WO2025201517A1 - Anti-cd93 antibody, and antigen-binding fragment thereof and use thereof - Google Patents

Abstract

Provided are an anti-CD93 antibody or an antigen-binding fragment thereof, and a preparation method therefor and a use thereof. The anti-CD93 antibody or the antigen-binding fragment thereof can specifically bind to a CD93 molecule, has strong immunoreactivity, and can effectively block the function of the CD93 molecule by means of the binding to the CD93 molecule, thereby effectively inhibiting tumor growth. Further provided is a humanized anti-CD93 antibody, which has strong binding affinity to CD93-expressing tumor cells and a good concentration-dependent effect, can effectively block the binding between CD93 and a ligand thereof, and can inhibit HUVEC tube formation and tumor growth, thereby being applicable to the treatment of CD93-associated diseases (such as angiogenic diseases and cancers).

Description

Anti-CD93 antibodies, antigen-binding fragments thereof, and uses thereof

Cross-references

This application claims priority to Chinese patent application No. 202410382220.9, filed on March 29, 2024, entitled “Anti-CD93 Antibodies, Antigen-Binding Fragments Thereof and Their Applications,” the entire contents of which are incorporated herein by reference.

Technical Field

The present application relates to the field of biomedicine technology, and specifically to anti-CD93 antibodies, antigen-binding fragments thereof, and applications thereof.

Background Art

CD93, also known as C1q R1, C1q R, and C1q RP, is a type I transmembrane glycoprotein. CD93 is a member of the type XIV lectin (CTLD) family, which also includes CLEC14A, THBD, and CD248. The extracellular domain of a typical type XIV protein contains a C-type lectin domain, a susi domain, an EGF-like domain, and a mucin-like region. CD93 is required for efficient endothelial cell migration and proper cell polarization in vitro, and also promotes angiogenesis within tumors. Therefore, blocking the function of the CD93 molecule has the potential to treat tumors and prevent further tumor progression.

Tumors refer to neoplasms formed by the proliferation of local tissue cells under the action of various tumorigenic factors. They are divided into two major categories: benign tumors and malignant tumors. Among them, malignant tumors (commonly known as cancer) have the characteristics of rapid growth, high invasiveness, and easy metastasis. They are extremely harmful to the human body and are one of the major diseases that seriously endanger human life. Tumors can be divided into hematological tumors and solid tumors according to their morphology. Currently, biological drug treatments for hematological tumors include antibody drugs, CAR-T, etc., but there is still a lack of effective drugs for most solid tumors. The use of immune checkpoint inhibitors (ICIs), such as those targeting PD-1/PD-L1 or CTLA4, has significantly changed the field of cancer treatment. However, a large number of patients still show extremely low response to ICIs. In terms of solid tumors, this may be related to the following two reasons: (1) the heterogeneity of the tumor cells themselves, and (2) the difficulty of drug exposure to tumor tissue. The huge differences between different patients in the latter can be largely explained by the complex tumor microenvironment (TME). It is reported that TME has a significant impact on clinical outcomes and response to treatment (Bagaev et al., 2021).

Tumor angiogenesis, due to an imbalance between angiogenic and angiogenic inhibitors, is characterized by its uncontrolled and immature nature. Blood vessels are in a state of continuous growth and remodeling, forming a distorted vascular system. When this balance is disrupted, abnormal angiogenesis can cause a tumor to transition from a benign state to a malignant state. Cancer cells then generate blood vessels at metastatic sites to sustain their growth. Angiogenesis is essential not only for cancer development and invasion of surrounding tissues, but also for metastasis. Without vascular support, tumors may undergo necrosis or even apoptosis. Continuous angiogenesis is the most prominent hallmark of cancer.

Currently, there is still no effective antibody drug targeting CD93 molecules in clinical practice to block its molecular function for the treatment of tumors and vascular proliferative diseases.

Summary of the Invention

Purpose of the Invention

In response to the problems or needs existing in the prior art, the purpose of the present application is to provide an anti-CD93 antibody or its antigen-binding fragment that can specifically bind to the CD93 receptor and has strong immunoreactivity to it, and can effectively block its molecular function by specifically binding to the CD93 molecule, as well as its preparation method and application.

Solution

To achieve the above objectives, the present application has obtained a monoclonal antibody specific for the CD93 receptor molecule through extensive screening. The monoclonal antibody can specifically bind to the CD93 molecule with high affinity and block its molecular function, thus having a tumor treatment effect.

Specifically, this application provides the following technical solutions:

In a first aspect, the present application provides an anti-CD93 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively have three HCDRs and three LCDRs selected from any one of the following heavy chain variable region and light chain variable region combinations:

(1) the heavy chain variable region shown in SEQ ID NO: 11, and the light chain variable region shown in SEQ ID NO: 12;

(2) The heavy chain variable region shown in SEQ ID NO: 13, and the light chain variable region shown in SEQ ID NO: 14.

In some embodiments, the heavy chain variable region of the antibody comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, whose amino acid sequences are shown in SEQ ID NO:4, SAS and SEQ ID NO:5, respectively.

In other embodiments, the heavy chain variable region of the antibody comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, whose amino acid sequences are shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, whose amino acid sequences are shown in SEQ ID NO:9, LAS and SEQ ID NO:10, respectively.

Optionally, the antibody is a murine antibody, a single domain antibody, a chimeric antibody, a human antibody or a humanized antibody;

Optionally, the antigen-binding fragment is Fab, Fab', F(ab') ₂ , Fv or a complementarity determining region fragment.

In a feasible embodiment, the antibody is a murine antibody or a chimeric antibody, wherein:

The amino acid sequence of the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 11. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 12. Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 12;

Alternatively, the amino acid sequence of the heavy chain variable region of the antibody includes the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region of the antibody includes the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14, preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 14.

Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., page 224, (4th edition)). Therefore, conservative modifications (in particular, conservative substitutions or replacements) of one or a few amino acids in the framework regions of the heavy and light chain variable regions can substantially retain the biological activity of the original sequence. Therefore, the derivative sequences defined by the percentage of sequence identity with the specified sequence are also within the scope of protection of this application.

When the antibody is a murine antibody, the murine antibody further comprises a murine heavy chain constant region and a murine light chain constant region, wherein the murine heavy chain constant region is selected from one of murine IgG1, IgG2a, IgG2b or IgG3 constant regions, preferably a murine IgG1 constant region; and the murine light chain constant region is a murine Ck constant region.

When the antibody is a chimeric antibody, the chimeric antibody further comprises a humanized antibody constant region.

In a second aspect, the present application provides a humanized anti-CD93 antibody or an antigen-binding fragment thereof, which is a humanized anti-CD93 antibody or an antigen-binding fragment thereof constructed on the basis of the anti-CD93 antibody or an antigen-binding fragment thereof as described in the first aspect above, using CDRs transplantation technology and/or CDR region mutation design.

In a preferred embodiment, the humanized anti-CD93 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region selected from any one of the following:

(1) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

(2) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

(3) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

(4) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19;

(5) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20;

(6) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20;

(7) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20;

(8) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20;

(9) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 21, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23;

(10) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23;

(11) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 21, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24;

(12) The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 22, and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24.

Furthermore, the heavy chain of the humanized anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1.

In a third aspect, the present application provides an ADC molecule comprising the antibody or antigen-binding fragment thereof as described in the first or second aspect above.

In a fourth aspect, the present application provides a bispecific or multispecific antibody molecule, which comprises the antibody or antigen-binding fragment thereof as described in the first or second aspect above.

In a fifth aspect, the present application provides a fusion protein comprising the antibody or antigen-binding fragment thereof as described in the first or second aspect above.

In a sixth aspect, the present application provides a polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described in the first or second aspect above. The polynucleotide is not limited by the method of its production and can be obtained using genetic engineering recombination technology or chemical synthesis methods.

In a possible embodiment, the polynucleotide is a set of polynucleotides.

In some preferred embodiments, the polynucleotide set comprises:

A nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 11 and a nucleotide sequence encoding the light chain variable region shown in SEQ ID NO: 12;

Alternatively, a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:13 and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:14.

Preferably, the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 11 includes the DNA sequence shown in SEQ ID NO: 25 or the RNA sequence corresponding thereto, and the nucleotide sequence encoding the light chain variable region shown in SEQ ID NO: 12 includes the DNA sequence shown in SEQ ID NO: 26 or the RNA sequence corresponding thereto;

Preferably, the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:13 includes the DNA sequence as shown in SEQ ID NO:27 or the corresponding RNA sequence, and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:14 includes the DNA sequence as shown in SEQ ID NO:28 or the corresponding RNA sequence.

