CN117866092A

CN117866092A - Anti-human CD73 antibody or antigen binding fragment thereof and application thereof

Info

Publication number: CN117866092A
Application number: CN202310659520.2A
Authority: CN
Inventors: 王双; 焦莎莎; 王荣娟; 张畅; 杨莹莹; 张锦超
Original assignee: Beijing Konuoxincheng Technology Co ltd
Current assignee: Beijing Konuoxincheng Technology Co ltd
Priority date: 2022-06-06
Filing date: 2023-06-06
Publication date: 2024-04-12

Abstract

The invention discloses an anti-human CD73 antibody, an antigen binding fragment and application thereof. The embodiment of the invention provides a series of anti-CD 73 functional antibodies, the binding epitope is positioned on the N-terminal domain of CD73, the binding epitope can specifically bind to the CD73 on the cell surface and inhibit the enzyme activity of the CD73, and the CD73 can be consumed through internalization, so that the enzyme activity is strong, the barb phenomenon is weak, and the clinical application value is wide.

Description

Anti-human CD73 antibody or antigen binding fragment thereof and application thereof

Technical Field

The present invention relates to the field of medicine, in particular to an anti-human CD73 antibody or an antigen-binding fragment thereof and application thereof, and more particularly to an anti-human CD73 antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody, a vector and a cell expressing the antibody, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and application of the antibody in preparing medicines.

Background

CD73, also known as extracellular-5 '-Nucleotidase (Ecto-5' -Nucleotidase), is a cell surface encoded by the NT5E gene and is the primary enzyme that catalyzes the formation of extracellular adenosine by AMP. Human CD73 precursor protein consists of 574 amino acid residues. The amino acid residue sequence at the 27 th to 549 th positions is mature CD73, 1 subunit with the molecular weight of about 70kDa is formed, a homodimer with biological activity is formed by the two subunits, and the subunits are connected through non-covalent bonds. CD73 comprises two domains of IgV and IgC that together form an open and a closed structure, the conversion of which is the structural basis for binding and catalyzing substrates, generating enzymatic activity.

The tumor microenvironment contains rich immunosuppressive molecules of adenosine, and the adenosine has inhibitory effects on various immune cells, especially on T cell proliferation, cytotoxicity and cytokine production, so that Antigen Presenting Cells (APC) are inhibited, proliferation of Treg cells is promoted, immunocompetence is inhibited, and MDSC and macrophage M2 polarization are stimulated. In addition, adenosine induces immunosuppressive cells such as Tregs and MDSCs, and in addition to these immunosuppressive functions, adenosine promotes proliferation, angiogenesis and metastasis of cancer cells. CD73 is a metabolic immune checkpoint that coordinates a critical homeostatic balance of extracellular adenosine levels. Adenosine production relies on the degradation of ATP by CD39 to produce Adenosine Monophosphate (AMP), then CD73 converts AMP to immunodepressed adenosine, blocks anti-tumor immune monitoring at T and NK cell, dendritic Cell (DC), myeloid Derived Suppressor Cell (MDSC) and Tumor Associated Macrophage (TAM) levels, and CD73 catalysis is the primary pathway for adenosine production.

Cancer is one of the diseases that currently causes the highest mortality rate in humans. CD73 is widely expressed in various tumor cells, and its expression and catalytic adenosine-forming activity are closely related to invasion and metastasis of tumors. Recent clinical and commercial success of anti-cancer anti-CD 73 antibodies has led to great interest in antibody-based therapies. Thus, anti-cancer antibodies for treating cancer are to be further studied.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of the present invention is to propose an anti-human CD73 antibody or antigen-binding fragment thereof, a method for preparing the same and use thereof.

CD73 is widely expressed in various tumor cells, and its expression and catalytic adenosine-forming activity are closely related to invasion and metastasis of tumors. Preclinical studies also fully demonstrate that CD73 can promote immune escape of tumors, blocking/consuming CD73 molecules can effectively activate anti-tumor immune responses, and change the suppressed immune microenvironment, thereby exerting anti-tumor effects. And can be combined with PD- (L) 1 monoclonal antibody and/or A2AR inhibitor to generate synergistic effect.

Several anti-CD 73 antibodies have been developed clinically, of which representative is MEDI9447 (abbreviated as 9447) from AZ company, which binds to the N-terminal domain of CD73 and inhibits the enzymatic activity of recombinant CD73 and cell surface CD73, but it is reported that this molecule requires a bivalent activity of enzyme inhibition, and thus an activity barb occurs at a higher concentration. Another representative molecule is Uiledlimab (TJD 5) of a celestial organism, which binds to the C-terminus of CD73, and the monovalent form inhibits the enzymatic activity of CD73, so that there is no barb effect of MEDI9447, but the enzymatic activity of TJD5 is relatively low and requires a higher concentration to achieve maximum inhibitory activity.

The embodiment of the invention provides a series of anti-CD 73 functional antibodies, the binding epitope is positioned on the N-terminal domain of CD73, the binding epitope can specifically bind to CD73 on the cell surface to inhibit the enzyme activity of the CD73, and the CD73 can be consumed through internalization, compared with 9447 of AZ company, the enzyme activity is slightly weaker than or basically equivalent to 9447, but the barb phenomenon is obviously weakened compared with 9447, compared with TJD5 of the astronomical pharmaceutical industry, the enzyme activity is stronger, and the clinical application value is wide.

