As shown in Figure 6, neither Ref1 (17B10) nor Ref2 (H3L3) antibodies could block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells, while hu37 could effectively block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells. 50 It is 745.6ng / mL.

[0292] Example 12: Binding of anti-hCD93 monoclonal antibodies to CHO hCD93 cells

[0293] CHOhCD93 cells (CHO-K1 cells stably expressing hCD93, hCD93 positive) were incubated with different concentrations of hCD93 monoclonal antibody hu37 (starting concentration of 3.3 μg / ml, 3-fold dilution, a total of 10 concentration gradients) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, cells were collected by flow cytometry (BD Fortessa) and fluorescent antibodies bound to the cell surface were detected. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad (Figure 1) and the EC was calculated.50 , positive controls were Ref1 (17B10) and Ref2 (H3L3) antibodies; negative control was anti-KLH hu-IgG1 antibody.

[0294] As shown in Figure 7 , hu37, Ref1 (17B10), and Ref2 (H3L3) antibodies were able to bind to hCD93 on the surface of CHO hCD93 cells with high affinity. 50 The binding activities of hu37, Ref1 (17B10) and Ref2 (H3L3) were comparable, being 144.2 ng / mL, 150.3 ng / mL and 128.6 ng / mL respectively.

[0295] Example 13: Anti-hCD93 monoclonal antibodies block the binding of IGFBP7 to CHO hCD93 cells

[0296] CHOhCD93 cells (CHO-K1 cells stably expressing hCD93, hCD93 positive) were incubated with different concentrations of hCD93 monoclonal antibody hu37 (starting concentration of 30 μg / ml, 3-fold dilution, a total of 10 concentration gradients) and 15 μg / ml hIGFBP7 (CD93 ligand) at 4 ° C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4 ° C for 30 minutes in the dark. Finally, the cells were collected by flow cytometry (BD Fortessa) and the fluorescent antibodies bound to the cell surface were detected. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent inhibition curve was fitted by GraphPad (Figure 2) and the IC was calculated. 50 , the positive controls were Ref1 (17B10) and Ref2 (H3L3) antibodies; the negative control was anti-KLH hu-IgG1 antibody.

[0297] As shown in Figure 8, neither Ref1 (17B10) nor Ref2 (H3L3) antibodies could block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells, while hu37 could effectively block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells. 50 It is 559.9ng / ml.

Claims

1. An anti-CD93 antibody or an antigen-binding fragment thereof, characterized in that: The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, or a HCDR1 having 1, 2 or 3 amino acid differences therefrom, a HCDR2 or a sequence having 1, 2 or 3 amino acid differences therefrom, and a HCDR3 or a sequence having 1, 2 or 3 amino acid differences therefrom; and / or the anti-CD93 antibody or antigen-binding fragment thereof comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 defined by Kabat. NO: 5, 13, 21, 28, 31, 32 or 36 LCDR1 or a sequence having 1, 2 or 3 amino acid differences with said LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with said LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with said LCDR3.

2. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1 of SEQ ID NO: 1 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the HCDR1, HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3; and / or, a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 5 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with the LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with the LCDR3; or The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1 of SEQ ID NO: 9 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the HCDR1, HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3; and / or a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 13 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with the LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with the LCDR3; or The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1 of SEQ ID NO: 17 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the HCDR1, HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3; and / or, the light chain variable region CDR (LCDR) it comprises is LCDR1 of SEQ ID NO: 21 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with the LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with the LCDR3; or The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1 of SEQ ID NO: 40 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the HCDR1, HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3; and / or, comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1 of SEQ ID NO: 32 defined by Kabat, or a sequence having 1, 2 or 3 amino acid differences with the LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with the LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with the LCDR3.

3. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region CDR (HCDR) and light chain variable region CDR (LCDR) comprised by the anti-CD93 antibody or antigen-binding fragment thereof are respectively HCDR1 or a sequence having 1, 2 or 3 amino acid differences with the HCDR1, HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3, and LCDR1 or a sequence having 1, 2 or 3 amino acid differences with the LCDR1, LCDR2 or a sequence having 1, 2 or 3 amino acid differences with the LCDR2, and LCDR3 or a sequence having 1, 2 or 3 amino acid differences with the LCDR3, in the heavy chain variable region and light chain variable region shown in each of the following groups 1 to 19, as defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably defined by Kabat:

4. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: In the anti-CD93 antibody or antigen-binding fragment thereof, HCDR1 comprises the amino acid sequence of SEQ ID NO: 2, 10 or 18, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, HCDR2 comprises the amino acid sequence of SEQ ID NO: 3, 11 or 19 or 57, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, HCDR3 comprises the amino acid sequence of SEQ ID NO: 4, 12 or 20, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and / or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 53, 54 or 55, or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, 15 or 23, or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8, 16, 24 or 56, or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

5. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein: The heavy chain variable region comprises: (I) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, SEQ ID NO: 4, respectively; or (II) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or (III) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; The light chain variable region comprises: (I) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 or SEQ ID NO: 8, respectively; or (II) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or 56, respectively.

6. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom. an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3 or 57 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 54 or 55 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 56 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom. Differences in amino acid sequence; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 57 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom; or The anti-CD93 antibody or antigen-binding fragment thereof comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.

7. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region of the anti-CD93 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 9, 17, 37-48, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1, 9, 17, 37-48, and / or the light chain variable region thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 5, 13, 21, 25-36, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 5, 13, 21, 25-36.

8. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (I) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; or (II) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:13, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:13; or (III) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:17, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:17; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:21, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:

21.

9. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-CD93 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36; or The anti-CD93 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD93 antibody or antigen-binding fragment thereof are selected from the amino acid sequence of the heavy chain variable region or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto and the amino acid sequence of the light chain variable region or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively, as shown in any of the following sequence numbers:

10. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-CD93 antibody or antigen-binding fragment thereof comprises: (I) a heavy chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 49 or 51, and a light chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 50 or 52; or (II) a heavy chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 49 or 51, and a light chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 50 or 52; or (III) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 49 and a light chain having an amino acid sequence as shown in SEQ ID NO: 50, or (IV) a heavy chain having an amino acid sequence as shown in SEQ ID NO:51 and a light chain having an amino acid sequence as shown in SEQ ID NO:

52.

11. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, characterized in that: The anti-CD93 antibody is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; preferably, the antigen-binding fragment is selected from: Fab; Fab′; F(ab′)2; Fv fragment; diabody; linear antibody; single-chain antibody molecule, such as sc-Fv; nanobodies and multispecific antibodies formed by antibody fragments.

12. The anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, characterized in that: The anti-CD93 antibody or antigen-binding fragment thereof is IgG1, IgG2, IgG3 or IgG4, preferably IgG1 subtype or IgG4 subtype.

13. An isolated anti-CD93 antibody or antigen-binding fragment thereof having at least one of the following properties: (I) binds to the same, completely overlapping, or partially overlapping epitope of human CD93 protein as the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12; (II) competing with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 for binding to an epitope of the human CD93 protein.

14. A polynucleotide molecule encoding the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the isolated antibody or antigen-binding fragment thereof according to claim 13.

15. A nucleic acid construct comprising the polynucleotide molecule according to claim 14; preferably, the nucleic acid construct is an expression vector, more preferably a eukaryotic expression vector.

16. A host cell comprising the polynucleotide molecule of claim 14 or the expression vector of claim 15 and / or expressing the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1-13; preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

17. A method for preparing the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13, characterized in that: The method comprises culturing the host cell of claim 16 under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

18. A single-chain antibody or a multispecific antibody, characterized in that: The single-chain antibody or multispecific antibody comprises the light chain variable region and the heavy chain variable region of the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13.

19. A pharmaceutical composition comprising the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13, the polynucleotide molecule according to claim 14, the nucleic acid construct according to claim 15, the host cell according to claim 16, the anti-CD93 antibody or antigen-binding fragment thereof prepared by the method according to claim 17, or the single-chain antibody or multispecific antibody according to claim 18, and a pharmaceutically acceptable carrier or excipient.

20. A drug combination comprising the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13, the polynucleotide molecule according to claim 14, the nucleic acid construct according to claim 15, the host cell according to claim 16, the anti-CD93 antibody or antigen-binding fragment thereof prepared by the method according to claim 17, the single-chain antibody or multispecific antibody according to claim 18, or the pharmaceutical composition according to claim 19, and one or more additional therapeutic agents; preferably, the therapeutic agent is one or a combination of anti-PD-1 antibody, PD-L1 antibody and anti-CTLA4 antibody.

21. Use of the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13, the polynucleotide molecule according to claim 14, the nucleic acid construct according to claim 15, the host cell according to claim 16, the anti-CD93 antibody or antigen-binding fragment thereof prepared by the method according to claim 17, the single-chain antibody or multispecific antibody according to claim 18, or the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating and / or preventing a CD93-mediated disease or condition; preferably, the disease or condition is cancer; more preferably, the cancer is characterized by having an abnormal tumor vasculature; further preferably, the cancer is selected from colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, brain tumor, bone cancer and melanoma.

22. A kit comprising the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13, the polynucleotide molecule according to claim 14, the nucleic acid construct according to claim 15, the host cell according to claim 16, the anti-CD93 antibody or antigen-binding fragment thereof prepared by the method according to claim 17, the single-chain antibody or multispecific antibody according to claim 18, the pharmaceutical composition according to claim 19, or the pharmaceutical combination according to claim 20.

23. A method for detecting the presence of CD93 in a sample, comprising the step of contacting the sample with the anti-CD93 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the isolated anti-CD93 antibody or antigen-binding fragment thereof according to claim 13, the anti-CD93 antibody or antigen-binding fragment thereof prepared by the method of claim 17, or a detection composition containing the anti-CD93 antibody or antigen-binding fragment thereof.

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