WO2025035451A1 - Anti-cd93 antibody, and composition and use thereof - Google Patents

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

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

Background Art

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

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

Currently, no anti-CD93 monoclonal antibody drugs have entered the preclinical stage, and new anti-CD93 antibodies are needed.

Summary of the invention

The present invention provides an anti-CD93 antibody or an antigen-binding fragment thereof, which has the function of blocking the binding of CD93 to IGFBP7. The anti-CD93 antibody or an antigen-binding fragment thereof provided by the present invention can be used as an independent therapy or in combination with other therapies/or other anticancer agents for the treatment of CD93-mediated diseases such as cancer.

In one aspect, the present invention provides an anti-CD93 antibody or an antigen-binding fragment thereof, wherein the anti-CD93 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, or a HCDR1 having 1, 2 or 3 amino acid differences with the HCDR1, a HCDR2 or a sequence having 1, 2 or 3 amino acid differences with the HCDR2, and a HCDR3 or a sequence having 1, 2 or 3 amino acid differences with the HCDR3; and/or the anti-CD93 antibody or an antigen-binding fragment thereof comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably SEQ ID NO: 1, 9, 17 or 39 defined by Kabat. 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. Different sequence.

In another aspect, the present invention provides an isolated anti-CD93 antibody or antigen-binding fragment thereof, which has at least one of the following properties: (I) binds to the same, completely overlapping, or partially overlapping epitope of human CD93 protein as the antibody or antigen-binding fragment thereof described in any embodiment of the present invention; (II) competes with the antibody or antigen-binding fragment thereof described in any embodiment of the present invention for binding to the epitope of human CD93 protein.

In yet another aspect, the present invention provides a polynucleotide molecule encoding an anti-CD93 antibody or antigen-binding fragment thereof described herein.

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

In yet 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.

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.

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

In yet 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.

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.

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

In yet another aspect, the present invention provides use of the antibodies or antigen-binding fragments thereof described herein, the polynucleotide molecules described herein, the nucleic acid constructs or expression vectors described herein, the host cells described herein, the multispecific antibodies described herein, the single-chain antibodies described herein, the pharmaceutical compositions described herein, or the pharmaceutical combinations described herein in the preparation of a medicament for treating and/or preventing a CD93-mediated disease or disorder.

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 method for treating and/or preventing a CD93-mediated disease or condition. The host cell described herein, the multispecific antibody described herein, the single-chain antibody described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein.

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

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.

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

A more detailed description of various aspects of this disclosure is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 : Graph showing the results of the cell-level binding experiment of anti-CD93 chimeric antibodies.

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

FIG3 : Graph showing the results of the cell-level binding experiment of anti-CD93 humanized antibodies.

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

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

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

Figure 7: Binding experiment results of anti-CD93 monoclonal antibody and CHO hCD93 cells.

Figure 8: Experimental results of anti-CD93 monoclonal antibody blocking the binding of IGFBP7 to CHO hCD93 cells.

DETAILED DESCRIPTION

definition

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.

In order to make the present invention more easily understood, certain technical terms are specifically defined as follows. Unless otherwise clearly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. For definitions and terms in this field, professionals can refer to Current Protocols in Molecular Biology. (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 common L-amino acids. As used herein (including the claims), singular forms include their corresponding plural forms unless the context clearly dictates otherwise.

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 that is 5% less than the specified numerical value and an upper limit that is 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.

The term "and/or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

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, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as "only one" or "exactly one" or when "consisting of..." is used in the claims, it will refer to only one number listed or one element of the list.

As used herein, the words "a" and "an" should be understood to mean "at least one" unless expressly stated to the contrary.

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 the amino acid sequences are aligned (and gaps introduced where necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

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 the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics are observed, measured or assessed using techniques known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays without limitation, 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).

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

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 its The term "isolated" does not imply the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.

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

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

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 (Chothia et al., 1989, Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948, 1997), Kabat (Kabat et al., Sequences of Proteins of Immunolobulins), and the like, based on the variability of antibody sequences. gical Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT))(1999 Nucleic Acids Research, 27, 209-212), and North based on affinity propagation clustering using a large number of crystal structures.

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

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 are different. When an antibody is defined by a specific CDR sequence defined in the present invention, the scope of the antibody also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose declared CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of a different scheme (such as a different assignment system or combination).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved 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 rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. 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 and multispecific antibodies formed from antibody fragments. When the binding activity of the antigen is expressed on a molar basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity. 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 the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies). The term "binding compound" refers to both antibodies and their binding fragments.

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 also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

The term "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. 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.

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.

The term "diabody" refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). 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.

The term "mouse antibody" or "hybridoma antibody" in the present disclosure refers to an anti-human CD93 antibody prepared according to the knowledge and skills in the art. Monoclonal antibodies are prepared by injecting a test subject with the CD93 antigen and then isolating hybridomas that express antibodies with the desired sequence or functional properties.