Thus, according to one aspect of the invention, there is provided an anti-human CD73 antibody or antigen-binding fragment thereof. According to an embodiment of the invention, the anti-human CD73 antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region (VH) comprising epitope regions (CDRs) 1, 2 and 3, and VH CDRs1, 2 and 3 comprise the amino acid sequences shown in the selected VH CDRs1, 2 and 3, respectively;

a light chain variable region (VL) comprising epitope regions (CDRs) 1, 2 and 3, and VL CDRs1, 2 and 3 comprise the amino acid sequences shown in the selected VH CDRs1, 2 and 3, respectively;

wherein the amino acid sequences of the selected VH CDRs1, 2 and 3 and the amino acid sequences of the selected VL CDRs1, 2 and 3 are selected from one of the following:

(1) The amino acid sequence of the selected VH CDR1 is shown in SEQ ID NO:20, the amino acid sequence of the selected VH CDR2 is shown in ftrnkvng htieyxasvkg, and the amino acid sequence of the selected VH CDR3 is shown in SEQ ID NO:22, the amino acid sequences of the selected VL CDRs1, 2 and 3 are respectively set forth in SEQ ID NOs: 17. 18 and 19, wherein X is N or S;

(2) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 26. 27 and 28, wherein the amino acid sequences of the selected VL CDRs1, 2 and 3 are set forth in SEQ ID NOs: 23. 24 and 25;

(3) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 30. 31 and 22, the amino acid sequences of the selected VL CDRs1, 2 and 3 are set forth in SEQ ID NOs: 29. 18 and 19.

According to the embodiment of the invention, the binding epitope of the anti-human CD73 antibody or the antigen binding fragment thereof is positioned in the N-terminal domain of the CD73, can specifically bind to the CD73 on the cell surface and inhibit the enzymatic activity of the CD73, and can consume the CD73 through internalization, so that the anti-CD 73 antibody can inhibit the tumor growth of tumor-bearing mice by in vivo administration, and has good anti-tumor therapeutic effect. .

According to an embodiment of the invention, the VH comprises an amino acid sequence having at least 90% identity to a selected VH sequence, the VL comprises an amino acid sequence having at least 90% identity to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of:

(1) The VH comprises SEQ ID NO:2 or 14, and the VL comprises the amino acid sequence set forth in SEQ ID NO:1 or 13;

(2) The VH comprises SEQ ID NO:4 or 16, and the VL comprises the amino acid sequence set forth in SEQ ID NO:3 or 15;

(3) The VH comprises SEQ ID NO:6, and the VL comprises the amino acid sequence set forth in SEQ ID NO:5.

According to an embodiment of the invention, the VH comprises SEQ ID NO:14, and the VL comprises the amino acid sequence set forth in SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

According to an embodiment of the invention, the VH comprises SEQ ID NO:16, and the VL comprises the amino acid sequence set forth in SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is a monoclonal antibody.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is a chimeric antibody.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human CD73 and is selected from Fv, fab, fab ', scFv and F (ab') ₂ 。

According to another aspect of the invention, an isolated nucleic acid is provided. According to an embodiment of the invention, the nucleic acid encodes the aforementioned anti-human CD73 antibody or antigen-binding fragment thereof.

According to another aspect of the invention, a carrier is provided. According to an embodiment of the invention, the vector comprises the aforementioned nucleic acid.

According to another aspect of the invention, an isolated cell is provided. According to an embodiment of the invention, the cell comprises the aforementioned vector.

According to another aspect of the present invention there is provided a method of preparing an anti-human CD73 antibody or antigen-binding fragment thereof as hereinbefore described. According to an embodiment of the invention, the method comprises:

(1) Culturing the aforementioned cells under suitable conditions; and

(2) The aforementioned anti-human CD73 antibody or antigen-binding fragment thereof is isolated and recovered.

According to another aspect of the present invention, a pharmaceutical composition is provided. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned anti-human CD73 antibody or antigen-binding fragment thereof; and a pharmaceutically acceptable carrier.

According to another aspect of the invention, there is provided an antibody-drug conjugate. According to an embodiment of the invention, the antibody-drug conjugate comprises the aforementioned antibody or antigen-binding fragment thereof covalently bound to a therapeutic agent.

According to an embodiment of the invention, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

According to a further aspect of the present invention there is provided the use of an anti-human CD73 antibody as hereinbefore described or an antigen-binding fragment thereof or a pharmaceutical composition as hereinbefore described in the manufacture of a medicament for the treatment of cancer.

According to an embodiment of the invention, the cancer is a solid tumor.

