CN112574313B - anti-CD73 antibodies and uses thereof - Google Patents

Abstract

The invention provides anti-CD73 antibodies and uses thereof. Specifically, the invention provides a high-affinity high-bioactivity CD73 antibody, which can be combined with CD73 with high affinity, can obviously inhibit CD73 protease activity, and can induce tumor cell internalization of CD73, so that the cancer-related diseases can be treated and/or prevented.

Description

anti-CD73 antibodies and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an anti-CD73 antibody and application thereof.

Background

CD73, cluster of differentiation 73, also called 5' -nucleotidase, is anchored to the cell membrane by GPI (glycosylphosphatidylinositol) and is structured as a dimer. CD73 plays an important role in tumor microenvironment, AMP can be converted into adenosine, adenosine is combined with a specific G protein coupled receptor, so that immune cells are abnormally infiltrated at tumor sites, the proliferation and migration of various cancers are regulated, the functions of a T cell-related immune system are mainly inhibited, and the CD73 is a marker for poor tumor prognosis.

In a number of studies in the past, it has been shown that inhibition of CD73 can prevent tumor development and metastasis. Both small molecule enzyme inhibitors of CD73 and large molecule antibodies showed anti-tumor effects. CD73 is expressed on the surface of various tumor cells, including lung cancer, melanoma, colon cancer, pancreatic cancer and other tumor cells. In addition, various immune cells are also affected by CD73, and CD73 is expressed on the surface of Tregs and participates in the inhibitory regulation of the Tregs on immune activation; secreted adenosine can bind to adenosine receptors on DC cells, thereby inhibiting DC function; high expression of CD73 on M2-type macrophages enhanced tumor metastasis.

The CD73 target has great potential in tumor treatment, and the CD73 antibody can also enhance the effects of the PD1 antibody and the CTLA4 antibody. However, no specific targeting antibody for CD73 is currently on the market. In addition, murine mabs have clinical limitations because they elicit human anti-mouse antibody responses (HAMA) when treated clinically. The antibody humanization technology can greatly reduce the immunogenicity of the murine monoclonal antibody.

Thus, in view of the role and function of CD73 in various types of related diseases, there is a need in the art to develop an anti-CD73 humanized antibody suitable for the treatment of patients.

Disclosure of Invention

The invention aims to provide an anti-CD73 humanized antibody with high affinity and high biological activity.

In a first aspect of the invention, there is provided an anti-CD73 antibody, said antibody comprising a light chain and a heavy chain,

and, the light chain variable region of the light chain comprises the following three light chain CDRs:

VL-CDR1 shown in SEQ ID NO. 8,

VL-CDR2 shown in SEQ ID NO. 9, and

VL-CDR3 shown in SEQ ID NO. 10;

wherein the heavy chain variable region of the heavy chain comprises the following three heavy chain CDRs:

VH-CDR1 shown in SEQ ID NO. 3,

VH-CDR2 shown in SEQ ID NO:4, and

VH-CDR3 shown in SEQ ID NO: 5.

In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, and more preferably 1) amino acid and capable of retaining the binding affinity of CD 73.

In another preferred embodiment, the light chain of said antibody comprises said three light chain CDRs and a light chain framework region for linking the light chain CDRs; and the heavy chain of said antibody comprises said three heavy chain CDRs and a heavy chain framework region for linking the heavy chain CDRs.

In another preferred embodiment, the light chain variable region further comprises an FR region of human or murine origin.

In another preferred embodiment, the light chain variable region is selected from the group consisting of: the light chain variable region with the sequence shown in SEQ ID NO. 6 or SEQ ID NO. 16.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.

In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.

In another preferred embodiment, the heavy chain variable region is selected from the group consisting of: the heavy chain variable region with the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 15.

In another preferred embodiment, the heavy chain of said antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.

In another preferred embodiment, the antibody is a humanized antibody.

In another preferred embodiment, the antibody binds to human CD73 protein with an affinity constant KD (M) of (0.5-10). times.10^-9Preferably (1-9). times.10^-9More preferably (5-6). times.10^-9。

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody comprises a monospecific, bispecific, or trispecific antibody.

In a second aspect of the present invention, there is provided a recombinant protein having:

(i) a light chain and/or a heavy chain, or an anti-CD73 antibody formed by said light chain and said heavy chain,

wherein the light chain variable region comprises the 3 light chain CDRs shown in SEQ ID NO 8, 9 and 10, and the heavy chain variable region comprises the 3 heavy chain CDRs shown in SEQ ID NO 3, 4 and 5;

and (ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, said recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to said element (i).