In a seventh aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the sixth aspect above, and, optionally, at least one expression control element operably linked to the polynucleotide.

In an eighth aspect, the present application provides a recombinant vector comprising the polynucleotide as described in the sixth aspect above, or the nucleic acid construct as described in the seventh aspect above.

The recombinant vector of the present application can be a cloning vector or an expression vector, for example, it can be a plasmid, a cosmid, a phage, etc.

In some preferred embodiments, the recombinant vector is an expression vector, preferably a eukaryotic expression vector.

In a ninth aspect, the present application provides a transformed host cell, wherein the polynucleotide as described in the sixth aspect, the nucleic acid construct as described in the seventh aspect, or the recombinant vector as described in the eighth aspect is transformed;

The host cell includes but is not limited to: prokaryotic cells, such as Escherichia coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell can also be a cell line, such as a 293T cell line.

In some feasible embodiments, the host cell is a bacterial, yeast or mammalian cell;

Optionally, the bacteria is Escherichia coli;

Optionally, the yeast is Pichia pastoris;

Optionally, the mammalian cells are Chinese hamster ovary cells or human embryonic kidney 293 cells.

In the tenth aspect, the present application provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof as described in the first or second aspect above, the ADC molecule as described in the third aspect above, the bispecific or multispecific antibody molecule as described in the fourth aspect above, the fusion protein as described in the fifth aspect above, the polynucleotide as described in the sixth aspect above, the nucleic acid construct as described in the seventh aspect above, the recombinant vector as described in the eighth aspect above and/or the transformed host cell as described in the ninth aspect above, and a pharmaceutically acceptable excipient, diluent or carrier.

In an eleventh aspect, the present application provides a reagent or kit for detecting the presence or expression level of a CD93 molecule, comprising the antibody or antigen-binding fragment thereof as described in the first or second aspect above and/or the transformed host cell as described in the ninth aspect above.

In certain preferred embodiments, the kit is a detection or diagnostic kit, wherein the antibody or antigen-binding fragment thereof described in the present application further comprises a detectable label; in certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof or anti-idiotypic antibody of the present application; preferably, the second antibody further comprises a detectable label; such detectable labels are well known to those skilled in the art and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), etc.

In the twelfth aspect, the present application provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first or second aspect above, the method comprising: allowing the transformed host cell as described in the ninth aspect above to express the antibody or antigen-binding fragment thereof 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 culture of the host cell.

In a thirteenth aspect, the present application provides the use of the antibody or antigen-binding fragment thereof as described in the first or second aspect above, the ADC molecule as described in the third aspect above, the bispecific or multispecific antibody molecule as described in the fourth aspect above, the fusion protein as described in the fifth aspect above, the polynucleotide as described in the sixth aspect above, the nucleic acid construct as described in the seventh aspect above, the recombinant vector as described in the eighth aspect above, the transformed host cell as described in the ninth aspect above and/or the pharmaceutical composition as described in the tenth aspect above in the preparation of a medicament for preventing and/or treating CD93-mediated diseases, disorders or conditions.

Possibly, the disease is a tumor and/or an angiogenic proliferative disease, preferably, the disease expresses CD93; and the treatment is to alleviate, relieve, ameliorate or inhibit the symptoms or progression of the disease, disorder or condition.

Preferably, the tumor is breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, hematological tumor, lymphoma, glioblastoma and/or melanoma.

Preferably, the angiogenic proliferative disease is selected from the group consisting of neovascular eye disease, psoriasis characterized by abnormal angiogenesis, psoriasis, rheumatoid arthritis or obesity.

In a fourteenth aspect, the present application provides a method for detecting the presence or expression level of a CD93 molecule, the method comprising using the antibody or antigen-binding fragment thereof as described in the first or second aspect above and/or the transformed host cell as described in the ninth aspect above.

In some preferred embodiments of this method, the antibody or antigen-binding fragment thereof further comprises a detectable label.

In other preferred embodiments of the method, the method further comprises: using a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof described in the present application.

The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive assay and the like.

In a fifteenth aspect, the present application provides a method for preventing and/or treating a CD93-mediated disease, disorder or condition, the method comprising: administering to a subject in need thereof a preventively and/or therapeutically effective amount of the antibody or antigen-binding fragment thereof as described in the first or second aspect above, the ADC molecule as described in the third aspect above, the bispecific or multispecific antibody molecule as described in the fourth aspect above, the fusion protein as described in the fifth aspect above, the polynucleotide as described in the sixth aspect above, the nucleic acid construct as described in the seventh aspect above, the recombinant vector as described in the eighth aspect above, the transformed host cell as described in the ninth aspect above and/or the pharmaceutical composition as described in the tenth aspect above.

Beneficial effects

The anti-CD93 antibodies or antigen-binding fragments provided herein are capable of specifically binding to CD93 molecules and exhibit strong immunoreactivity to them. By binding to CD93 molecules, they can effectively block their molecular functions, for example, blocking their binding to ligands IGFBP7 and MMRN2, thereby effectively inhibiting tumor growth. Furthermore, the anti-CD93 humanized antibodies provided herein not only have strong binding ability to CD93-expressing tumor cells, with a good concentration-dependent effect, but also effectively block their binding to ligands such as IGFBP7, inhibit HUVEC tube formation, and inhibit tumor growth, thereby being useful in the treatment of CD93-related diseases (e.g., tumors and vascular proliferative diseases).

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

Figure 1 shows the binding ability of the anti-CD93 antibody of the present application to its antigen as determined by ELISA, as described in Example 4; wherein, Figure A is the binding curve of the gradient-dilution anti-CD93 antibody of the present application to the human CD93-his protein antigen; Figure B is the binding curve of the gradient-dilution anti-CD93 antibody of the present application to the monkey CD93-his protein antigen; Figure C shows the binding ability of the anti-CD93 antibody of the present application to the CD93-EGF-his protein antigen at two concentrations; Figure D shows the binding ability of the anti-CD93 antibody of the present application to the CD93-unrelated antigen DDR1-his, with a positive antibody as a control.

Figure 2 shows the concentration-dependent blocking effect of the anti-CD93 antibodies C132 and C164 of the present application on the binding of CD93 molecules to ligands as determined by ELISA, as described in Examples 5 and 6; wherein, Figure A shows that the anti-CD93 antibodies C132 and C164 blocked the binding of CD93 molecules to the ligand IGFBP-7, and Figure B shows that the anti-CD93 antibodies C132 and C164 blocked the binding of CD93 molecules to the ligand MMRN2.

Figure 3 shows the binding ability of the CD93-overexpressing B16F10 stably transfected cell line B16F10-CD93 and the SNU-1 cell line to IGFBP7, as detected by flow cytometry, as described in Example 7; wherein, Panel A shows the positive rate of the B16F10-CD93 cell line binding to IGFBP7, Panel B shows the MFI value of the B16F10-CD93 cell line binding to IGFBP7, Panel C shows the positive rate of the SNU-1 cell line binding to IGFBP7, and Panel D shows the MFI value of the SNU-1 cell line binding to IGFBP7.

Figure 4 shows the binding ability of the SNU-1 cell line to the anti-CD93 antibody C132 detected by flow cytometry, as described in Example 8; wherein, Figure A shows the positive rate of the SNU-1 cell line binding to the antibody C132, and Figure B shows the MFI value of the SNU-1 cell line binding to the antibody C132.

FIG5 shows the results of anti-CD93 antibodies C132 and C164 blocking HUVEC tube formation at different co-incubation times, as described in Example 10.

Figure 6 shows the inhibitory effect of the humanized antibody of the present application on HUVEC tube formation, as described in Example 10; wherein, Figure A shows the blocking results of HUVEC tube formation when the humanized antibody of the present application is co-incubated with cells for 4 hours, Figure B shows the blocking results of HUVEC tube formation when the humanized antibody of the present application is co-incubated with cells for 8 hours, Figure C shows the blocking results of HUVEC tube formation when the humanized antibody of the present application is co-incubated with cells for 20 hours, and Figure D shows the concentration-dependent blocking results of HUVEC tube formation when the humanized antibody of the present application is co-incubated with cells for 4 hours.

Figure 7 shows the effect of the humanized antibodies of the present application on gene transcription of HUVEC cells under different culture conditions, as described in Example 11; wherein, Figure A shows HUVEC cells in the passage state, and Figure B shows HUVEC cells in the tube-forming condition.

