Anti-cd73 antibodies and uses thereof

Abstract

The present application relates to an antibody, antigen binding fragment or variant thereof specifically binding to CD73, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRH1 with the sequence shown as SEQ ID NO. 1, CDRH2 with the sequence shown as SEQ ID NO. 2, and CDRH3 with the sequence shown as SEQ ID NO. 3, and the light chain variable region comprises CDRL1 with the sequence shown as SEQ ID NO. 4, CDRL2 with the sequence shown as SEQ ID NO. 5, and CDRL3 with the sequence shown as SEQ ID NO. 6. The anti-CD73 antibody of the present application has high binding affinity; and has strong activity in inhibiting CD73 enzyme activity at the level of soluble protein and cell. In addition, the binding of these antibodies can induce the intracellular internalization of CD73 tumor cells, resulting in further reduction of CD73 activity on the cell surface. Compared with the reference antibody, the antibody of the present application shows more excellent anti-tumor activity in vitro and in vivo, making it an excellent candidate drug molecule for therapeutic and diagnostic use.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to anti-CD73 antibodies and their uses. Background Technology

[0002] CD73 possesses enzymatic activity in cancer development and metastasis. CD73 is upregulated in many cancer cell types and tumors, including colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, leukemia, melanoma, glioma, thyroid cancer, prostate cancer, and breast cancer (Jin et al. Cancer Res 2010; 70:2245-2255 and Stagg et al. PNAS 2010; 107:1547-1552). CD73 expression has been shown to be associated with tumor angiogenesis, invasion, and metastasis. CD73 overexpression impairs adaptive antitumor immune responses and promotes tumor growth and metastasis. It has been reported that adenosine can inhibit T-cell immune killing, enabling tumors to escape immune responses, and CD73 is a key enzyme catalyzing adenosine production, helping to convert ATP, which has immune-activating effects, into adenosine. Tumor growth is also impaired in CD73-deficient mice, and these effects have been confirmed to be mainly attributed to reduced adenosine production in these mice.

[0003] Therefore, researchers have begun to actively explore the potential applications of CD73 inhibitors in cancer treatment (see, for example, M. Al-Rashida et al., Eur. J. Med. Chem., 115(2016): 484-494 and the references cited therein). However, there is still a need to develop anti-CD73 antibodies with better affinity and / or activity. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention proposes an antibody that specifically binds to CD73, its antigen-binding fragment, or a variant thereof, 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 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 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.

[0005] In some embodiments, the antibody, its antigen-binding fragment, or a variant thereof as described above, has the heavy chain variable region sequence as shown in SEQ ID NO.7; and the light chain variable region sequence as shown in SEQ ID NO.8.

[0006] In some embodiments, the antibody or its variants as described above further include a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from one or more of IgG, IgM, IgA, IgE or IgD; and the light chain constant region is selected from the κ or λ chain.

[0007] In some embodiments, the IgG series antibodies are selected from one or more of IgG1, IgG2 and IgG4.

[0008] In some embodiments, the antibody or its antigen-binding portion is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0009] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

[0010] In some embodiments, the CD73 is selected from primate CD73.

[0011] In some embodiments, the primates are selected from humans and cynomolgus monkeys.

[0012] The present invention further proposes a fusion protein comprising an antibody, an antigen-binding fragment thereof, or a variant thereof, as described in any of the preceding claims.

[0013] The present invention further proposes one or more isolated nucleic acid molecules that encode an antibody, an antigen-binding fragment thereof, or a variant thereof, as described above, or a fusion protein as described above.

[0014] The present invention further proposes one or more carriers comprising one or more isolated nucleic acid molecules as described above.

[0015] The present invention further proposes a cell comprising one or more isolated nucleic acid molecules as described above or one or more carriers as described above.

[0016] In some embodiments, the cells described above are further CAR-T or CAR-NK cells comprising one or more isolated nucleic acid molecules as described above or one or more vectors as described above.