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

The term "humanized antibody" refers to an antibody form containing sequences from 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 the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

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 sugar chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat sugar chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

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

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. 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, such as Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

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

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

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

The term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the _KD for binding to the protein or cell is 1.0× ^10-6 M or higher, more preferably 1.0× ^10-5 M or higher, more preferably 1.0× ^10-4 M or higher, 1.0× ^10-3 M or higher, more preferably 1.0× ^10-2 M or higher.

The term "high affinity" for IgG antibodies refers to a _KD for an antigen of 1.0× ^10-6 M or less, preferably 5.0× ^10-8 M or less, more preferably 1.0× ^10-8 M or less, 5.0× ^10-9 M or less, and more preferably 1.0× ^10-9 M or less. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a _KD of ^10-6 M or less, preferably ^10-7 M or less, and more preferably ^10-8 M or less.

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, 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. Pat. 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 specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated. 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 bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

"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 include a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the invention.

"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 with bacterial replication origins and episomal mammalian vectors). After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors".

The term "expression vector" as used herein refers to a vector that is capable of replicating and expressing a target cell when transformed, transfected or transduced into a host cell. The expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host when necessary.

"Activation," "stimulation," and "treatment" as used for 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 otherwise or clearly dictates. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response/reaction," such as the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

As used herein, the term "treatment" or "treating" of any disease or condition refers to improving the disease or condition (i.e., slowing down or preventing or reducing the progression of the disease or at least one of its clinical symptoms) in one embodiment. 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 in both aspects. Unless explicitly described herein, methods for assessing the treatment and/or prevention of a disease are generally known in the art.

"Subject" includes any human or non-human animal. 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 cynomolgus monkeys.

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

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

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

The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. 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.

Anti-CD93 antibodies and antigen-binding fragments thereof

The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CD93. The term "anti-CD93 antibody", "anti-CD93", "CD93 antibody" or "antibody that binds to CD93" refers to an antibody that is capable of binding to a CD93 protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD93.

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

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

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, and/or comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 5, 13, 21, 28, 31, 32 or 36 defined by Kabat.

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1 defined by Kabat, and/or comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 5 defined by Kabat.

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 9 defined by Kabat, and/or the light chain variable region CDR (LCDR) comprised thereof comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 13 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 13 defined by Kabat.

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 17 defined by Kabat, and/or, the light chain variable region CDR (LCDR) comprised thereof is LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 21 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably defined by Kabat.

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 40 defined by Kabat, and/or the light chain variable region CDR (LCDR) comprised thereof comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 32 defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 32 defined by Kabat.

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

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region CDR (HCDR) of HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 1, 9, 17 or 39 defined by Kabat, and/or comprises a light chain variable region CDR (LCDR) of LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 5, 13, 21, 28, 31, 32 or 36 defined by Kabat.

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

In some embodiments, in the anti-CD93 antibody or antigen-binding fragment thereof of the present invention, its heavy chain variable region comprises: (I) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, and SEQ ID NO: 4, respectively, or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 or 57, and SEQ ID NO: 4, respectively; or (II) HDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. CDR1, HCDR2 and HCDR3, or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or (III) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively. CDR3; and/or its light chain variable region comprises: (I) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (II) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively. CDR3, or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (III) LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 or 56, respectively, or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 or 56, respectively.

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 shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

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 invention further comprises an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 15, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 16, or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom.

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 shown in SEQ ID NO: 18 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20 The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

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 shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or 57 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 or 58. The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

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 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom. The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 54 or 55 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

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 shown in SEQ ID NO: 18 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20 The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

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 shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having 6 amino acid differences with the amino acid sequence, The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with this amino acid sequence.

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 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom, and the LCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences therefrom. The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence.

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:

The heavy chain variable region comprises:

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

(II) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or

(III) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are 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 having amino acid sequences as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7, or SEQ ID NO: 8, respectively; or

(II) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; 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.

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 contains the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8.

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 shown in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

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 shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 24.

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 contains the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3 or 57, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 53, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8.

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 contains the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 54 or 55, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16.

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 contains the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 56.

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 contains the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 57, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 8.

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 shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 55, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.

In some embodiments, the heavy chain variable region of the anti-CD93 antibody or its antigen-binding fragment of the present invention comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 17, 37-48, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 17, 37-48, and/or, its light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 13, 21, 25-36, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 5, 13, 21, 25-36.

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:

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

(II) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:9, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:13, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:13; or

(III) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 17; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 21, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 21.

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment described in the present invention comprises: a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

In some embodiments, the anti-CD93 antibody or its antigen-binding fragment described in the present invention comprises: a heavy chain variable region, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

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 shown in any of the following sequence numbers or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto and the amino acid sequence of the light chain variable region or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively. Amino Acid Sequence:

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

(I) a heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NO: 49 or 51, and a light chain comprising an amino acid sequence as shown in any one of SEQ ID NO: 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 NO: 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 NO: 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.

