CN117567613A - anti-CD 73 antibodies and uses thereof - Google Patents

Abstract

The present invention relates to an antibody, antigen-binding fragment thereof, or variant thereof, that specifically binds CD73, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDRH1, the sequence of which is shown as SEQ ID NO. 1; CDRH2, the sequence of which is shown as SEQ ID NO. 2; and CDRH3, the sequence of which is shown as SEQ ID NO. 3; the light chain variable region comprises: CDRL1, the sequence of which is shown in SEQ ID NO. 4; CDRL2, the sequence of which is shown in SEQ ID NO. 5; and CDRL3, the sequence of which is shown in SEQ ID NO. 6. The anti-CD 73 antibodies of the present application have high binding affinity for primate CD73 proteins; and has potent activity of inhibiting CD73 enzyme activity at both the soluble protein level and the cellular level. Furthermore, binding of these antibodies can induce tumor cell internalization of CD73, resulting in further reduction of CD73 activity on the cell surface. The antibodies of the invention exhibit superior in vivo and in vitro antitumor activity compared to the known reference antibody MEDI9447, making them excellent candidate drug molecules for therapeutic and diagnostic use.

Description

anti-CD 73 antibodies and uses thereof

Technical Field

The present invention relates to the field of biological medicine, in particular to anti-CD 73 antibodies and uses thereof.

Background

In the Tumor Microenvironment (TME), substances are present that inhibit immune cells, which may lead to Tumor cell escape from killing of immune cells. TME is a very complex system, consisting of a variety of cells, cytosol, enzymes, cytokines, metabolites, etc., and has the characteristics of significant hypoxia, low pH and high pressure. One of the important immunosuppressive mechanisms in this context is mediated by the CD39-CD 73-Adenosine (Adenosine) metabolic signaling pathway. It has been reported in the literature that adenosine can inhibit the immune killing effect of T cells, so that tumors can escape immunologically, while CD73 is a key enzyme catalyzing the production of adenosine, which helps to convert ATP with immune activating effect into adenosine.

CD73 is also called extracellular-5 '-Nucleotidase (Ecto-5' -Nucleotidase), is a member of the extracellular Nucleotidase family, and is a cell surface enzyme widely expressed on the surfaces of endothelial cells and lymphocytes of human bodies, such as Treg and the like. CD73 has been reported to be highly expressed in a variety of different tumor tissues, including colorectal, esophageal, pancreatic, lung, ovarian, bladder, blood, melanoma, glioma, thyroid, prostate, breast, etc. (Jin et al cancer Res2010;70:2245-2255 and Stagg et al PNAS 2010; 107:1547-1552).

CD73 antibodies are currently being developed as potential anti-cancer drugs by several biopharmaceutical companies worldwide, but none have been approved for sale to date. There is still a strong need to develop anti-CD 73 antibodies with better affinity and/or activity.

Disclosure of Invention

In view of the technical problems in the prior art, the present invention proposes an antibody, an antigen-binding fragment thereof or a variant thereof that specifically binds to CD73, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: CDRH1, the sequence of which is shown as SEQ ID NO. 1; CDRH2, the sequence of which is shown as SEQ ID NO. 2; and CDRH3, the sequence of which is shown as SEQ ID NO. 3; the light chain variable region comprises: CDRL1, the sequence of which is shown in SEQ ID NO. 4; CDRL2, the sequence of which is shown in SEQ ID NO. 5; and CDRL3, the sequence of which is shown in SEQ ID NO. 6.

In some embodiments, an antibody, antigen binding fragment thereof, or variant thereof as described above, is X in SEQ ID No.3 ₁ D (aspartic acid), E (glutamic acid), S (serine), or K (lysine); x in SEQ ID NO.3 ₂ Is G (glycine), A (alanine), W (tryptophan), F (phenylalanine), V (valine), L (leucine), I (isoleucine), P (proline) or M (methionine or methionine), preferably G (glycine) or A (alanine).

In some embodiments, the sequence of the CDRH3 of the antibody, antigen binding fragment thereof, or variant thereof as described above is set forth in SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, or SEQ ID No. 10.

In some embodiments, the sequence of the heavy chain variable region in an antibody, antigen-binding fragment thereof, or variant thereof as described above is set forth in SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, or SEQ ID No. 14; and the sequence of the light chain variable region is shown in SEQ ID NO. 15.

In some embodiments, an antibody, antigen-binding fragment thereof, or variant thereof as described above, further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from one or more of IgG, igM, igA, igE or IgD; the light chain constant region is selected from kappa or lambda chains.

In some embodiments, wherein the IgG series antibody is selected from one or more of IgG1, igG2, and IgG 4.

In some embodiments, wherein the antibody or antigen binding portion thereof is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In some embodiments, wherein the antigen binding fragment is selected from the group consisting of: fab fragments, fab' fragments, F (ab) ₂ Fragments, fv fragments and ScFv.

In some embodiments, wherein the CD73 is selected from primate CD73.

In some embodiments, the primate is selected from the group consisting of a human, a cynomolgus monkey.

The invention further proposes a fusion protein comprising an antibody, an antigen-binding fragment thereof or a variant thereof according to any one of the above.

The invention further provides one or more isolated nucleic acid molecules encoding an antibody, antigen-binding fragment thereof, or variant thereof, as defined in any one of the above, or a fusion protein as defined above.

The invention further proposes one or more vectors comprising one or more isolated nucleic acid molecules as described above.

The invention further proposes a cell comprising one or more isolated nucleic acid molecules as described above or one or more vectors as described above.

In some embodiments, the cell as described above is further a CAR-T or CAR-NK cell comprising one or more isolated nucleic acid molecules as described above or one or more vectors as described above.

The invention further proposes a method for producing an antibody, antigen-binding fragment thereof or variant thereof as defined in any one of the preceding claims or a fusion protein as defined in any one of the preceding claims, comprising culturing a cell as defined in any one of the preceding claims under conditions that enable expression of the antibody, antigen-binding fragment thereof or variant thereof or fusion protein as defined in any one of the preceding claims.

The invention further proposes a composition comprising an antibody, antigen-binding fragment or variant thereof according to any one of the above, a fusion protein as described above, one or more isolated nucleic acid molecules as described above, one or more vectors and/or cells as described above, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the use of an antibody, antigen-binding fragment thereof or variant thereof of any one of the above, a fusion protein as described above, one or more isolated nucleic acid molecules as described above, one or more vectors as described above, and/or a cell as described above in the manufacture of a medicament for the prevention and/or treatment of cancer or tumor.

In some embodiments, the drug is a cell therapy drug as described above.

In some embodiments, the cancer or tumor is a CD73 expression positive cancer or tumor as described above.

In some embodiments, the cancer or tumor is selected from colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, blood cancer, melanoma, glioma, thyroid cancer, prostate cancer, and breast cancer as described above.

In some embodiments, the use of an antibody, antigen-binding fragment thereof, or variant thereof, or fusion protein as defined above, in the manufacture of a reagent or medicament that specifically binds to the N-terminal domain of CD73 protein.

In some embodiments, the use of an antibody, antigen-binding fragment thereof, or variant thereof, or fusion protein as defined above, of any one of the above, in the preparation of a reagent for determining the presence and/or amount of CD73 in a sample.

The invention further proposes a pharmaceutical composition comprising: the antibody, antigen-binding fragment or variant thereof of any one of the above, fusion protein as described above, one or more isolated nucleic acid molecules as described above, or one or more vectors and/or cells as described above.

The anti-CD 73 antibodies of the present application have high binding affinity for primate CD73 proteins; and has potent activity of inhibiting CD73 enzyme activity at both the soluble protein level and the cellular level. Furthermore, binding of these antibodies can induce tumor cell internalization of CD73, resulting in further reduction of CD73 activity on the cell surface. The antibodies of the invention exhibit superior in vivo and in vitro antitumor activity compared to the known reference antibody MEDI9447, making them excellent candidate drug molecules for therapeutic and diagnostic use.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.