[0017] The present invention further proposes a method for generating an antibody, an antigen-binding fragment thereof, or a variant thereof, or a fusion protein as described above, comprising culturing the cells described above under conditions that enable the antibody, its antigen-binding fragment thereof, or a variant thereof, or the fusion protein described above to be expressed.

[0018] The present invention further proposes a composition comprising an antibody as described above, an antigen-binding fragment thereof or a variant thereof, a fusion protein as described above, one or more isolated nucleic acid molecules as described above, one or more carriers as described above and / or cells as described above, and optionally a pharmaceutically acceptable excipient.

[0019] In some embodiments, the use of antibodies, antigen-binding fragments thereof, or variants thereof, fusion proteins, one or more isolated nucleic acid molecules, one or more carriers, and / or cells as described above in the preparation of medicaments for the prevention and / or treatment of cancer or tumors.

[0020] In some embodiments, the drug described above is a cell therapy drug.

[0021] In some embodiments, the cancer or tumor described above is a CD73-positive cancer or tumor.

[0022] In some embodiments, the cancer or tumor described above is selected from colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, leukemia, melanoma, glioma, thyroid cancer, prostate cancer, and breast cancer.

[0023] In some embodiments, the use of an antibody, an antigen-binding fragment thereof, or a variant thereof, or a fusion protein as described above in the preparation of a reagent or pharmaceutical product that specifically binds to the N-terminal domain of the CD73 protein.

[0024] In some embodiments, the use of antibodies, antigen-binding fragments thereof, or variants thereof, or fusion proteins as described above, in the preparation of reagents for determining the presence and / or amount of CD73 in a sample.

[0025] The present invention further proposes a pharmaceutical composition comprising: an antibody as described above, an antigen-binding fragment thereof or a variant thereof, a fusion protein as described above, one or more isolated nucleic acid molecules as described above, or one or more carriers as described above and / or cells as described above.

[0026] The anti-CD73 antibodies of this application possess high binding affinity and exhibit potent inhibitory activity against CD73 enzyme activity at both the soluble protein and cellular levels. Furthermore, binding to these antibodies induces CD73 internalization in tumor cells, leading to a further reduction in CD73 activity on the cell surface. Compared to reference antibodies, the antibodies of this application demonstrate superior in vitro and in vivo antitumor activity, making them excellent candidate drug molecules for therapeutic and diagnostic applications.

[0027] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive.

[0028] All patents, published patent applications and non-patent publications mentioned in this application are expressly incorporated herein by reference in their entirety, as if specifically and individually indicated by reference to each patent or publication. Attached Figure Description

[0029] The novel features of this application are specifically set forth in the appended claims. A better understanding of the features and advantages of this application will be obtained by referring to the following detailed description of illustrative embodiments employing the principles of this application, along with the accompanying drawings (also referred to herein as “figures” and “FIGs”), wherein the drawings are:

[0030] Figure 1 This invention demonstrates the species cross-reactivity of the anti-CD73 antibody P2A1 according to an embodiment of the present invention; wherein CHO-huCD73 shows the binding of P2A1 to human CD73 antigen, CHO-cynoCD73 shows the binding of P2A1 to cynomolgus monkey CD73 antigen, and CHO-mCD73 shows the binding of P2A1 to mouse CD73 antigen.

[0031] Figure 2 The diagram shows the binding curve of P2A1 to the reference antibody MEDI-9447 on CHOK1-huCD73 cells according to an embodiment of the present invention; wherein the reference antibody MEDI-9447 serves as a positive control; and the negative control is an isotype control antibody that determines that it does not bind to CD73.

[0032] Figure 3 The diagram illustrates the protein-level enzyme activity inhibition of P2A1 and the reference antibody MEDI-9447 according to an embodiment of the present invention; wherein, the horizontal axis represents the amount of added P2A1, the reference antibody MEDI-9447, or the negative control; and the vertical axis represents the enzyme activity inhibition rate.