According to an embodiment of the invention, the cancer is melanoma.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a schematic representation of the results of inhibition of recombinant CD73 enzyme activity by an anti-CD 73 hybridoma antibody according to one embodiment of the invention;

FIG. 2 shows a schematic representation of the results of binding curves of anti-CD 73 antibodies to BXPC3 cells according to one embodiment of the invention;

FIG. 3 shows a schematic representation of the results of binding of humanized pre-and post-antibodies to cell surface CD73 according to one embodiment of the invention;

FIG. 4 shows an inhibition curve of anti-CD 73 antibody versus SKOV-3 cell surface CD73 enzyme activity according to an embodiment of the present invention;

FIG. 5 shows an inhibition curve of anti-CD 73 antibodies against A375 cell surface CD73 enzyme activity according to one embodiment of the invention;

FIG. 6 is a graph showing the results of inhibition of CD73 enzyme activity by an anti-CD 73 antibody against MDA-MB-231 cell surface according to one embodiment of the present invention;

FIG. 7 shows a Fortebio assay of the competitive effect of hz13B8 on MEDI9447 binding to CD73 according to one embodiment of the present invention

FIG. 8 shows a schematic diagram of the outcome of a Fortebio assay MEDI9447 competition for hz13B8 binding to CD73 according to one embodiment of the present invention;

FIG. 9 shows a graphical representation of the results of internalization of cell surface CD 73-mediated antibodies in accordance with one embodiment of the invention;

FIG. 10 shows a schematic representation of the growth curve of PBMC immunoreconstruction tumor-bearing melanoma A375 tumors according to one embodiment of the present invention;

figure 11 shows a schematic representation of the body weight growth curve of PBMC immunore-established tumor-bearing melanoma a375 tumor mice according to one embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.

Anti-human CD73 antibodies and antigen binding fragments thereof

The present invention provides antibodies and antigen binding fragments thereof that specifically bind CD 73. The anti-human CD73 antibody or the antigen binding fragment thereof according to the embodiment of the invention binds to an epitope located at the N-terminal domain of CD73, can specifically bind to cell surface CD73, inhibits the enzymatic activity thereof, can deplete CD73 by internalization, and can inhibit proliferation of tumor cells. Several murine anti-CD 73 antibodies, such as 13B8, 13E2, and 17E7, and humanized antibodies thereto, are provided in embodiments of the invention.

The CDR sequences of 13B8 and 13B8 derived antibodies (e.g., humanized antibodies) comprise the CDRs of the heavy chain variable domain, SEQ ID NOS: 20-22, and the CDRs of the light chain variable domain, SEQ ID NOS: 17-19, as defined by the Kabat coding.

Similarly, the CDR sequences of the 13E2 and 13E2 derived antibodies comprise the CDRs of the heavy chain variable domain, SEQ ID NOS: 26-28, and the CDRs of the light chain variable domain, SEQ ID NOS: 23-25, as defined by the Kabat coding.

Similarly, the CDR sequences of 17E7 and 17E7 derived antibodies comprise the CDRs of the heavy chain variable domain, SEQ ID NOS 30, 31 and 22, and the CDRs of the light chain variable domain, SEQ ID NOS 29, 18 and 19, as defined by the Kabat coding.

The embodiment of the invention also provides amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized antibody. Because there are different methods of humanizing mouse antibodies (e.g., the sequences may be replaced with different amino acids), the heavy and light chains of an antibody may have more than one form of humanized sequence. The amino acid sequence of the heavy chain variable region of the humanized 13B8 (hz 13B 8) antibody is shown in SEQ ID NO. 14. The amino acid sequence of the light chain variable region of humanized 13B8 is shown in SEQ ID NO. 13.

Similarly, the amino acid sequence of the heavy chain variable region of the humanized 13E2 (hz 13E 2) antibody is shown in SEQ ID NO. 16. The amino acid sequence of the light chain variable region of the humanized 13E2 (hz 13E 2) antibody is shown in SEQ ID NO. 15.

In some embodiments, an antibody may have a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to the selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to the selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to the selected VL CDR3 amino acid sequence.

In some embodiments, an antibody or antigen binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO. 20 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 21 or 32 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 22 having 0, 1 or 2 amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen binding fragment described herein may comprise a light chain variable domain comprising one, two, or three of the following CDRs: 17 of SEQ ID NO. 17 having 0, 1 or 2 amino acid insertions, deletions or substitutions; 18 of SEQ ID NO. with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 19 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the following CDRs: 26 of SEQ ID NO. 26 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 27 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 28 having 0, 1 or 2 amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen binding fragment described herein may comprise a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO. 23 with 0, 1 or 2 amino acid insertions, deletions or substitutions; 24 of SEQ ID NO. 24 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 25 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the following CDRs: 30 of SEQ ID NO. 30 having 0, 1 or 2 amino acid insertions, deletions or substitutions; 31 of SEQ ID NO. 31 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 22 having 0, 1 or 2 amino acid insertions, deletions or substitutions.

In some embodiments, an antibody or antigen binding fragment described herein may comprise a light chain variable domain comprising one, two, or three of the following CDRs: 29 of SEQ ID NO. 29 with 0, 1 or 2 amino acid insertions, deletions or substitutions; 18 of SEQ ID NO. with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO. 19 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

Among them, it is noted that insertions, deletions and substitutions may be within the CDR sequence, or at one or both ends of the CDR sequence.