All patents, published patent applications, and non-patent publications mentioned in this application are expressly incorporated herein by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features believed characteristic of the application are set forth with particularity in the appended claims. A better understanding of the features and advantageous aspects of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present application are utilized, and the accompanying drawings (also referred to herein as "figure") and the accompanying drawings (FIG):

FIG. 1 shows the species cross-reactivity of an anti-CD 73 antibody P1G11 according to one embodiment of the invention; wherein CHO-huCD73 shows binding of P1G11 to human CD73 antigen, CHO-cynoCD73 shows binding of P1G11 to cynomolgus monkey CD73 antigen, CHO-mCD73 shows binding of P1G11 to murine CD73 antigen;

FIG. 2 shows a CHOK1-huCD73 cell binding curve of P1G11 and reference antibody MEDI-9447 according to one embodiment of the present invention; wherein reference antibody MEDI-9447 serves as a positive control; the negative control is a isotype control antibody that is determined to not bind CD 73;

FIG. 3 shows protein level enzyme activity inhibition of P1G11 and reference antibody MEDI-9447 according to one embodiment of the invention; wherein the abscissa indicates the amount of added P1G11, reference antibody MEDI-9447 or negative control; the ordinate indicates the enzyme activity inhibition rate;

FIG. 4 shows inhibition of cellular level enzyme activity of P1G11 and reference antibodies TJD5, MEDI-9447 according to one embodiment of the invention; wherein the abscissa indicates the amount of added P1G11, reference antibody MEDI-9447 or negative control; the ordinate indicates the enzyme activity inhibition rate;

FIG. 5 shows the binding dissociation constants of P1G11 and human CD73 according to one embodiment of the invention;

FIG. 6 shows endocytosis detection of P1G11 with reference antibodies TJD5, MEDI-9447 according to one embodiment of the invention;

FIG. 7 shows a Mixed Lymphocyte Reaction (MLR) assay of P1G11 with reference antibodies TJD5, MEDI-9447 according to one embodiment of the invention;

FIG. 8 shows the detection of P1G11 reversal of AMP-mediated T cell proliferation, in accordance with one embodiment of the present invention;

FIG. 9 shows a graph of relative change in A375 cell xenograft tumor volume according to one embodiment of the invention;

FIG. 10A shows a CHOK1-huCD73 Fluorescence Activated Cell Sorting (FACS) assay of P1G11 and P1G11 variants according to one embodiment of the invention;

FIG. 10B shows a CHOK1-cynoCD73 Fluorescence Activated Cell Sorting (FACS) assay of P1G11 and P1G11 variants according to one embodiment of the invention;

FIG. 10C shows protein level enzyme activity inhibition of P1G11, P1G11 variants with reference antibody MEDI-9447 according to one embodiment of the invention; and

FIG. 10D shows the detection of reverse AMP-mediated T cell proliferation of P1G11, P1G11 variants, and reference antibody MEDI-9447, according to one embodiment of the invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration specific embodiments in which the application may be practiced. In the drawings, like reference numerals describe substantially similar components throughout the different views. Various specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to practice the teachings of the present application. It will be understood that many variations, changes, and substitutions may be made by those skilled in the art without departing from the present application, and that various alternatives to the embodiments of the present application described herein may be employed.

As used herein, the term "antibody" generally refers to an immunoglobulin molecule that consists of two pairs of identical polypeptide chains, each pair of polypeptide chains having one "light" (L) chain and one "heavy" (H) chain. The light chains of antibodies can be divided into kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V _H ) And a heavy chain constant region (C) _H ) Composition is prepared. The heavy chain constant region consists of three domains (C _H 1、C _H 2 and C _H 3) Composition is prepared. Each light chain consists of a light chain variable region (V _L ) And a light chain constant region (C _L ) Composition is prepared. The light chain constant region consists of one domain C _L Composition is prepared. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). V (V) _H And V _L Regions can also be subdivided into regions of high variability termed Complementarity Determining Regions (CDRs) interspersed between regions that are more conserved termed Framework Regions (FR). Each V _H And V _L The arrangement from the N-terminal to the C-terminal consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V _H And V _L ) The antibody binding sites are formed separately. The distribution of amino acids into regions or domains follows the Kabat sequence (KabatSequences of Proteins of Immunological Interest) (National Institutes ofHealth, bethesda, md. (1987 and 1991)) or Chothia of immunologically relevant proteins&Lesk (1987) J.mol.biol.196:901-917, chothia et al, (1989) Nature 342:878-883. The amino acid positions described in the present invention are based on an on-line comparison of the abysation tool (http:// www.bioinf.org.uk/abysation/index. Html) and do not represent the actual positions in the amino acid sequence. The term "antibody" is not limited by any method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG 1, igG2, igG3, or IgG4 isotypes), igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of a full-length antibody that retain the ability to bind to the same antigen (e.g., CD 73) to which the antibody binds and compete for antigen-specific binding with the intact antibody. Antigen binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen binding sites include Fab, fab ', F (ab') 2, F (ab) 2, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

As used herein, the term "variant" generally refers to a protein that differs from the parent molecule (e.g., polypeptide) by at least one amino acid. A variant may refer to the molecule itself, a composition comprising the molecule. When such a molecule is a polypeptide or a protein, it may also refer to the amino acid sequence of the molecule. In some cases, a variant differs from its parent molecule (e.g., protein) by the addition, deletion, or substitution of one or more amino acids, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids. In some cases, a variant may have at least about 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) sequence homology to the amino acid sequence of its parent molecule.

For example, in the present application, several amino acid substitutions in the CDRH3 sequence of P1G11 form a P1G11 variant. In some embodiments, wherein X in SEQ ID NO.3 ₁ D (aspartic acid), E (glutamic acid), S (serine), or K (lysine); x in SEQ ID NO.3 ₂ Is G (glycine), A (alanine), W (tryptophan), F (phenylalanine), V (valine), L (leucine), I (isoleucine), P (proline) or M (methionine or methionine), preferably G (glycine) or A (alanine). Further, the sequence of CDRH3 is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10.

As used herein, the term "CD73" generally refers to the protein CD73 or a nucleic acid molecule encoding the same. CD73 is an extracellular-5' -nucleotidase, a member of the extracellular nucleotidase family. Adenosine 5' -monophosphate (amp) is the major substrate for cd73, and its hydrolysis product is adenosine. Adenosine is ubiquitous in the body and is an important regulator of purinergic cell signaling critical to many physiological and pathophysiological processes.

As used herein, the term "binding specificity" generally refers to the ability of one substance to specifically bind to another substance, and not readily bind randomly to any other substance. Such as the ability to specifically bind to (e.g., immunoreact with) a given target (while not binding or substantially not binding to a non-target). For example, one protein may bind specifically to another protein due to its specific structure. The targeting moiety may exhibit binding specificity for a corresponding tumor antigen. The antibodies (or antigen binding fragments or variants thereof) of the present application may be monospecific and comprise one or more binding sites that specifically bind to a target, or may be multispecific (e.g., bispecific or trispecific) and comprise two or more binding sites that specifically bind to the same or different targets.

As used herein, the term "substantially free" generally means little or no binding of a particular substance. For example, very little or no (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%).

As used herein, the term "KD" generally refers to the equilibrium dissociation constant, which is a specific type of equilibrium constant that measures the tendency of a larger object to reversibly separate (dissociate) into smaller components, such as when a complex separates into its constituent components. The dissociation constant is the inverse of the association constant. Equilibrium dissociation constant kd=kd/Ka, KD unit is M (mol/L). Wherein kd is the dissociation constant, the value is less than 1, and the units of Kd are S ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Kd represents the proportion of dissociated two (hereinafter referred to as AB) per unit time to the initial AB before dissociation, whereby it can be seen that Kd can represent the speed of dissociation reaction, a larger Kd represents a faster dissociation and a smaller Kd represents a slower dissociation; KD is an equilibrium dissociation constant that can indicate the degree of dissociation of two in equilibrium, with a larger KD indicating more dissociation and weaker affinity between the two; smaller KD indicates less dissociation, representing stronger affinity between the two.

In the specific case of antibodies (abs) that bind to an antigen (Ag), the term affinity constant generally refers to the association constant. This chemical equilibrium is also the ratio of the binding rate (kford) and dissociation rate (kback) constants. Both antibodies may have the same affinity, but one antibody may have a high binding and dissociation rate constant, while the other antibody may have a low binding and dissociation rate constant.

As used herein, the term "dissociation constant", i.e., pKa, is a polar parameter of a solute having a certain degree of dissociation in an aqueous solution. The dissociation constant gives a quantitative measure of the acidity or basicity of the molecule, with an increase in Ka and an increase in acidity for the proton donor; ka decreases and for proton acceptors its basicity increases. Definition pKa is a specific type of equilibrium constant. The dissociation constant pKa is the negative logarithm of Ka. The greater Ka, the smaller the pKa.

