CN117946274A

CN117946274A - Anti-CD 73 antibody and application thereof

Info

Publication number: CN117946274A
Application number: CN202410136146.2A
Authority: CN
Inventors: 王海彬; 周雅琼; 高栋
Original assignee: Haizheng Biopharmaceutical Co ltd
Current assignee: Haizheng Biopharmaceutical Co ltd
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2024-04-30
Also published as: CN112111008B; WO2020253568A1; CN112111008A

Abstract

The invention belongs to the field of biological medicine, and provides preparation and use of an anti-CD 73 antibody or an antigen binding fragment thereof, wherein the antibody comprises H CDR1, H CDR2 and H CDR3, and L CDR1, L CDR2 and L CDR3; the sequence of H CDR1 is shown as SEQ ID NO. 1, SEQ ID NO. 9 or SEQ ID NO. 17, the sequence of H CDR2 is shown as SEQ ID NO. 2, SEQ ID NO. 10 or SEQ ID NO. 18, the sequence of H CDR3 is shown as SEQ ID NO. 3, SEQ ID NO. 11 or SEQ ID NO. 19, the sequence of L CDR1 is shown as SEQ ID NO. 5, SEQ ID NO. 13 or SEQ ID NO. 21, the sequence of L CDR2 is shown as SEQ ID NO. 6, SEQ ID NO. 14 or SEQ ID NO. 22, and the sequence of L CDR3 is shown as SEQ ID NO. 7, SEQ ID NO. 15 or SEQ ID NO. 23. The anti-CD 73 antibody can specifically bind to CD73 and inhibit the enzymatic activity of the CD 73.

Description

Anti-CD 73 antibody and application thereof

The present application is a divisional application of Chinese patent application with the application number 201910532994.4, the application date 2019, 6-month 19 and the invention name of 'anti-CD 73 antibody' and application.

Technical Field

The invention belongs to the field of biological medicine, and relates to preparation and use of an antibody.

Background

CD73, also known as extracellular-5' -nucleotidase, is a glycoprotein anchored to the plasma membrane by Glycosyl Phosphatidylinositol (GPI), and is widely expressed in endothelial cells and lymphocytes of the human body, helping to convert ATP with immune activating effect into adenosine with immune suppressing effect. Adenosine as a signaling molecule can mediate its biological effects by a variety of receptors (including A1, A2A, A2B and A3), e.g., binding of adenosine to adenosine receptor A2AR (adenosine receptor AR) can inhibit T cell immunity, thereby helping cancer cells escape the T cell "chase-kill". In addition to inhibiting immune cell function, more and more studies indicate that CD73 can also directly stimulate tumor cell proliferation, migration and invasion, and tumor angiogenesis. It can be seen that targeting CD73 has the advantage of "double engraving" in comparison to other targets that modulate immune responses.

Preclinical studies have shown that targeting CD73 can produce good anti-tumor effects, alter tumor immune microenvironment by increasing T lymphocytes and tumor leukocyte populations, and also reduce tumor Myeloid Derived Suppressor Cells (MDSCs), shift macrophages in responsive rats toward an activated (anti-tumor) phenotype. And combining CD73 blocking therapy with other immune molecule modulators (e.g., PD- (L) 1 antibodies) can significantly enhance anti-tumor effects such as CD73 mab, which is also an attractive option for enhancing the anti-tumor product lines of the present enterprise.

Analysis of clinical tumor samples shows that high expression of CD73 is a potential biomarker, is expressed on a plurality of different tumors, and is closely related to poor prognosis of various types of tumors, including breast cancer, lung cancer, ovarian cancer, renal cancer, gastric cancer, head and neck cancer and the like. In addition, the treatment schemes of PD-1/CD73 monoclonal antibody, radiotherapy and chemotherapy and the like can cause the increase of the expression of CD73 in the tumor microenvironment, and then the treatment is greatly discounted by suppressing the immune response through the adenosine signal, namely the acquired drug resistance. Thus, CD 73-targeting therapeutic strategies have potential for clinical application as single agents or combination therapies.

In view of the still urgent need for improvements in the treatment of targeted diseases (e.g., cancer), methods of modulating tumor progression through a variety of mechanisms, as well as methods of modulating CD73 activity and related therapeutic agents, are highly desirable.

Disclosure of Invention

It is an object of the present invention to provide an anti-CD 73 antibody or antigen-binding fragment thereof that specifically recognizes and binds CD73 and has desired functional properties including the ability to bind to CD73 with high affinity and inhibit the enzymatic activity of CD 73. The antibodies described herein are useful for inhibiting tumor growth, reducing adenosine production, stimulating an immune response, and detecting CD73 protein in a sample.

In a first aspect, the invention provides an anti-CD 73 antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising H CDR1, H CDR2 and H CDR3 and a light chain variable region comprising L CDR1, L CDR2 and L CDR3;

The sequence of the H CDR1 is shown as SEQ ID NO. 1, SEQ ID NO. 9 or SEQ ID NO. 17, or the sequence of the H CDR1 has more than 95 percent, such as 96 percent, 97 percent, 98 percent and 99 percent of sequence identity with the sequence of the SEQ ID NO. 1, the SEQ ID NO. 9 or the SEQ ID NO. 17;

the sequence of the H CDR2 is shown as SEQ ID NO. 2, SEQ ID NO. 10 or SEQ ID NO. 18, or the sequence of the H CDR2 has more than 95 percent, such as 96 percent, 97 percent, 98 percent and 99 percent of sequence identity with the sequence of the SEQ ID NO. 2, the SEQ ID NO. 10 or the SEQ ID NO. 18;

the sequence of the H CDR3 is shown as SEQ ID NO. 3, SEQ ID NO. 11 or SEQ ID NO. 19, or the sequence of the H CDR3 has more than 95 percent, such as 96 percent, 97 percent, 98 percent and 99 percent of sequence identity with SEQ ID NO. 3, SEQ ID NO. 11 or SEQ ID NO. 19;

The sequence of the L CDR1 is shown as SEQ ID NO. 5, SEQ ID NO. 13 or SEQ ID NO. 21, or the sequence of the L CDR1 has more than 95 percent, such as 96 percent, 97 percent, 98 percent and 99 percent of sequence identity with SEQ ID NO. 5, SEQ ID NO. 13 or SEQ ID NO. 21;

the sequence of the L CDR2 is shown as SEQ ID NO. 6, SEQ ID NO. 14 or SEQ ID NO. 22, or the sequence of the L CDR2 has more than 95 percent, such as 96 percent, 97 percent, 98 percent and 99 percent of sequence identity with SEQ ID NO. 6, SEQ ID NO. 14 or SEQ ID NO. 22;

the sequence of the L CDR3 is shown as SEQ ID NO. 7, SEQ ID NO. 15 or SEQ ID NO. 23, or the sequence of the L CDR3 has more than 95 percent, such as 96 percent, 97 percent, 98 percent and 99 percent of sequence identity with the SEQ ID NO. 7, SEQ ID NO. 15 or SEQ ID NO. 23.