Embodiments of the invention also provide antibodies or antigen-binding fragments thereof that bind CD 73. The antibody or antigen binding fragment thereof comprises: a heavy chain variable region (VH) comprising or consisting of an amino acid sequence having at least 90%, 95% or 98% identity to a selected VH sequence; and a light chain variable region (VL) comprising or consisting of an amino acid sequence having at least 90%, 95% or 98% identity to the selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO. 2 or 14 and the selected VL sequence is SEQ ID NO. 1 or 13. In some embodiments, the selected VH sequence is SEQ ID NO. 4 or 16 and the selected VL sequence is SEQ ID NO. 3 or 15. In some embodiments, the selected VH sequence is SEQ ID NO. 6 and the selected VL sequence is SEQ ID NO. 5.

Fragments of antibodies are suitable for use in the provided methods, so long as they retain the desired affinity and specificity of the full length antibody. Thus, fragments of antibodies that bind to CD73 will retain the ability to bind to CD 73. Fv fragments are antibody fragments which comprise complete antigen recognition and binding sites. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in close association, which association may be covalent in nature, such as in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, the six CDRs, or a subset thereof, confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, but typically has less affinity than the entire binding site.

Single chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

The Fab fragment comprises the variable and constant domains of the light chain and the variable and first constant domains of the heavy chain (CH 1). F (ab') 2 antibody fragments comprise a pair of Fab fragments which are typically covalently linked near their carboxy-terminus by a hinge cysteine between them. Other chemical couplings of antibody fragments are also known in the art.

Nucleic acids, vectors and cells

Embodiments of the invention also provide nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds CD73 (e.g., human CD 73).

Embodiments of the invention also provide recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotides and/or the polynucleotide-containing vectors), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, a polynucleotide of interest is localized for expression in the vector by being operably linked to regulatory elements such as promoters, enhancers and/or polyadenylation tails, located at or near or flanking the integration site of the polynucleotide of interest in the vector or in the genome of the host cell, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (useful for producing recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

Embodiments of the invention provide host cells transformed with the vectors described above. The host cell may be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is E.coli (Escherichia coli). Preferably, the eukaryotic cell is selected from: primordial biological cells, animal cells, plant cells, and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. A preferred fungal cell is Saccharomyces cerevisiae.

Methods of making anti-CD 73 antibodies

Isolated fragments of human CD73 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies may be raised in an animal by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. The animals may be injected with the antigenic peptide or protein multiple times.

Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic formulations may contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides (e.g., fragments of human CD 73). The formulation may further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

Pharmaceutical composition, use and method of treatment

The present embodiments also provide pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the embodiments of the present invention. Two or more (e.g., two, three, or four) of any of the antibodies or antigen binding fragments described herein can be present in any combination in a pharmaceutical composition. The pharmaceutical composition may be formulated in any manner known in the art.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or efficacy of the antibody.

In one aspect, the invention provides the use of an anti-human CD73 antibody or antigen binding fragment thereof as described above in the manufacture of a medicament for the treatment of cancer. According to an embodiment of the invention, the cancer comprises melanoma.

The present invention provides that one or more antibodies or antigen binding fragments thereof may be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides methods for treating cancer in a subject, reducing the rate at which tumor volume increases over time in a subject, reducing the risk of metastasis, or reducing the risk of additional metastasis in a subject. In some embodiments, the treatment may stop, slow, delay or inhibit the progression of the cancer. In some embodiments, the treatment may result in a decrease in the number, severity, and/or duration of one or more symptoms of cancer in the subject.

In one aspect, the disclosure features a method comprising administering to a subject in need thereof (e.g., a subject having, or identified as having, or diagnosed with, a cancer, such as melanoma) a therapeutically effective dose of an antibody or antigen binding fragment thereof disclosed herein.

In some embodiments, the compositions and methods disclosed herein are useful for treating a patient at risk of cancer. Patients with cancer may be identified using a variety of methods known in the art.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are not manufacturer specific and are conventional products commercially available, for example, from Sigma company.

Example 1: preparation of anti-human CD73 hybridoma antibodies

Balb/c mice were immunized with human CD73-mFc recombinant protein (SEQ ID NO: NM-002526.3, 27-547 aa), and the obtained clones were subjected to serum titer detection with the coated recombinant human CD73-his recombinant protein (SEQ ID NO: NM-002526.3, 27-547 aa), and hybridoma cell fusion and positive clone screening after fusion requirements were met.

The obtained positive clones were subjected to enzyme activity blocking analysis at the recombinant protein level. Specifically, rhCD73-his was diluted to 0.3ug/ml, mixed with different concentrations of CD73 Ab and incubated at 37℃for 20min; AMP at a final concentration of 0.1mM and ATP at a final concentration of 0.03mM were added, mixed and incubated at 37 ℃ for 30min; luciferase-containing CellTiter-Glo (Promega) was added and the spectrophotometric readings were taken. The percentage of enzyme activity was assessed as follows: the activity of residual CD73 was calculated as: (cd73+ab+atp+amp) - (atp+amp)/(cd73+atp+amp) - (atp+amp) x 100. The enzyme activity blocking assays were performed on the purified positive hybridoma supernatants, respectively, and as shown in FIG. 1, clones 17E7, 13B8 and 13E2 were shown to have a clear enzyme inhibition activity.