As used herein, the term "monoclonal antibody" generally refers to an antibody that is a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that occur in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), the singular determinant modifier "monoclonal" of each monoclonal antibody against an antigen is not to be construed as requiring antibody production by any particular method. For example, the monoclonal antibodies may be prepared by hybridoma technology or produced in bacterial, eukaryotic, or plant cells by using recombinant DNA methods, and may also be obtained from phage antibody libraries using techniques such as those described by Clackson et al, nature,352:624-628 (1991) and Marks et al, mol.biol.,222:581-597 (1991).

As used herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to a corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portion is homologous to the antibody sequences from another species (e.g., human). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, and the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region with which it is combined. Meanwhile, since the constant region of a chimeric antibody may be derived from human, the chimeric antibody may be less likely to elicit an immune response upon injection than an antibody using a constant region of non-human origin.

In the present application, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing immunogenicity of a xenogeneic antibody when introduced into humans, while maintaining the full antigen binding affinity and specificity of the antibody. For example, CDR grafting (Jones et al, nature 321:522 (1986)) and variants thereof may be used; non-human binding domains are humanised by technical means including "remodeling" (reshaping), (Verhoeyen, et al, 1988Science 239:1534-1536;Riechmann,et al, 1988Nature 332:323-337;Tempest,et al), "high addition" (hyperchimerisation), "Queen, et al, 1989Proc Natl AcadSci USA 86:10029-10033; co, et al, 1991Proc Natl Acad Sci USA 88:2869-2873; co, et al, 1992J Immunol 148:1149-1154) and" veneering "(Mark, et al," "Derivation of therapeutically active humanized and veneered anti-CD18 ntibody," In: metcalf B W, dalton B J, eds.cell addition: moleculardefinition to therapeutic potential New York: plenum Press, 1994:291-312), surface reconstruction (US patent US 5639641). If other regions, such as hinge and constant region domains, are also derived from non-human sources, these regions may also be humanized.

As used herein, the term "fully human antibody" generally refers to an antibody having an antibody region therapeutic derived from a fully human amino acid sequence, wherein antigen specificity has been selected in vivo by using genetically modified mice or by antibody engineering methods of binding screening. Fully human antibodies and humanized antibodies have a lower risk of inducing an immune response in humans than do mouse or chimeric antibodies.

As used herein, the term "bispecific antibody" generally refers to an artificial protein capable of binding to two different types of antigens simultaneously. The main types of manufacturing methods are quadromas (quadromas), chemical conjugation and gene recombination. The IgG-like format retains the conventional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab sites bind to different antigens. Each heavy and light chain pair is from a unique mAb. The Fc region made from two heavy chains forms the third binding site. non-IgG-like forms include chemically linked Fab, consisting only of Fab regions, and various types of divalent and trivalent single chain variable fragments (scFvs). There are also fusion proteins that mimic the variable domains of two antibodies. Bispecific antibodies have higher cytotoxic potential and bind to antigens that are expressed relatively poorly at lower effective doses. In addition, targeting more than one molecule can be used to circumvent modulation of parallel pathways and avoid resistance to treatment.

As used herein, the term "Fab fragment" generally refers to a portion of an immunoglobulin molecule (such as an antigen binding fragment). Fab fragments may comprise a portion of one light chain and heavy chain, with a single antigen binding site. Fab fragments can be obtained by papain digestion of immunoglobulin molecules. For example, a Fab fragment may consist of one constant domain and one variable domain for each heavy and light chain. The variable domain may contain a paratope (antigen binding site) comprising a set of complementarity determining regions at the amino terminus of the immunoglobulin molecule. Papain can be used to cleave immunoglobulin molecules into two Fab fragments and one Fc fragment. Pepsin cleaves below the hinge region, forming a F (ab') ₂ Fragments and a pFC' fragment. Bivalent F (ab) ₂ Or F (ab') ₂ The fragment has two antigen binding regions linked by disulfide bonds. F (ab) ₂ Or F (ab') ₂ Reduction of the fragment yields 2 monovalent Fab or Fab' fragments with free sulfhydryl groups available for conjugation to other molecules.

The term "Fv fragment" as used herein generally refers to the smallest fragment that can be made by enzymatic cleavage of antibodies of the IgG and IgM classes. Fv fragment having the sequence of V _H And V _L Zone makingBut they lack C _H 1 and C _L A zone. V by non-covalent interactions _H And V _L The chains are held together in the Fv fragment.

As used herein, the term "ScFv" generally refers to a single chain antibody fragment. ScFv may refer to a recombinant single chain polypeptide molecule in which the light and heavy chain variable regions of an antibody are linked by a peptide linker. Single chain antibodies (ScFv) typically do not include portions of the Fc region of the antibody that are involved in effector function and are therefore naked antibodies, although methods are known to add such regions to known ScFv molecules, if desired. See Helfrich et al Arapid and versatile method for harnessingScFv antibody fragments with various biological functions.J Immunol Methods237:131-145 (2000) and de Haard et al Creating and engineering human antibodies for improved Drug Delivery Reviews 31:5-31 (1998).

As used herein, the term "IgG" generally refers to an antibody of one subtype. Each IgG has two antigen binding sites. Representing approximately 75% of human serum antibodies, igG is the most common type of antibody found in the circulation. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma (IgG 1, igG2, igG3, igG 4).

As used herein, the term "modification" generally refers to any manipulation of the peptide backbone (e.g., amino acid sequence) of a polypeptide or any post-translational modification (e.g., glycosylation). For example, the modification is compared to the sequence of the corresponding wild-type polypeptide. Modifications may be substitutions, additions and/or deletions of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

As used herein, the term "fusion protein" may generally refer to an amino acid sequence of a polypeptide comprising or consisting of an amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to a previous polypeptide or domain thereof).

As used herein, the term "one or more isolated nucleic acid molecules" or "nucleic acid molecules" generally refers to polymeric forms of nucleotides (whether deoxyribonucleotides or ribonucleotides or analogs thereof) of any length, either isolated from the natural environment or synthesized.

In the present application, the term "sequence homology" generally means that the amino acid sequences of homologous proteins have a clear similarity.

In this application, the term "epitope" generally refers to an antigenic determinant, i.e., a portion of a molecule that is recognized by the immune system (e.g., by an antibody). For example, an epitope is a discrete three-dimensional site on an antigen that is recognized by the immune system. Epitopes are typically composed of chemically active surface groups of molecules (e.g., amino acids or sugar side chains) and typically have specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be classified into conformational epitopes and non-conformational epitopes (linear epitopes) according to structure. Conformational epitopes differ from non-conformational epitopes in that the former loses binding in the presence of denaturing solvents, while the latter does not. Epitopes that are located only on the surface of an antigenic substance and that bind readily to antigen recognizing receptors or antibodies may be referred to as functional epitopes; epitopes that are internal to the molecule and that are non-immunogenic may be referred to as cryptic epitopes. Epitopes can be made up of contiguous residues or discontinuous residues that are closely related by folding of the antigenic polymer. Epitopes formed by consecutive amino acids in proteins are typically maintained upon exposure to denaturing solvents, whereas epitopes formed by discontinuous amino acids are typically lost upon such exposure.

As used herein, the term "vector" or "vector(s)" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material elements carried in the vector may be expressed in a host cell by transforming, transducing or transfecting the host cell with the vector. Embodiments of the vector include plasmids; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), jaundice viruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40 virus). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also comprise an origin of replication. The vector may also include components that facilitate its entry into the cell, such as viral particles, liposomes, or protein shells, but not just these.

As used herein, the term "cell" or "host cell" generally refers to a cell into which a vector is introduced, including many cell types, such as prokaryotic cells, such as Escherichia coli (Escherichia coli) and bacillus subtilis (Bacillus subtilis), fungal cells, such as yeast cells or Aspergillus (Aspergillus) cells, insect cells, such as S2 drosophila cells or Sf9, or animal cells, such as fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells or human cells. In the present application, the term "recombinant host cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a particular cell but also the progeny of such a cell.

As used herein, the term "conditions that enable expression" generally refers to conditions that enable expression of an antibody, antigen-binding fragment thereof, or variant thereof of the present application. In some embodiments, the conditions that enable expression include, but are not limited to, incubation time, temperature, and medium, and may depend on the cell type, and may be readily determined by one of ordinary skill in the art. In some embodiments, during the production of the antibodies, antigen-binding fragments thereof, or variants thereof of the present application, the cells are grown in culture and in any device useful for growing cultures, including fermentors. Cells may be grown as a monolayer or attached to a surface. Alternatively, cells may be grown in suspension. Cells can be grown in serum-free medium.