In one embodiment, the heavy chain variable region of the antibody comprises:

h CDR1, its amino acid sequence is shown in SEQ ID NO 1,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO. 2, and

H CDR3 with the amino acid sequence shown in SEQ ID NO 3.

In one embodiment, the heavy chain variable region of the antibody comprises:

h CDR1, the amino acid sequence of which is shown as SEQ ID NO 9,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO 10, and

H CDR3 with the amino acid sequence shown in SEQ ID NO. 11.

In one embodiment, the heavy chain variable region of the antibody comprises:

h CDR1, the amino acid sequence of which is shown as SEQ ID NO. 17,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO. 18, and

H CDR3, the amino acid sequence of which is shown in SEQ ID NO. 19.

In one embodiment, the light chain variable region of the antibody comprises:

L CDR1 with the amino acid sequence shown in SEQ ID NO 5,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 6, and

L CDR3 has the amino acid sequence shown in SEQ ID NO. 7.

In one embodiment, the light chain variable region of the antibody comprises:

l CDR1 with the amino acid sequence shown in SEQ ID NO 13,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 14, and

The amino acid sequence of the L CDR3 is shown as SEQ ID NO. 15.

In one embodiment, the light chain variable region of the antibody comprises:

L CDR1 with the amino acid sequence shown in SEQ ID NO. 21,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 22, and

L CDR3 has the amino acid sequence shown in SEQ ID NO. 23.

In one embodiment, the antibody is A7, the heavy chain variable region of which comprises:

h CDR1, its amino acid sequence is shown in SEQ ID NO 1,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO. 2, and

H CDR3 with the amino acid sequence shown in SEQ ID NO 3;

the light chain variable region comprises:

L CDR1 with the amino acid sequence shown in SEQ ID NO 5,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 6, and

L CDR3 has the amino acid sequence shown in SEQ ID NO. 7.

In one embodiment, the antibody is B11, the heavy chain variable region of which comprises:

h CDR1, the amino acid sequence of which is shown as SEQ ID NO 9,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO 10, and

H CDR3 with the amino acid sequence shown as SEQ ID NO. 11;

the light chain variable region comprises:

l CDR1 with the amino acid sequence shown in SEQ ID NO 13,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 14, and

The amino acid sequence of the L CDR3 is shown as SEQ ID NO. 15.

In one embodiment, the antibody is B22, the heavy chain variable region of which comprises:

h CDR1, the amino acid sequence of which is shown as SEQ ID NO. 17,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO. 18, and

H CDR3 with the amino acid sequence shown as SEQ ID NO. 19;

the light chain variable region comprises:

L CDR1 with the amino acid sequence shown in SEQ ID NO. 21,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 22, and

L CDR3 has the amino acid sequence shown in SEQ ID NO. 23.

In one embodiment, the anti-CD 73 antibody A7 comprises a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 4, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID No. 4.

In one embodiment, the anti-CD 73 antibody A7 comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 8, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID No. 8.

In one embodiment, the anti-CD 73 antibody B11 comprises a heavy chain variable region, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 12 or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 12.

In one embodiment, the anti-CD 73 antibody B11 comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 16, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 16.

In one embodiment, the anti-CD 73 antibody B22 comprises a heavy chain variable region, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 20 or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 20.

In one embodiment, the anti-CD 73 antibody B22 comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 24, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 24.

In one embodiment, the anti-CD 73 antibody comprises a heavy chain constant region (CH), wherein the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO. 26, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 26.

In one embodiment, the anti-CD 73 antibody comprises a light chain constant region (CL), wherein the amino acid sequence of the light chain constant region is as shown in SEQ ID NO. 25 or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 25.

In one embodiment, the anti-CD 73 antibody or antigen-binding fragment thereof is capable of specifically recognizing and binding CD73, wherein the affinity is 1 x 10 ^-9 M or less, preferably 5 x 10 ^-10 M or less, more preferably 1 x 10 ^-10 M or less.

In one embodiment, the anti-CD 73 antibody or antigen-binding fragment thereof binds human CD73 with a KD of about 0.1nm to 10nm or less as measured by fortebio octet analysis.

In one embodiment, the anti-CD 73 antibody or antigen-binding fragment thereof binds human CD73 with EC ₅₀ of 0.1nm to 10nm or less as measured by FACS.

In another aspect, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence region encoding the antibody or antigen binding fragment thereof of any one of claims 1-4.

In one embodiment, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, or SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, or SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51.

In a further preferred embodiment, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO:32 and SEQ ID NO:36, or SEQ ID NO:40 and SEQ ID NO:44, or SEQ ID NO:48 and 52.

In another aspect, the invention also provides an expression vector comprising a nucleic acid molecule of the invention.

In another aspect, the invention also provides a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.

In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition, the antibody is A7, B11, B22, or a combination thereof.

In another aspect, there is provided a method of making an anti-CD 73 antibody comprising: culturing the host cell of the invention, and isolating the antibody from the host cell.

In another aspect, there is provided the use of an anti-CD 73 antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a neoplastic disease.

In another aspect, the invention provides a method of treating a neoplastic disease in a subject comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof of the invention or a pharmaceutical composition of the invention.

In another aspect, the invention relates to an antibody of the invention or an antigen binding fragment thereof or a pharmaceutical composition of the invention for use in the treatment of a neoplastic disease.

In one embodiment, the neoplastic disease described herein is a CD73 positive tumor. In another embodiment, the CD 73-positive tumor is lung cancer (e.g., non-small cell lung cancer), ovarian cancer, uterine/cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, renal cancer, bladder cancer, breast cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, hematological malignancy, head and neck cancer, melanoma, myeloma, gastric cancer, sarcoma, and virus-related cancers.

In another aspect, the invention provides a method of reducing the level of adenosine in a CD73 expressing tumor cell comprising contacting the cell with an anti-CD 73 antibody or antigen binding fragment thereof of the invention.

In another aspect, the invention provides a method of inhibiting the growth of a tumor cell expressing CD73 comprising contacting the cell with an anti-CD 73 antibody or antigen-binding fragment thereof of the invention.

In another aspect, the invention provides a method of stimulating an antigen-specific T cell response comprising administering to an individual in need thereof an anti-CD 73 antibody or antigen-binding fragment thereof of the invention.

In another aspect, the invention provides a method of stimulating an immune response in an individual comprising administering to an individual in need thereof an anti-CD 73 antibody or antigen-binding fragment thereof of the invention.

In some embodiments, the above methods or uses further comprise administering other anti-tumor therapeutic means, such as administration of a chemotherapeutic agent, an antibody targeting other tumor-specific antigens, or radiation therapy.

In the present invention, the CD73 is human or murine CD73, the sequence of which is well known. For example, the sequence of human CD73 subtype 1 is shown in SEQ ID NO. 27 and the sequence of human CD73 subtype 2 is shown in SEQ ID NO. 28.

The anti-CD 73 antibodies of the invention are capable of specifically binding to CD73, endocytosing CD73 into cells, such as tumor cells, by antibody-mediated internalization of CD73, thereby inhibiting the enzymatic activity of CD73, activating T cells, and thus inhibiting tumor growth.