[0033] Figure 4 This diagram illustrates the cellular-level enzyme activity inhibition of P2A1 with reference antibodies TJD5 and MEDI-9447 according to an embodiment of the present invention; wherein the horizontal axis represents the amount of added P2A1, reference antibody MEDI-9447, or negative control; and the vertical axis represents the enzyme activity inhibition rate.

[0034] Figure 5 This shows the binding dissociation constant of P2A1 with human CD73 according to an embodiment of the present invention;

[0035] Figure 6 The endocytic activity assay of P2A1 with reference antibodies TJD5 and MEDI-9447 according to an embodiment of the present invention is shown.

[0036] Figure 7 This invention demonstrates the detection of a mixed lymphocyte reaction (MLR) of P2A1 with reference antibodies TJD5 and MEDI-9447 according to an embodiment of the present invention.

[0037] Figure 8 The detection of P2A1 reversing AMP-mediated T cell proliferation according to an embodiment of the present invention; and

[0038] Figure 9 This shows a curve illustrating the relative change in tumor volume from A375 cell xenografts according to an embodiment of the present invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] In the following detailed description, reference may be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that many variations, modifications, and substitutions can be made by those skilled in the art without departing from the present application, and various alternatives to the embodiments of the present application described herein can be adopted.

[0041] This invention provides CD73 antibodies, in both soluble and cell membrane surface forms, that exhibit high binding affinity for human and cynomolgus monkey CD73 proteins; and possess potent inhibitory activity against CD73 enzyme activity at both the soluble protein and cellular levels. Furthermore, binding to these antibodies induces CD73 internalization in tumor cells, leading to a further reduction in CD73 activity on the cell surface. Compared to Medimmune's MEDI9447, a known antibody with similar properties (binding to the N-terminal domain), the antibodies of this invention demonstrate superior in vitro and in vivo antitumor activity, making them excellent candidate drug molecules for therapeutic and diagnostic applications.

[0042] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chain of an antibody can be classified as κ and λ light chains. The heavy chain can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also includes "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V... H ) and heavy chain constant region (C H It consists of three structural domains (C). The heavy chain constant region consists of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions called complementary determination regions (CDRs), which are interspersed among more conservative regions called framing regions (FRs). Each V H and V L Arranged from N-terminus to C-terminus in the following order, consisting of 3 CDRs and 4 FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V) of each heavy / light chain pair... H and V LThese amino acids form antibody binding sites. The distribution of amino acids to regions or domains follows the definitions of Kabat sequences of proteins of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, and Chothia et al. (1989) Nature 342:878-883. The amino acid positions described in this invention are based on online comparisons using the abysis tool (http: / / www.bioinf.org.uk / abysis / index.html) and do not represent actual positions in the amino acid sequence. The term "antibody" is not limited to any antibody production method. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0043] 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., CD73) to which the antibody binds and competes with the intact antibody for antigen-specific binding. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. 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, biantibodies, and peptides that contain at least a portion of an antibody sufficient to confer peptide-specific antigen-binding ability.

[0044] As used herein, the term "variant" generally refers to a protein that differs from its parent molecule (e.g., a polypeptide) by at least one amino acid. A variant can refer to the molecule itself, or a composition containing that molecule. When such a molecule is a polypeptide or a protein, it can also refer to the amino acid sequence of the molecule. In some cases, a variant differs from its parent molecule (e.g., a 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, the 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 higher) sequence homology with the amino acid sequence of its parent molecule.

[0045] As used herein, the term "CD73" generally refers to the protein CD73 or the nucleic acid molecule encoding it. CD73 is an extracellular 5'-nucleotidase, a member of the extracellular nucleotidase family. Adenosine 5'-monophosphate (AMP) is the major substrate of CD73, and its hydrolysis product is adenosine. Adenosine is ubiquitous in the body and is an important regulator of purinergic cell signaling, which is crucial for many physiological and pathophysiological processes.