As used herein, the term "cancer" generally refers to a group of diseases involving abnormal cell growth that have the potential to invade or spread to other parts of the body. Cancer is fundamentally a disease of regulation of tissue growth. In order to transform normal cells into cancer cells, genes that regulate cell growth and differentiation must be altered. The genes affected fall into two broad categories. Oncogenes are genes that promote cell growth and proliferation. Tumor suppressor genes are genes that inhibit cell division and survival. Malignant transformation can occur through the formation of novel oncogenes, inappropriate overexpression of normal oncogenes, or under-expression or inactivation of tumor suppressor genes. In general, to transform normal cells into cancer cells, multiple gene changes are required. Cancers are classified by cell type, including carcinomas, sarcomas, lymphomas and leukemias, germ cell tumors, and blastomas.

As used herein, the term "tumor" refers to a neoplasm (neogram) formed by local tissue cell proliferation of an organism under the influence of various tumorigenic factors, because such neoplasms are often in the form of occupied massive projections, also known as neoplasms (neoplasm). Liver cancer and pancreatic cancer are both called "cancer king", and are tumors with extremely high malignancy. It was found that tumor cells can undergo metabolic changes different from normal cells, while tumor cells themselves can adapt to changes in metabolic environment by switching between glycolysis and oxidative phosphorylation (OXPHOS). In 2019, cancer Cell has recently published an article, and researchers have found that the use of metformin in a fasted state can significantly inhibit tumor growth, and suggest that the PP2A-gsk3β -MCL-1 pathway may be a new target for tumor treatment.

As used herein, the term "T cell" generally refers to a type of lymphocyte (a subtype of leukocyte) that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes (such as B cells and natural killer cells) by the presence of T cell receptors on the cell surface. They are called T cells because they mature from thymic cells in the thymus. Most human T cells rearrange their alpha and beta chains at the cell receptor and are called alpha beta T cells (αβt cells) which are part of the adaptive immune system. Specialized γδ T cells (a small fraction of T cells in humans, more common in ruminants) have invariant T cell receptors, have limited diversity, can efficiently present antigens to other T cells, and are considered to be part of the innate immune system.

As used herein, the term "aspartate isomerisation" is a spontaneous non-enzymatic post-translational modification of the isomerised aspartate (isoAsp) in which a methylene group is inserted in the protein backbone, i.e. the side chains of aspartate are simultaneously reduced by a methylene group, thus causing a change in the structure of the protein and thus in the function of the protein. However, not all aspartic acids will isomerise. According to one embodiment of the present application, a particular combinatorial hot spot empirically predicts the risk of isomerization, e.g., DG is a high risk isomerization site. Thus, in some embodiments, D and/or G are mutated.

It will be appreciated by those skilled in the art that amino acid mutations are not arbitrary mutations, particularly in the CDR regions, and that amino acid basicity, polarity and hydrophobicity are generally a combination of factors in the mutation process. In this application, 3 common mutations were tried, and in fact, more mutation types could be tried.

As used herein, the term "CHOK1" or "CHO" is a hamster ovary cell, wherein CHOK1 is a sub-strain of hamster ovary cells, both commonly used experimental cells. In this application, CHOK1 and CHO are interchangeable.

As used herein, the term "a375 cells" refers to human melanoma cells. The source of the species of the cell may be of human, murine or other species.

As used herein, the terms "MEDI9447", "BMS986179" and "TJD5" are currently known anti-CD 73 antibodies, which serve as reference antibodies for the anti-CD 73 antibody P1G11 of the present application.

As used herein, the term "secondary antibody" refers to a fluorescent antibody, such as anti-human IgG-PE, that recognizes P1G11 and a reference antibody.

As used herein, the term "pharmaceutically acceptable excipient" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.

As used herein, the term "EC50" refers to the half maximal effect concentration (concentration for 50%of maximal effect,EC50) to the concentration that causes 50% of the maximal effect. EC50 is a drug safety indicator. The meaning is as follows: resulting in an effective drug concentration in 50% of the individuals. LD50/ED50, TD50/ED50, TC50/EC50, etc. are collectively referred to as therapeutic indices, and are safety indicators for a class of drugs, generally the larger the value, the safer the drug. It should be noted that these indices reflect only the relationship between the therapeutic effect and acute toxicity, and not chronic toxicity or allergy.

As used herein, the term "IC50" refers to the half-inhibitory concentration, or half-inhibitory rate, which is a very important datum in an indirect competition ELISA standard curve.

As used herein, the term "about" generally refers to an approximation of a given value that may be reasonably inferred based on ordinary skill in the art, including equivalent values and approximations due to experimental and/or measurement conditions of the given value. For example, it may refer to a value that is no more than 10% higher or lower than the value to which the term is modified. For example, the term "about 5. Mu.g/kg" refers to a range of 4.5. Mu.g/kg to 5.5. Mu.g/kg. As another example, "about 1 hour" means a range of 48 minutes to 72 minutes.

As used herein, the term "effective amount" generally refers to a dosage sufficient to provide a sufficiently high concentration to impart a beneficial effect to its recipient. The particular therapeutically effective dosage level of any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the particular component, the route of administration, the rate of clearance, the duration of treatment, the age, weight, sex, diet and general health of the subject, and other related factors.

The present application provides an antibody, antigen binding fragment thereof, or variant thereof, which is 1X 10 ^-9 Below M (e.g., KD of no more than about 1X 10 ^-9 M, not higher than about 9X 10 ^-10 M, not higher than about 8×10 ^-10 M, not higher than about 7X 10 ^-10 M is not higher than about 6×10 ^-10 M, not higher than about 5X 10 ^-10 M, not higher than about 4X 10 ^-10 M, not higher than about 3X 10 ^-10 M, not higher than about 2X 10 ^-10 M is not higher than 1×10 ^-10 M or not higher than about 2X 10 ^-11 M or below) binds to CD73 protein. That is, the antibody, antigen-binding fragment thereof, or variant thereof specifically binds to CD73 protein. Further, CD73 protein is from murine, primate and human. Further, the CD73 protein is selected from the group consisting of: human D73, murine CD73, and monkey CD73. In this application, the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to CD73 protein is designated P1G11.

In the present application, P1G11 includes heavy and light chains. Further, the heavy chain comprises a heavy chain variable region and a heavy chain constant region; the light chain includes a light chain variable region and a light chain constant region.

In some embodiments, the heavy chain variable region of P1G11 of the present application comprises CDRH1, the sequence of which is shown in SEQ ID No.1, CDRH2, the sequence of which is shown in SEQ ID No.2, and CDRH3, the sequence of which is shown in SEQ ID No. 3; and the light chain variable region comprises CDRL1, the sequence of which is shown as SEQ ID NO.4, CDRL2, the sequence of which is shown as SEQ ID NO.5, and CDRL3, the sequence of which is shown as SEQ ID NO. 6.

In some embodiments, the heavy chain variable region of P1G11 of the present application has a sequence as set forth in SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, or SEQ ID No. 14; and the sequence of the light chain variable region is shown in SEQ ID NO. 15.

Further, in some embodiments, the heavy chain constant region of P1G11 of the present application is selected from one or more of IgG (IgG 1, igG2, or IgG 4), igM, igA, igE, or IgD; the light chain constant region is selected from kappa or lambda chains.

In some embodiments, the antibodies or antigen binding portions thereof of P1G11 of the present application are selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In some embodiments, the antigen binding fragment of P1G11 of the present application is selected from the group consisting of: fab fragments, fab' fragments, F (ab) ₂ Fragments, fv fragments and ScFv.

Further, P1G11 of the present application can specifically recognize and bind primate CD73 proteins with little recognition of rodent CD73. Wherein primates include, but are not limited to, humans, cynomolgus monkeys. Rodents include, but are not limited to, mice and rats. The CD73 protein described herein may be a human CD73 protein or a monkey CD73 protein. For example, the CD73 protein may not be a mouse CD73 protein, or may not be a rat CD73 protein. In certain embodiments, the antibodies, antigen-binding fragments or variants thereof described herein do not substantially bind to the mouse CD73 protein or the rat CD73 protein.

In some embodiments, P1G11 has a binding capacity for CD73 that is superior to reference antibody MEDI9447, a known anti-CD 73 antibody. In some embodiments, the binding activity of P1G11 to primate CD73 protein is dose dependent. In some embodiments, protein level enzyme activity inhibition of P1G11 is superior to reference antibody MEDI9447, while the onset of P1G11 function as enzyme activity inhibition is lower, with a certain dose dependency.