In a third aspect of the invention, there is provided an antibody preparation comprising:

(a) an antibody according to the first aspect of the invention; and

(b) a vector, said vector comprising: a buffer, sterile water, and optionally a surfactant.

In a fourth aspect of the invention, there is provided a kit comprising an antibody preparation according to the third aspect of the invention, and a container for holding the antibody preparation.

In a fifth aspect of the invention there is provided a CAR construct comprising a scFv segment of an antigen binding region which is a binding region which specifically binds to CD73 and which scFv segment has a light chain variable region comprising 3 light chain CDRs as set out in SEQ ID NOs 8, 9 and 10 and a heavy chain variable region comprising 3 heavy chain CDRs as set out in SEQ ID NOs 3, 4 and 5.

In a sixth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the fifth aspect of the invention.

In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.

In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).

In a seventh aspect of the present invention, there is provided an antibody drug conjugate comprising:

(a) an antibody moiety selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In an eighth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the seventh aspect of the invention, or a combination thereof, wherein the active ingredient is for:

(a) preparing a detection reagent or a kit;

(b) preparing a medicament or preparation for preventing and/or treating CD73 related diseases; and/or

(c) Preparing a medicament or a preparation for preventing and/or treating cancer or tumor.

In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.

In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the fifth aspect of the invention, an immune cell according to the sixth aspect of the invention, an antibody drug conjugate according to the seventh aspect of the invention, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred embodiment, the pharmaceutical composition is used for treating tumors.

In another preferred embodiment, the tumor is a tumor highly expressing CD 73.

In a tenth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) an antibody according to the first aspect of the invention; or

(2) A recombinant protein according to the second aspect of the invention;

(3) a CAR construct according to the fifth aspect of the invention.

In an eleventh aspect of the invention there is provided a vector comprising a polynucleotide according to the tenth aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a twelfth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the eleventh aspect of the invention into which has been integrated a polynucleotide according to the tenth aspect of the invention.

In a thirteenth aspect of the invention, there is provided an in vitro non-diagnostic method for detecting CD73 protein in a sample, said method comprising the steps of:

(1) contacting said sample in vitro with an antibody according to the first aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD73 protein in the sample.

In a fourteenth aspect of the present invention, there is provided an in vitro method for detecting (including diagnostic or non-diagnostic) CD73 protein in a sample, said method comprising the steps of:

(1) contacting said sample in vitro with an antibody according to the first aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD73 protein in the sample.

In a fifteenth aspect of the present invention, there is provided a method of treating a CD 73-associated disease, the method comprising:

administering to a subject in need thereof an antibody according to the first aspect of the invention, a drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the magnitude of the affinity of CQ134 for recombinant hCD73 protein.

FIG. 2 shows the detection of the binding of CQ134 to recombinant monkey CD73 by ELISA.

FIG. 3 shows the binding of CQ134 to human CD73 positive cells (SK-OV-3 cells).

FIG. 4 shows the binding of CQ134 to monkey CD73 positive cells (CHOK 1-cynoCD73 cells).

FIG. 5 shows the effect of CD73 antibodies (CQ134 and MEDI-9447) on the activity of rhCD73 protein.

FIG. 6 shows the effect of CQ134 on CD73 activity on human SK-OV-3 cells.

FIG. 7 shows the effect of CQ134 on CD73 activity on CHOK1-cynoCD73 cells.

Fig. 8 shows the detection of the binding of the humanized antibody CQ289 to recombinant human CD73 by ELISA.

Fig. 9 shows the detection of the binding of the humanized antibody CQ289 to the recombinant monkey CD73 by ELISA.

FIG. 10 shows the binding of humanized antibody CQ289 to SK-OV-3 cells.

Fig. 11 shows the effect of humanized antibody CQ289 on the activity of rhCD73 protein.

FIG. 12 shows the effect of humanized antibody CQ289 on CD73 activity on human SK-OV-3 cells.

Fig. 13 shows the results of cell internalization mediated by the CD73 antibody (humanized antibodies CQ289 and MEDI-9447).

Fig. 14 shows that humanized antibody CQ289 inhibited the proliferation of human melanoma cell a375 in mice.