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 characteristics: (1) the epitope of human CD93 protein bound by it is the same as, completely overlaps with, or partially overlaps with the anti-CD93 antibody or antigen-binding fragment thereof described herein; (2) the epitope of human CD93 protein bound by it is the same as, completely overlaps with, or partially overlaps with the anti-CD93 antibody or antigen-binding fragment thereof described herein; The fragments compete for binding to an epitope of the human CD93 protein.

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.

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.

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

In some embodiments, the antibodies described herein are monoclonal antibodies.

In some embodiments, the antigen-binding fragment is selected from: Fab; Fab'; F(ab')2; Fv fragment; diabody; linear antibody; single-chain antibody molecule, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.

Any suitable method for producing antibodies can be used to produce antibodies of the present invention. Any suitable form of CD93 can be used as an immunogen (antigen) for producing antibodies. 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 monoclonal anti-human CD93 antibodies of murine origin can be produced by methods known in the art.

Antibodies derived from rodents (e.g., mice) may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use leads to the body's immune response to the therapeutic antibodies, which at least results in loss of therapeutic efficacy and, in severe cases, potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of intact rodent variable regions in chimeric antibodies may still cause harmful immunogenicity in patients. Transplanting 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).

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

In some embodiments, the chimeric CD93 antibody of the present invention can be prepared by effectively linking the heavy and light chain variable regions of the immunoglobulin derived from hybridomas to the human IgG constant region (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy chains and chimeric light chains. In some embodiments, the constant region contained in the chimeric antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.

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

The humanized antibodies of the present invention can be prepared by inserting mouse CDR regions into human germline framework regions using methods known in the art, such as Winter et al. U.S. Pat. No. 5,225,539 and Queen et al. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

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

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.

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 (such as 1-10, 1-5 or 1-3) amino acid deletions, insertions and/or substitutions.

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 activity of binding to the CD93 protein or fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic agent and/or therapeutic agent targeting CD93. In other words, the antibody or antigen-binding fragment obtained by mutation, compared with the antibody or antigen-binding fragment before mutation, at least retains the binding affinity of the antibody or antigen-binding fragment before mutation to the CD93 protein or fragment thereof.

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.

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

In some embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced with cysteine residues.

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

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

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

Polynucleotide and Antibody Expression

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 the 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.

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.

In yet another aspect, the present invention provides an expression vector comprising a polynucleotide molecule or a 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.

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.

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 the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including those obtainable from American Type Culture Many immortalized cell lines of the American Culture Collection (ATCC) are available. These especially include Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

In one embodiment, the present invention provides a method for preparing an anti-CD93 antibody, wherein the method comprises, when an expression vector is introduced into a mammalian host cell, culturing the host cell for a sufficient time to allow the antibody to be expressed in the host cell, or more preferably allowing the antibody to be secreted into the culture medium in which the host cell grows, thereby preparing the antibody. The antibody can be recovered from the culture medium using standard protein purification methods.

It is likely that antibodies expressed by different cell lines or expressed in transgenic animals 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 unlikely to be immunogenic because their sugar structures are normal components of native human serum IgG.

Pharmaceutical compositions and kits

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 thereof provided by the present invention can be integrated with suitable carriers, excipients and other agents in the formulation for combined administration, thereby providing improved transfer, delivery, tolerance, etc.

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

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

The pharmaceutical composition or preparation of the present invention may also include one or more other active ingredients, which are required for the specific indications to be treated, preferably those active ingredients that do not adversely affect the complementary activities of each other. In some embodiments, other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics or various known anti-tumor or anti-cancer agents, and the active ingredients are appropriately combined in an amount effective for the intended use. In some embodiments, the pharmaceutical composition of the present invention also includes a composition of a polynucleotide molecule encoding an anti-CD93 antibody.

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

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.

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

Medical uses and treatments

Any anti-CD93 antibody or antigen-binding fragment thereof provided herein can be used in a method of treatment. It should also be understood that when discussing "antibodies", compositions comprising antibodies or antigen-binding fragments thereof are also included. The anti-CD93 antibodies or antigen-binding fragments thereof of the present invention can be used in a therapeutic or preventive method described in any embodiment of the present invention in a therapeutically effective amount or a preventively effective amount.

In another aspect, the present invention provides use of the antibodies or antigen-binding fragments thereof described herein, the polynucleotide molecules or nucleic acid constructs described herein, the expression vectors described herein, the host cells described herein, or the multispecific antibodies or single-chain antibodies described herein, or the pharmaceutical compositions or pharmaceutical combinations described herein in the preparation of drugs for treating and/or preventing CD93-mediated diseases or disorders.