FIG8 shows the concentration-dependent effect curve of the cell positive rate (A) and mean fluorescence intensity (B) of the humanized antibody huC164 of the present invention binding to SNU-1 detected by flow cytometry, as described in Example 12.

FIG9 shows the concentration-dependent effect curve of the humanized antibody huC132 of the present invention in blocking the binding of CD93-mFc to MMRN2.

Figure 10 shows the binding and dissociation curves of the humanized antibodies of the present invention and antigens detected by biomembrane interferometry (BLI), as described in Example 12; wherein, Figure A is the result of the humanized antibody HuC132-3, and Figure B is the result of the humanized antibody HuC164-2.

Figure 11 shows the Tm and Tagg values of the humanized antibodies of the present invention, as described in Example 12; wherein, Figure A shows the particle size distribution of the test substance, and Figure B shows the Tm and Tagg values of the test antibody measured in BCM and SLS266 at elevated temperatures of 25-95 degrees Celsius, calculated according to the software embedded formula.

Figure 12 shows the inhibitory effect of the fully mouse anti-CD93 antibody of the present application on B16F10-CD93 tumor formation in C57BL/6J mice, as described in Example 13; wherein, Figure A shows the mouse tumor volume detection results, Figure B shows the mouse weight change curve, and Figure C shows the mouse survival curve.

Figure 13 shows the tumor inhibitory effect of the humanized anti-CD93 antibody of the present application on the hPBMC immune-reconstructed NPG mouse tumor transplantation model, as described in Example 13; wherein, Figure A shows the mouse tumor volume detection results, Figure B shows the change curve of tumor volume in the antibody-treated group and the control group, Figure C shows the mouse survival curve, Figures D-H show the marker positive ratio curve in the mouse blood, and Figure I shows the immunohistochemistry and immunofluorescence analysis of mouse tumor tissue.

DETAILED DESCRIPTION

Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

The practice of the present application 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 it easier to understand this application, some 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 skilled in the art to which this application belongs.

The term "CD93" is also referred to as C1q R1, C1qR, and C1qRP. These names are used interchangeably and include variants, isoforms, species homologs, and analogs of human CD93 that share at least one common epitope with CD93. The complete CD93 sequence can be found in GenBank under accession number Q9NPY3.

In this disclosure, the three-letter and one-letter codes for amino acids are as described in J. biol. chem, 243, p3558 (1968).

In this disclosure, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of the heavy chains of immunoglobulins differ, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class of Ig, there are further subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five classes of Ig can have either kappa or lambda chains.

In the present disclosure, the antibody light chain may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

In the present disclosure, the antibody heavy chain may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) consists of three CDRs and four FRs, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are HCDR1, HCDR2, and HCDR3.

In the present disclosure, the antibodies include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies.

The term "murine antibody," as used herein, refers to a monoclonal antibody against human CD93 prepared according to the knowledge and skill in the art. This antibody is prepared by injecting a subject with the CD93 antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional properties. In a preferred embodiment of the present disclosure, the murine CD93 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or variants thereof.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes murine-specific monoclonal antibodies. The variable region genes are then cloned from the murine hybridoma cells. The constant region genes of the human antibody are then cloned as needed. The murine variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the CD93 chimeric antibody further comprises a light chain constant region of a human κ chain, λ chain, or variants thereof. The antibody heavy chain of the CD93 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprises a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant using amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations).

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. This can overcome the heterologous reaction induced by chimeric antibodies due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

CDR grafting may result in a reduction in the affinity of the resulting CD93 antibody or antigen-binding fragment for the antigen due to framework residues that contact the antigen. Such interactions may be the result of somatic hypermutation. Therefore, it may still be necessary to graft such donor framework amino acids into the framework of the humanized antibody. Amino acid residues from non-human CD93 antibodies or antigen-binding fragments thereof that participate in antigen binding can be identified by examining the sequence and structure of the murine monoclonal antibody variable region. Residues in the CDR donor framework that differ from the germline can be considered relevant. If the closest germline cannot be determined, the sequence can be compared to a subtype consensus sequence or a consensus sequence of murine sequences with a high percentage of similarity. Rare framework residues are considered to be the result of somatic hypermutation and thus play an important role in binding.

"HuMAb," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably and may be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigenic stimulation and may be produced by any method known in the art. In certain techniques, elements of the human heavy and light chain loci are introduced into cell lines of an organism derived from an embryonic stem cell line, wherein the endogenous heavy and light chain loci in these cell lines are targeted for disruption. These cell lines contain targeted disruption of the endogenous heavy and light chain loci. The transgenic organism can synthesize human antibodies specific for human antigens, and the organism can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene or chromosomal transfection methods and phage display technology, or by in vitro activated B cells, all of which are known in the art.

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD93). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody. In some embodiments, the antigen-binding fragment of the present disclosure is Fab, F(ab') ₂ , Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv) or peptide comprising CDRs, etc.

Fab is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity among the fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond. In some embodiments, the Fab of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure that specifically recognizes human CD93 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain. In addition, the Fab can be produced by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab.

F(ab') ₂ is an antibody fragment with a molecular weight of approximately 100,000, antigen-binding activity, and comprising two Fab regions linked at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin. In some embodiments, the F(ab') ₂ of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure that specifically recognizes human CD93 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with pepsin. Furthermore, the F(ab') ₂ can be produced by linking the Fab' regions described below with a thioether bond or a disulfide bond.

Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab') _2. Furthermore, the Fab' fragment can be produced by inserting a DNA encoding the Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.

The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The scFv of the present disclosure can be produced by the following steps: obtaining cDNAs encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human CD93 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing a DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the scFv.

Bispecific antibodies are antibody fragments in which scFv or Fab are dimerized, and possess bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different. Bispecific and multispecific antibodies are antibodies that can simultaneously bind to two or more antigens or antigenic determinants and contain scFv or Fab fragments.

dsFv is obtained by linking polypeptides in which one amino acid residue in each of VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue substituted with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).

The CDR-containing peptide is constructed by including one or more regions in the CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.

The CDR-containing peptides of the present disclosure can be produced by the following steps: constructing DNA encoding the CDRs of the VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human CD93 and binds to the amino acid sequence or three-dimensional structure of the extracellular region, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the peptide. The CDR-containing peptides can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.

The terms "CDR," "complementarity determining region," and "hypervariable region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al. (1997) JMB 273:927-937). -948) and ImMunoGenTics (IMGT) numbering rules (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered The CDRs in human VH are numbered as 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3) according to the Chothia convention; and the amino acid residues in VL are numbered as 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) according to the Kabat and Chothia conventions. The CDR regions of antibodies are determined using the program IMGT/DomainGap Align.

The term antibody "framework region", "skeleton region" or "FR", "FR region" as used herein refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about 10-8 M, such as less than about 10-9 M, 10-10 M, 10-11 M, or less.

The term "KD" or "Kd" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure bind to CD93 with a dissociation equilibrium constant (KD) of less than about ^10-7 M, such as less than about 10-8 ^M or 10-9 ^M , as measured, for example, in a BIACORE instrument using surface plasmon resonance (SPR) technology.

When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., CD93 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIAs (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Competitive inhibition is measured by measuring the amount of label that is bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically interfere with each other in binding. Additional details on methods for determining competitive binding are provided in the Examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence, after aligning the amino acid sequences and, if necessary, introducing gaps to achieve maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining amino acid sequence identity percentage, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, a vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, a vector is a viral vector, in which another DNA segment can be connected to a viral genome. Vectors disclosed herein can autonomously replicate in the host cell into which they have been introduced (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after introducing the host cell, thereby replicating (e.g., non-additional mammalian vectors) with the host genome.

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as those described in the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human CD93 or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the present invention utilize genetic engineering methods to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website at https://www.imgt.org/ by comparing the IMGT human antibody variable region germline gene database and MOE software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) and NSO cells.

The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred existing technology, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human CD93. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture medium secreting the antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

"Administer," "give," or "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "give," or "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," or "treat" also means treating, for example, a cell in vitro and ex vivo, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptom. Although the embodiments of the present disclosure (e.g., treatment methods or products) may not be effective in alleviating every symptom of the target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

"Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative replacements are set forth in the table "Exemplary Amino Acid Conservative Replacements" below.