[0046] 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 to any other substance randomly. This includes the ability to specifically bind (e.g., to an immune response) to a given target (while not binding to or substantially not binding to non-targets). For example, a protein may bind specifically to another protein due to its specific structure. Targeting portions may exhibit binding specificity to corresponding tumor antigens. The antibodies (or their antigen-binding fragments or variants) of this application may be monospecific and contain one or more binding sites that specifically bind to a target, or they may be multispecific (e.g., bispecific or trispecific) and contain two or more binding sites that specifically bind to the same or different targets.

[0047] As used in this article, the term “substantially not” generally means very little or almost no binding of a particular substance. For example, very little or almost none (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%).

[0048] As used herein, the term "KD" generally refers to the equilibrium dissociation constant, a specific type of equilibrium constant that measures the tendency of a larger substance to reversibly separate (dissociate) into smaller components (e.g., when a complex separates into its component molecules). The dissociation constant is the reciprocal of the association constant. The equilibrium dissociation constant KD = Kd / Ka, and the unit of KD is M (mol / L). Here, kd is the dissociation constant, which is less than 1, and the unit of Kd is s. -1 Kd represents the proportion of the two dissociated components (hereinafter referred to as AB) to the initial AB before dissociation per unit time. It can be seen that Kd can indicate the speed of the dissociation reaction. The larger the Kd, the faster the dissociation, and the smaller the Kd, the slower the dissociation. KD is the equilibrium dissociation constant, which can indicate the degree of dissociation of the two components in equilibrium. The larger the KD, the more dissociation there is and the weaker the affinity between the two components. The smaller the KD, the less dissociation there is and the stronger the affinity between the two components.

[0049] In the specific case of antibody (Ab) binding to antigen (Ag), the term affinity constant usually refers to the association constant. This chemical equilibrium is also the ratio of the binding rate (kforward) to the dissociation rate (kback) constant. Two antibodies can have the same affinity, but one antibody can have a high binding and dissociation rate constant, while the other antibody can have a low binding and dissociation rate constant.

[0050] As used in this article, the term "dissociation constant," or pKa, is a polar parameter of a solute in aqueous solution that has a certain degree of dissociation. The dissociation constant provides a quantitative measure of the acidity or basicity of a molecule; an increase in Ka indicates increased acidity for a proton donor, while a decrease in Ka indicates increased basicity for a proton acceptor. pKa is defined as a specific type of equilibrium constant. The dissociation constant pKa is the negative logarithm of Ka. The larger Ka is, the smaller pKa is.

[0051] As used herein, the term "monoclonal antibody" generally refers to a collection of substantially homologous antibodies, meaning that the individual antibodies comprising the group are identical except for the possibility of naturally occurring mutations present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site directly. Furthermore, unlike polyclonal antibody preparations which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. The modifier "monoclonal" is not interpreted as requiring the antibody to be produced by any special method. For example, said monoclonal antibodies can be prepared by hybridoma technology or by using recombinant DNA methods in bacterial, eukaryotic, or plant cells. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0052] As used herein, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a particular species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may be derived from the variable regions of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant regions are homologous to antibody sequences from another species (e.g., human). For instance, to obtain a chimeric antibody, the variable regions can be generated using non-human B cells or hybridoma cells, with the combined constant regions derived from humans. The variable regions have the advantage of being easy to prepare, and their specificity is unaffected by the source of the combined constant regions. Furthermore, because the constant regions of chimeric antibodies can be derived from humans, the likelihood of a chimeric antibody eliciting an immune response upon injection is lower than with antibodies using constant regions derived from non-human sources.

[0053] In this application, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of the xenobiotic antibody when introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity. For example, CDR transplantation (Jones et al., Nature 321:522(1986)) and its variants can be used; including “reshaping” (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 19919:266-271), and “hyperchimerization” (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol). Techniques such as veneering (Mark, et al., “Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.” In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), and surface reconstruction (US Patent US5639641) are used to humanize non-human-derived binding domains. If other regions, such as hinge regions and constant region structural domains, are also derived from non-human sources, these regions can also be humanized.