Further, the epitope of P1G11 that binds to CD73 protein is the N-terminal domain.

Variants described herein may be selected from the group consisting of: 1) A protein or polypeptide having one or more amino acids substituted, deleted or added to said antibody or said antigen binding fragment thereof; and 2) a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the antibody or antigen binding fragment thereof.

In some embodiments, one or more mutations (e.g., one or more, such as one or several amino acid substitutions) may also be included in the amino acid sequences of the light and/or heavy chains of P1G11 herein. For example, the antibody, antigen binding fragment or variant thereof may comprise one or more mutations (e.g., one or more, such as one or several amino acid substitutions) at one or more sites of the complementarity determining region CDRH3 of the heavy chain variable region thereof.

In some embodiments, the sequence of CDRH3 of the present application is shown as SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

In this application, the mutated CD73 antibody, or antigen-binding fragment or variant thereof, still has the ability to specifically bind to the primate CD73 protein. In some embodiments of the present invention, in some embodiments, primates include, but are not limited to, humans, verruceae, large verruceae, dactylicaceae, lazy monkey, cynomolgaceae, suspension monkey, marmoset, macaque, gibbon and chimpanzee; further, the primate is a human and a cynomolgus monkey.

In another aspect, the present application also provides for isolated one or more nucleic acid molecules. The one or more nucleic acid molecules may encode an antibody, antigen-binding fragment or variant thereof described herein. For example, each of the one or more nucleic acid molecules may encode the entire antibody, antigen-binding fragment or variant thereof, or may encode a portion thereof (e.g., CDRH1-3, CDRL1-3, V) _L 、V _H One or more of a light chain or a heavy chain).

At least one of the nucleic acid molecules described herein may be codon optimized. For example, the methods of codon optimization include, but are not limited to: eliminating rare codons, adjusting GC content, increasing stability of mRNA, adjusting secondary structure of mRNA, rationally designing linkers, and adjusting start codon environment.

The nucleic acid molecules described herein may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.

In the present application, nucleic acids encoding the antibodies, antigen binding fragments or variants thereof may be prepared by a variety of methods known in the art, including, but not limited to, overlap extension PCR using restriction fragment procedures or using synthetic oligonucleotides, see Sambrook et al Molecular Cloning, albometer manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y.,1989; and Ausube et al Current Protocols in Molecular Biology, greene Publishing and Wiley-Interscience, new York N.Y.,1993.

In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. One or more nucleic acid molecules described herein may be operably linked to the expression control element.

The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. For example, the expression vector may be T-easy.

In another aspect, the present application provides a host cell that can comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like. For example, the host cell may be CHO-K1.

In another aspect, the present application provides methods of making the antibodies, antigen-binding fragments or variants thereof. The method may comprise culturing the host cell described herein under conditions such that the antibody, antigen-binding fragment or variant thereof is expressed. For example, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.

In certain cases, the methods may further comprise the step of isolating and/or purifying the antibody or antigen binding fragment thereof. For example, protein G-sepharose or protein A-sepharose can be used for affinity chromatography, and antibodies or antigen binding fragments thereof described herein can also be purified and isolated by gel electrophoresis and/or high performance liquid chromatography, and the like. Protein a affinity purification may also be used, for example.

In another aspect, the present application provides a pharmaceutical composition that may comprise an antibody, antibody-binding fragment or variant thereof, the nucleic acid molecule, the vector, the host cell, and optionally a pharmaceutically acceptable adjuvant described herein.

The pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or nonionic surfactants, and the like.

In this application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration via a subcutaneous depot.

The pharmaceutical composition can be used for inhibiting tumor growth. For example, the pharmaceutical compositions of the present application may inhibit or delay the progression or progression of a disease, may reduce tumor size (even substantially eliminate tumors), and/or may reduce and/or stabilize a disease state.

The pharmaceutical compositions described herein may comprise a therapeutically effective amount of the antibody or antigen-binding fragment thereof. The therapeutically effective amount is that amount which is required to be able to prevent and/or treat (at least partially treat) a disorder or condition (e.g., cancer) and/or any complications thereof in a subject suffering from or at risk of developing the disorder or condition.

In another aspect, the application provides the use of the antibody, antigen binding fragment or variant thereof in the manufacture of a medicament for the prevention or treatment of a tumor.

In another aspect, the present application provides said antibody, antigen binding fragment or variant thereof for use in the prevention or treatment of a tumor.

In another aspect, the present application provides a method of preventing or treating a tumor comprising administering an antibody, antigen-binding fragment or variant thereof, the molecular nucleic acid, the vector, the host cell, and/or the pharmaceutical composition described herein to a subject in need thereof.

Use of P1G11, or a fusion protein comprising P1G11, of the present application for the preparation of a reagent for determining the presence and/or amount of CD73 in a sample.

Pharmaceutical compositions comprising the P1G11, fusion proteins comprising P1G11, one or more isolated nucleic acid molecules encoding P1G11 or one or more vectors comprising nucleic acid molecules encoding P1G11 of the present application and/or cells comprising the above antibodies, fusion proteins, nucleic acid molecules, vectors.

In this application, the tumor may include a CD73 positive tumor. For example, the CD73 positive tumor may be selected from the group consisting of: colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, blood cancer, melanoma, glioma, thyroid cancer, prostate cancer, and breast cancer.

Without intending to be limited by any theory, the following examples are presented merely to illustrate the manner in which the products, methods, or systems of the present application function and are not intended to limit the scope of the invention.

Examples

Example 1 production of anti-CD 73 antibody P1G11

A plurality of different immunization strategies (DNA immunization, protein immunization, cell immunization) are adopted to immunize mice of different species, including Balb/c or SJL and other varieties. The single cell photoconductive system (Beacon) is used for screening the positive mouse B cells combined with the human CD73, and the candidate molecule P1G11 capable of specifically recognizing the human CD73 is obtained through sequence extraction and sequencing.

The amino acid sequence of the variable region of the antibody was analyzed according to the Kabat sequence determination method. Heavy chain variable region V of P1G11 _H Comprising heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3; light chain variable region V _L Comprising light chain complementarity determining regions CDRL1, CDRL2 and CDRL3. In this example, the sequence of CDRH1 is: the sequences of NYGIN (SEQ ID NO. 1) and CDRH2 are as follows: DIYPGSGNTYYNEKFKG (SEQ ID NO. 2), CDRH3 has the sequence: EGGFYX ₁ X ₂ YLDY (SEQ ID NO. 3), wherein X ₁ D (aspartic acid), E (glutamic acid), S (serine), or K (lysine); x in SEQ ID NO.3 ₂ G (glycine), a (alanine), W (tryptophan), F (phenylalanine), V (valine), L (leucine), I (isoleucine), P (proline) or M (methionine or methionine), preferably G (glycine) or a (alanine); further, the sequence of CDRH3 is: EGGFYDGYLDY (SEQ ID NO. 7), or CDRH3 has the sequence: EGGFYDAYLDY (SEQ ID NO. 8), or CDRH3 has the sequence: EGGFYEGYLDY (SEQ ID NO. 9), or CDRH3 has the sequence: EGGFYSGYLDY (SEQ ID NO. 10); the sequence of CDRL1 is: KANQDVSTAVA (SEQ ID NO. 4), CDRL2 has the sequence: SASHRY (SEQ ID NO. 5), and CDRL3 has the sequence: QEHYSSPRT (SEQ ID NO. 6).

Further, the heavy chain variable region sequence of P1G11 is:

QVQLQQSGAELARPGASVKLSCKASGYTFTNYGINWVKQRTGQGLE WIGDIYPGSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCAR EGGFYDGYLDYWGQGTTLTVSS(SEQ ID NO.11)；

the light chain variable region sequence of P1G11 is:

DIVMTQSHKFMSTSVGDRVSITCKANQDVSTAVAWYQQKPGQSPKLLI YSASHRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQEHYSSPRTFGG GTKLEIK(SEQ ID NO.15)。

according to the Kabat sequence determination method, the constant region amino acid sequence of the anti-CD 73 antibody P1G11 was analyzed as follows: heavy chain constant region C _H The amino acid sequence of (2) is:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

light chain constant region C _L The amino acid sequence of (2) is:

AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC。

further, the full length of the amino acid sequence of the heavy chain of P1G11 is:

QVQLQQSGAELARPGASVKLSCKASGYTFTNYGINWVKQRTGQGLEWIGDIYPGSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCAREGGFYDGYLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

the full length of the amino acid sequence of the light chain of P1G11 is:

DIVMTQSHKFMSTSVGDRVSITCKANQDVSTAVAWYQQKPGQSPKLLIYSASHRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQEHYSSPRTFGGGTKLEIKAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

example 2 Cross-reactivity of species of P1G11

According to realityThe antibody sequence of example 1, X ₁ D, X of a shape of D, X ₂ For the case of G, P1G11 binding to CHOK1 overexpressing different species CD73 was detected by FACS and its species cross-reactivity was assessed.