In yet 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 use in treating and/or preventing a CD93-mediated disease or condition.

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

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.

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.

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, etc.

Methods for diagnosis and detection

In yet 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 may be used for diagnostic purposes, or for non-diagnostic purposes.

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.

The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of 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 only by way of illustration, 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. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety.

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

Example

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

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

The cDNA sequence encoding the CD93 extracellular domain was amplified by conventional PCR technology, and the amplified fragment was cloned into a self-constructed eukaryotic expression plasmid system (MX2-mFc, containing the mouse IgG2a Fc domain) using conventional cloning technology, which contains a puromycin selection system, thereby generating a recombinant fusion protein expression plasmid hCD93 ECDmFC. The hCD93 ECD mFC recombinant protein was expressed and purified using expression cells 293E.

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

Example 2. Preparation of mouse hybridoma cells

2.1. Immunization of animals

CD93-Fc recombinant protein was used as an antigen to immunize 5 BALB/c mice (purchased from Simonsen Laboratories of Gilroy, female BALB/c, 8 weeks old). After the initial immunization (50ug/mouse), booster immunization (25ug/mouse) was performed every 1 or 2 weeks for a total of 6 immunizations.

2.2 Cell Fusion

On the fourth day after the last booster immunization, the inguinal lymph nodes, popliteal lymph nodes and spleen of the mice were taken, and the lymphocyte-rich suspension was obtained after grinding in physiological saline, and it was fused with mouse myeloma cells Sp2/0 according to the conventional electroporation method. The fusion product was cultured in DMEM complete medium containing 1:50 HAT (hypoxanthine, methotrexate and thymidine) for 5 days to screen the successfully fused cells (hybridoma cells). Then it was changed to DMEM complete medium containing 1:50 HT (hypoxanthine and thymidine) until the end of the screening.

The DMEM complete medium had a ratio of: 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U/mL penicillin-streptomycin + 1:100 OPI (oxaloacetate, pyruvate and insulin), and the incubator conditions were 8% CO ₂ , 37°C.

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

Among 9600 different polyclonal hybridoma cells, 181 hybridoma cells were screened by ELISA, and the antibodies secreted by them could specifically bind to hCD93. Among these 181 hybridoma cells with positive binding, antibodies expressed by 15 hybridoma cells could block the binding of IGFBP7 to hCD93 expressed on the cell surface in FACS. In the subsequent ELISA blocking experiment, 7 of the polyclonal cell lines were determined to be able to block the binding of hCD93 to IGFBP7.

The above ELISA includes: 96-well MaxiSorp plates (Thermo Fisher Nunc) were coated with 2ug/mL hCD93 (mouse Fc tag, Junmeng) and then blocked with 1% BSA. Igfbp7 with a final concentration of 0.5ug/ml was incubated with hybridoma culture supernatant on the microplate for 30 minutes. The mixture was then transferred to the MaxiSorp plate and incubated for 30 minutes. Detection was performed with horseradish peroxidase-labeled goat anti-human secondary antibody.

The above FACS includes: incubating the self-constructed human CD93-overexpressing CHO CD93 cells with hIGFBP7mFc protein solution (0.5μg/ml) prepared with staining buffer (PBS+1% FBS) and anti-CD93 hybridoma antibodies of different concentrations at 4°C for 30 minutes. Then wash away the unbound antibodies with staining buffer, and then incubate the cells with 1% (v/v) sheep anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch) prepared with staining buffer at 4°C in the dark for 30 minutes. Finally, collect the cells with flow cytometry to detect the fluorescent antibodies bound to the cell surface. FlowJo was used to analyze the raw data to obtain the MFI value, and GraphPad Prism software was used to perform a four-parameter curve fitting to obtain the IC50 value.

Seven blocking monoclonal cell lines were obtained through subclone screening. The antibodies secreted by them were purified and analyzed. mRNA was extracted from monoclonal hybridoma cells, and the variable regions of the antibodies were sequenced. The seven sequences were determined to be 1H2.3, 2A6.5, 2D9.4, 3D9.7, 3E2.7, 4B5.3 and 4C5.4.

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

The DNA coding sequence corresponding to the variable region of the anti-CD93 mouse antibody was determined using a degenerate primer PCR method. Candidate hybridoma cells were cultured, the cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. The first-strand cDNA was synthesized using this as a template, and then the first-strand cDNA was used as a subsequent template to PCR amplify the corresponding variable region DNA coding sequence. The primer sequences used in the amplification reaction are complementary to the first framework region and constant region of the variable region of the antibody (Larrick, J.W. et al., 1990, Scand. J. Immunol., 32, 121-128 and Coloma, J.J. et al., (1991) BioTechniques, 11, 152-156). In a 50μl reaction system, add cDNA 1μl, 10×PCR buffer 5μl, upstream and downstream primers 1μl (25pmol), dNTP 1μl, 25mmol PL MgCl2 1μl, H2O 39μl, pre-denature at 95℃ for 10min, add Taq enzyme 1μl, enter temperature cycle, and perform PCR amplification. The reaction conditions are 94℃ denaturation for 1min, 58℃ annealing for 1min, 72℃ extension for 15s, a total of 32 cycles, and then keep at 72℃ for 10min. Recover and purify the PCR product. Sequence the amplified product to obtain the amino acid sequence of the heavy chain variable region and light chain variable region of the anti-human CD93 mouse antibody.