Drawings

FIG. 1 shows the flow-through analysis (FACS) binding curves of the A7, B11 and B22 antibodies to human CD73 positive Calu-6 cells (human lung adenocarcinoma cell line).

FIG. 2 shows a graph of inhibition of CD73 enzyme activity. After the A7, B11 and B22 antibodies are treated, the AMP generated by the ATP treated by the Calu-6 cells is reduced, and the surface CD73 enzyme activity is inhibited, wherein the inhibition effect of the A7 and B11 antibodies is better than that of the B22 antibodies.

FIG. 3 shows the percentage of endocytosis of CD73 on the surface of Calu-6 cells treated with the A7, B11 and B22 antibodies, showing antibody-mediated endocytosis of CD73 by the antibodies labeled in Calu-6 cells over time.

Fig. 4 shows an antibody charge isomer profile, wherein a is antibody A7, B is antibody B11, and C is antibody B22.

Fig. 5 shows SEC profiles of antibodies, where a is antibody A7, B is antibody B11, and C is antibody B22.

Detailed Description

Other features and advantages of the invention are set forth in the following detailed description and examples, which should not be construed as limiting. The content of the invention is defined by the scope of the claims.

Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "at least one" or "one or more" may mean 1,2,3, 4,5, 6, 7, 8 or more.

As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally or partially or fully synthetically (e.g., recombinantly) produced, including any fragment thereof comprising at least a portion of the variable region of an immunoglobulin molecule that retains the binding specificity of a full-length immunoglobulin. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies encompass antibody fragments. As used herein, the term antibody thus includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, fab fragments, fab ' fragments, F (ab ') 2 fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd ' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotype (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b). In a preferred embodiment, the antibody of the invention is a human antibody.

As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds an antigen, and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as an antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, fab ', F (ab ') 2, single chain Fv (scFv), fv, dsFv, diabodies, fd, and Fd ' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology,Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov). the fragments may include multiple chains linked together, e.g., by disulfide bonds and/or by peptide linkers.

As used herein, "conventional antibody" refers to an antibody comprising two heavy chains (which may be labeled H and H ') and two light chains (which may be labeled L and L') and two antigen binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding capacity (e.g., heavy chains including but not limited to VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region (e.g., light chains including but not limited to VL chains and VL-CL chains). Each heavy chain (H and H ') is paired with a light chain (L and L', respectively).

As used herein, a full length antibody is an antibody having two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH 4) and two full length light chains (VL-CL) and a hinge region, e.g., an antibody naturally produced by B cells by antibody secretion and an antibody synthetically produced with the same domains.

As used herein, dsFv refers to Fv with engineered intermolecular disulfide bonds that stabilize VH-VL pairs.

As used herein, fab fragments are antibody fragments obtained by digestion of full length immunoglobulins with papain, or fragments of the same structure synthetically produced, e.g., by recombinant methods. The Fab fragment comprises a light chain (comprising VL and CL) and another chain comprising the variable domain of the heavy chain (VH) and one constant region domain of the heavy chain (CH 1).

As used herein, a F (ab') ² fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at a pH of 4.0-4.5, or a fragment having the same structure, e.g., synthetically produced by recombinant methods. The F (ab') ² fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises an additional few amino acids, including cysteines forming disulfide bonds linking the two fragments.

As used herein, a Fab 'fragment is a fragment comprising half (one heavy and one light chain) of the F (ab') ² fragment.

As used herein, an scFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The linker length is such that the two variable domains bridge substantially undisturbed. Exemplary linkers are (Gly-Ser) n residues interspersed with some Glu or Lys residues to increase solubility.

As used herein, a variable domain or variable region is a particular Ig domain of an antibody heavy or light chain that comprises amino acid sequences that vary between different antibodies. Each light chain and each heavy chain has one variable region domain VL and VH, respectively. The variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region comprises a CDR, which is part of an antigen binding site domain, and a Framework Region (FR).

As used herein, "antigen binding domain" and "antigen binding site" are synonymously used to refer to a domain within an antibody that recognizes and physically interacts with the cognate (cognate) antigen. The natural conventional full length antibody molecule has two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. Conventional antigen binding sites comprise loops linking antiparallel β chains within the variable region domain. The antigen binding site may comprise other portions of the variable region domain. Each conventional antigen binding site comprises 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. The hypervariable regions are also known as Complementarity Determining Regions (CDRs).

As used herein, "hypervariable region," "HV," "complementarity determining region," and "CDR," and "antibody CDR" are used interchangeably and refer to one of a plurality of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain contains 3 CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain comprises 3 CDRs, designated L CDR1 (or VL CDR 1), L CDR2 (or VL CDR 2), and L CDR3 (or VL CDR 3); the heavy chain variable region domain comprises 3 CDRs, designated H CDR1 (or VH CDR 1), H CDR2 (or VH CDR 2), and H CDR3 (or VH CDR 3). The 3 CDRs in the variable region are discontinuous along the linear amino acid sequence but are contiguous in the folded polypeptide. The CDRs are located within loops connecting parallel chains of the β -sheet of the variable domain. As described herein, CDRs are known to those of skill in the art and can be identified based on Kabat or Chothia numbering (see, e.g., Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242, and Chothia, c.et al (1987) j.mol.biol.196:901-917).

As used herein, a Framework Region (FR) is a domain located within the variable region domain of an antibody within the β -sheet; the FR region is relatively more conserved than the hypervariable region in terms of amino acid sequence.

As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises an amino acid sequence that is relatively more conserved than the amino acid sequence of a variable region domain. In conventional full length antibody molecules, each light chain has a single light chain constant region (CL) domain, while each heavy chain comprises one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3, and CH4. Full length IgA, igD and IgG isotypes comprise CH1, CH2, CH3 and hinge regions, while IgE and IgM comprise CH1, CH2, CH3 and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thus helping to interact with the antigen and turn the antibody arm. The antibody constant regions may serve effector functions such as, but not limited to, clearing antigens, pathogens, and toxins that the antibody specifically binds, such as by interacting with various cells, biomolecules, and tissues.

As used herein, a functional region of a VH domain is at least a portion of a complete VH domain that retains at least part of the binding specificity of the complete VH domain (e.g., by retaining one or more CDRs of the complete VH domain) such that the functional region of the VH domain binds antigen alone or in combination with another antibody domain (e.g., a VL domain) or region thereof. The functional region of an exemplary VH domain is a region comprising CDR1, CDR2, and/or CDR3 of the VH domain.

As used herein, a functional region of a VL domain is at least a portion of an intact VL domain that retains at least a portion of the binding specificity of the intact VL domain (e.g., by retaining one or more CDRs of the intact VL domain) such that the functional region of the VL domain binds antigen alone or in combination with another antibody domain (e.g., VH domain) or region thereof. The functional region of an exemplary VL domain is a region comprising CDR1, CDR2, and/or CDR3 of the VL domain.