The corresponding stably transformed cell lines expressing human, monkey, and murine CD73 antigen, including CHOK1-huCD73, CHOK1-cynoCD73, CHOK1-mCD73, were cultured and treated according to the SOP procedure of cell culture. Cells harvested after digestion were centrifuged at 300g for 5 min at room temperature, and after discarding the supernatant, washed 2 times with FACS buffer (pbs+2% fbs). 2-5×10 in 96-well plate ⁵ Cells/well, after centrifugation of the supernatant, cells were resuspended in diluted antibody solution, 100 uL/well, mixed well and incubated at 4℃for 1 hour. The supernatant antibody solution was discarded after centrifugation at 300g for 5 min at 4℃and the cells were washed 2 times with FACS buffer, and the corresponding secondary antibody solution, 100 uL/well, was added and incubated at 4℃for 1 hour. After centrifugation at 300g for 5 min at 4℃and discarding the supernatant antibody solution, cells were washed 2 times with FACS buffer and finally resuspended with PBS, signal values were detected on a BD flow cytometer.

FIG. 1 shows species cross-reactivity of P1G11 according to one embodiment of the present application. As a result, as shown in FIG. 1, P1G11 recognizes and binds to CD73 of primate humans and cynomolgus monkeys, and does not recognize CD73 of rodent mice.

Example 3 binding Activity of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ For the case of G, EC50 was measured by FACS detection of P1G11 binding to cell surface huCD73 at various concentrations and its binding activity was evaluated.

The stably transformed cell line CHOK1-huCD73 expressing human CD73 antigen was cultured and processed as described above by flow cytometry. Cells harvested after digestion were centrifuged at 300g for 5 min at room temperature, and after discarding the supernatant, washed 2 times with FACS buffer (pbs+2% fbs). Put into a 96-well plate (2-5). Times.10 ⁵ Cells/well, after centrifugation of the supernatant, cells were resuspended in diluted antibody solution at different concentration gradients, 100 uL/well, mixed and incubated at 4℃for 1 hour. Centrifugation at 300g for 5 min at 4℃and discarding the supernatant antibody solution using FACS bufferThe cells were washed 2 times, and the corresponding secondary antibody solution, 100 uL/well, was added and incubated at 4℃for 1 hour. After centrifugation at 300g for 5 min at 4℃and discarding the supernatant antibody solution, cells were washed 2 times with FACS buffer and finally resuspended with PBS, signal values were detected on a BD flow cytometer.

FIG. 2 shows a CHOK1-huCD73 cell binding curve of P1G11 and reference antibody MEDI-9447 according to one embodiment of the present application. Table 1 shows CHOK1-huCD73 cell binding parameters of P1G11 and reference antibody MEDI-9447 according to one embodiment of the present application. As shown in FIG. 2 and FIG. 1, P1G11 has a strong binding activity to human CD73 on the cell surface. P1G11 was shown to bind efficiently to the cell surface of CHOK1-huCD73 over-expressed human CD73 and was dose dependent and its binding capacity was superior to that of reference antibody MEDI-9447.

TABLE 1 CHOK1-huCD73 cell binding parameters of P1G11 and reference antibody MEDI-9447

	P1G11	MEDI-9447	Negative control
				Top	226471	198582	4597
EC50(nM)	1.164	1.98	NA
				Fitting degree (R) 2 )	0.9993	0.9992	0.9998

Example 4 protein level enzyme activity inhibition function of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ In the case of G, the biological function of P1G11 was evaluated by detecting the protein level enzyme activity inhibition function.

A5 Xsolution of the antigenic protein (final concentration 300 ng/ml) was diluted in TM buffer and 50 uL/well of the dilution was added to a 96-well plate. A proportion of 5 Xantibody dilution was diluted in TM buffer, 50 uL/well of dilution was added to 96-well plates, and after mixing, incubated at 37℃for 30 minutes. 10 XAMP (final concentration of 200 uM) was diluted in TM buffer, 25 uL/well of the dilution was added to 96 well plates and incubated at 37℃for 30 minutes. 50uL of supernatant was placed in a new 96-well plate, 2 XATP solution (final concentration 65 uM) diluted with TM buffer was added, 50 uL/well was added to 50uL of supernatant, and then 100 uL/well of CellTiter Glo Reagent was added. Mix and incubate for 10 minutes at room temperature and read the luminescence signal.

FIG. 3 shows protein level enzyme activity inhibition of P1G11 and reference antibody MEDI-9447 according to one embodiment of the present application. As a result, as shown in FIG. 3, P1G11 was similar to reference antibody MEDI-9447, and the occurrence of reduced activity of inhibiting enzyme activity at the protein level at high concentrations of antibody ("hook effect") was likely associated with the binding epitope. As shown in FIG. 3 below, P1G11 has a lower concentration of active ingredients that exert an enzyme activity inhibition function than the reference antibody MEDI-9447, and has a better protein-level enzyme activity inhibition activity.

Example 5 cellular level enzyme activity inhibition function of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ In the case of G, for example, the inhibition of the function by enzyme activity at the cellular levelCan be detected and the biological function of P1G11 can be evaluated.

A5 Xcell suspension (3200 CHOK1-hCD 73/well) was diluted in TM buffer and 50 uL/well of dilution was added to 96-well plates. A proportion of 5 Xantibody dilution was diluted in TM buffer, 50 uL/well of dilution was added to 96-well plates, and after mixing, incubated at 37℃for 30 minutes. 10 XAMP (final concentration of 200 uM) was diluted in TM buffer, 25 uL/well of the dilution was added to 96 well plates and incubated at 37℃for 30 minutes. 50uL of supernatant was placed in a new 96-well plate, 2 XATP solution (final concentration 65 uM) diluted with TM buffer was added, 50 uL/well was added to 50uL of supernatant, and then 100 uL/well of CellTiter Glo Reagent was added. Mix and incubate for 10 minutes at room temperature and read the luminescence signal.

FIG. 4 shows inhibition of cellular level enzyme activity of P1G11 and reference antibodies TJD5, MEDI-9447 according to one embodiment of the present application. Table 2 shows the level of inhibition of enzyme activity at the cellular level of P1G11 and reference antibodies TJD5, MEDI-9447 according to one embodiment of the present application. As shown in fig. 4 and table 2, both P1G11 and reference antibody MEDI-9447 can inhibit the enzymatic activity of CD73 overexpressed by cells, and have a certain dose dependency, but P1G11 has a smaller IC50 and a better enzymatic activity inhibition function.

TABLE 2 inhibition of enzyme activity at cellular level of P1G11 and reference antibodies TJD5, MEDI-9447

	Vertex value	IC50
			P1G11	78.745	0.386
MEDI9447	81.135	0.417
			TJD5	99.332	0.669

Example 6 affinity detection of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ For the case of G, the affinity of P1G11 was detected by a protein molecular interaction analysis platform (Biacore).

CM5 chips were amino-conjugated with a quantity of anti-human IgG antibodies, and the quantity of antibodies was captured by capture, with different concentrations of human CD73-his flowing through the chip as an analyte, and their affinities were detected.

FIG. 5 shows the binding dissociation constants of P1G11 and human CD73 according to one embodiment of the present application. Table 3 shows the binding dissociation constants of P1G11 and human CD73 according to one embodiment of the present application. The results are shown in FIG. 5 and Table 3, which demonstrate that antibody P1G11 has a higher affinity for CD73 target protein, up to 1.78X10 ^-11 M。

TABLE 3 dissociation constants for binding of P1G11 to human CD73

Example 7 endocytosis assay of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ For G, endocytic activity of P1G11 and reference antibody was detected by FACS.