Consensus sequences were searched in germline and rearranged Ig variable region sequence databases using NCBI Ig-Blast (http://www.ncbi.nlm.nih.gov/projects/igblast/). Complementarity determining region (CDR) amino acid sequences were identified by sequence annotation based on the Kabat (Wu, T.T and Kabat, E.A. 1970 J. Exp. Med., 132: 211-250) and IMGT systems (Lefranc M.-P. 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/).

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

Table 1: Correspondence between mouse antibodies and chimeric antibodies

Example 5. Construction of anti-CD93 chimeric antibody

Considering the expression amount, activity, and type of antibodies expressed by hybridoma cells, the light chain and The heavy chain variable region was used to construct an anti-CD93 chimeric antibody.

The coding sequences of the heavy chain constant region IgG4 and the light chain constant region κ were cloned from human B lymphocytes (from Beijing Institute of Hematology) and introduced into the pCDNA3.1 plasmid to form vectors HXT1S-KDL and HXT2, respectively. The heavy chain and light chain variable region coding sequences of the aforementioned anti-CD93 mouse antibody were synthesized by tsingke, digested by BSPQI, and subcloned into HXT4S and HXT2, respectively. Various chimeric heavy chain and light chain expression plasmids were mixed and paired to transfect expression cells (CHOK118, Suzhou Junmeng), and 49 anti-human CD93 chimeric antibodies were obtained, and their numbers and pairing methods are shown in Table 2. Subsequent experimental materials were extracted after transfection of cells with this series of plasmids.

Table 2: Numbering of chimeric antibodies and the origin of their heavy and light chain variable regions

Example 6. Screening of chimeric antibodies

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

CHO-CD93 cells overexpressing human CD93 (CHO-K1 cells were purchased from ATCC cell bank, number CCL-61. Based on this cell line, a stable cell line overexpressing CD93 was independently constructed, and the amino acid sequence of CD93 is from NCBI accession number NP_036204.2) were incubated with different concentrations of anti-CD93 chimeric antibodies (starting concentration of 50 μg/ml, 3-fold dilution, a total of 10 concentration gradients) at 4°C for 30 min, and then unbound cells were washed with staining buffer (PBS + 1% FBS). The cells were incubated with the fluorescent secondary antibody goat anti-human IgG PE (Southern Biotech, Cat#2040-09) prepared in the staining buffer at 4°C for 30 minutes in the dark. Finally, the cells were collected by flow cytometry (BD, Canto II model) to detect the fluorescent antibody bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad and the EC50 was calculated.

As shown in FIG. 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.

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

6.2 Anti-CD93 chimeric antibody blocks the binding of IGFBP7 protein to human CD93-expressing cells

The cells overexpressing human CD93, CHO-CD93, were incubated with hIGFBP7mFc protein solution (Junshi's own expression, Lot#20200318, working concentration 10 μg/ml) prepared with staining buffer (PBS+1% FBS), and different concentrations of anti-CD93 chimeric antibodies (starting concentration of 50 μg/ml, 3-fold dilution, 10 concentration gradients) at 4°C for 30 minutes. Unbound antibodies were then washed away with staining buffer, and the cells were incubated with 1% (v/v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch, Cat#115-175-071) prepared with staining buffer at 4°C in the dark for 30 minutes. Finally, the cells were collected using a flow cytometer (BD, CantoII model) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed with FlowJo to obtain the MFI value, and the IC50 value was obtained by performing a four-parameter curve fitting with GraphPad Prism software.

As shown in FIG. 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.

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

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

C1-VH:TAB1913-1HC

CDR1:DYFMY(SEQ ID NO:2);

CDR2: YITNGGGGTYYPDTVKG (SEQ ID NO: 3);

CDR3:DYYGSGAWFAY(SEQ ID NO:4);

C1-VL:TAB1913-1LC

CDR1: RSSQSLIHRNGDTYLH (SEQ ID NO: 6);

CDR2:KVSNRFS(SEQ ID NO:7);

CDR3: SQSTHVPPWT (SEQ ID NO: 8);

C33-VH:TAB1913-5HC

CDR1:DYALH(SEQ ID NO:10);

CDR2: FINTDYGNTHFNQKFKG (SEQ ID NO: 11);

CDR3:NYYGNSWGYAMDY (SEQ ID NO: 12);