"Affinity" or "binding affinity" KD is often determined by measuring the equilibrium association constant (ka) and equilibrium dissociation constant (KD) and calculating the quotient of KD divided by ka (kd=kd/ka). As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with an alloantigen through non-covalent interactions between the antibody and the antibody binding site of the antigen. The antigen may be an isolated antigen or be present in, for example, a tumor cell.

In one embodiment of the invention, an antibody or antigen binding fragment thereof of the invention can bind a target antigen (e.g., CD73, such as human CD 73) with an affinity of 2x 10 ^-9 M or less, 1 x 10 ^-9 M or less, 9 x 10 ^-10 M or less, 8 x 10 ^-10 M or less, 7 x 10 ^-10 M or less, 6 x 10 ^-10 M or less, 5 x 10 ^-10 M or less, 4 x 10 ^-10 M or less, 3 x 10 ^-10 M or less, 2x 10 ^-10 M or less, 1 x 10 ^-10 M or less.

Affinity can be readily determined using conventional techniques, for example by equilibrium dialysis; the general procedure listed by the manufacturer was utilized by using a BIAcore 2000 instrument; performing a radioimmunoassay by using the radiolabeled target antigen; or by other methods known to the skilled person.

An "isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is not associated with components that accompany it in its natural state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) is not found in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system that differs from the naturally derived cell of the polypeptide will be "isolated" from its naturally associated components. The protein may also be isolated such that it is substantially free of naturally associated components, i.e., using protein purification techniques well known in the art.

In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art, and can generally be made without altering the biological activity of the resulting molecule. In general, one skilled in the art recognizes that single amino acid substitutions in the non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings pub. Co., p.224).

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen binding fragments.

Sequence "identity" has art-recognized meanings and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity may be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (see, e.g., ：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991). although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrello, H. & Lipman, d., SIAM J APPLIED MATH 48:1073 (1988)).

As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, coomassie blue staining after gel electrophoresis, lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell that is used to receive, hold, replicate, and amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, heLa cells, HEK cells such as HEK 293 cells.

As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those comprising nucleic acids encoding polypeptides. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, for amplifying the nucleic acid or for expressing/displaying the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. Vectors for artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

As used herein, vectors also include "viral vectors" or "viral vectors". The vector of the virus is an engineered virus that is operably linked to an exogenous gene to transfer (as a vehicle or shuttle) the exogenous gene into the cell.

As used herein, an "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that when introduced into an appropriate host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of a host cell.

As used herein, "treating" an individual with a disease or condition means that the symptoms of the individual are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or disease progression. Treatment also includes any antibody or antigen-binding fragment thereof provided, and any pharmaceutical use of the compositions provided herein.

As used herein, "therapeutic effect" refers to the effect resulting from treatment of an individual that alters, generally improves or ameliorates symptoms of, or cures a disease or condition.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or disorder. Also, as used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., prevent or delay the occurrence or recurrence of a disease or symptom, reducing the likelihood of occurrence or recurrence of a disease or symptom. The fully prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "individual" refers to a mammal, such as a human.

Antibodies of the invention

The present invention provides an antibody directed against CD73, i.e., an anti-CD 73 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically recognizes and binds CD73.

In one embodiment, the antibody or antigen binding fragment thereof is capable of specifically recognizing and binding CD73, wherein the affinity (KD) is 1 x 10 ^-9 M or less, preferably 5 x 10 ^-10 M or less, more preferably 1 x 10 ^-10 M or less.

In another embodiment, the antibody or antigen binding fragment thereof has at least one of the following characteristics:

1) Inhibition of CD73 enzymatic activity; 2) Internalizing CD73 into cells, such as tumor cells, by antibody-mediated internalization of CD 73; 3) Activating T cells; 4) Inhibit tumor growth.

In some embodiments, the antibodies of the invention, or antigen binding fragments thereof, are capable of specifically binding to CD73 (e.g., human CD 73) and inhibiting CD73 enzymatic activity, reducing the production of adenosine.

In some embodiments, the tumors targeted include, but are not limited to, those described below with respect to neoplastic disease. In other embodiments, the antibodies or antigen binding fragments thereof of the invention are capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.

In specific embodiments, the anti-CD 73 antibody comprises an anti-CD 73 antibody A7, an anti-CD 73 antibody B11, an anti-CD 73 antibody B22, or a combination thereof.

Antibody A7

In one aspect, the invention provides an antibody A7 or antigen binding fragment thereof directed against CD73,

Wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein

The heavy chain variable region comprises:

H CDR1 comprising the amino acid sequence shown in SEQ ID NO. 1,

H CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, and

H CDR3 comprising the amino acid sequence set forth in SEQ ID No. 3;

the light chain variable region comprises:

l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 5,

L CDR2 comprising the amino acid sequence shown in SEQ ID NO. 6, and

L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 7.

In another embodiment, the anti-CD 73 antibody A7 comprises a heavy chain variable region (VH),

Wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 4.

In another embodiment, the anti-CD 73 antibody A7 comprises a light chain variable region (VL),

Wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 8.

In yet another embodiment, the anti-CD 73 antibody A7 comprises a heavy chain constant region (CH),

Wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO. 26 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 26.

In yet another embodiment, CD73 antibody A7 comprises a light chain constant region (CL),

Wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 25 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 25.

Antibody B11

In another aspect, the invention provides an antibody B11 or antigen-binding fragment thereof directed against CD73,

The heavy chain variable region comprises:

H CDR1 comprising the amino acid sequence shown in SEQ ID NO 9,

H CDR2 comprising the amino acid sequence shown in SEQ ID NO. 10, and

H CDR3 comprising the amino acid sequence shown in SEQ ID NO. 11;

the light chain variable region comprises:

L CDR1 comprising the amino acid sequence shown in SEQ ID NO. 13,

L CDR2 comprising the amino acid sequence shown in SEQ ID NO. 14, and

L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 15.

In one embodiment, anti-CD 73 antibody B11 comprises a heavy chain variable region (VH),

Wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 12.

In one embodiment, anti-CD 73 antibody B11 comprises a light chain variable region (VL),

The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 16 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 16.

In a preferred embodiment, anti-CD 73 antibody B11 comprises a heavy chain constant region (CH),

In yet another embodiment, the anti-CD 73 antibody B11 comprises a light chain constant region (CL),

Antibody B22

In a further aspect, the invention provides an antibody B22 or antigen binding fragment thereof directed against CD73,

The heavy chain variable region comprises:

H CDR1 comprising the amino acid sequence shown in SEQ ID NO:17,

H CDR2 comprising the amino acid sequence shown in SEQ ID NO. 18, and

H CDR3 comprising the amino acid sequence shown in SEQ ID NO. 19;

the light chain variable region comprises:

l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 21,

L CDR2 comprising the amino acid sequence shown in SEQ ID NO. 22, and

L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 23.

In another embodiment, the anti-CD 73 antibody B22 comprises a heavy chain variable region,

Wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 20.

In another embodiment, the anti-CD 73 antibody B22 comprises a light chain variable region,

Wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 24 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 24.