100uL was added to a 96-well plate at a concentration of 2X 10 ⁶ Per mL of the CHOK1-huCD73 cell suspension, 300g, centrifuged at 4℃for 5 minutes, and the supernatant was discarded. 100uL of CD73 Tab antibody was added to each well to give a final concentration of 20ug/mL, and incubated at 4 ℃30 minutes. Cells were washed 2 times with FACS buffer to remove unbound excess antibody. Endocytosis was performed for 1, 2 and 4 hours at 4 ℃ and 37 ℃ after resuspension of the cells using 100ul FACS buffer, respectively. Cells at different time points and temperatures were fixed with 2% paraformaldehyde in ice for 15 min, the supernatant was washed off, and then the corresponding secondary antibody solution, 100 uL/well, was added separately and incubated at 4℃for 1 hour. After centrifugation at 300g for 5 min at 4℃and discarding the supernatant antibody solution, cells were washed 2 times with FACS buffer and finally resuspended with PBS, signal values were detected on a BD flow cytometer.

FIG. 6 shows endocytosis detection of P1G11 with reference antibodies TJD5, MEDI-9447 according to one embodiment of the present application. Table 4 shows the endocytosis assay of P1G11 with reference antibodies TJD5, MEDI-9447 according to one embodiment of the present application. As shown in FIG. 6 and Table 4, the endocytosis of P1G11 is equivalent to that of reference antibody MEDI-9447, and is significantly superior to that of reference antibody TJD5, and can reduce CD73 on the cell surface and exert better biological activity.

TABLE 4 endocytosis assay of P1G11 with reference antibodies TJD5, MEDI-9447

Example 8 Mixed Lymphocyte Reaction (MLR) assay of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ In the case of G, the in vitro immune activation function of P1G11 was evaluated by MLR assay.

Adjusting DC (stimulator) cell concentration to 1×10 ⁶ Per ml, 10ug/ml mitomycin C (mitomycin C) was added to inhibit DC proliferation and incubated at 37℃for 45 minutes. After centrifugation at 500g for 5 minutes, the cells were washed 2 times with 10mL of PBS. According to the kit handbook (easy Sep) ^TM The Human CD4+ T Cell Isolation kit separates and purifies CD4+ T cells (response) from PBMC. Adjustment of CD4+ T cell density to 2X 10 with X-VIVO15 Medium ⁶ Per ml, the density of DC cells was adjusted to 4X 10 ⁵ Per ml,1:1 equal volume of mixed cd4+ T cells and DC cells (DC: cd4+t=1:20). Adding in100 uL/well of mixed cell suspension to 96-well plate, 50 uL/well of 5 Xanti-PD 1 (final concentration 10 nM), 50 uL/well of 5 XAMP (final concentration 40 uM) and 50 uL/well of 5 Xgradient diluted antibody, 37 ℃ after 120 hours incubation, 500g,5min centrifugation, collect supernatant, according to IFN-gamma HTRF kit (Cisbio, CAT#62 HIFUGGEG) instructions to detect the content of IFN-gamma in the supernatant.

FIG. 7 shows MLR detection of P1G11 with reference antibodies TJD5, MEDI-9447 according to one embodiment of the present application. As shown in fig. 7, P1G11 antagonizes AMP-mediated immunosuppression, which is manifested by promotion of cd4+ T cell interferon release. After the P1G11 with a certain concentration is added, the release of interferon from CD4+ T cells can be obviously promoted, the dosage dependence is realized, the interferon release promoting capability is better than that of the reference antibodies TJD5 and MEDI-9447, and the biological activity of the P1G11 is proved.

Example 9 detection of the reversal of AMP-mediated T cell proliferation by P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ For the case of G, P1G11 was tested to reverse AMP-mediated T cell proliferation.

By easy Sep ^TM Human cd4+ T cell extraction kit (Stemcell, cat# 17952) cd4+ T cells were extracted from PBMCs. The extracted CD4+ T cells were mixed with 30mL of PBS, centrifuged at 500g for 5 minutes, and the CD4+ T cells were resuspended with 0.1% BSA/PBS to adjust the cell density to 2X 10 ⁶ /ml. T cells were mixed with an equal volume of CFSE (4 uM) and incubated for 10 min at 37 ℃. Staining was terminated by adding 40% by volume of FBS and incubated at 37 ℃ for 10 minutes. Washed 2 times with T cell medium (10%FBS in RPMI1640), 500g,5 minutes. T cell density was adjusted to 1X 10 ⁶ Per ml, anti-CD 2/CD3/CD28 beads (beads) (1 bead: 2 cells) were added. T cell suspensions were transferred to 96 well cell culture plates (Corning # 3799), 100 uL/well. 50uL 4 Xgradient diluted antibody and 50uL 4 XAMP (final concentration 800uM or 1000 uM) were added to each well. The cells were incubated at 37℃in a 5% carbon dioxide incubator for 96 hours, centrifuged at 400g for 5 minutes, the supernatant was removed, resuspended in 100uL PBS and the CFSE signal was detected on a BD flow cytometer.

FIG. 8 shows the detection of P1G11 reversal of AMP-mediated T cell proliferation, according to one embodiment of the present application. Table 5 is an assay for reversing AMP-mediated T cell proliferation of P1G11 according to one embodiment of the present application. As shown in fig. 8 and table 5, P1G11 can reverse AMP-mediated T cell proliferation. After a certain concentration of P1G11 is added, the proliferation of T cells can be obviously promoted, and the T cell proliferation promoting capacity is a certain dose dependency, and is equivalent to that of a reference antibody MEDI-9447.

TABLE 5 detection of P1G11 reverse AMP-mediated T cell proliferation

Example 10 epitope Classification of P1G11

To further clarify the epitope bound by P1G11 to CD73, the antibody sequence according to example 1 was followed by X ₁ D, X of a shape of D, X ₂ For the G example, the P1G11 antibody was subjected to competition FACS with reference antibodies MEDI9447, BMS986179 and TJD5 known to bind to epitopes of different regions of CD73 to define the antigen binding region.

The corresponding antigen expressing stably transformed cell lines were cultured and treated according to the SOP procedure of cell culture. Cells harvested after digestion were centrifuged at 300g for 5 min at room temperature, and after discarding the supernatant, washed 2 times with FACS buffer (pbs+2% fbs). Put into a 96-well plate (2-5). Times.10 ⁵ Cells per well, after centrifugation of the supernatant, cells were resuspended in 40ug/ml unlabeled antibody solution, 100 uL/well, and after mixing, incubated at 4℃for 30 min. Without washing, cells were resuspended in fluorescence-labeled antibody solution, 100 uL/well, mixed well, and incubated at 4℃for 45 min. After centrifugation at 300g for 5 min at 4℃and discarding the supernatant antibody solution, the cells were washed 3 times with FACS buffer and finally resuspended with PBS, the signal values were detected on a BD flow cytometer.

Table 6 epitope Classification of P1G11

Table 6 shows epitope classification of P1G11 according to one embodiment of the present application. As shown in table 6, P1G11 overlapped the binding epitope of BMS986179, unlike the binding epitope of MEDI9447 and TJD5, MEDI9447 overlapped the binding epitope of BMS986179, demonstrating that the epitope of P1G11 that binds CD73 antigen is the N-terminal domain.

EXAMPLE 11 in vivo efficacy of P1G11

The antibody sequence according to example 1, in X ₁ D, X of a shape of D, X ₂ For the example of G, the in vivo efficacy of P1G11 and the cognate reference antibody MEDI-9447 test in a375 cell xenograft tumor model was evaluated.

Will contain 4X 10 ⁶ 0.2mL of cell suspension of individual A375 cells (cell suspension basal medium DMEM=200. Mu.L) was inoculated subcutaneously into the right back of each BALB/c nude mouse. On day 21 post inoculation, the group dosing was started when the average tumor volume reached 99.16mm 3. Each group of 8 mice is dosed with 10mg/kg three times a week, and if the weight is reduced by more than 15%, the dosing should be stopped immediately until the weight is recovered to more than 98% of the original weight, and normal dosing is resumed; if the weight loss exceeds 20%, the animals are euthanized.

Fig. 9 shows the relative change in volume of a375 cell xenograft tumor according to one embodiment of the present application. Table 7 shows the relative changes in A375 cell xenograft tumor volume according to one embodiment of the present application. As shown in fig. 9 and table 7, the tumor inhibition rate of P1G11 was much better than MEDI9447.