Detailed Description

The present inventors have extensively and intensively studied and, through a large number of screenings, have unexpectedly obtained an anti-CD73 humanized antibody having excellent affinity. Experiments have shown that the CD73 antibodies of the invention are capable of high affinity binding to CD 73. Through the binding experiment and affinity detection of the humanized antibody, the antibody specifically binds to human and monkey CD73 protein and CD73 positive cells and has the effect of inhibiting the activity of CD73 protease. The antibodies of the invention may also induce tumor cell internalization of CD 73. In mouse experiments, the antibodies of the invention showed superior antitumor activity to MEDI-9447 of AstraZeneca (AZ)/Medmimmune. The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of the CD73 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, comprising any one of the CD73 antibodies of the invention and compositions thereof. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.

Antibodies

As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or different classes called immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are referred to as a, d, e, g, and m, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3, and IgG 4. Light chains are classified as kappa or lambda chains by differences in the constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 FR Regions (FRs) which are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, which are sequentially arranged from amino terminus to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain, the light chain hypervariable region (LCDR), designated LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain, the hypervariable region of the Heavy Chain (HCDR), are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDR2-3), or in accordance with Kabat and chothia numbering convention (HCDR 1). The four FR regions in the native heavy and light chain variable regions are in a substantially b-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a b-folded structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The term "antigen-binding fragment," as used herein, refers to a Fab fragment, Fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen-binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.

As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.

As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell transfected with a vector by splicing a V region gene of a murine antibody to a C region gene of a human antibody into a chimeric gene. Not only retains the high specificity and affinity of the parent mouse antibody, but also ensures that the humanized Fc segment can effectively mediate the biological effect function.

As used herein, the term "humanized antibody", is a variable region engineered version of a murine antibody of the invention, having CDR regions derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; that is, the CDR sequence of the mouse antibody is grafted to the framework sequences of different types of human germline antibodies. Because the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

Table a.

anti-CD73 antibodies

As used herein, the term "CD 73" generally refers to natural or recombinant human CD73, as well as non-human homologs of human CD 73.

The present invention provides a highly specific and high affinity antibody to CD73 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.

In the invention, a high-quality human CD73 antigen is selected to immunize a mouse, a mouse immune cell is taken to construct a phage library, a special phage display technology is adopted, a single-chain antibody (scfv) is displayed on the surface of the phage, a CD73 antigen is subjected to multiple rounds of screening to obtain an antibody sequence, the antibody sequence is constructed on a eukaryotic expression vector of an hIgG1 frame in a recombinant construction mode, and a mammal cell expresses to obtain an anti-CD73 full-length antibody (namely, a human-mouse chimeric antibody is obtained).

In a preferred embodiment of the present invention, the obtained human murine chimeric anti-CD73 full length antibody is CQ134 antibody protein. The obtained CQ134 antibody can bind to the tumor-indicated CD73 molecule and inhibit CD73 activity on human ovarian adenocarcinoma cells. Has the potential of treating various CD73 over-expression tumors.

Preferably, the CDRs of the heavy chain variable region (VH) are selected from the group consisting of:

VH-CDR1 shown in SEQ ID NO. 3,

VH-CDR2 shown in SEQ ID NO:4, and

VH-CDR3 shown in SEQ ID NO. 5; and/or

The CDRs of the light chain variable region (VL) are selected from the group consisting of:

VL-CDR1 shown in SEQ ID NO. 8,

VL-CDR2 shown in SEQ ID NO. 9, and

VL-CDR3 shown in SEQ ID NO. 10.

Wherein, any one of the above amino acid sequences further comprises a derivative sequence with CD73 binding affinity, which is added, deleted, modified and/or substituted with at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and still more preferably a fully humanized antibody.

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab')2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.

Among them, the animal is preferably a mammal such as a mouse.

The antibodies of the invention may be murine, chimeric, humanized, CDR grafted and/or modified antibodies targeting human CD 73.

In a preferred embodiment of the present invention, any one or more of the above-mentioned SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, or a sequence having a CD73 binding affinity thereof, which has been subjected to addition, deletion, modification and/or substitution of at least one amino acid, is located in a CDR region of a heavy chain variable region (VH).

In a preferred embodiment of the present invention, any one or more of the above-mentioned SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, or a sequence thereof having a CD73 binding affinity, which has been subjected to addition, deletion, modification and/or substitution of at least one amino acid, is located in a CDR region of a light chain variable region (VL).