C33-VL:TAB1913-5LC

CDR1:RSNKSLLHSNGLTYLY(SEQ ID NO:14);

CDR2: QMSNLAS(SEQ ID NO: 15);

CDR3: AQNLEVPWT (SEQ ID NO: 16);

C49-VH:TAB1913-7HC

CDR1: SYWMQ (SEQ ID NO: 18);

CDR2: AIYPGDGDTRYTQKFKG (SEQ ID NO: 19);

CDR3: HYYGYDMDS (SEQ ID NO: 20);

C49-VL:TAB1913-7LC

CDR1: RASQSISNNLH (SEQ ID NO: 22);

CDR2: FTSQSIS(SEQ ID NO: 23);

CDR3: QQSNGWPYT (SEQ ID NO: 24).

Example 7: Humanization of antibody variable regions

For humanization of antibody variable regions, firstly, the NCBI (http://www.ncbi.nlm.nih.gov/igblast/) website The human immunoglobulin gene database in the .NET library is used to search for human germline IgG genes that are homologous to the cDNA sequences of mouse antibodies, and then the amino acid sequences and precise boundaries of the variable region CDRs are defined using the Kabat numbering system or the IMGT numbering system. In principle, human IGHVs with high homology to mouse antibodies are selected as humanization templates, and humanization of the antibody variable regions is performed by CDR grafting.

According to the sequence of the mouse antibody obtained above, humanization transformation is carried out. In short, the humanization transformation process involves the following steps: A. Compare the gene sequence of the mouse antibody with the gene sequence of the human embryonic system antibody to find out the sequence with high homology; B. Analyze and examine the HLA-DR affinity and select the human embryonic system framework sequence with low affinity; C. Use computer simulation technology to apply molecular docking to analyze the variable region and its surrounding framework amino acid sequence to examine its spatial three-dimensional binding mode. By calculating the electrostatic force, van der Waals force, hydrophilicity and entropy value, analyze the key amino acid individuals in the mouse antibody gene sequence that can interact with human CD93 and maintain the spatial structure, graft it back to the selected human embryonic system gene framework, and on this basis, standardize the framework region amino acid sites that must be retained to synthesize humanized antibodies. On this basis, a variety of humanized antibody variable regions were obtained, and the designed humanized anti-CD93 antibody variable regions were self-paired and combined to obtain 48 humanized anti-CD93 antibodies, whose numbers and amino acid sequences of light chain variable regions and heavy chain variable regions are specifically shown below.

TAB1913-1LC-1

TAB1913-1LC-2

TAB1913-1LC-3

TAB1913-1LC-4

TAB1913-5LC-1

TAB1913-5LC-2

TAB1913-5LC-3

TAB1913-5LC-4

TAB1913-7LC-1

TAB1913-7LC-2

TAB1913-7LC-3

TAB1913-7LC-4

TAB1913-1HC-1

TAB1913-1HC-2

TAB1913-1HC-3

TAB1913-1HC-4

TAB1913-5HC-1

TAB1913-5HC-2

TAB1913-5HC-3

TAB1913-5HC-4

TAB1913-7HC-1

TAB1913-7HC-2

TAB1913-7HC-3

TAB1913-7HC-4

Example 8. Construction of anti-CD93 humanized antibody

Various chimeric heavy chain and light chain expression plasmids were mixed and paired to transfect expression cells, and 48 anti-human CD93 humanized antibodies were obtained, and their numbers and pairing methods are shown in Table 5. Subsequent experimental materials were extracted from cells transfected with this series of plasmids.

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

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

Hu37-HC:

Hu37-LC:

hu92-HC:

hu92-LC:

Example 9. Screening of humanized anti-CD93 antibodies

9.1 Binding of anti-CD93 humanized antibodies to human CHO-CD93 expressing cells

CHO-CD93 cells overexpressing human CD93 (CHO-K1 cells were purchased from ATCC cell bank, numbered CCL-61. Based on this cell line, a stable cell line overexpressing CD93 was independently constructed, and the amino acid sequence of CD93 is from NCBI accession number NP_036204.2) were incubated with different concentrations of anti-CD93 humanized antibody (starting concentration was 10 μg/ml, 3-fold dilution, a total of 10 concentration gradients) at 4°C for 30 min, and then the unbound antibody was washed away with staining buffer (PBS + 1% FBS), and then incubated with 1% (v/v) fluorescent secondary antibody goat anti-human IgG PE prepared in staining buffer (Southern Biotech, Cat#2040-09) was incubated at 4°C in the dark for 30 minutes. Finally, the cells were collected by flow cytometry (BD, Canto II model) to detect the fluorescent antibodies bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad and the EC50 was calculated. C1 was used as the positive control of the experiment.

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 are all able to bind with high affinity to human CD93 highly expressed on the surface of CHO-CD93 cells.