In yet another embodiment, the anti-CD 73 antibody B22 comprises a heavy chain constant region (CH),

In yet another embodiment, CD73 antibody B22 comprises a light chain constant region (CL),

Neoplastic disease

Blocking CD73 by the antibodies of the invention may enhance the immune response to cancer cells in a patient. CD73 converts ATP with immune activating effects to adenosine with immune suppressing effects. Adenosine as a signaling molecule can mediate its biological effects by a variety of receptors (including A1, A2A, A2B and A3), e.g., binding of adenosine to adenosine receptor A2AR (adenosine receptor AR) can inhibit T cell immunity, thereby helping cancer cells escape the T cell "chase-kill". Tumor cells can also express CD73 and release adenosine, thereby reducing antitumor activity. Targeting CD73 can produce good anti-tumor effects, alter tumor immune microenvironment by increasing T lymphocytes and tumor leukocyte populations, and reduce tumor Myeloid Derived Suppressor Cells (MDSCs).

The antibodies or antigen binding fragments thereof of the invention may be used to treat neoplastic diseases. Preferred neoplastic diseases (or cancers) that can be prevented and/or treated using the antibodies or antigen binding fragments thereof of the invention include cancers that generally respond to immunotherapy. Non-limiting examples of cancers that can be treated include, but are not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine fibroids, and osteosarcoma. Examples of other cancers include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the foregoing. The antibodies of the invention are also useful in the treatment of metastatic cancers, particularly those expressing CD 73. In yet another embodiment, the neoplastic disease (or cancer) is lung cancer, ovarian cancer, colorectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer or osteosarcoma, particularly colorectal cancer or non-small cell lung cancer. In one embodiment, the neoplastic disease is a CD73 positive tumor.

Nucleic acid, vector and antibody production method

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence region encoding an antibody or antigen binding fragment thereof of the invention as described above. In some embodiments, the nucleotide sequence of the nucleotide sequence region may be codon optimized for the host cell used for expression. For example, the nucleotide sequence of the nucleotide sequence region includes SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, or SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO.41, SEQ ID NO. 42, SEQ ID NO. 43, or SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51; further preferably, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO:32 and SEQ ID NO:36, or SEQ ID NO:40 and SEQ ID NO:44, or SEQ ID NO:48 and 52.

The invention also provides an expression vector comprising at least one of the nucleic acid molecules of the invention described above.

In one aspect, the invention also provides a host cell transformed with at least one of the nucleic acid molecules or expression vectors of the invention described above.

Yet another aspect of the invention relates to a method of producing an anti-CD 73 antibody or antigen-binding fragment thereof, the method comprising: (i) Culturing a host cell of the invention under conditions suitable for expression of the nucleic acid molecule or expression vector, and (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by said nucleic acid molecule or expression vector.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In one embodiment, the antibodies include anti-CD 73 antibody A7, anti-CD 73 antibody B11, anti-CD 73 antibody B22, or a combination thereof, particularly anti-CD 73 antibody B11.

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. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, immunoconjugate, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) Oil-soluble antioxidants such as ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents.

The prevention of the presence of microorganisms may be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide in the composition. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion in the composition of delayed absorption agents, for example, monostearates and gelatins.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except insofar as they are incompatible with the active compound, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, the dispersants are prepared by incorporating the active compound into a sterile carrier which contains a basic dispersion medium and the other required ingredients enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) from a previously sterile-filtered solution thereof to yield a powder of the active ingredient plus any additional desired ingredient.

The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form is generally the amount of the composition that produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient, on a 100% basis, in combination with a pharmaceutically acceptable carrier.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as needed for the emergency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specific description of dosage unit forms of the invention is limited to and directly depends on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art for formulating such active compounds for use in the treatment of sensitivity in individuals.

For administration of antibody molecules, the dosage range is about 0.0001 to 100mg/kg, more typically 0.01 to 20mg/kg of the recipient body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly dosing, biweekly dosing, tricyclically dosing, weekly dosing, monthly dosing, 3 months dosing, 3-6 months dosing, or slightly shorter initial dosing intervals followed by longer post dosing intervals. In one embodiment, the dosage used may be 1200mg administered every three weeks. The mode of administration may be intravenous drip.

Alternatively, the tumor-directed antibody molecule may be administered as a sustained release formulation, in which case less frequent administration is required. Dosages and frequencies will vary depending on the half-life of the antibody molecule in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over a long period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient exhibits a partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

The actual dosage level of the active ingredient in the pharmaceutical composition may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response to the particular patient, composition and mode of administration without toxicity to the patient. The dosage level selected will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention being used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and/or materials being used in combination with the particular compositions being used, the age, sex, weight, condition, general health and medical history of the patient undergoing treatment, and like factors well known in the medical arts.

An "effective amount" of an antibody or antigen-binding fragment thereof of the invention preferably results in a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of the disease asymptomatic phase, or prevention of injury or disability due to suffering from the disease. For example, for the treatment of a tumor, an "effective amount" of an antibody or antigen-binding fragment thereof of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it may be assessed by examining the ability to inhibit cell growth, which inhibition may be determined in vitro by assays well known to those of skill in the art. An effective amount of an antibody or antigen-binding fragment thereof of the invention is capable of reducing tumor size or otherwise alleviating a symptom in a subject, such as preventing and/or treating metastasis or recurrence. Such amounts may be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The antibodies of the invention, or antigen-binding fragments thereof, or the pharmaceutical compositions of the invention, may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will appreciate that the route and/or mode of administration will vary depending upon the desired result. Preferred routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the antibodies of the invention or antigen-binding fragments thereof or the pharmaceutical compositions of the invention may be administered by a non-parenteral route, such as topical, epidermal or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

The active compounds can be prepared with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Many methods of preparing such formulations are patented or generally known to those skilled in the art.

The antibodies or antigen binding fragments thereof of the invention in the pharmaceutical compositions may also be conjugated to a therapeutic moiety such as a cytotoxin, radioisotope or biologically active protein.

Cytotoxins include any agent that is detrimental to cells (e.g., killer cells). Examples include, but are not limited to: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thioglycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax-dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogues or homologs.

Therapeutic agents useful for conjugation also include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, amamide), alkylating agents (e.g., nitrogen mustard, chlorambucil, phenylalanine nitrogen mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptavidin, mitomycin C and cisplatin (II) (DDP) cisplatin), anthranilotics (e.g., daunorubicin (formerly known as daunorubicin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly known as actinomycin), bleomycin, mithramycin and Aflatoxin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies of the present invention include sesquialter mycin, spinosad, maytansinoid, auristatin, and derivatives thereof.

Cytotoxins may be conjugated to antibodies of the invention using linker techniques used in the art. Examples of types of linkers that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Alternatively, for example, a linker within the lysosomal compartment that is susceptible to cleavage by low pH or by a protease, such as a protease preferentially expressed in tumor tissue, such as a cathepsin (e.g., cathepsin B, C, D).

Antibodies of the invention may also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioisotopes. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177. Methods of preparing radioactive antibody conjugates are well known in the art.