[0054] As used herein, the term "fully human antibody" generally refers to an antibody therapeutic having antibody regions derived from a fully human amino acid sequence, wherein antigen specificity has been selected in vivo using genetically modified mice or through antibody engineering methods involving binding screening. Compared to mouse or chimeric antibodies, fully human antibodies and humanized antibodies have a lower risk of inducing an immune response in humans.

[0055] As used herein, the term "bispecific antibody" generally refers to an artificial protein capable of binding simultaneously to two different types of antigens. The main types of manufacturing methods include quadromas, chemical conjugation, and genetic recombination. IgG-like forms retain the structure of a conventional monoclonal antibody (mAb) with two Fab arms and an Fc region, in addition to binding to two different antigens at two Fab sites. Each heavy chain and light chain pair originates from a unique mAb. The Fc region, formed from the two heavy chains, forms a third binding site. Non-IgG-like forms include chemically linked Fabs, consisting only of Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs). There are also fusion proteins that mimic the variable domains of two antibodies. Bispecific antibodies possess high cytotoxic potential and bind to antigens expressed relatively weakly at lower effective doses. Furthermore, targeting more than one molecule can be used to circumvent parallel pathway regulation and avoid resistance to treatment.

[0056] As used herein, the term "Fab fragment" generally refers to a portion of an immunoglobulin molecule (such as an antigen-binding fragment). A Fab fragment may comprise a portion of a light chain and a heavy chain, having a single antigen-binding site. Fab fragments can be obtained by digesting immunoglobulin molecules with papain. For example, a Fab fragment may consist of a constant domain and a variable domain for each heavy and light chain. The variable domain may contain a complementary site (antigen-binding site) containing a set of complementarity-determining regions at the amino terminus of the immunoglobulin molecule. Papain can be used to cleave an immunoglobulin molecule into two Fab fragments and one Fc fragment. Pepsin cleaves below the hinge region, resulting in an F(ab')2 fragment and a pFc' fragment. The divalent F(ab)2 or F(ab')2 fragment has two antigen-binding regions linked by disulfide bonds. Reduction of the F(ab)2 or F(ab')2 fragment yields two monovalent Fab or Fab' fragments, which have free thiol groups that can be used for conjugation with other molecules.

[0057] As used in this article, the term "Fv fragment" generally refers to the smallest fragment produced by the enzymatic cleavage of IgG and IgM antibodies. The Fv fragment has a structure consisting of V... H and V L Antigen binding sites are formed in the region, but they lack the CH1 and CL regions. Through non-covalent interactions, V... H and V L The chains are joined together in the Fv fragment.

[0058] As used herein, the term “ScFv” generally refers to a single-chain antibody fragment. ScFv can also refer to a recombinant single-chain polypeptide molecule in which the variable regions of the antibody’s light and heavy chains are linked by peptide linkers. Single-chain antibodies (ScFvs) typically do not include the portion of the antibody’s Fc region involved in effector functions, and are therefore naked antibodies, although methods are known to be available for adding such regions to known ScFv molecules (if desired). See Helfrich et al., A rapid and versatile method for harnessing ScFv antibody fragments with various biological functions. J Immunol Methods 237:131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31:5-31 (1998).

[0059] As used in this article, the term "IgG" generally refers to a subtype of antibody. Each IgG has two antigen-binding sites. Representing approximately 75% of human serum antibodies, IgG is the most common type of antibody found in circulation. Recognized immunoglobulin genes include κ, λ, α, and γ (IgG1, IgG2, IgG3, and IgG4).