Table 1. Exemplary conservative amino acid substitutions

"Effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease, and an effective amount also means an amount sufficient to allow or facilitate diagnosis. For preventive uses, beneficial or desired results include eliminating or reducing risk, alleviating severity, or delaying the onset of a disease, including biochemical, histological, and/or behavioral symptoms of an intermediate pathological phenotype presenting during the development of the disease, its complications, and the disease. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related diseases of the present invention or improving one or more symptoms of the disease, reducing the dose of other agents required for treating the disease, enhancing the efficacy of another agent, and/or delaying the progression of the target antigen-related disease of the present invention in the patient. The effective amount for a particular patient or veterinary subject can vary according to the following factors: for example, the disease to be treated, the patient's overall health, the method, route, and dosage of administration, and the severity of side effects. The effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

“Exogenous” refers to substances produced outside the body of an organism, cell, or human body, depending on the circumstances. “Endogenous” refers to substances produced inside the body of a cell, organism, or human body, depending on the circumstances.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Generally speaking, comparison is performed when two sequences are aligned to obtain the maximum percentage of homology. For example, comparison can be performed using the BLAST algorithm, where the algorithm parameters are selected to provide the maximum match between each sequence over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410; Gish, W. et al. (1993) Nature Genet. 3:266-272; Madden, T.L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those skilled in the art.

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that possess the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

As used herein, the term "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of specific portions of nucleic acids, RNA and/or DNA, are amplified as described, for example, in U.S. Patent No. 4,683,195. Generally, sequence information from the ends of the target region or beyond is required so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be identical to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). As used herein, PCR is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes using a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific portion of a nucleic acid.

The term "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically/pharmaceutically acceptable salts or prodrugs, together with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering an antibody or antigen-binding fragment. A carrier can be an antiadhesive, a binder, a coating, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, an antibacterial or antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

Furthermore, the present disclosure includes an agent for treating a disease associated with CD93-positive cells, the agent comprising the anti-CD93 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.

There is no limitation on the disease associated with CD93, as long as it is a disease associated with CD93. For example, the therapeutic response induced by the molecules of the present disclosure can be achieved by binding to human CD93 and then inhibiting the binding of CD93 to its ligand MMRN2 or CD93, or killing tumor cells that overexpress CD93, or increasing the infiltration of drugs or immune effector molecules or immune cells into tumor tissue. Therefore, when in preparations and formulations suitable for therapeutic applications, the molecules of the present disclosure are very useful for people who suffer from tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.

Furthermore, the present disclosure relates to methods for immunodetection or measurement of CD93, reagents for immunodetection or measurement of CD93, methods for immunodetection or measurement of cells expressing CD93, and diagnostic agents for diagnosing diseases associated with CD93-positive cells, comprising as an active ingredient the monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes human CD93 and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof.

In the present disclosure, the method for detecting or measuring the amount of CD93 may be any known method, including, for example, immunodetection or measurement methods.

Immunoassay or determination method is a method for detecting or determining the amount of antibody or antigen using a labeled antigen or antibody. Examples of immunoassay or determination methods include radiolabeled immunoantibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescent immunoassays, protein immunoblotting, physicochemical methods, etc.

The above-mentioned diseases associated with CD93-positive cells can be diagnosed by detecting or measuring cells expressing CD93 using the monoclonal antibodies or antibody fragments disclosed herein.

To detect cells expressing the polypeptide, known immunoassay methods can be used, preferably immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. In addition, fluorescent antibody staining using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present disclosure, there is no particular limitation on the living sample used for detecting or measuring CD93, as long as it has the possibility of containing cells expressing CD93, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

Depending on the desired diagnostic method, the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain reagents for performing an antigen-antibody reaction or reagents for detecting the reaction. Reagents for performing an antigen-antibody reaction include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assays, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the labeled second antibody.

The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are provided only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. Those skilled in the art may make various modifications and replacements to the present application without departing from the purpose and spirit of the present application.

Experimental methods in the examples disclosed herein that do not specify specific conditions generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Technology and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents without specific sources were purchased from conventional commercial sources.

Herein, antibodies are designated as SF02-C followed by a numerical designation, sometimes abbreviated as C followed by a numerical designation, representing the same light and heavy chain sequence pair. Humanized antibodies are designated by adding huC or HC to the prefix and a -number to the suffix to distinguish them. For the CD93 antibodies constructed and used in the Examples, unless otherwise stated, the VH and VL antibodies were grafted onto a HuIgG1 backbone.

In this article, the quantitative data obtained were plotted and statistically analyzed using GraphPad Prism software after being processed in Microsoft Excel. In the data graphs obtained from in vivo experiments, where applicable, the data used for graphing was the mean plus the sample standard deviation. The statistical method between groups at two or more consecutive time points used two-way analysis of variance. A p-value less than 0.05 was considered to be statistically different, a p-value less than 0.01 was considered to be a significant statistical difference, a p-value less than 0.001 was considered to be an extremely significant statistical difference, and a p-value greater than 0.05 was considered to be no difference. The Log-rank (Mantel-cox) test was used to compare the survival curve data.

Example 1: Sources of primary proteins and control antibodies used

In the examples herein, the main proteins used for animal immunization and detection were purchased from Saifu Xinsheng. The main proteins and product numbers are shown in Table 2 below. The amino acid positions of the human CD93 protein sequence refer to the sequence of the accession number mentioned above, the amino acid positions of human IGFBP7 and MMRN2 refer to the sequences of protein database accession numbers Q16270 and Q9H8L6, respectively, the amino acid positions of the mouse CD93 protein sequence refer to the sequence of protein database accession number NP_034870.1, the amino acid positions of the monkey CD93 protein sequence refer to the sequence of protein database accession number F7C9U4, and the amino acid positions of the protein sequence of the unrelated protein DDR1 refer to the sequence of protein database accession number Q08345.

Table 2. Commercial and structural information of major proteins

In addition to commercial antibodies (e.g., the isotype controls shown in Table 2), the VH and VL sequences of the 7F3 antibody (a fully human antibody) are derived from patent PCT/US2021/052446, and bevacizumab (hereinafter referred to as "Avastin") uses the light and heavy chain sequences published in Drugbank (shown in SEQ ID NO: 29 and 30, respectively). The antibody sequence was synthesized by codon reverse engineering to construct the required antibody IgG1 form. The expression and purification were performed using protein A according to conventional methods. The protein concentration was measured using a UV spectrophotometer (nanoDrop), and the protein size was detected using SDS-PAGE. ELISA binding or flow cytometry was used to determine whether the target protein met the requirements for further experiments.

The sequence of bevacizumab is as follows, and it was named avastin in this study.

>"Bevacizumab light chain"

>"Bevacizumab heavy chain"

Example 2: Mouse immunization, immune phage library construction, and antibody panning, screening, and sequence identification

The recombinant CD93-hFc protein in Table 2 of Example 1 was used as an antigen and mixed with Freund's adjuvant to prepare an oil emulsion, which was then used to immunize 6-8 week old Balb/C mice (purchased from Vital River). The immunization protocol was as follows: 100 μg/mouse, multi-point immunization, once every 2 weeks, for a total of 4 immunizations. When the serum titer was greater than 500,000, the mice were euthanized, fresh spleens were obtained, spleen RNA was extracted, and the first-strand cDNA was synthesized by reverse transcription. VH and VK fragments were amplified using mouse VH and VK specific primers, respectively. After gel recovery, VH and VK were spliced into scFv format using a universal linker, digested, and inserted into the phage display plasmid pCoMB3X. The phage-displayed mouse antibody scFv library was obtained by electroporation of TG1 engineered bacteria. After panning and screening with the coated antigen, phages with the two monoclonal antibodies (i.e., SF02-132 and SF02-164, also known as C132 and C164) with the best antigen binding affinity were obtained. These phages were purified and sequenced to obtain the VH and VL DNA sequences of these two monoclonal antibodies (as shown in Table 3).

Table 3. Obtained VH and VL nucleotide sequences

Furthermore, the amino acid sequences of VH and VL of the two monoclonal antibodies were obtained (see Table 4), and their CDR sequences were identified (see Table 5).