Example 2: preparation of anti-human CD73 chimeric antibody and control antibody

The 17E7, 13B8 and 13E2 hybridoma cells selected in example 1 were subjected to extraction of total RNA of the cells according to the procedure of TRIzol kit (Cat: 15596026, invitrogen); reverse transcription of hybridoma Total RNA into cDNA Using M-MuLV reverse transcriptase (Cat: M0253S, NEB) followed by amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V _H The sequence was cloned into a T vector for DNA sequencing and translated into amino acids. The result shows that the light and heavy chain variable region sequences of 13B8 are shown as SEQ ID NO. 1 and SEQ ID NO. 2; 13E2 has the amino acid sequence shown in SEQ ID NO. 3 and SEQ ID NO. 4; the amino acid sequences of the 17E7 light and heavy chain variable region are shown as SEQ ID NO. 5 and SEQ ID NO. 6.

SEQ ID NO. 1 13B8 VL (CDRs 1, 2 and 3 are SEQ ID NO. 17, 18 and 19 respectively)

DIVMTQSPSSLAVSVGEKVTLSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKSLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK

SEQ ID NO. 2 13B8 VH (CDRs 1, 2 and 3 are SEQ ID NO. 20, 21 and 22 respectively)

EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYSMNWVRQPPGKALEWLGFTRNKVNGHTIEYNASVKGRFTISRDNSQSILYLQMNDLRSEDSATYYCARDIDYDYDEAWFAYWGQGTLVTVSA

SEQ ID NO. 3 13E2 VL (CDRs 1, 2 and 3 are SEQ ID NO. 23, 24 and 25 respectively)

DIKMTQSPSSMYASLGERVTLTCKASQDINTYLSWFQQKPGKTPKTLIYRANLLVDGV PSRFSGSGSGQEYSLTISSLEYEDMGIYHCLQYDEFPYTFGGGTKLEIK

SEQ ID NO. 4 13E2 VH (CDRs 1, 2 and 3 are SEQ ID NO. 26, 27 and 28 respectively)

QVQLQQSGAELAKPGASVKMSCKASGYSFTSYWMHWVKQRPGQGLEWIGYINPTTGYTEYNQKFKDKATLTADRSSTTAYMQLSSLTSEDSAVYYCARIYYGSLAVDSWGQGTSVTVSS

SEQ ID NO. 5 17E7 VL (CDRs 1, 2 and 3 are SEQ ID NO. 29, 18 and 19 respectively)

DIVMTQSPSSLPVSVGEKVTMSCKSSQSLLYSNNQKNYLAWYQQKPGQSPQLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK

SEQ ID NO. 6 17E7 VH (CDRs 1, 2 and 3 are SEQ ID NO. 30, 31 and 22 respectively)

EVKLVESGGGLVQPGDSLRLSCATSGFTFSDYSMSWVRQPPGKALEWLGFIRNKVNGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDIDYDYDEAWFAYWGQGTLVTVSA

The light and heavy chain variable region sequences of the control antibodies MEDI9447 (with the sequence reference patent US 9938356B 2) and TJD5 (with the sequence reference patent CN 109476755A) are fully synthesized, the light and heavy chain sequences are cloned into eukaryotic transient expression vectors filled with human light chain constant region (with the amino acid sequence shown in SEQ ID NO: 7) coding genes and heavy chain constant region (with the amino acid sequence shown in SEQ ID NO: 8) coding genes respectively, control antibody light chain and heavy chain recombinant expression plasmids are obtained, and HEK293 cells (293 fectin, cat:12347019, gibco) are transfected for recombinant expression. And 5-6 days after cell transfection, taking a culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain a control antibody. The MEDI9447 amino acid sequence is derived from patent US 9938356B 2, the light chain amino acid sequence is shown in SEQ ID NO:9, the heavy chain amino acid sequence is shown in SEQ ID NO:10.TJD5 sequence is from patent CN109476755A, the light chain amino acid sequence is shown as SEQ ID NO. 11, and the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 12. The chimeric antibody expression vectors of 13B8, 13E2 and 17E7 are constructed and expressed in a recombinant mode in the same method, and the purified products of the chimeric antibodies ch13B8, ch13E2 and ch17E7 proteins are obtained and then are identified later.

SEQ ID NO. 7 light chain constant region amino acid sequence

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 8 heavy chain constant region amino acid sequence

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:9MEDI-9447VL

QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGTAPKLLIYLDNLRLSGV PDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSHPGWTFGGGTKLTVL

SEQ ID NO:10MEDI-9447VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAYSWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGYGRVDEWGRGTLVTVSS

SEQ ID NO:11TJD-5VL

EIVLSQSPATLSLSPGERATLSCRASSRVNYMHWYQQKPGQSPRPWISATSNLASGVPA RFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKVEIK

SEQ ID NO:12TJD-5VH

EVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWNWIRQPPGKKLEWMGYINYGGSNGYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARDYDAYYEALDDWGQGTTVTVSS

Example 3: chimeric anti-CD 73 antibody assay

FACS detection of binding Activity of antibodies to cell surface human CD73 protein

Adjusting the number of BXPC-3 cells in logarithmic growth phase to 2.multidot.10 ⁵ Sample, the anti-CD 73 antibodies to be detected (chimeric antibodies ch13E2, ch13B8 and ch17E7, with MEDI9447 and TJD5 as controls) were diluted to 33nM respectively, then diluted 3-fold in a gradient for 11 concentrations, incubated with BXPC-3 cells at 4℃for 1h, washed well, FITC-labeled goat anti-human IgG antibody (Cat: F9512, sigma) was added, incubated for 30min, and after washing well, the average fluorescence intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMN COULTER) to detect the binding capacity of the chimeric antibodies to human CD73 on the cell surface. The results are shown in FIG. 2 and the EC50 s are shown in Table 1. The binding activity of the three chimeric antibodies was substantially equivalent to that of the control antibody MEDI9447, which was much higher than that of the control antibody TJD5.