[0060] 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, a modification is compared to the sequence of the corresponding wild-type polypeptide. A modification can be the substitution, addition, and / or deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

[0061] As used herein, the term "fusion protein" can generally refer to the amino acid sequence of a polypeptide that is directly or indirectly (e.g., through a linker) fused to the amino acid sequence of a heterologous polypeptide (i.e., a polypeptide independent of the preceding polypeptide or its domains), or a polypeptide composed of such a polypeptide.

[0062] As used herein, the term “one or more isolated nucleic acid molecules” or “nucleic acid molecule” generally refers to a polymeric form of any length of nucleotide (whether deoxyribonucleotide or ribonucleotide or analogue) isolated from its natural environment or synthesized artificially.

[0063] In this application, the term "sequence homology" generally refers to the significant similarity of the amino acid sequences of homologous proteins.

[0064] In this application, the term "epitaph" generally refers to an antigenic determinant, that is, a portion of a molecule that is recognized by the immune system (e.g., by antibodies). For example, an epitope is a discontinuous three-dimensional site on an antigen recognized by the immune system. Epitopes typically consist of chemically active surface groups of a molecule (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural features and specific charge characteristics. Based on their structure, epitopes can be classified into conformational epitopes and non-conformational epitopes (linear epitopes). The difference between conformational and non-conformational epitopes is that the former loses binding in the presence of denaturing solvents, while the latter does not. Epitopes located only on the surface of antigenic substances and readily binding to antigen recognition receptors or antibodies can be called functional epitopes; epitopes located inside the molecule and lacking immunogenicity can be called latent epitopes. Epitopes can consist of continuous residues or be formed by adjacent discontinuous residues due to the folding of antigen polymers. In proteins, epitopes formed by continuous amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by discontinuous amino acids are usually lost after such exposure.

[0065] As used herein, the term "vector" or "one or more vectors" generally refers to a nucleic acid medium into which a polynucleotide encoding a protein can be inserted and expressed. Genetic elements carried in a vector can be expressed in a host cell by transformation, transduction, or transfection with the vector. Vector implementation schemes include: plasmids; phage particles; granules; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as λ 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, and multivacuolar papillomaviruses (such as SV40 virus). Vectors may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication. Vectors may also include components that facilitate their entry into the cell, such as viral particles, liposomes, or protein shells, but not only these substances.

[0066] As used herein, the term "cell" or "host cell" generally refers to the cell in which the vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells or 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 this application, the term "recombinant host cell" generally refers to the cell in which the recombinant expression vector is introduced. The recombinant host cell includes not only a specific type of cell but also its progeny.

[0067] As used herein, the term "conditions enabling expression" generally refers to conditions that enable the expression of the antibody, its antigen-binding fragment, or variant thereof of this application. In some embodiments, the conditions enabling expression include, but are not limited to, incubation time, temperature, and culture medium, and may depend on the cell type and can be readily determined by those skilled in the art. In some embodiments, in the process of generating the antibody, its antigen-binding fragment, or variant thereof of this application, cells are grown in a culture and in any apparatus (including fermenters) that can be used to grow the culture. Cells may be grown as a monolayer or attached to a surface. Alternatively, cells may be grown in suspension. Cells may be grown in a serum-free culture medium.

[0068] As used in this article, the term "cancer" generally refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer is fundamentally a disease of tissue growth regulation. For normal cells to transform into cancer cells, genes regulating cell growth and differentiation must be altered. The affected genes fall into two main categories: oncogenes, which promote cell growth and proliferation; and tumor suppressor genes, which inhibit cell division and survival. Malignant transformation can occur through the formation of novel oncogenes, inappropriate overexpression of normal oncogenes, or underexpression or inactivation of tumor suppressor genes. Typically, multiple gene alterations are required to transform normal cells into cancer cells. Cancers are classified by cell type, including carcinomas, sarcomas, lymphomas and leukemias, germ cell tumors, and blastomas.