TABLE 7 relative changes in tumor volume for 375 cell xenograft

Example 12 Hot spot removal of P1G11

After sequence analysis, the antibody sequence according to example 1, when X ₁ D, X of a shape of D, X ₂ In the case of G, the CDRH3 of P1G11 is aspartic acidThe site of structuring, and thus its hot spot removal, is engineered to avoid the impact of aspartic acid isomerisation on the structure and function of the antibody. The hot spot removal retrofit variants are shown in table 8.

Amino acid substitutions of Table 8P1G11

The results of characterization of the hotspot-removed variants are shown in figures 10A-10D and tables 9-10. Wherein, FIG. 10A shows CHOK1-huCD73 Fluorescence Activated Cell Sorting (FACS) detection of P1G11 and P1G11 variants according to one embodiment of the present invention; FIG. 10B shows a CHOK1-cynoCD73 Fluorescence Activated Cell Sorting (FACS) assay of P1G11 and P1G11 variants according to one embodiment of the invention; FIG. 10C shows protein level enzyme activity inhibition of P1G11, P1G11 variants with reference antibody MEDI-9447 according to one embodiment of the invention; FIG. 10D shows the detection of reverse AMP-mediated T cell proliferation of P1G11, P1G11 variants, and reference antibody MEDI-9447, according to one embodiment of the invention. Table 9 is a CHOK1-huCD73 Fluorescence Activated Cell Sorting (FACS) assay for P1G11 and P1G11 variants according to one embodiment of the invention; table 10 shows the CHOK1-cynoCD73 Fluorescence Activated Cell Sorting (FACS) assays for P1G11 and P1G11 variants according to one embodiment of the invention. P1g11_v _H The (DG_SG) variant has the least influence on the activity, and the binding activity and the protein level enzyme activity inhibition function of the variant are not different from those of the original sequence of the P1G 11.

TABLE 9 CHOK1-huCD73 Fluorescence Activated Cell Sorting (FACS) assay for P1G11 and P1G11 variants

	EC50	Span of
			P1G11_V1(DG_DA)	2.07	265051
P1G11_V1(DG_EG)	1.856	263157
			P1G11_V1(DG_SG)	1.783	262766
P1G11	2.326	265570
			Negative control

TABLE 10 CHOK1-cynoCD73 Fluorescence Activated Cell Sorting (FACS) assay for P1G11 and P1G11 variants

	EC50	Span of
			P1G11_V1(DG_DA)	2.752	268281
P1G11_V1(DG_EG)	2.5	267197
			P1G11_V1(DG_SG)	2.543	267575
P1G11	2.707	267232
			Negative control

In summary, the present invention provides CD73 antibodies, either in soluble form or in cell membrane surface form, which have high binding affinity for human CD73 protein and cynomolgus monkey CD73 protein; and has potent activity of inhibiting CD73 enzyme activity at both the soluble protein level and the cellular level. Furthermore, binding of these antibodies can induce tumor cell internalization of CD73, resulting in further reduction of CD73 activity on the cell surface. Compared to MEDI9447 of known mediimune, which has similar properties (binds to the N-terminal domain) to the antibody of the present invention, the antibody of the present invention exhibits superior in vivo and in vitro antitumor activity, making it an excellent candidate drug molecule for therapeutic and diagnostic use.

The above embodiments are provided for illustrating the present invention and not for limiting the present invention, and various changes and modifications may be made by one skilled in the relevant art without departing from the scope of the present invention, therefore, all equivalent technical solutions shall fall within the scope of the present disclosure.

Claims (24)

1. An antibody, antigen-binding fragment thereof, or variant thereof, that specifically binds CD73, comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises:

CDRH1, the sequence of which is shown as SEQ ID NO. 1;

CDRH2, the sequence of which is shown as SEQ ID NO. 2; and

CDRH3, the sequence of which is shown as SEQ ID NO. 3; and

the light chain variable region comprises:

CDRL1, the sequence of which is shown in SEQ ID NO. 4;

CDRL2, the sequence of which is shown in SEQ ID NO. 5; and

CDRL3, the sequence of which is shown in SEQ ID NO. 6.

2. The antibody, antigen-binding fragment thereof, or variant thereof of claim 1, wherein X in SEQ ID No.3 ₁ D (aspartic acid), E (glutamic acid), S (serine), or K (lysine); x in SEQ ID NO.3 ₂ Is G (glycine), A (alanine), W (tryptophan), F (phenylalanine), V (valine), L (leucine), I (isoleucine), P (proline) or M (methionine or methionine), preferably G (glycine) or A (alanine).

3. The antibody, antigen-binding fragment thereof, or variant thereof of claim 2, wherein the sequence of CDRH3 is as set forth in SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, or SEQ ID No. 10.

4. The antibody, antigen-binding fragment thereof, or variant thereof of claim 1, wherein: the sequence of the heavy chain variable region is shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14; and the sequence of the light chain variable region is shown in SEQ ID NO. 15.

5. The antibody, antigen-binding fragment thereof, or variant thereof of claim 1, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from one or more of IgG, igM, igA, igE or IgD; the light chain constant region is selected from kappa or lambda chains.

6. The antibody, antigen binding fragment thereof, or variant thereof of claim 5, wherein the IgG series antibody is selected from one or more of IgG1, igG2, and IgG 4.

7. The antibody, antigen-binding fragment thereof, or variant thereof of any one of claims 1-6, wherein the antibody or antigen-binding portion thereof is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

8. The antibody, antigen-binding fragment thereof, or variant thereof of claim 1, wherein the antigen-binding fragment is selected from the group consisting of: fab fragments, fab' fragments, F (ab) ₂ Fragments, fv fragments and ScFv.

9. The antibody, antigen-binding fragment thereof, or variant thereof of claim 1, wherein the CD73 is selected from primate CD73.

10. The antibody, antigen-binding fragment thereof, or variant thereof of claim 9, wherein the primate is selected from the group consisting of a human, a cynomolgus monkey.

11. A fusion protein comprising an antibody, antigen-binding fragment thereof, or variant thereof according to any one of claims 1-10.

12. One or more isolated nucleic acid molecules encoding an antibody, antigen-binding fragment thereof or variant thereof according to any one of claims 1-10, or a fusion protein according to claim 11.

13. One or more vectors comprising one or more isolated nucleic acid molecules according to claim 12.

14. A cell comprising one or more isolated nucleic acid molecules according to claim 12 or one or more vectors according to claim 13.

15. The cell of claim 14, further being a CAR-T or CAR-NK cell comprising one or more isolated nucleic acid molecules according to claim 12 or one or more vectors according to claim 13.

16. A method for producing an antibody, antigen-binding fragment thereof or variant thereof according to any one of claims 1-10 or a fusion protein according to claim 11, comprising culturing a cell according to claim 14 or 15 under conditions that enable expression of the antibody, antigen-binding fragment thereof or variant thereof according to any one of claims 1-10 or the fusion protein according to claim 11.

17. A composition comprising an antibody, antigen-binding fragment thereof or variant thereof according to any one of claims 1-10, a fusion protein according to claim 11, one or more isolated nucleic acid molecules according to claim 12, one or more vectors according to claim 13 and/or cells according to claim 14 or 15, and optionally a pharmaceutically acceptable excipient.

18. Use of an antibody, antigen-binding fragment or variant thereof according to any one of claims 1-10, a fusion protein according to claim 11, one or more isolated nucleic acid molecules according to claim 12, one or more vectors according to claim 13 and/or cells according to claim 14 or 15 for the preparation of a medicament for the prevention and/or treatment of cancer or tumors.

19. The use of claim 18, wherein the medicament is a cytotherapeutic medicament.

20. The use of claim 18, wherein the cancer or tumor is a CD73 expression positive cancer or tumor.

21. The use of claim 20, wherein the cancer or tumor is selected from colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, blood cancer, melanoma, glioma, thyroid cancer, prostate cancer, and breast cancer.

22. Use of an antibody, antigen-binding fragment thereof or variant thereof according to any one of claims 1-10, or a fusion protein according to claim 11, in the preparation of a reagent or pharmaceutical product that specifically binds to the N-terminal domain of CD73 protein.

23. Use of an antibody, antigen-binding fragment thereof or variant thereof according to any one of claims 1-10, or a fusion protein according to claim 11, in the preparation of a reagent for determining the presence and/or amount of CD73 in a sample.

24. A pharmaceutical composition comprising: an antibody, antigen-binding fragment or variant thereof according to any one of claims 1-10, a fusion protein according to claim 11, one or more isolated nucleic acid molecules according to claim 12 or one or more vectors according to claim 13 and/or cells according to claim 14 or 15.