In a more preferred embodiment of the invention, the VH CDR1, CDR2 and CDR3 are respectively and independently selected from any one or more of SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, or sequences with CD73 binding affinity which are added, deleted, modified and/or substituted by at least one amino acid; VL CDR1, CDR2 and CDR3 are respectively and independently selected from any one or more sequences of SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, or sequences with CD73 binding affinity obtained by adding, deleting, modifying and/or substituting at least one amino acid.

In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

In a preferred embodiment of the present invention, there is provided an antibody against human CD73(CQ 134):

CQ134 light chain variable region (SEQ ID NO: 6):

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKR

CQ134 heavy chain variable region (SEQ ID NO: 1):

QVQLQQSGAELARPGASVKLSCKASGYTFTGYWMQWVRQRPGQGLEWIGAIYPGDGDTRYTQKFKGKAILTADKSSSTAYMQLSSLASEDSAVYYCATRGDWDYWYFDVWGAGTTVTVSS

hIgG1 constant region amino acid sequence (SEQ ID NO: 11):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

kappa chain constant region amino acid sequence (SEQ ID NO: 13):

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

the invention also provides a nucleotide sequence for coding the amino acid:

CQ134 VH nucleotide sequence (SEQ ID NO: 2):

CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTGGCTACTGGATGCAGTGGGTACGACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGAGATGGTGATACTAGGTACACTCAGAAGTTCAAGGGCAAGGCCATATTGACTGCAGATAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAACTCGGGGGGACTGGGACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTTTCCTCG

CQ134 VL nucleotide sequence (SEQ ID NO: 6):

GACATTCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGT

the selected hIgG1 constant region nucleotide sequence was (SEQ ID NO: 12):

GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

the selected kappa chain constant region nucleotide sequence is (SEQ ID NO: 14):

ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG。

humanized anti-CD73 antibodies

In 1986, Jones et al firstly transplanted the heavy chain CDR of the murine monoclonal antibody to the framework region of the heavy chain of the human antibody, and then assembled with the light chain of the murine monoclonal antibody into a complete antibody and kept the affinity similar to that of the original murine monoclonal antibody, thereby providing a thought for the development of antibody humanization technology. Queen et al succeeded in constructing a humanized antibody against CD25 in 1989 by a CDR grafting method in which a human antibody Eu framework region was humanized and amino acids of a murine antibody were retained at partial sites of the framework region to maintain affinity. In 1992 Presta et al reported a successful humanization method by CDR grafting using human antibody subgroup consensus (consensus sequence) as a template. Pedersen et al, 1994, reported humanization of antibodies using surface remodeling (resurfacing). Hsiao et al, 1994, reported humanization methods for CDR grafting with human antibody Germine sequence framework regions. Jespers et al succeeded in constructing a humanization method by a method using a phage library (Shuffling library) in 1994.

The present invention also provides a highly specific and high affinity humanized antibody against CD73(CQ 289):

CQ289 VH amino acid sequence (SEQ ID NO: 15):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMQWVRQAPGQGLEWIGAIYPGDGDTRYTQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCATRGDWDYWYFDVWGRGTLVTVSS

CQ289 VL amino acid sequence (SEQ ID NO: 16):

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIK

in another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

The antibody of the present invention may be a double-chain or single-chain antibody, and may preferably be a fully humanized antibody.

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab')₂Or other known antibody derivatives in the art, as well as any of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subclassesOne or more of them.

The antibodies of the invention may be humanized antibodies, CDR grafted and/or modified antibodies targeting CD 73.

In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

Preparation of antibodies

Any method suitable for producing monoclonal antibodies may be used to produce the CD73 antibodies of the invention. For example, an animal may be immunized with a linked or naturally occurring CD73 protein or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of CD73 may be used as an immunogen (antigen) for the production of non-human antibodies specific for CD73, which antibodies are screened for biological activity. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, baculovirus vectors, plasmids and non-viral vectors.

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

An exemplary method for preparing the CD73 antibody of the invention is described in example 1.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

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

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Antibody-drug conjugates (ADC)

The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.

Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

The antibody of the invention may be conjugated to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).

Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.

Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker degrades at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.

Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and attachment is achieved by the reactive group. Thiol-specific reactive groups are preferred and include: for example maleimide compounds, haloamides (for example iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiolsulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.

The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.

Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.

Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of a drug moiety and a linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.