[0069] As used in this article, the term "tumor" refers to a new growth formed by the proliferation of local tissue cells under the influence of various tumorigenic factors. Because this new growth often presents as a space-occupying, mass-like protrusion, it is also called a neoplasm. Liver cancer and pancreatic cancer are both known as the "kings of cancers," being highly malignant tumors. Research has found that tumor cells exhibit metabolic changes different from normal cells, and that tumor cells themselves can adapt to changes in the metabolic environment through the switching between glycolysis and oxidative phosphorylation (OXPHOS). In 2019, a recent article published in Cancer Cell found that metformin used in a fasting state can significantly inhibit tumor growth and proposed that the PP2A-GSK3β-MCL-1 pathway may be a new target for cancer treatment.

[0070] As used in this article, the term "T cell" generally refers to a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T cells are distinguished from other lymphocytes, such as B cells and natural killer cells, by T cell receptors present on their cell surface. They are called T cells because they mature from thymic cells in the thymus. Most T cells rearrange their α and β chains on cell receptors and are called alpha-beta T cells (αβ T cells), which are part of the adaptive immune system. Specialized γδ T cells (a small subset of T cells in the human body, more common in ruminants) have invariant T cell receptors, limited diversity, are capable of effectively presenting antigens to other T cells, and are considered part of the innate immune system.

[0071] As used herein, the terms "CHOK1" or "CHO" refer to hamster ovary cells, with CHOK1 being a substrain of hamster ovary cells; both are commonly used experimental cells. In this application, CHOK1 and CHO are used interchangeably.

[0072] As used in this article, the term "A375 cell" refers to human melanoma cells. These cells can be of human, mouse, or other species origin.

[0073] As used herein, the terms “MEDI9447”, “BMS986179”, and “TJD5” are known anti-CD73 antibodies used as reference antibodies for the anti-CD73 antibody P2A1 of this application.

[0074] As used in this article, the term "secondary antibody" refers to a fluorescent antibody, such as anti-human IgG-PE, that recognizes P2A1 and a reference antibody.

[0075] As used in this article, the term "pharmaceutically acceptable excipient" generally refers to any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0076] As used in this article, the term "EC50" refers to the half-maximal concentration (CMC), which is the concentration at which a drug produces a 50% maximum effect. EC50 is a drug safety indicator. It means the drug concentration that is effective in 50% of individuals. LD50 / ED50, TD50 / ED50, TC50 / EC50, etc., are collectively referred to as therapeutic indices, which are safety indicators for a class of drugs; generally, the higher the value, the safer the drug. It should be noted that these indicators only reflect the relationship between therapeutic effect and acute toxicity, and do not reflect chronic toxicity or allergenicity.

[0077] As used in this article, the term "IC50" refers to the half-inhibition concentration, or half-inhibition rate, which is a very important data in the indirect competitive ELISA standard curve.

[0078] As used herein, the term "about" generally refers to an approximation of a given value that can be reasonably inferred based on ordinary techniques in the art, including equivalent and approximate values ​​resulting from the experimental and / or measurement conditions of that given value. For example, it can refer to a value that is no more than 10% higher or lower than the value modified by the term. For example, the term "about 5 μg / kg" refers to a range of 4.5 μg / kg to 5.5 μg / kg. As another example, "about 1 hour" means a range of 48 minutes to 72 minutes.

[0079] As used herein, the term "effective dose" generally refers to a dose sufficient to provide a high enough concentration to impart a beneficial effect to the recipient. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the condition being treated, the severity of the condition, the activity of the specific component, the route of administration, clearance, duration of treatment, the subject's age, weight, sex, diet, and general health condition, as well as other relevant factors.