Table 4. VH and VL sequences of antibodies

Table 5. CDR sequences of antibodies

Example 3: Construction, expression and purification of intact antibodies

The VL and VH sequences in Table 2 above were amplified separately from the phage vectors. The 5' end of the amplification primer had a 12 bp homology arm. The amplified VL and VH were constructed into the eukaryotic expression vectors pTT5-L (recombinant expression plasmid containing the human kappa constant region) and pTT5-H1 (recombinant expression plasmid containing the human IgG1 heavy chain constant region) or into pTT5-mL (recombinant expression plasmid containing the mouse kappa constant region) and pTT5-M1 (recombinant expression plasmid containing the mouse IgG1 heavy chain constant region) through the homology arm. The plasmids were extracted and the corresponding light and heavy chain plasmids were co-transfected into 293F cells. The transfected cells were cultured at 37°C, 5% _CO2 , and 120 rpm for 5 days. The culture medium was then collected and purified using a Protein A column. Protein A purification procedures were as follows: the column was equilibrated with PBS (50 mM PB, 0.15 M NaCl) at pH 7.2 at a flow rate of 150 cm/h over 6 column volumes. The 293F cell culture supernatant was adjusted to pH 7.2 at a flow rate of 150 cm/h. The column was then re-equilibrated with equilibration buffer at a flow rate of 150 cm/h over 6 column volumes. Elution was then performed in a single step with 50 mM citric acid-sodium citrate at pH 3.5 over 8 column volumes, and the eluted peak was collected. Through this procedure, purified murine and human-mouse chimeric forms of the two antibodies were obtained.

Example 4: Determination of Antibody Antigen Binding Ability by ELISA

96-well plates were coated with CD93-EGF2-his or CD93-his antigen (as shown in Table 2 of Example 1). Specifically, the antigen was diluted to 1 μg/ml with PBS, and 100 μl of the dilution was added to the 96-well plate at 4°C overnight. The plates were washed three times with TBST in a plate washer. Blocked with 5% milk at 37°C for 1-2 hours, the plates were washed three times with TBST in a plate washer, and the test antibody samples (including the human-mouse chimeric antibodies C132 and C164 obtained in Example 3 and control antibodies, with isotype antibodies as negative controls and 7F3 antibodies as positive controls) were diluted to 10 μg/ml. 150 μl was added to the first row of wells, diluted three-fold, and incubated at 37°C for 1 hour. The plates were then washed six times with TBST in a plate washer. Subsequently, according to the antibody instructions, anti-human FC-AP secondary antibody (Southern Biotech, 9040-04) was diluted 1:2500, mixed thoroughly, and added to each well at 100 μl. The cells were incubated at 37°C for 1 hour, and washed six times using a TBST plate washer. pNPP (50 μl/w) was added, and the cells were incubated at 37°C for 10 minutes. The reaction was terminated with 3M NaOH. OD410 readings were taken. The concentration-dependence curve of antigen binding for the example antibody of this application is shown in Figure 1A. Due to the low homology between human and mouse CD93 molecules, the antibody did not cross-bind with mouse CD93; however, it did cross-bind with monkey rhesus-CD93-mFc (Table 2) (Figure 1B). Furthermore, the antibody's ability to bind to truncated CD93 was tested, and binding to CD93-EGF2-his (Table 2) was found, indicating that the antibody binds to the EGF-like region of CD93 (see Figure 1C). At the same time, in order to detect the specificity of the obtained antibodies for binding to CD93, we coated the irrelevant antigen DDR1-his (Table 2) antigen, and incubated the antibody with the antigen at 30 μg/mL and 3 μg/mL, respectively. The signal of the bound antibody was detected using an anti-human secondary antibody. The results are shown in Figure 1D (wherein the PC group is a DDR1-positive antibody group, and the antibody was purchased from Sino Biological, catalog number 10730-MM05T). Figure 1D shows that the two human-mouse chimeric antibodies C132 and C164 of the present application have no binding ability to non-CD93 antigens.

The above results indicate that the human-mouse chimeric antibodies C132 and C164 of the present application both have the ability to specifically bind to CD93.

Example 5: ELISA assay to detect the ability of the present antibody to block the binding of CD93 to IGFBP7

In this example, ELISA was used to detect whether the antibodies C132 and C164 of the present application could block the binding of CD93 to IGFBP7.

First, 100 μl of 1 μg/mL IGFBP7-mFc (as shown in Table 2 of Example 1) was coated onto an ELISA plate and blocked overnight at 4°C. CD93-his-Biotin (Table 2) and different concentrations of the two human-mouse chimeric antibodies constructed in Example 3 were mixed and added to the ELISA plate. The 7F3 group served as a positive control, and the Isotype group served as a negative control. After incubation at 37°C for 1 hour, the plate was washed with PBST, and detection was performed with SA-HRP (horseradish peroxidase-labeled streptavidin, Genscript, M00091). After incubation at 37°C for 1 hour, the plate was washed with PBST, and the color reaction was developed with TMB. The color reaction was terminated with 2M sulfuric acid, and the plate was read at 450 nm using a microplate reader. The blocking ratio was calculated using the formula (1-OD _{experimental group} /OD _{control group} ) * 100%. A higher blocking ratio indicates a better blocking effect. The results are shown in Figure 2A. Figure 2A shows the concentration-dependent blocking effect of C132 and C164 antibodies on the binding of CD93 to IGFBP7. The results indicate that the human-mouse chimeric antibodies C132 and C164 of the present application can effectively block the binding of CD93 to its ligand IGFBP7.

Example 6: ELISA assay to detect the ability of the present antibody to block the binding of CD93 to MMRN2

In this example, ELISA was used to detect whether the antibodies C132 and C164 of the present application could block the binding of CD93 to MMRN2.

First, 100 μl of 1 μg/mL his-SUMO-MMRN2 (Table 2) was coated onto an ELISA plate and blocked overnight at 4°C. 1 μg/mL CD93-his-Biotin (Table 2) was mixed with different concentrations of the two human-mouse chimeric antibodies constructed in Example 3 and added to the ELISA plate. The 7F3-hIgG1 group was set as a positive control, and the Isotype group was set as a negative control. After incubation at 37°C for 1 hour, the plate was washed with PBST, and SA-HRP (horseradish peroxidase-labeled streptavidin) was added for detection. After incubation at 37°C for 1 hour, the plate was washed with PBST, and the color reaction was developed with TMB. The color reaction was terminated with 2M sulfuric acid, and the plate was read at 450 nm using a microplate reader. The blocking ratio was calculated according to the formula (1-OD _{experimental group} /OD _{control group} ) * 100%. A higher blocking ratio indicates a better blocking effect. The results are shown in Figure 2B. The results in Figure 2B show the concentration-dependent blocking effects of C132 and C164 antibodies on the binding of CD93 to MMRN2. This result indicates that the human-mouse chimeric antibodies C132 and C164 of the present application can effectively block the binding of CD93 to its ligand MMRN2.

Example 7: Construction of CD93 overexpressing cell line

In this example, a stable transposon cell line pool was constructed using a transposon system. Construction was commissioned by Beijing Renyuan Xinsheng Biotechnology Co., Ltd. Briefly, PCR was used to add new restriction enzyme sites, AsisI/claI, to both ends of the human CD93 expression sequence. This was then ligated into a prefabricated CMV-PGK-Puro dual-expression plasmid framework, placing the CD93 sequence under the regulation of the PGK promoter. Junction PCR was then performed to identify the constructed donor plasmid. The Sleeping Beauty transposase plasmid and the constructed CD93 transposase donor plasmid were then introduced into B16F10 (mouse melanoma) cells using an ECM830 cell electroporator. After electroporation, cells were seeded into 6-well culture dishes at a certain dilution ratio and cultured in a 37°C incubator with complete medium containing 1.5 μg/mL puromycin. After the cells in the control blank plasmid group died, the transfected plasmid group was cultured and clustered clones were formed. Subcloning and subculture were performed. After 4-5 days of growth, the wells with a confluence of 70% were selected for subculture. At the same time, some cells were collected to extract DNA, and the gene integration of the mixed cell pool was identified by PCR. The constructed cells were passaged, and the cell pool was subjected to flow cytometry analysis to analyze the ability of the cells to bind to IGFBP7-hFc. Isotype antibodies were used as negative controls, and untransfected B16F10 cell lines were set as negative controls for cells. FITC-488-labeled anti-human antibodies were used as secondary antibodies (Southern biotech, 2045-02). The cells were analyzed using a BD Accuri C6 Plus flow cytometer.

The results showed that the ability of the constructed B16F10-CD93 cell line to bind IGFBP-7 increased with increasing protein concentration, as evidenced by both the cell positivity rate (Figure 3A) and MFI values (Figure 3B). No binding was detected in the B16F10 control cells, and no binding was detected with the isotype control antibody on either cell line. Furthermore, the binding ability of SNU-1 cells (human gastric cancer cells), a cell line with high CD93 expression, to IGFBP-7-hFc (Table 2) was also tested. The results showed that IGFBP-7 also bound to SNU-1 cells in a concentration-dependent manner, with both the cell positivity rate and MFI values (see Figures 3C and D), whereas no binding was detected with the isotype control antibody. These results demonstrate that we have successfully constructed a cell line overexpressing CD93 and that CD93 expressed on the cell membrane can bind to its ligand, IGFBP7.