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

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

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

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

6.2 Anti-CD93 humanized antibody blocks the binding of IGFBP7 protein to human CD93 expressing cells

CHO-CD93 cells overexpressing human CD93 were incubated with hIGFBP7mFc protein solution (Junshi's own expression, Lot#20200318, working concentration 10μg/ml) prepared with staining buffer (PBS+1% FBS) and different concentrations of anti-CD93 humanized antibody (starting concentration of 50μg/ml, 3-fold dilution, 10 concentration gradients) at 4°C for 30 minutes. Unbound antibodies were then washed away with staining buffer, and the cells were incubated with 1% (v/v) goat anti-mouse IgG Fc fluorescent secondary antibody (Jackson ImmunoResearch, Cat#115-175-071) prepared with staining buffer at 4°C in the dark for 30 minutes. Finally, cells were collected using a flow cytometer (BD, CantoII model) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the IC50 value was obtained by four-parameter curve fitting using GraphPad Prism software. C1 was used as the positive control in the experiment.

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.

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

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

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

Example 10. Binding of anti-hCD93 monoclonal antibodies to CD93 protein

Use PBS (manufacturer: Hyclone, catalog number: SH30256.01) to dilute human CD93 (His Tag) (manufacturer: Suzhou Junmeng) to 2.0 μg / mL, add 100 μl / well to the ELISA plate, and let it stand in a 37 ° C constant temperature incubator for 90 minutes; wash the plate; add 200 μl / well 2% BSA to the plate, and incubate in a 37 ° C constant temperature incubator for 90 minutes; wash the plate; dilute the antibodies (hu37, 7B10 and H3L3; 7B10 and H3L3 used in this article are 7B10 and H3L3 disclosed in WO2022067262A1, respectively) to 10000 ng / ml with 2% BSA, and dilute to 0.056 ng / ml in a 3-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; the horseradish peroxidase (HRP)-coupled goat anti-human antibody IgG (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 developing solution TMB (manufacturer: Sigma, catalog number: T2885) 0.1 mg/ml, 100 μl/well, avoiding bubbles, and coloring at 37°C in the dark for 15 min; finally, 2M hydrochloric acid solution was added to terminate the reaction, 100 μl/well, avoiding bubbles, and the ELISA reader reading was completed within 10 min (detection wavelength 450 nm; reference wavelength 620 nm), and the four-parameter logarithmic regression (4PL) model was used to fit the EC ₅₀ .

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

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

Use PBS (manufacturer: Hyclone, catalog number: SH30256.01) to dilute human CD93 (His Tag) (manufacturer: Suzhou Junmeng) to 2μg/mL, add 100μl/well to the ELISA plate, and place it in a 37℃ constant temperature incubator for coating for 90 minutes; wash the plate; add 200μl/well 2% BSA to the plate, and incubate it in a 37℃ constant temperature incubator for 90 minutes; wash the plate; dilute human IGFBP7 (mFc Tag) (manufacturer: Suzhou Junmeng) to 0.3μg/mL with 2% BSA, and then dilute the sample to 100000ng/ml with 0.3μg/mL human IGFBP7 (mFc Tag), and dilute it to 4.19ng/ml in 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; the horseradish peroxidase (HRP)-coupled goat anti-mouse antibody IgG (manufacturer: Sigma, catalog number: A2554) (Fc specificity) 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, avoiding bubbles, and coloring at 37°C in the dark for 15 min; finally, 2M hydrochloric acid solution was added to terminate the reaction, 100 μl/well, avoiding bubbles, and the ELISA reader reading was completed within 10 min (detection wavelength 450 nm; reference wavelength 620 nm), and the IC50 was fitted using a four-parameter logarithmic regression (4PL) model.

As shown in Figure 6, neither Ref1 (17B10) nor Ref2 (H3L3) antibodies can block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells, while hu37 can effectively block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells, with an _IC50 of 745.6 ng/mL.

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

CHOhCD93 cells (CHO-K1 cells stably expressing hCD93, hCD93 positive) were incubated with different concentrations of hCD93 monoclonal antibody hu37 (starting concentration of 3.3 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) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad (Figure 1) and the EC ₅₀ was calculated. The positive controls were Ref1 (17B10) and Ref2 (H3L3) antibodies; the negative control was anti-KLH hu-IgG1 antibody.

As shown in Figure 7, hu37, Ref1 (17B10) and Ref2 (H3L3) antibodies can all bind to hCD93 on the surface of CHO hCD93 cells with high affinity, with _EC50 of 144.2 ng/mL, 150.3 ng/mL and 128.6 ng/mL, respectively. The binding activities of hu37, Ref1 (17B10) and Ref2 (H3L3) are comparable.