The antibodies of the invention may also be conjugated to proteins having a desired biological activity and may be used to modify a particular biological response. Such biologically active proteins include, for example: toxins having enzymatic activity or active fragments thereof, such as abrin, ricin a, pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon-gamma; or a biological response modifier, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), interleukin-10 ("IL-10"), granulocyte-macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other immune factors such as IFN, etc.

Combination therapy

The antibodies or pharmaceutical compositions of the invention may be administered in combination with a chemotherapeutic agent or an antibody targeting other tumor antigens. The antibodies of the invention and the chemotherapeutic agent or antibodies targeting other tumor antigens may all be administered at one time or separately. When administered separately (in the case of using mutually different administration regimens), they can be administered continuously without interruption or at predetermined intervals. The antibodies of the invention or the pharmaceutical compositions of the invention may also be combined with radiation therapy, for example comprising administering ionizing radiation to the patient prior to, during and/or after the administration of the antibodies or pharmaceutical compositions of the invention.

Kit for detecting a substance in a sample

Also included within the scope of the invention are kits comprising an antibody or antigen-binding fragment thereof of the invention, and instructions for use. Kits generally include a label that indicates the intended use and/or method of use of the kit contents. The term label includes any written or recorded material provided on or with or otherwise with the kit.

Advantageous effects

The antibody provided by the invention has very strong affinity to a target antigen, inhibits the enzyme activity of the target antigen, reduces the generation of adenosine, has potential T cell activation and anti-tumor activity, and has excellent stability and lower side effect.

A further understanding of the present application may be obtained by reference to the specific examples which are set forth to illustrate, but are not intended to limit the scope of the present application. It will be apparent that various modifications and variations can be made to the present application without departing from the spirit of the application, and therefore, such modifications and variations are also within the scope of the application as claimed. The proportions used herein include percentages, by weight unless otherwise indicated.

Reagents and instrumentation used in the examples are all commercially available.

Example 1: production of human anti-CD 73 antibodies

Peripheral blood of 106 adult healthy persons was collected, and PBMC (peripheral blood mononuclear cells) cells were isolated by lymphocyte separation liquid, and 2.7X10 ⁹ cells were collected in total. The Trizol method extracts total RNA, which is reverse transcribed into cDNA. Referring to the method in recombinant antibody (science Press, 2005), the conventional PCR method amplifies variable region genes of different antibody subtypes, the antibody variable region genes are cloned into pDF vector which is also subjected to enzyme digestion by using conventional molecular biology technology, and E.coli XL1-Blue (Agilent Technology) is transformed by an electrotransformation method, so as to finally obtain a large-capacity antibody library of 1E+10pfu. After the 2YT culture solution is subjected to expansion culture, helper phage VCSM13 (BioVector NTCC Inc.) is added for infection, and a primary phage antibody library is obtained.

After blocking recombinant human CD73 (R & D company product) coated immune tubes (Maxisorp immune tubes, thermo Nunc) with 5% nonfat milk powder, the phage antibody library was added and incubated at 37℃for 2h; discarding unbound phage, washing with PBS-T wash for 5 times, and washing off nonspecifically adsorbed phage; phage were eluted by adding 1mL of elution buffer (0.1 mol/L glycine-HCl, pH 2.2) and neutralized with 2mol/L Tris solution; adding a log-phase XL1-Blue bacterium (Agilent Technology), a 2YT culture medium and an auxiliary phage VCSM13 for amplification and enrichment; the procedure was repeated 3-4 times, and freshly prepared log-phase XL1-Blue plates were infected with eluted phages and incubated overnight at 37℃to obtain monoclonal antibodies (see: PHAGE DISPLAY, humana Press for specific procedures and reagents).

Recombinant CD73 was incubated with 96-well plates at 4 ℃ for 16 hours and then blocked with BSA solution. Approximately 400 (5 96-well plates) individual phage clones from the phage library that had significant enrichment after screening were selected and incubated in 500 μl of 2YT broth supplemented with carbenicillin for 24 hours, the supernatant was centrifuged and the supernatant was added to the CD73 coated 96-well plate and incubated for 1h. After PBST washing, incubation with HRP-conjugated anti-phage antibody (anti-M13 HRP) was performed, and the final substrate was developed to determine the amount of phage bound to the 96-well plate. By using the experiment, 8 candidate antibodies with relatively high affinity and specific sequences are initially selected for further detection and screening.

E.coli expression is carried out on the 8 screened candidate antibodies, and the preserved monoclonal bacterial liquid is prepared according to the following steps of 1:100 were inoculated with 50ml of 2YT-Tet medium, cultured overnight for 12-16 h, the cells were collected, the pellet was collected by centrifugation and resuspended in 10ml of PBS, the cells were sonicated, the supernatant was centrifuged, and the supernatant was analyzed for affinity of CD73 Fab antibodies by fortebio, thereby performing affinity ranking (Table 1).

TABLE 1 Fab antibody affinity measured using fortebio anti-Fab sensor chip

Antibody cloning	Ka(1/Ms)	Kd(1/s)	KD(M)
				A7	3.647×10⁶	1.35×10^-3	3.7×10^-10
B11	6.517×10⁶	1.16×10^-3	1.78×10^-10
				B22	2.56×10⁶	1.263×10^-3	4.93×10^-10
A5	1.79×10⁶	2.63×10^-3	1.47×10^-9
				A10	1.51×10⁶	1.34×10^-3	8.9×10^-10
B17	1.49×10⁶	2.37×10^-3	1.59×10^-9
				B53	4.16×10⁵	3.86×10^-3	9.3×10^-9
A65	1.8×10⁵	6.19×10^-4	3.434×10^-9

Example 2: antibody expression

Clones A7, B11 and B22 were selected for sequencing and optimizing the coding genes, and the genes of the cloning variable regions A7 (SEQ ID NO:32, SEQ ID NO: 36), B11 (SEQ ID NO:40, SEQ ID NO: 44), B22 (SEQ ID NO:48, SEQ ID NO: 52) and the gene of the human IgG constant region (light chain constant region sequence: SEQ ID NO:53; heavy chain constant region sequence: 54) were fused and cloned into the expression vector pCDNA3.4 (Thermo Fisher). The eukaryotic expression vector obtained was transiently transfected into an Expi-CHO cell (Thermo Fisher). Culturing in serum-free culture medium for about 12 days, and purifying the culture supernatant with protease A medium to obtain the target antibody. The A7, B11 and B22 obtained were identical to the target sequences by sequencing.

Example 3: binding of anti-CD 73 antibodies to CD73 positive cells

Binding curves were generated using Calu-6 (endogenously expressed CD73, a human lung adenocarcinoma cell line, purchased from the national academy of sciences cell bank), DMS114 (CD 73 negative, a human small cell lung cancer cell line, purchased from the guangzhou Sakukuai biotechnology limited) as the cell to be detected, using Alexa Fluor 488 goat anti-human IgG (h+l) (Thermo Fisher) as the second antibody, using the following flow assay technique

One cell was divided into 6 parts at 6 concentrations of each antibody, with 5+10E5 cells per part. The initial concentration of anti-CD 73 antibody was 5. Mu.g/ml, 6 gradients were diluted with a 6-fold gradient of 1640 medium, added to cells, 300. Mu.l/serving of cells, and incubated at 37℃for 1h. After washing, add 1: secondary antibody at 1000 dilution was stained for 45min. After washing, fluorescence values were detected using a flow analyzer (MerkGuava).