Antibody formulations

The antibody has different stability in different preparation buffers, and is represented by the change of charge heterogeneity, degradation, polymerization and the like of antibody molecules, and the change of the quality properties is related to the physicochemical properties of the antibody, so that the preparation buffers suitable for the antibody need to be screened according to the physicochemical properties of different antibodies in the development process of antibody drugs. The currently commonly used antibody preparation buffer systems include phosphate buffer, citric acid buffer, histidine buffer, and the like, and according to the antibody properties, saline ions with different concentrations or excipients such as sorbitol, trehalose, sucrose, and the like, and a proper amount of surfactants such as tween 20 or tween 80 and the like are added to maintain the stability of the antibody.

The antibody drug combination preparation can effectively inhibit side reactions such as aggregation precipitation, hydrolysis, oxidation, deamidation and the like of the humanized antibody, and can effectively improve the stability of the product under the conditions of pressurization (high temperature, strong light irradiation, freeze thawing and the like), acceleration and long-term refrigeration.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or an active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition of the invention can be directly used for binding CD73 protein molecules, and thus can be used for preventing and treating CD73 related diseases. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Detection use and kit

The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.

In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention include

(1) The antibody of the invention has excellent biological activity and specificity;

(2) compared with a murine antibody and a chimeric antibody, the humanized antibody has better safety;

(3) the antibody has higher affinity through competitive screening;

(4) the antibody of the invention is derived from a phage display technology, and has stronger diversity than a hybridoma technology.

(5) The antibody of the invention has obvious inhibition effect on the enzymatic activity of CD 73.

The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

EXAMPLE 1 Generation of anti-human CD73 monoclonal antibodies

This example describes the sequence of mouse-derived anti-human CD73 single-chain antibody obtained by immunization of mouse and phage display. Balb/C mice were immunized with recombinant human CD73 protein (C446, Novoprotein), after the first immunization, booster immunization was performed every 14 days for 4 times, and mouse serum was taken for antibody titer evaluation. And (4) evaluating the effective mouse, taking B cells, extracting RNA (ribonucleic acid) for reverse transcription, and constructing a phage display library. Packing the recombinant human CD73 protein according to 1-5 microgram/ml, adding the displayed phage, screening the phage library, collecting the screened phage which is not washed away, infecting host bacteria to obtain a first round of screening library, and carrying out two-round and three-round screening according to the method. After screening is completed, the screening is carried out by phase-ELISA, and the Anti-CD73 candidate sequence is obtained by sequencing the positive identification. CQ134 VH and VL were constructed separately on eukaryotic expression vectors containing hIgG1-kappa constant region, transfected into freestyle 293F cells, cultured for 3-7 days, and the supernatants were collected and purified by protein A column to obtain CQ134 antibody protein.

CQ134 VH amino acid sequence (SEQ ID NO: 1)

QVQLQQSGAELARPGASVKLSCKASGYTFTGYWMQWVRQRPGQGLEWIGAIYPGDGDTRYTQKFKGKAILTADKSSSTAYMQLSSLASEDSAVYYCATRGDWDYWYFDVWGAGTTVTVSS

CQ134 VL amino acid sequence (SEQ ID NO:6)

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKR

hIgG1 constant region amino acid sequence (SEQ ID NO: 11)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

kappa chain constant region amino acid sequence (SEQ ID NO:13)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Wherein the underlined regions are CDRs (defined by IMGT, as follows):

TABLE 1 murine anti-CD73 antibody CDR sequences

Example 2 affinity detection of CQ134 antibodies

This example describes the affinity assay of CQ134 with human and monkey recombinant CD73 proteins, as well as with human CD73 positive cells (SK-OV-3, human ovarian adenocarcinoma cells) and monkey CD73 cell line (CHOK 1-cynoCD 73).

(1) Affinity with human CD73 protein

The affinity of CQ134 and recombinant hCD73 protein was determined by Biacore, CQ134 was solidified and CD73 protein was diluted in a two-fold gradient (2.5 nM-40 nM), and the data obtained in an affinity diagram are shown in FIG. 1 and Table 2.

TABLE 2 CQ134 affinity to human CD73 protein

That is, the affinity of CQ134 to rhCD73 was found to be 5.238X 10^-9M。

(2) Affinity with monkey CD73 protein

The recombinant monkey CD73 protein was coated at 3. mu.g/ml, CQ134 was added in a gradient dilution, and the binding of CQ134 to recombinant monkey CD73 was detected by ELISA, and the results are shown in FIG. 2.

EC50 for CQ134 was calculated to be 0.3211. mu.g/ml.