Example 13: Anti-hCD93 monoclonal antibody blocks the binding of IGFBP7 to CHO hCD93 cells

CHOhCD93 cells (CHO-K1 cells stably expressing hCD93, hCD93 positive) were incubated with different concentrations of hCD93 monoclonal antibody hu37 (starting concentration of 30 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 in the dark for 30 minutes. Finally, the cells were collected by flow cytometry (BD Fortessa) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent inhibition curve was fitted by GraphPad (Figure 2) and the IC ₅₀ was calculated. The positive controls were Ref1 (17B10) and Ref2 (H3L3) antibodies; the negative control was anti- KLH hu-IgG1 antibody.

As shown in Figure 8, both Ref1 (17B10) and Ref2 (H3L3) antibodies could not block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells, while hu37 could effectively block the binding of IGFBP7 to hCD93 on the surface of CHO hCD93 cells with an _IC50 of 559.9 ng/ml.

Claims (23)

An anti-CD93 antibody or an antigen-binding fragment thereof, characterized in that the anti-CD93 antibody or the 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, 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/or, the anti-CD93 antibody or its antigen-binding fragment comprises a light chain variable region CDR (LCDR) defined by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1 of SEQ ID NO: 5, 13, 21, 28, 31, 32 or 36 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. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-CD93 antibody or its antigen-binding fragment 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, the light chain variable region CDR (LCDR) comprised by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1 of 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 its antigen-binding fragment 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, the light chain variable region CDR (LCDR) comprised by Kabat, Chothia, AbM, Contact, North or IMGT, preferably LCDR1 of 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 its antigen-binding fragment 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, the light chain variable region CDR (LCDR) comprised 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. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, characterized in that the heavy chain variable region CDR (HCDR) and light chain variable region CDR (LCDR) contained in 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, preferably HCDR1 defined by Kabat, Chothia, AbM, Contact, North or IMGT, in the heavy chain variable region and light chain variable region defined by Kabat:

The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, characterized in that in the anti-CD93 antibody or antigen-binding fragment thereof, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, 10 or 18, or an amino acid sequence having 1, 2 or 3 amino acid differences with such amino acid sequence, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, 11 or 19 or 57, or an amino acid sequence having 1, 2 or 3 amino acid differences with such amino acid sequence, HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4, 12 or 20, or an amino acid sequence having 1, 2 or 3 amino acid differences with such amino acid sequence, 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 the above 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 the above 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 the above amino acid sequence. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, characterized in that 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 having 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 whose amino acid sequences are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or (III) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are 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 having amino acid sequences as shown in SEQ ID NO: 6 or 53 or 54 or 55, SEQ ID NO: 7, or SEQ ID NO: 8, respectively; or (II) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; 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. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-CD93 antibody or its antigen-binding fragment comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence; or The anti-CD93 antibody or its antigen-binding fragment comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12 an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an amino acid sequence, said LCDR1 contains the amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an amino acid sequence, said LCDR2 contains the amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an amino acid sequence, and said LCDR3 contains the amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an 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: 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 its antigen-binding fragment comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or 57 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence; or The anti-CD93 antibody or its antigen-binding fragment comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12 an amino acid sequence or an amino acid sequence having 1, 2 or 3 amino acid differences with such an amino acid sequence, wherein LCDR1 contains the amino acid sequence shown in SEQ ID NO: 54 or 55 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an amino acid sequence, wherein LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an amino acid sequence, and wherein LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with such an 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: 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 its antigen-binding fragment comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence; or The anti-CD93 antibody or its antigen-binding fragment comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 55 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 1, 2 or 3 amino acid differences with the amino acid sequence. The anti-CD93 antibody or its antigen-binding fragment as described in claim 1 is characterized in that the heavy chain variable region of the anti-CD93 antibody or its antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 17, 37-48, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 17, 37-48, and/or, its light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 13, 21, 25-36, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 5, 13, 21, 25-36. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, characterized in that 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 shown in SEQ ID NO:9, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:13, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:13; or (III) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 17; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 21, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 21. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-CD93 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36; or The anti-CD93 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48; and a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36; 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:


The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, characterized in that the anti-CD93 antibody or antigen-binding fragment thereof comprises: (I) a heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NO: 49 or 51, and a light chain comprising an amino acid sequence as shown in any one of SEQ ID NO: 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 NO: 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 NO: 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. 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. 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. 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. 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. A nucleic acid construct comprising the polynucleotide molecule of claim 14; preferably, the nucleic acid construct is an expression vector, more preferably a eukaryotic expression vector. 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 to 13; preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell. 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 according to 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. A single-chain antibody or a multispecific antibody, characterized in that the single-chain antibody or the multispecific antibody comprises the light chain variable region and the heavy chain variable region of the anti-CD93 antibody or its antigen-binding fragment as described in any one of claims 1 to 12 or the isolated anti-CD93 antibody or its antigen-binding fragment as described in claim 13. 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. 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. 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 cancer Squamous cell carcinoma, brain tumors, bone cancer and melanoma. 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. 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.