The results shown in FIG. 1 indicate that all anti-CD 73 antibodies bind to cells naturally expressing CD73 (Calu-6). None of the three antibodies bound cells that did not express CD73 (DMS 114) (data not shown). The bound EC ₅₀ obtained for each antibody is shown in table 2.

Table 2: EC ₅₀ value of antibody binding to Calu-6 cells

Example 4: antibody affinity analysis

In this example, fortebio was used to determine antibody affinity. AHC (anti-Fc) sensor chips (Fortebio, 18-5060) were used in this experiment to bind ligand, i.e., igG antibody, followed by analyte. anti-CD 73 antibodies were diluted with 0.02% pbst buffer and antibodies A7, B11, B22 were adsorbed as ligands on the same buffer using an AHC chip. Recombinant His-tagged CD73 (offshore protein technologies Co., ltd., C446) was diluted to 8. Mu.g/ml, 4. Mu.g/ml, 1.6. Mu.g/ml, 0.64. Mu.g/ml, 0.256. Mu.g/ml and 0.102. Mu.g/ml with PBST as analyte, and affinity analysis experiments were performed. The capture time was set at 180s per concentration cycle of analyte; ligand binding time 180s with analyte; dissociation time 2400s. The raw data are imported into analysis software, zero concentration control is deducted, the reference channel is deducted to eliminate volume effect, a 1:1 binding mode fitting graph in a kinetic analysis method is used, and the affinity data of the antigen and the antibody are calculated by regression of the analysis software according to a binding curve. Experimental results indicate that all of the candidate antibodies A7, B11 and B22 can recognize the target antigen with high affinity (Table 3).

Table 3: affinity assay results for antibodies A7, B11, B22

Antibody cloning	Ka(1/Ms)	Kd(1/s)	KD(M)
				A7	1.435×10⁶	1.23×10^-4	8.6×10^-11
B11	1.361×10⁶	1.07×10^-4	7.89×10^-11
				B22	1.190×10⁶	1.063×10^-4	8.93×10^-11

Example 5 Calu-6 inhibition of cell surface CD73 enzymatic Activity

Calu-6 (endogenously expressing CD73, a human lung adenocarcinoma cell line) was used as the cell to be examined. Four monoclonal antibody solutions of A7, B11, B22, and IgG control (irrelevant antibodies) were diluted to 10 concentration gradients at an initial concentration of 30 μg/ml, 5-fold concentration gradient, with 3 multiplex wells per concentration. Calu-6 cells were digested and plated into U-bottom 96-well plates, 10000 cells/well. The antibody was then added to the cell plate, 10. Mu.l/well, placed on a 96-well plate thermostatted shaker, incubated at 37℃for 30min,150rpm/min. AMP (20 μm) and ATP (100 mM) were measured according to AMP: ATP 1640 medium = 1:1:8 ratio dilution configuration, add 10 μl per well to cell plate, incubate with a constant temperature shaker at 37 ℃ for 2h. After centrifugation, 30. Mu.l of the cell culture supernatant was added to a white-bottomed 96-well plate, and 30. Mu. L CELLTITER-Glo 2.0assay (promega/G9241) was added thereto, followed by mixing, and the microplate reader was read with a luminescence reader.

As shown in FIG. 2, the inhibition of cell surface CD73 by the antibodies of the present invention was enhanced as the concentration of the antibodies increased. Table 4 calculates IC ₅₀ for inhibition of enzyme activity by the antibodies.

Table 4: IC for inhibiting cell surface CD73 enzyme activity by antibody ₅₀

Antibody μg/ml	A7	B11	B22
				IC₅₀	0.0537	0.0336	0.08017

Example 6: CD73 endocytic activity

Endocytosis was detected using Calu-6 (endogenously expressed CD73, human lung adenocarcinoma cell line) as the cell to be detected, using Alexa Fluor 488 goat anti-human IgG (h+l) (Thermo Fisher) as the secondary antibody, using the following analytical technique.

Three monoclonal antibodies B11, A7 and B22 were incubated with three equal amounts of Calu-6 cells, respectively, at room temperature for 30min, then the different monoclonal antibody cell groups were aliquoted, centrifuged for 1h,2h and 4h, antibody supernatants were discarded, cells were washed, and 1: secondary antibody diluted at 1000 was stained for 30min. After washing, fluorescence values were detected using a flow analyser (Merk).

FIG. 3 shows that three mabs have close endocytosis effects on the surface of Calu-6 cells, increasing endocytosis over time, 2h approaching 4h endocytosis, and 4h endocytosis saturation.

Example 7: detection of antibody molecule charge isomers

Capillary isoelectric focusing electrophoresis apparatus (Protein SIMPLE ICE), capillary column (Protein Simple FC-coated)

Sample solution: and (3) taking a proper amount of system applicability sample and a test sample, adding ultrapure water to dilute to 1mg/ml, and oscillating and uniformly mixing for later use.

A proper amount of blank solution (dilution multiple is the same as that of the test sample) and sample solution are prepared according to the table 5, 150 μl of the blank solution and sample solution are added into an inner cannula, the inner cannula is placed into a centrifuge tube, and the centrifuge tube is centrifuged at 12000rpm for 1min, so that a blank control solution, a system applicability solution and a test sample solution are obtained.

TABLE 5 sample injection solution formulation method

Reagent(s)	Volume of	Final volume percent
			1% Methylcellulose	70μl	35％
3 To 10 portions of ampholyte	7.0μl	3.5％
			Amphoteric electrolyte 8-10.5	3.0μl	1.5％
The marker is as follows: pi=7.05, pi=9.50	0.5. Mu.l each, 1.0. Mu.l total	0.25% Each
			1Mg/ml sample solution/blank	50μl	25％
40MM arginine	69μl	34.5％

And (3) sequentially injecting 1 needle of a blank control solution, 3 needles of a system applicability solution, 1 needle of each solution of the to-be-tested sample, and finally injecting the system applicability solution, and operating the experiment according to the conditions of the table 6. And (3) performing correction analysis by using the self-contained operation software of the instrument and using the pI values of the two markers and the corresponding pixel position values of the two markers, and automatically calculating the corresponding pI values of the samples.

Table 6: isoelectric focusing conditions

Method parameters	Status of
		Focus Period 1Time	1.00min
Focus Period 1Voltage	1500V
		Focus Period 2Time	9.00min
Focus Period 2Voltage	3000V

Table 7 shows the ratios of the basic peak, the main peak and the acidic peak of the sample, and shows that the concentration of the main peak of the antibody accords with the analysis result of typical antibody drug property. FIG. 4 shows isoelectric point and purity profiles of samples, also consistent with the results of a typical antibody patency analysis.