Example 8: Flow cytometry to detect the binding ability of antibodies to cells

Cell lines B16F10-CD93, B16F10 cells (no CD93 expression) stably transfected with a full-length human CD93 expression vector, or test cell lines (Table 5) were plated and grown to a confluence of 80% to 90%. The cells were digested with a citrate solution at 37°C for approximately 5 to 10 minutes. The cells were resuspended in PBS and counted, centrifuged at 900g for 5 minutes at 4°C, and resuspended in a solution containing BSA at a concentration of 5*10^6 cells/ml. 200ng of the human-mouse chimeric antibody C164 or C132 obtained in Example 3 was incubated with 100ul of 5× ¹⁰⁵ cells for 30 minutes at room temperature. An isotype antibody group was provided as a negative control. The cells were washed three times with 1% BSA 10mM PBS and resuspended with 100ul of FITC secondary antibody diluted 200 times in 1% BSA 10mM PBS at 4°C for 30 minutes. The cells were washed three times with 10 mM PBS containing 1% BSA, and then resuspended for detection.

The chimeric antibodies C164 and C132 both showed some binding ability to the CD93-overexpressing cell line B16F10-CD93 or CD93 ⁺ tumor cell lines, such as U937 (human histiocytic lymphoma) and SNU-1, demonstrating a high cellular positivity rate. However, the positivity rate was very low for B16F10 or ^CD93- cell lines (such as RPMI-8826 and HeLa). The cellular positivity rates for each antibody are shown in Table 6.

Table 6. Antibody positive rate (%)

Furthermore, antibody C132 was diluted in a gradient concentration to test its binding ability to the tumor cell line SNU-1. The flow cytometry results are shown in Figure 4 . As can be seen in Figure 4 , both the cell positivity rate ( Figure 4A ) and the MFI value ( Figure 4B ) showed a concentration-dependent effect of the antibody, while the isotype control antibodies were all negative.

The above results show that the two antibodies of the present application can maintain high binding ability with CD93 high-expressing cell lines of different tumor types, but show no binding ability with low-expressing cell lines. At the same time, these antibodies can specifically bind to CD93 high-expressing lines and have the potential to be used in the treatment of tumors.

Example 9: Acquisition of humanized antibodies of the present invention

When mouse antibodies are used to treat human diseases, xenoimmunogenicity can reduce drug safety. To adapt the drug molecules for human drug development, this example humanized the mouse antibodies C132 and C164 screened above and expressed them.

Specifically, the sequences of the heavy and light chain variable regions (VH and VL) were compared with human antibody sequences in the Protein Data Bank (PDB) to establish a homology model. The framework regions of the four humanized VH sequences of C132 and C164 each adopted the framework region of the IGHV1-69-2*01 genotype, the framework region of the humanized VL sequence of huC132K1 adopted the IGKV1-9*03 framework, and the framework region of the humanized VL sequence of huC132K2, HuC164-K1, and HuC164-K2 adopted the IGKV1-39*01 framework. The CDRs of the heavy and light chains of the mouse mAb were transplanted into the human framework region according to the Kabat number scheme. At the same time, to maximize the binding activity of the antibody, individual hot spots in the human framework were back mutated. The VH and VL sequences of ScFv from the same source were mutated, recombined and renamed, as shown in Table 7. The humanized heavy and light chain variable region sequences are shown in Table 8.

Table 7. Humanized antibodies and their light and heavy chain compositions

The sequences of the humanized antibody light and heavy chains are shown in Table 8 below.

Table 8. Humanized variable region sequences

The humanized VH and VL were fused to the constant regions of the human IgG1 heavy chain and κ light chain, respectively, to construct antibody light and heavy chain expression vectors. After plasmid extraction, transient transfection was performed in 293F cells, and the activity of the purified humanized antibody was analyzed using ELISA combined with flow cytometry analysis, as described below.

Example 10: Antibody Biological Activity Test - HUVEC Tube Formation Inhibition

HUVEC cells (GPC0114, source: China Center for Type Culture Collection) at passage number P11 or lower were cultured in HUVEC-specific culture medium (CM-0122, Wuhan Punosai) supplemented with FBS (164210-50) and 1% streptomycin (PB180120). When the cells reached 70-80% confluence, they were starved for 3-6 hours with antibiotic-free Ham's F-12K medium (PM150910, Wuhan Punosai). HUVECs were counted and seeded at ¹⁰ cells/well in 96-well plates coated with Matrigel. Appropriate concentrations of test antibodies or control antibodies were added as needed. Microscopic observation and photography were performed at the indicated times.

In this example, the biological activities of the two human-mouse chimeric antibody molecules (i.e., chimeric antibody molecules of mouse VH-VL and human Fc) C132 and C164 prepared in Example 3 and the 12 humanized antibodies described in Example 9 were tested. The results are shown in Figures 5 and 6, respectively.

In the test of human-mouse chimeric antibody molecules, isotype antibodies were used as negative controls. In the humanized antibody test, we used the expression product of the bevacizumab sequence at the same concentration as a positive control for inhibiting angiogenesis. At the same time, the human-mouse chimeric antibody form of C164 was used as a control for the parental antibody, and a group without antibody addition (adding an equal volume of PBS) was set as a blank control.

The results showed that at a concentration of 20ug/mL, the human-mouse chimeric antibodies C132 and C164 could be observed to weaken the HUVEC tube-forming ability within the indicated time (Figure 5), indicating that the human-mouse chimeric antibodies C132 and C164 had a relatively significant inhibitory effect on the tube-forming ability of HUVEC at different co-incubation time points; and, as shown in Figure 6, humanized antibodies with different light and heavy chain combinations also showed significant in vitro tube-forming inhibition, among which HuC132-3, HuC132-1, etc. were particularly significant after being incubated with HUVEC for 3h (Figure 6A), 8h (Figure 6B), and 20h (Figure 6C). Further, after incubating HuC132-3 and HuC164-2 antibodies with HUVEC for 4h at different concentration gradients, a significant inhibitory effect on cell tube formation was observed (Figure 6D). The results of this example indicate that the antibody molecules of the present application have a significant inhibitory effect on angiogenesis.

Example 11: Analysis of the difference in gene expression between HUVEC cells cultured under subculture and tube formation conditions using CD93 antibodies

In this example, the effects of CD93 antibody on the expression levels of three cancer-related genes (ALDH1A3, BNIP3, MAFA) in HUVEC cells under subculture and tube formation conditions were detected.

Specifically, humanized anti-CD93 antibodies HuC132-3 and HuC164-2 were incubated with passaged HUVEC cells and HUVECs in the tube-forming conditions of Example 10 for 24 hours, and then mRNA was extracted and reverse transcribed into cDNA. The expression levels of three cancer-related genes, ALDH1A3, BNIP3, and MAFA, were detected by qPCR. The mRNA extraction protocol was based on the RNA-easy Isolation Reagen kit (R701-1, Norvegian). The concentration and purity of the extracted RNA were determined using a UV spectrophotometer (thermo Nano drop), and reverse transcription was performed according to the kit protocol ( II Q RT SuperMix qPCR, +g DNA wiper, R233-01, Novozymes). Then, according to the standard protocol, 20 μl of reaction solution (reaction system see Table 9) was prepared in a 250 μl PCR tube using qPCR reagents (ChamQ Universal SYBR qPCR Master Mix, Q711-02, Novozymes). The corresponding primers (primer sequences see Table 10) were added, with three replicates set per group. Amplification was performed in a real-time fluorescence PCR instrument according to the reaction program shown in Table 11, and data analysis was performed according to standard procedures.

The results showed that under the passage state (Figure 7A) and tube formation conditions (Figure 7B), HuC132-3 and HuC164-2 had different effects on the three cancer-promoting genes. Under tube formation conditions, both antibody molecules showed the effect of reducing the expression of the three genes, indicating that the humanized antibody molecules of the present application may affect the formation of new blood vessels by affecting the expression of cancer-related genes.

Table 9. Solutions added to the reaction mixture

Table 10. Primer sequences

Table 11. qRT-PCR reaction program

Example 12: Expression and characterization of humanized antibodies in eukaryotic systems

In order to test the structural stability of humanized antibodies and whether they meet the properties that can be used in future drug production, we preliminarily measured the protein transient expression efficiency, antibody Tm and Tagg values of a series of antibodies.