(3) Binding to human CD73 positive cells

Take 4X 10⁵Each cell SK-OV-3 was incubated for 1h with a three-fold gradient (0.009-20. mu.g/ml) of diluted CQ134 protein, washed 3 times with PBS, and subjected to Anti-hFC-APC (purchased from Jackson immunology) flow-on-machine detection. The results are plotted as an S-curve in FIG. 3.

EC50 for CQ134 was calculated to be 1.883. mu.g/ml.

(4) Binding to monkey CD73 positive cells

Constructing monkey CD73 full length on NEO resistance-containing eukaryotic expression vector, transfecting CHO-K1 cell, screening with G418 to obtain CHOK1-cynoCD73 cell strain, and taking 4 × 10⁵CHOK1-cynoCD73 cells were plated with Anti-hFC-APC (purchased from Jackson immunology) and assayed on a flow machine. The results are plotted as an S-curve in FIG. 4.

Calculated EC50 for CQ134 was 0.022 μ g/ml.

Example 3 CQ134 mediated inhibition of CD73 enzyme Activity assay

This example shows mainly that CQ134 inhibits CD73 protein and cellular enzymatic activity:

(1) inhibition of CD73 protease activity

CD73 protein is diluted to a working concentration of 5 mug/ml, CD73 antibody diluted by a triple gradient (0.001-10 mug/ml) is added, the mixture is incubated at 37 ℃ for 15min, a mixture of 1mM ATP and AMP is added, the mixture is incubated at 37 ℃ for 30min, Cell Titer-Glo detection reagent (purchased from promega) is added in the same volume, a self-luminescence value is read by an enzyme reader, and the activity change of rhCD73 is obtained by calculating the enzyme activity by taking the non-added antibody as 100 percent.

The results show that: CQ134 has better protease activity inhibition than control molecule MEDI-9447.

(2) Inhibition of CD73 enzyme activity on human SK-OV-3 cells

Take 5X 10⁴SK-OV-3 cells, adding three times gradient (0.009-6.67 u g/ml) diluted antibody, 37 ℃ incubation for 15min, adding 1mM AMP 37 ℃ incubation for 2h, adding 1mM ATP, immediately adding Cell Titer-Glo detection reagent, enzyme reader reading the self luminescence value, with no antibody added as 100% of enzyme activity, to obtain CD73 activity change on cells FIG. 6.

Calculated IC50 for CQ134 was 0.3022. mu.g/ml.

(3) Inhibition of CD73 enzyme activity on CHOK1-cynoCD73 cells

Take 5X 10⁴CHOK1-cynoCD73 cells, three times of the diluted antibody (0.003-20 mu g/ml) is added, the cells are incubated at 37 ℃ for 15min, 1mM AMP is added and incubated at 37 ℃ for 2h, 1mM ATP is added, Cell Titer-Glo detection reagent is immediately added, a microplate reader reads the self-luminescence value, and the change of the activity of CD73 on the cells is obtained by calculating by taking the activity of 100% without the added antibody as the enzyme, and a graph 7 is shown.

Calculated IC50 for CQ134 was 0.5547. mu.g/ml.

Example 4 CQ134 humanization

Analyzing a human frame closest to CQ134 by using structural simulation and rational design to obtain a series of humanized antibodies, and evaluating to obtain an optimal humanized molecule CQ 289. CQ289 VH and VL were constructed on vectors containing hIgG1 and kappa light chain constant regions, respectively, and transfected with Freestyle 293F to obtain purified CQ289 molecule.

CQ289 VH amino acid sequence (SEQ ID NO: 15)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMQWVRQAPGQGLEWIGAIYPGDGDTRYTQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCATRGDWDYWYFDVWGRGTLVTVSS

CQ289 VL amino acid sequence (SEQ ID NO: 16)

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIK

Wherein the underlined regions are CDRs (defined as IMGT), CQ289 is a humanized antibody protein of CQ134, and the CDR regions of CQ289 are identical to those of chimeric antibody CQ 134.

Example 5 CQ289 affinity evaluation

This example shows that the humanized CD73 antibody has affinity for recombinant human and monkey CD73 proteins,

(1) affinity to SK-OV-3 cells which are CD73 positive cells. In particular, the method comprises the following steps of,

the recombinant human CD73 protein was coated at 3. mu.g/ml, and the humanized CQ289 was added in a gradient dilution, and the binding of the antibody to recombinant human CD73 was detected by ELISA, the results are shown in FIG. 8

Calculated EC50 for MEDI-9447 was 0.1667. mu.g/ml, CQ289 was 0.1062. mu.g/ml.