Table 7: acid-base peak to main peak ratio of sample

Name of the name	Basic peak	Major peak	Acid peak
				A7	11.86	80.89	7.25
B11	1.35	86.77	11.88
				B22	50.42	43.65	5.94

Example 8: size exclusion chromatography analysis

Liquid chromatographic column: TOSOH TSK-GEL G3000 SWXL 7.8.300mm 5 μm;

Chromatographic conditions: the mobile phase is 0.02M sodium dihydrogen phosphate-0.2M sodium chloride buffer, pH7.4;

the sample is diluted to 5mg/ml with ultrapure water, 20 μl of the sample is taken for loading detection, the wavelength is 280nm, the flow rate is 0.5ml/min, and the sample is operated for 30min at room temperature.

Table 8 shows the polymer, monomer, fragment ratios of each sample as determined by size exclusion, consistent with typical antibody formation analysis results. Figure 5 shows a specific SEC chromatogram consistent with the results of a typical antibody drug formulation assay.

Table 8: component ratio measured by antibody molecular exclusion

Name of the name	Polymer body	Monomer(s)	Fragments
				A7	0.459	99.310	0.231
B11	0.075	99.842	0.084
				B22	0.250	99.640	0.110

Example 9: determination of monoclonal antibody purity by capillary gel electrophoresis

Instrument: capillary electrophoresis apparatus Agilent Technology 7100, capillary Agilent Technology having an inner diameter of 50 μm, running Buffer Beckmann coulter Co., ltd., SDS-MW Gel Buffer-proprietary formulation, pH8,0.2% SDS;

Solution preparation: sample buffer (100 mM Tris-HCl, pH9.0,1% SDS); 0.1M hydrochloric acid wash; 800mM iodoacetamide solution. Run solution formulation (table 9).

Table 9: run solution formulation table

Note that: sequentially placing sample bottles with serial numbers 1-12 on positions 1-12 (or positions 15-26) of a sample tray of a capillary electrophoresis apparatus

CE-SDS reduction electrophoresis sample preparation: including test solutions and blank solutions.

A proper amount of sample is taken, the sample buffer solution and mercaptoethanol are added for dilution to 1.0mg/ml, wherein the volume ratio of the mercaptoethanol is 5%, and after uniform mixing, water bath is carried out for 15min at 70 ℃; taking out, cooling to room temperature, centrifuging for 1min at 6000 rpm, transferring 75 μl of the solution into a sample bottle, ensuring no bubble generation, and replacing the sample with sample buffer to obtain a blank control solution.

Preparing a CE-SDS non-reducing electrophoresis sample, namely a sample solution: taking a sample, adding a sample buffer solution and 800mM iodoacetamide solution to dilute to 1.0mg/ml, wherein the volume ratio of the 800mM iodoacetamide solution is 5%, and after uniform mixing, carrying out water bath at 70 ℃ for 5min; taking out, cooling to room temperature, centrifuging for 1min at 6000 rpm, and transferring 75 μl of the solution into a sample bottle to ensure no bubbles are generated. The sample buffer was used instead of the sample to obtain an empty control solution.

The detection method is shown in Table 10.

Table 10: overview of method parameters

Non-reducing electrophoresis: the peak content of immunoglobulin monomers, the sum of the peak content of low molecular fragments, and the content of HIL peaks were reported as calculated by area normalization.

And (3) reduction electrophoresis: the reported light chain content, heavy chain content and immunoglobulin heavy and light chain content were summed as calculated by area normalization.

The results are shown in Table 11: the content of the complete antibody in A7 in non-reducing CE-SDS is 82%, the content of the complete antibody in B11 is 85%, the content of the complete antibody in non-reducing CE-SDS is close to the content of the complete antibody in B11, and the content of the fragment HIL is 14-18%; HC content in the reduced CE-SDS was 64%.

Table 11: CE-SDS results table

Sequence listing

A7

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B11

B22

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B11

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B22

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Claims

1. An anti-CD 73 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region of the anti-CD 73 antibody or antigen-binding fragment thereof comprises:

h CDR1, its amino acid sequence is shown in SEQ ID NO 1,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO. 2, and

H CDR3 with the amino acid sequence shown in SEQ ID NO 3;

The light chain variable region of the anti-CD 73 antibody or antigen-binding fragment thereof comprises:

L CDR1 with the amino acid sequence shown in SEQ ID NO 5,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 6, and

L CDR3 with the amino acid sequence shown as SEQ ID NO. 7; or alternatively

h CDR1, the amino acid sequence of which is shown as SEQ ID NO. 17,

H CDR2, the amino acid sequence of which is shown in SEQ ID NO. 18, and

H CDR3 with the amino acid sequence shown as SEQ ID NO. 19;

L CDR1 with the amino acid sequence shown in SEQ ID NO. 21,

L CDR2 having an amino acid sequence as shown in SEQ ID NO. 22, and

L CDR3 has the amino acid sequence shown in SEQ ID NO. 23.

2. The anti-CD 73 antibody or antigen-binding fragment thereof according to claim 1, wherein,

The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 4, or has at least 85%, at least 90%, at least 95% or more sequence identity with SEQ ID NO. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8, or has at least 85%, at least 90%, at least 95% or more sequence identity with SEQ ID NO. 8; or alternatively

The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 20, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 20; the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 24, or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 24.

3. The anti-CD 73 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-CD 73 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

4. An anti-CD 73 antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the heavy chain constant region has an amino acid sequence as shown in SEQ ID No. 26 or has at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID No. 26; and

The amino acid sequence of the light chain constant region is shown as SEQ ID NO. 25 or has at least 85%, at least 90%, at least 95% or more sequence identity with SEQ ID NO. 25.

5. A nucleic acid molecule comprising a nucleotide sequence region encoding the anti-CD 73 antibody or antigen-binding fragment thereof of any one of claims 1-4; preferably, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, or SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51; further preferably, the nucleotide sequence of the nucleotide sequence region comprises SEQ ID NO:32 and SEQ ID NO:36, or SEQ ID NO:48 and 52.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. A pharmaceutical composition comprising the anti-CD 73 antibody or antigen-binding fragment thereof of any one of claims 1-4 and a pharmaceutically acceptable carrier.

9. A method of making the anti-CD 73 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising: culturing the host cell of claim 7, and isolating the antibody from the host cell.

10. Use of an anti-CD 73 antibody or antigen-binding fragment thereof according to any one of claims 1-4 or a pharmaceutical composition according to claim 8 for the manufacture of a medicament for the treatment of a neoplastic disease; preferably, the neoplastic disease is a CD73 positive tumor; preferably, the CD 73-positive tumor is lung cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colon cancer, rectal cancer, renal cancer, bladder cancer, breast cancer, germ cell cancer, bone cancer, liver cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, skin cancer, central nervous system neoplasms, lymphomas, hematological malignancies, head and neck cancer, glioma, melanoma, myeloma, gastric cancer, sarcoma, and virus-associated cancers.