Table 1: EC50 value of anti-CD 73 antibody and BXPC3 cell binding activity

Antibodies to	ch13E2	ch13B8	ch17E7	MEDI9447	TJD5
						EC50(nM)	0.1639	0.2606	0.4489	0.242	7.414

Fotebio assay of affinity of antibodies to recombinant CD73 antigen

Antibody affinity was determined using an Octet QKe system instrument from Fortebio, using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe capture antibody Fc fragment method. The antibody was diluted to 4ug/mL in PBS buffer and passed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120s. As mobile phase, 60nM eukaryotic transiently expressed human CD73-his recombinant protein (SEQ ID NO: NM-002526.3, 27-547 aa) was used, with a binding time of 300s and a dissociation time of 300s. After the experiment is completed, the response value of the blank control is deducted, and the software is used for carrying out 1:1Langmuir binding pattern fitting, the kinetic constants of antigen-antibody binding were calculated. The results are shown in Table 2, where the three chimeric antibodies have slightly lower affinity than the control antibody MEDI-9447, but significantly higher affinity than the control antibody TJD5.

Table 2: anti-CD 73 antibody binding dissociation constant

Sample of	KD(M)	kon(1/Ms)	kdis(1/s)
				MEDI9447	6.35E-10	1.65E+05	1.05E-04
TJD5	7.57E-09	5.07E+04	3.84E-04
				ch13B8	1.07E-09	1.43E+05	1.53E-04
ch17E7	1.34E-09	1.10E+05	1.47E-04
				ch13E2	1.20E-09	1.17E+05	1.40E-04

Example 4:13B8 and 13E8 antibody humanization

1. Humanization of murine monoclonal antibodies 13B8 and 13E2

Comprehensive analysis of the light and heavy chain sequences of the 13B8 and 13E2 murine antibodies was performed, a human antibody, the germline library (http:// ww2. Src-lmb. Cam. Ac. Uk/vbase/alignments2.Php#VHEX; http:// ww2. Src-lmb. Cam. Ac. Uk/vbase/alignments2. Php#VHEX), was selected, the most similar human antibody, the template, was used to humanize the 13B8 and 13E2 light and heavy chain variable regions, the sequences after 13B8 humanization were shown as SEQ ID NO:13 and SEQ ID NO:14, and the sequences after 13E2 humanization were shown as SEQ ID NO:15 and SEQ ID NO:16, respectively.

SEQ ID NO 13hz13B8 VL (CDRs 1, 2 and 3 are SEQ ID NO 17, 18 and 19 respectively)

DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYLQKPGQSPQLLIYWAST RESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYSYPYTFGQGTKLEIK

SEQ ID NO. 14hz13B8VH (CDRs 1, 2 and 3 are SEQ ID NO. 20, 32 and 22 respectively)

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYSMNWVRQAPGKGLEWVGFTRNKVNGHTIEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDIDYDYDEAWFAYWGQGTLVTVSS

15hz13E2 VL (CDRs 1, 2 and 3 are SEQ ID NO:23, 24 and 25, respectively)

DIQMTQSPSSLSASVGDRVTITCKASQDINTYLSWFQQKPGKAPKLLIYRANLLVDGV PSRFSGSGSGTEEFLTISSLQPEDFATYHCLQYDEFPYTFGQGTKLEIK

16hz13E2 VH (CDRs 1, 2 and 3 are SEQ ID NO:26, 27 and 28 respectively)

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGYINPTTGYTEYNQKFKDRVTMTRDTSTSTAYMELSSLRSEDTAVYYCARIYYGSLAVDSWGQGTLVTVSS

2. Recombinant expression of humanized monoclonal antibodies

Fully synthesizing humanized-designed hz13B8 and hz13E2 light and heavy chain variable region sequences, cloning the humanized hz13B8_VH and hz13E2_VH into a eukaryotic transient expression vector pKN041 (a Kenuo self-constructed expression vector containing a human IgG1 constant region coding gene) upstream of a human IgG1 heavy chain constant region coding gene by enzyme digestion; humanized hz13B 8-VL and hz13E 2-VL are cloned into eukaryotic transient expression vectors pKN019 (Kenuo hong self-constructed expression vectors containing Ck coding genes) upstream of coding genes of human light chain Ck by enzyme digestion, humanized hz13B8 and hz13E2 light and heavy chain recombinant expression vectors are constructed, and hz13B8 and hz13E2 light and heavy chain plasmids are transferred into HEK293 cells for recombinant expression according to the operation instructions of transfection reagents 293fectin (Cat: 12347019, gibco). And 5-6 days after cell transfection, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain humanized antibody samples of hz13B8 and hz13E 2.