(2) Affinity with monkey CD73 protein

The recombinant monkey CD73 protein was coated at 3. mu.g/ml, and the humanized CQ289 diluted in gradient was added, and binding of the antibody to the recombinant monkey CD73 was detected by ELISA, the results are shown in FIG. 9

Calculated as EC50 for MEDI-9447 was 0.4041. mu.g/ml, CQ289 was 0.3219. mu.g/ml.

(3) Binding to human CD73 positive cells

Take 4X 10⁵The individual cells SK-OV-3, adding gradient dilution of humanized antibody protein, after 1h incubation, using PBS washing 3 times, adding Anti-hFC-APC (from Jackson immunology), flow-on machine detection. The results are plotted as an S-curve in FIG. 10.

CQ289 has better binding to SK-OV-3 cells than MEDI-9447, which has EC50 of 0.1205. mu.g/ml and CQ289 of 0.02351. mu.g/ml.

Comparing the binding activity of CQ134, CQ289, and MEDI-9447 (table 3), the results show that the humanized CQ289 antibody had lower EC50 and stronger binding activity to human CD73 positive cells compared to the positive control MEDI-9447.

TABLE 3 detection of binding Activity of antibodies to monkey CD73, human CD73 positive cells (SK-OV-3) and monkey CD73 positive cells (CHOK 1-cynoCD 73)

Example 6 evaluation of CQ289 enzyme Activity inhibition

This example shows primarily that humanized antibody CQ289 inhibits CD73 protein and cellular enzyme activity. In particular, the method comprises the following steps of,

(1) inhibition of CD73 protease activity

CD73 protein is diluted to a working concentration of 5 mug/ml, a humanized antibody CQ289 which is diluted in a gradient way is added, the mixture solution of 1mM ATP and AMP is added and incubated at 37 ℃ for 15min, a Cell Titer-Glo detection reagent (purchased from promega) is added in an equal volume, a self-luminescence value is read by an enzyme-labeling instrument, and the activity change of rhCD73 is calculated by taking the activity of the enzyme without the addition of the antibody as 1, so that a rhCD73 activity change graph 11 is obtained.

Calculated IC50 for CQ289 was 5.282. mu.g/ml.

(2) Inhibition of CD73 enzyme activity on human SK-OV-3 cells

Take 5X 10⁴And adding the antibody diluted in a gradient manner into SK-OV-3 cells, incubating at 37 ℃ for 15min, adding 1mM AMP, incubating at 37 ℃ for 2h, adding 1mM ATP, immediately adding a Cell Titer-Glo detection reagent, reading a self-luminescence value by an enzyme-labeling instrument, and calculating by taking the antibody not added as enzyme activity 1 to obtain a CD73 activity change graph 12 on the cells.

Calculated IC50 for CQ289 was 0.1776. mu.g/ml.

Compared with CQ134 inhibition of CD73 enzyme activity on human SK-OV-3 cells (IC 50=0.3022 μ g/ml), humanized CQ289 has more significant inhibition effect on CD73 enzyme activity on human SK-OV-3 cells (IC 50=0.1776 μ g/ml).

Example 7 CD73 antibody-mediated cellular internalization

Take 3X 10⁵And adding 0.2 mu g of CD73 antibody into each cell, incubating at 37 ℃, incubating according to the time of 0h, 2h and 3h, adding the same amount of corresponding CD73 antibody after the incubation is finished, adding Anti-Hfc-APC flow antibody after the incubation is carried out for 1h, carrying out up-flow cytometry detection after the incubation and elution, and calculating the relative fluorescence intensity (MFI) of the cells along with the change of time by taking the relative fluorescence intensity (MFI) of 0h as 1, wherein the result is shown in figure 13.

The results show that: CQ289 was comparable to MEDI9447 mediated cellular internalization results.

Example 8 CQ289 antibody inhibits the proliferation of human melanoma cells A375 in mice

NSG mice were used to construct a mouse model NSG-A375 of human melanoma cells (A375) and vaccinated human PBMC rats and non-vaccinated human PBMC control mice were dosed every other day and tumor volumes were measured. Experimental mice were set with vehicle PBS administration blank control, MEDI-9447 low dose (0.5 mpk) administration, MEDI-9447 high dose (3 mpk) administration, CQ289 high dose (3 mpk) and low dose (0.5 mpk) groups, and the results are shown in FIG. 14. Wherein mpk (Milligrams Per Kilograms) is milligram kg.