Briefly, the prepared plasmids containing the antibody's light and heavy chains were co-transfected into suspended 293 cells using a transfection reagent. The cell culture volume was no higher than 10 mL. After 72 hours of incubation, the supernatant was harvested and purified using an AKTA Purifier 100 protein A affinity chromatography column. The protein concentration was measured using a nano-drop assay, and protein purity was analyzed using SDS-PAGE. The results showed that the humanized antibody molecule of this application expressed at a level of no less than 90 mg/L under transient expression conditions, with a purity exceeding 98%, meeting the expression efficiency requirements for future drug development.

The binding ability of humanized antibodies HuC164-1, 2, 5, and 6 to tumor cell lines expressing CD93 was detected by flow cytometry, and the results are shown in Figure 8. Figure 8 shows that the humanized antibodies of the present application retain the ability to bind to tumor cell lines expressing CD93, and their binding positivity (Figure 8A) and MFI value (Figure 8B) demonstrate that this binding has a good concentration-dependent effect.

The blocking ability of humanized antibodies HuC132-1, 2, 3, 4, 5, 6, 7, and 8 against the ligand IGFBP7 was also tested by flow cytometry, and the results are shown in FIG9 ; FIG9 shows that the humanized antibodies of the present application also retain the blocking ability against the ligand IGFBP7.

In order to test the affinity of the antibody to the antigen, the affinity constants of the humanized antibodies HuC132-3 and HC164-2 to the antigen were respectively detected using the Octet instrument. According to the Octet detection instrument Octet RED96e, CD93-his was immobilized on the NI-NTA sensor, and the antibody was diluted 2-fold from a concentration of 50ug/mL to a minimum concentration of 1.56ug/mL, and the measurement was performed according to the standard procedure. The data obtained showed that the affinity constants were all less than ^10-12 M. The binding and dissociation curves of the antigen and the antibody are shown in Figure 10, where Figure A is the result of HuC132-3 and Figure B is the result of HuC164-2. Figure 10 shows that the humanized antibodies of the present application have excellent affinity for the antigen.

The characterization and identification of antibodies was performed using the Uncle automatic analyzer from Unchained lab. According to the instrument manual, 9ul of antibody sample was added to the Uni tube, with 2 replicates for each sample. According to the standard procedure, the heating temperature was set to 0.5 degrees, starting from 25 degrees and ending at 95 degrees, and the Tm and Tagg values were tested to analyze the denaturation and aggregation process of the protein during the heating process. The dynamic light scattering of the samples was tested at 25 degrees and 95 degrees respectively to obtain the particle size and particle size distribution of the antibody molecules in the antibody samples. Table 12 shows the PDI and particle size of the humanized antibodies HuC132-3, HC164-2, and HC164-6 at 25 degrees. Figure 11A shows the particle size distribution of the antibody samples at 25 degrees. Figure 11B shows the denaturation curves, fluorescence, and SLS266 values of the antibody samples (2 replicates for each sample).

Table 12. Antibody characterization data

The above results indicate that the humanized antibody molecule of the present application has excellent stability.

Example 13: CD93 monoclonal antibody effectively inhibits tumor growth in mice

(1) Inhibition of mouse melanoma growth by fully mouse antibodies

First, we tested the tumorigenicity of the B16F10-CD93 overexpressing cell line in C57BL/6J mice. Briefly, 1×10 ⁶ freshly cultured B16F10-CD93 overexpressing cells were subcutaneously implanted into the dorsal region of the mice. Five to seven healthy C57BL/6J mice, aged 6-8 weeks, were used per group. On the day of tumor inoculation, mice were administered a 15 mg/kg dose via the tail vein in a volume of no more than 100 μl. The experimental groups received C132 and C164, fully murine antibodies (constructed as described in Example 3), and 7F3-mIgG1, a murine-human chimeric antibody (i.e., the VH and LV of the humanized 7F3 molecule were grafted onto a mouse IgG1 backbone). The model control group received saline. Dosing was twice weekly for six consecutive doses. Tumor growth was measured twice weekly, and tumor volume was calculated. To reduce the immune response of mice to human CD93 expressed in the tumor cell line, cyclosporine A was administered twice, on days 7 and 10 after tumor cell implantation. Animals were euthanized in accordance with animal welfare regulations when tumors grew to a volume exceeding 2500 ^mm3 or a diameter greater than 20 mm.

Figures 12A and 12B show the mouse tumor volume detection results and the mouse weight change curve, respectively. The results show that the antibody molecules C164 and C132 of the present application inhibited the formation of mouse tumors to a certain extent. Compared with the model control group, the C132 administration group had a statistically significant difference (p < 0.001), and the C164 administration group had a significant difference (p = 0.0021). Although the difference did not reach a statistically significant difference, the tumor inhibition effect of C132 was better than that of the positive control antibody 7F3 (Figure 12A); the body weight of mice in each administration group was not significantly different from that of the vehicle control group (Figure 12B), indicating that the antibody molecule had a small effect on the weight of mice. In addition, we defined the standard of mouse death as a mouse tumor volume greater than 1500 mm ³ , and thus drew a mouse survival curve, as shown in Figure 12C. It can be seen from the survival curve that the C132 antibody effectively prolonged the survival of mice (p < 0.05).

(2) Inhibition of humanized monoclonal antibodies on the growth of human lymphoma

First, purchased NPG immunodeficient mice (Beijing Weitongda Biotechnology Co., Ltd.) were used for immune reconstitution. When PBMC was inoculated, the mice were 5-6 weeks old and weighed about 20 g.

Frozen hPBMCs were gently and rapidly thawed in a 37°C water bath. 4 mL of complete culture medium (89% RPMI-1640 + 10% FBS + 1% penicillin-streptomycin) was added and mixed thoroughly. The cells were centrifuged (1000 rpm/min for 5 min). The supernatant was discarded and the cells were resuspended in an appropriate amount of complete culture medium (89% RPMI-1640 + 10% FBS + 1% penicillin-streptomycin). The cells were counted and adjusted to a concentration of 2.5 × 10 ⁷ cells/mL for transfusion. Human PBMCs were transfused via the tail vein at a concentration of 5 × 10 ⁶ cells/animal in 0.2 mL/animal. The ratios of mCD45 ⁺ and hCD45 ⁺ cells in the peripheral blood of mice were regularly tested using anti-mouse CD45 antibodies (BioLegend, 103114) and anti-human CD45 antibodies (BioLegend, 304028), respectively. The test results are shown in Figures 13D&E. The results show that the proportion of mCD45 ⁺ T cells decreased over time, while the proportion of hCD45 ⁺ T cells increased over time. This result is consistent with the trend of changes in peripheral blood cell composition in immune-reconstituted animals and suggests that the experimental system is stable and reliable.

One tube of frozen U937 cells was thawed in a 37°C water bath and centrifuged at 1000 rpm/min for 5 minutes. The supernatant was discarded and the cells were resuspended in an appropriate amount of complete culture medium (89% RPMI-1640 + 10% FBS + 1% penicillin-streptomycin) and cultured in a flask at 37°C in a 5% CO2 incubator. When the cells reached 60-80% confluency, they were passaged according to their growth characteristics until each cell line expanded to a minimum of 1.2 × 108 cells. On day 5 (M5) of PBMC transfusion, U937 cells in the logarithmic growth phase were harvested and centrifuged at 1000 rpm/min for 5 minutes. The supernatant was discarded and the cells were resuspended in PBS to a concentration of 2 × ¹⁰⁷ cells/mL. Tumor cells were subcutaneously inoculated into the right side of the back of the animal, near the axilla, at a dose of 2 × ¹⁰⁶ cells/animal in 0.1 mL/animal. After inoculation, the growth of tumor cells in vivo was observed and measured twice a week, and the tumor volume was calculated.