EXAMPLE 5 comparison of binding Activity of humanized antibodies to chimeric antibodies

1. Affinity analysis

Antibody affinity was determined using an Octet QKe system instrument from Fortebio, using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe capture antibody Fc fragment method. In the assay, the antibody to be tested was diluted to 4ug/mL with PBS buffer and passed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120s. Human CD73-His recombinant protein (SEQ ID NO: NP-002344.2, 1aa-274 aa) was used as mobile phase at concentrations of 90, 60, 30, 15 and 7.5nM, respectively. The binding time was 300s and the dissociation time was 300s. After the experiment is completed, the response value of the blank control is deducted, and the software is used for carrying out 1:1Langmuir binding pattern fitting, the kinetic constants of antigen-antibody binding were calculated. The affinities of hz13B8 and hz13E2 after humanization are shown in table 3, and the results show that both hz13B8 and hz13E2 maintain comparable affinity levels to chimeric antibodies.

TABLE 3 Fortebio determination of affinity of antibodies before and after humanization

Sample of	KD(M)	kon(1/Ms)	kdis(1/s)
				ch13E2	9.58E-10	2.40E+05	2.30E-04
hz13E2	6.62E-10	2.87E+05	1.90E-04
				ch13B8	8.17E-10	2.40E+05	1.96E-04
hz13B8	9.17E-10	3.02E+05	2.77E-04

The binding capacity of humanized and chimeric antibodies to human CD73 on the cell surface was examined by FACS, the method was the same as in example 3, except that the antibodies to be tested were humanized and chimeric antibodies, the results are shown in FIG. 3, the EC50 is shown in Table 4, and the results show that the binding activity before and after humanization was substantially equivalent, far higher than that of the control antibody TJD5.

Table 4: EC50 value of anti-CD 73 antibody and BXPC3 cell binding activity

Abs	hz13B8	ch13B8	hz13E2	ch13E2	TJD5
						EC50(nM)	0.6822	0.5637	0.2293	0.3274	15.04

EXAMPLE 6 analysis of the enzymatic blocking Activity of humanized antibodies

1. Analysis of SKOV-3 cell surface CD73 enzymatic Activity blocking

SKOV3 cell digests were counted to 1×10e5, mixed with different concentrations of CD73 Ab (400 nM start, 6-fold dilution of 6 gradients) and incubated at 37 ℃ for 30min, where CD73 Ab included the antibodies to be tested hz13B8 and hz13E2, positive control antibodies MEDI-9447 and TJD5, and the irrelevant antibody NC-IgG1; AMP was added at a final concentration of 0.1mM, mixed and incubated at 37 ℃ for 30min; ATP was added at a final concentration of 0.016mM, and after mixing, 1/3 volume CellTiter-Glo (promega, G7571) was added and read. The formula of the inhibition treatment of the CD73 enzyme activity is as follows: [ (cd73 ab+cell+amp+atp) - (amp+atp) ]/[ (cell+amp+atp) - (amp+atp) ]x100%.

As a result, as shown in FIG. 4, the hz13B8 enzyme inhibitor activity was substantially equivalent to that of the control antibody 9447 (MEDI-9447), and significantly higher than that of the control antibody TJD5; the activity of hz13E2 enzyme is weaker than that of hz13B8 and is equivalent to TJD5.

2. Analysis of blocking of A375 and MDA-MB-231 cell surface CD73 enzymatic Activity

The enzyme activity blocking effect of hz13B8 on the cell surface CD73 of two other tumor cells A375 and MDA-MB-231 was analyzed in the same manner as described above, and the results are shown in FIGS. 5 to 6 and Table 5. Hz13B8 has obvious inhibition activity on the activity of CD73 enzyme on the surfaces of two tumor cells, and the activity is slightly weaker than that of a control antibody 9447, but is obviously better than that of the control antibody TJD5. Notably, on a375 cells of lower CD73 antigen density, 9447 showed activity reversal of barbs at high concentrations, but neither hz13B8 nor TJD5.

TABLE 5 IC50 values for inhibition of cell surface CD73 enzymatic activity by anti-CD 73 antibodies

Example 7: analysis of competition of hz13B8 with MEDI-9447 epitope

Hz13B8 and MEDI-9447 were identical to antibodies binding to the N-terminal domain of CD73 and the epitope competition relationship of Hz13B8 with both antibodies of MEDI-9447 was analyzed using Fortebio. Specifically, hz13B8 and MEDI9447 were biotinylated, respectively. And biotinylated hz13B8 and MEDI9447 were captured using SA chips at an antibody concentration of 5ug/ml. 200nM of CD73-His was used as mobile phase, binding time 240s, and further 60nM of non-biotinylated analytical antibody was used as mobile phase, binding time 300s, and dissociation time 300s.

The experimental results showed (fig. 7 and 8) that both hz13B8 and MEDI9447 had no epitope competition, and that binding of one molecule did not affect binding of the other molecule to the target antigen, suggesting that both bind to the N-terminal domain of CD73, but the epitopes were not identical.