The results show that: CQ289 inhibited tumor growth better than MEDI-9447 at both low and high doses.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Wujiang Yoashan protein science and technology Co Ltd

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Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe

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caggttcagc tgcagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagttg 60

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cctggacagg gtctggaatg gattggggct atttatcctg gagatggtga tactaggtac 180

actcagaagt tcaagggcaa ggccatattg actgcagata aatcctccag cacagcctac 240

atgcaactca gcagcttggc atctgaggac tctgcggtct attactgtgc aactcggggg 300

gactgggact actggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtttcctcg 360

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Gly Tyr Trp Met Gln

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys

1 5 10 15

Gly

<210> 5

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Arg Gly Asp Trp Asp Tyr Trp Tyr Phe Asp Val

1 5 10

<210> 6

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 7

<211> 324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gacattcaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa acgt 324

<210> 8

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Gln Gly Asn Thr Leu Pro Trp Thr

1 5

<210> 11

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 12

<211> 993

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gggtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa tga 993

<210> 13

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 14

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acggtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240

cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300

ttcaacaggg gagagtgtta g 321

<210> 15

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Arg Gly Asp Trp Asp Tyr Trp Tyr Phe Asp Val Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

Claims (14)

1. An anti-CD73 antibody, wherein the antibody comprises a light chain and a heavy chain,

and, the light chain variable region of the light chain comprises the following three light chain CDRs:

VL-CDR1 shown in SEQ ID NO. 8,

VL-CDR2 shown in SEQ ID NO. 9, and

VL-CDR3 shown in SEQ ID NO. 10;

wherein the heavy chain variable region of the heavy chain comprises the following three heavy chain CDRs:

VH-CDR1 shown in SEQ ID NO. 3,

VH-CDR2 shown in SEQ ID NO:4, and

VH-CDR3 shown in SEQ ID NO: 5.

2. The antibody of claim 1, wherein the light chain of said antibody comprises said three light chain CDRs and a light chain framework region for linking the light chain CDRs; and the heavy chain of said antibody comprises said three heavy chain CDRs and a heavy chain framework region for linking the heavy chain CDRs.

3. A recombinant protein, said recombinant protein having:

(i) a light chain and a heavy chain, or an anti-CD73 antibody formed by said light chain and said heavy chain,

wherein the light chain variable region comprises the 3 light chain CDRs shown in SEQ ID NO 8, 9 and 10, and the heavy chain variable region comprises the 3 heavy chain CDRs shown in SEQ ID NO 3, 4 and 5;

and (ii) optionally a tag sequence to facilitate expression and/or purification.

4. An antibody preparation, comprising:

(a) the antibody of claim 1; and

(b) a vector, said vector comprising: buffer, sterile water.

5. A kit comprising the antibody preparation of claim 4 and a container holding said antibody preparation.

6. A CAR construct wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to CD73 and has a light chain variable region comprising 3 light chain CDRs as set forth in SEQ ID NOs 8, 9 and 10 and a heavy chain variable region comprising 3 heavy chain CDRs as set forth in SEQ ID NOs 3, 4 and 5.

7. A recombinant immune cell expressing an exogenous CAR construct of claim 6.

8. An antibody drug conjugate, comprising:

(a) an antibody moiety selected from the group consisting of: the antibody of claim 1, or the recombinant protein of claim 3, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

9. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 3, the CAR construct of claim 6, the immune cell of claim 7, the antibody drug conjugate of claim 8, or a combination thereof, the active ingredient for:

(a) preparing a detection reagent or a kit;

(b) preparing a medicament or preparation for preventing and/or treating CD73 related diseases; and/or

(c) Preparing the medicine or preparation for preventing and/or treating the cancer or tumor related to the anti-human CD73 antibody.

10. A pharmaceutical composition, comprising:

(i) an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 3, the CAR construct of claim 6, the immune cell of claim 7, the antibody drug conjugate of claim 8, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

11. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the antibody of claim 1; or

(2) The recombinant protein of claim 3;

(3) the CAR construct of claim 6.

12. A vector comprising the polynucleotide of claim 11.

13. A genetically engineered host cell comprising the vector or genome of claim 12 having the polynucleotide of claim 11 integrated therein.

14. An in vitro non-diagnostic method for detecting CD73 protein in a sample, said method comprising the steps of:

(1) contacting the sample with the antibody of claim 1 in vitro;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD73 protein in the sample.

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