According to the immune reconstitution situation, animals with a tumor volume of about 100 mm ³ and relatively uniform were screened for enrollment and randomly divided into 3 groups according to the tumor volume: a model control group, a 7F3-hIgG1 group, and a HuC132-3 group, with 10 mice in each group. Dosing began on the day of grouping, with a dose of 20 mg/kg and a dosing volume of no more than 100 ul/mouse/time, 2 times/week, for 5 consecutive doses. After administration, the growth of tumor cells in the body was observed and measured twice a week, and the tumor volume was calculated. At the same time, the positive rate of immune cells in the immune reconstructed mice was detected on days 4, 10, and 17 after administration. The experimental results showed that compared with the model control group, the tumor volume of the mice in the HuC132-3 administration group was significantly reduced, and the difference was statistically significant (p < 0.001), and the tumor inhibition effect was better than that of the control antibody 7F3 (see Figure 13A). Calculation of the tumor volume ratio between the test and control groups showed that the T/C value of the HuC132-3 group dropped below 10% by the end of the experiment (see Figure 13B). A mouse death criterion of tumor volume greater than 1500 ^mm3 was defined as the standard for mouse death. Survival curves were plotted, as shown in Figure 13C. The huC132-3 antibody effectively prolonged mouse survival. At the same time, fluorescently labeled antibodies, including anti-human CD3 antibody (Biolegend, 300406), anti-human CD4 antibody (BioLegend, 300514), and anti-human CD8 antibody (Biolegend, 344706), were used to perform immune cell analysis on the peripheral blood of mice using a Beckman flow cytometer (Beckman, DxFLEX). The results showed that there was no difference in the positive rates of the above-mentioned various immune cell markers among the mice groups, and there was no statistically significant difference (p>0.05) (Figure 13F-H), indicating that the proportions of hCD3 ⁺ , hCD4 ⁺ , and hCD8 ⁺ T cells in the blood of animals in the test substance treatment group were comparable to those in the control group, with no statistical difference, indicating that our immune reconstitution was successful and that the administration of HuC132-3 played a tumor-suppressing role.

The tumor tissues of the mice harvested after the experiment were sealed, fixed, dewaxed, and antigen retrieval was performed. After blocking with the blocking solution, the samples were incubated according to the dilution multiple of the instructions for hCD8 antibody reagent (Abcam, ab237710), rinsed, and then incubated with HRP-labeled goat anti-rabbit secondary antibody (Abcam, ab205718). After rinsing, the samples were photographed under a microscope for analysis. The staining results are shown in the upper figure of Figure 13I, which shows that CD8 ⁺ T cells are widely distributed in the tumor tissues of the Hu132-3-treated mice, and are stronger than those in the 7F3-treated group and the model control group. The tumor tissues of the model control group and the huC132-3 group animals at the end of the experiment were double-stained with hCD8 and CD31 fluorescent immunostaining, where the CD31 antibody was labeled with FITC488 and the hCD8 antibody was labeled with Cy3.5. The results are shown in the lower figure of Figure 13I, which also reproduces the widespread distribution of CD8 ⁺ T cells in the tumor tissues. Both staining results indicated that the huC132-3 antibody may inhibit tumor growth by enhancing the efficiency of CD8 ⁺ T cells infiltrating into tumors.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Industrial Applicability

The anti-CD93 antibodies or antigen-binding fragments thereof provided herein can efficiently and specifically bind to CD93 molecules, and by binding to CD93 molecules, can effectively block their molecular functions, thereby effectively inhibiting tumor growth. In particular, the humanized anti-CD93 antibodies provided herein not only have a strong binding ability to tumor cells expressing CD93, and have a good concentration-dependent effect, but can also effectively block the binding of CD93 to its ligands, and can inhibit HUVEC tube formation and inhibit tumor growth, thereby being used for the treatment of CD93-related diseases (such as vascular proliferative diseases and cancer). Therefore, the anti-CD93 antibodies or antigen-binding fragments thereof of the present application have excellent industrial prospects.

Claims (23)

An anti-CD93 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively have three HCDRs and three LCDRs selected from any one of the following heavy chain variable region and light chain variable region combinations: (1) the heavy chain variable region shown in SEQ ID NO: 11, and the light chain variable region shown in SEQ ID NO: 12; (2) The heavy chain variable region shown in SEQ ID NO: 13, and the light chain variable region shown in SEQ ID NO: 14. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 4, SAS, and SEQ ID NO: 5, respectively; Alternatively, the heavy chain variable region comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:9, LAS and SEQ ID NO:10, respectively. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody is a murine antibody, a single domain antibody, a chimeric antibody, a human antibody, or a humanized antibody; And/or, the antigen-binding fragment is Fab, Fab', F(ab') ₂ , Fv or a complementarity determining region fragment. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody is a murine antibody or a chimeric antibody, wherein: The amino acid sequence of the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 11. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 12. Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 12; Alternatively, the amino acid sequence of the heavy chain variable region of the antibody includes the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region of the antibody includes the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14, preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 14. The anti-CD93 antibody or antigen-binding fragment thereof according to claim 4, characterized in that: When the antibody is a murine antibody, the murine antibody further comprises a murine heavy chain constant region and a murine light chain constant region, the murine heavy chain constant region is selected from one of murine IgG1, IgG2a, IgG2b or IgG3 constant regions, and the murine light chain constant region is a murine Ck type constant region; When the antibody is a chimeric antibody, the chimeric antibody further comprises a humanized antibody constant region. A humanized anti-CD93 antibody or antigen-binding fragment thereof, which is constructed based on the anti-CD93 antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 using CDRs grafting technology and/or CDR region mutation design. The humanized anti-CD93 antibody or antigen-binding fragment thereof according to claim 6, characterized in that the humanized anti-CD93 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region selected from any one of the following: (1) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; (2) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; (3) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; (4) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; (5) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; (6) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; (7) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; (8) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; (9) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 21, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; (10) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; (11) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 21, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24; (12) The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 22, and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24. The humanized anti-CD93 antibody or antigen-binding fragment thereof according to claim 7, characterized in that the heavy chain of the humanized anti-CD93 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. An ADC molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. A bispecific or multispecific antibody molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. A polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. The polynucleotide according to claim 12, wherein the polynucleotide is a polynucleotide group, and the polynucleotide group comprises: A nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 11 and a nucleotide sequence encoding the light chain variable region shown in SEQ ID NO: 12; Alternatively, a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:13 and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:14. The polynucleotide according to claim 13, wherein the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 11 comprises the DNA sequence shown in SEQ ID NO: 25 or the RNA sequence corresponding thereto, and the nucleotide sequence encoding the light chain variable region shown in SEQ ID NO: 12 comprises the DNA sequence shown in SEQ ID NO: 26 or the RNA sequence corresponding thereto; Alternatively, the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO:13 includes the DNA sequence as shown in SEQ ID NO:27 or the RNA sequence corresponding thereto, and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:14 includes the DNA sequence as shown in SEQ ID NO:28 or the RNA sequence corresponding thereto. A nucleic acid construct comprising the polynucleotide according to any one of claims 12 to 14, and, optionally, at least one expression control element operably linked to the polynucleotide. A recombinant vector comprising the polynucleotide according to any one of claims 12 to 14, or the nucleic acid construct according to claim 15. A transformed host cell, wherein the polynucleotide according to any one of claims 12 to 14, the nucleic acid construct according to claim 15 or the recombinant vector according to claim 16 is transformed. The transformed host cell according to claim 17, wherein the host cell is a bacterial, yeast or mammalian cell; Optionally, the bacteria is Escherichia coli; Optionally, the yeast is Pichia pastoris; Optionally, the mammalian cells are Chinese hamster ovary cells or human embryonic kidney 293 cells. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the ADC molecule according to claim 9, the bispecific or multispecific antibody molecule according to claim 10, the fusion protein according to claim 11, the polynucleotide according to any one of claims 12 to 14, the nucleic acid construct according to claim 15, the recombinant vector according to claim 16 and/or the transformed host cell according to claim 17 or 18, and a pharmaceutically acceptable excipient, diluent or carrier. A reagent or kit for detecting the presence or expression level of a CD93 molecule, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and/or the transformed host cell according to claim 17 or 18. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the method comprising: allowing the transformed host cell according to claim 17 or 18 to express the antibody or antigen-binding fragment thereof 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 culture of the host cell. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the ADC molecule according to claim 9, the bispecific or multispecific antibody molecule according to claim 10, the fusion protein according to claim 11, the polynucleotide according to any one of claims 12 to 14, the nucleic acid construct according to claim 15, the recombinant vector according to claim 16, the transformed host cell according to claim 17 or 18, and/or the pharmaceutical composition according to claim 19 in the preparation of a medicament for preventing and/or treating a CD93-mediated disease, disorder or condition. The use according to claim 22, characterized in that the disease is a tumor and/or an angiogenic disease; and/or the treatment is to alleviate, relieve, improve or inhibit the symptoms or progression of the disease, disorder or condition; Preferably, the tumor is breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, hematological tumor, lymphoma, glioblastoma and/or melanoma; Preferably, the angiogenic proliferative disease is selected from the group consisting of neovascular eye disease, psoriasis characterized by abnormal angiogenesis, psoriasis, rheumatoid arthritis or obesity.