Example 8: analysis of hz13B8 internalization Activity

SKOV-3 and a431 cells naturally expressing human CD73 in the logarithmic growth phase were grown at 4×10 ⁵ cells/well and humanized antibodies hz13B8, hz13E2, MEDI-9447 and TJD5 were diluted to 10. Mu.g/mL and added to the cells separately. A group of electric heating constant temperature incubator with 37 ℃ and a group of refrigerator with 4 ℃ are used as negative control; negative control was incubated for 1 hour, washed 3 times with PBS, 1:200 dilution of goat anti-human IgG Fc-FITC secondary antibody was added, incubated for 30 minutes at 4℃and 3 times with PBS, FACS was used to detect the Mean Fluorescence Intensity (MFI) of cells, 37℃was incubated for 3 hours, 1:200 dilution of goat anti-human IgG Fc-FITC secondary antibody (Cat: F9512, sigma) was added, incubation for 30 minutes at 4℃was performed 3 times with PBS, and FACS was used to detect the Mean Fluorescence Intensity (MFI) of cells. The internalization efficiency of the antibody was calculated according to the formula: internalization% = 100- (MFI of 37 ℃ incubation sample x MFI of control sample incubated at 100/4 ℃).

The results of the experiment are shown in FIG. 9, and indicate that hz13B8, hz13E2 and MEDI-9447 internalize to different extents on both cells, but no internalization was observed with TJD5.

Example 9: evaluation of in vivo antitumor efficacy of hz13B8

Human melanoma A375 cells were inoculated subcutaneously into the right anterior hypochondrium of male NCG mice (available from Jiangsu Jiuyaokang Biotechnology Co., ltd., 6-8 weeks old) and PBMC cells (from normal human peripheral blood supplied from Kangsheng) were inoculated 1 day before tumor cell inoculation in a 2X 10 manner ⁶ Tail intravenous injection into mice, tumor growth to 50mm ³ The left and right time group administration is respectively as follows: NC-hIgG1 (Group 1), hz13B8 (Group 2), TJD5 (Group 3) and MEDI-9447 (Group 4), the NC-hIgG1, hz13B8, MEDI-9447 and TJD5 antibodies were administered to 6 mice each, by: 10mg/kg, ip, tiw.times.9. Tumor volume and body weight were measured weekly and tumor-bearing mice body weight and tumor volume changes were recorded as a function of time of administration. At the end of the experiment, tumor-bearing mice were euthanized, and the peeled tumors were weighed and photographed. Calculation of tumor growth inhibition rate TGI _TV (%) and statistically analyzed. TGI _TV (％)＝(1－T/C)×100％。

Experimental results show that the growth of the tumors in the hz13B8, TJD5 and MEDI-9447 groups is significantly slower than that of the hIgG4 group (p < 0.05), and the TGI reaches 50%,39% and 48%, respectively, indicating that the hz13B8 of the present invention can significantly inhibit the growth of the tumors. The body weight measurement results are shown in fig. 11, and no obvious difference is seen among groups, which indicates that the mice have good tolerance to the anti-CD 73 antibody, and the hz13B8 of the embodiment of the invention has little or no toxic or side effect.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An anti-human CD73 antibody or antigen-binding fragment thereof, comprising:

a light chain variable region (VL) comprising epitope regions (CDRs) 1, 2 and 3, and VL CDRs1, 2 and 3 comprise the amino acid sequences shown in the selected VL CDRs1, 2 and 3, respectively;

2. The antibody or binding fragment thereof of claim 1, wherein the VH comprises an amino acid sequence having at least 90% identity to a selected VH sequence and the VL comprises an amino acid sequence having at least 90% identity to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of:

3. The antibody or binding fragment thereof of claim 1, wherein the VH comprises SEQ ID NO:14, and the VL comprises the amino acid sequence set forth in SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

4. The antibody or binding fragment thereof of claim 1, wherein the VH comprises SEQ ID NO:16, and the VL comprises the amino acid sequence set forth in SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

5. The antibody or binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof is a monoclonal antibody.

6. The antibody or binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody.

7. The antibody or binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human CD73 and is selected from Fv, fab, fab ', scFv and F (ab') ₂ 。

8. An isolated nucleic acid encoding the anti-human CD73 antibody or antigen-binding fragment thereof of any one of claims 1-7.

9. A vector comprising the nucleic acid of claim 8.

10. An isolated cell comprising the vector of claim 9.

11. A method of making the anti-human CD73 antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising:

(1) Culturing the cell of claim 10 under suitable conditions; and

(2) Isolating and recovering said anti-human CD73 antibody or antigen-binding fragment thereof.

12. A pharmaceutical composition comprising:

the anti-human CD73 antibody or antigen-binding fragment thereof of any one of claims 1-7; and

a pharmaceutically acceptable carrier.

13. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7 covalently bound to a therapeutic agent.

14. The antibody-drug conjugate of claim 13, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

15. Use of an anti-human CD73 antibody or antigen-binding fragment thereof according to any one of claims 1-6, a pharmaceutical composition according to claim 12, or an antibody-drug conjugate according to claims 13-14 in the manufacture of a medicament for the treatment of cancer.

16. The use according to claim 15, wherein the cancer is a solid tumor.

17. The use according to claim 15, wherein the cancer is melanoma.