[0080] This application provides an antibody, its antigen-binding fragment, or a variant thereof, which is expressed in 1×10-1... -9 M is below (e.g., the value of KD is not higher than approximately 1×10). -9 M, not higher than approximately 9×10 -10 M, not higher than approximately 8×10 -10 M, not higher than approximately 7×10 -10 M, not higher than approximately 6×10 -10 M, not higher than approximately 5×10 -10 M, not higher than approximately 4 × 10 -10 M, not higher than approximately 3×10 -10M, not higher than approximately 2×10 -10 M, not higher than 1×10 -10 M or not higher than approximately 2 × 10 -11 The antibody (with a KD value of M or less) binds to the CD73 protein. That is, the antibody, its antigen-binding fragment, or a variant thereof specifically binds to the CD73 protein. Further, the CD73 protein is derived from mice, primates, and humans. Further, the CD73 protein is selected from the group consisting of human D73, mouse CD73, and monkey CD73. In this application, the antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the CD73 protein is named P2A1.

[0081] In some embodiments, the heavy chain variable region of P2A1 of this application includes CDRH1, whose sequence is shown in SEQ ID NO.1, CDRH2, whose sequence is shown in SEQ ID NO.2, and CDRH3, whose sequence is shown in SEQ ID NO.3; and the light chain variable region includes CDRL1, whose sequence is shown in SEQ ID NO.4, CDRL2, whose sequence is shown in SEQ ID NO.5, and CDRL3, whose sequence is shown in SEQ ID NO.6.

[0082] In some embodiments, the sequence of the heavy chain variable region of P2A1 in this application is shown in SEQ ID NO.7; and the sequence of the light chain variable region is shown in SEQ ID NO.8.

[0083] Furthermore, in some embodiments, the heavy chain constant region of P2A1 in this application is selected from one or more of IgG (IgG1, IgG2 or IgG4), IgM, IgA, IgE or IgD; the light chain constant region is selected from the κ or λ chain.

[0084] In some embodiments, the antibody or antigen-binding portion of P2A1 of this application is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0085] In some embodiments, the antigen-binding fragment of P2A1 in this application is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

[0086] Furthermore, the P2A1 of this application can specifically recognize and bind to the CD73 protein of primates, while recognizing almost no CD73 protein of rodents. Primates include, but are not limited to, humans and cynomolgus monkeys. Rodents include, but are not limited to, mice and rats. The CD73 protein described in this application can be human CD73 protein or monkey CD73 protein. For example, the CD73 protein may not be mouse CD73 protein, or may not be rat CD73 protein. In some embodiments, the antibody, its antigen-binding fragment, or variants described in this application substantially do not bind to mouse or rat CD73 protein.

[0087] In some embodiments, P2A1 exhibits superior binding affinity to CD73 compared to the reference antibody MEDI9447—a known anti-CD73 antibody. In some embodiments, the binding activity of P2A1 to primate CD73 protein is dose-dependent. In some embodiments, P2A1 can better exert its enzyme activity inhibitory function, demonstrating superior protein-level enzyme activity inhibitory activity.

[0088] Furthermore, P2A1 binds to the N-terminal domain of the antigenic epitope of the CD73 protein.

[0089] The variants described in this application may be selected from the group consisting of: 1) proteins or polypeptides in which one or more amino acids have been substituted, deleted, or added to the antibody or its antigen-binding fragment; and 2) proteins or polypeptides 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 higher) sequence homology to the antibody or its antigen-binding fragment. In some embodiments, one or more random mutations (e.g., one or more, such as one or more amino acid substitutions) may also be included in the amino acid sequence of the light chain and / or heavy chain of P2A1 in this application. In this application, the mutated CD73 antibody or its antigen-binding fragment or variant still has the ability to specifically bind to primate CD73 protein. In some embodiments, primates include, but are not limited to, humans, lemurs, aye-tailed primates, slow lorises, tarsiers, sulcimers, marmosets, macaques, gibbons, and orangutans; further, the primates are humans and cynomolgus monkeys.

[0090] In another aspect, this application also provides isolated one or more nucleic acid molecules. These one or more nucleic acid molecules may encode the antibody, its antigen-binding fragment, or a variant described in this application. For example, each of the one or more nucleic acid molecules may encode the complete antibody, its antigen-binding fragment, or a variant, or may encode a portion thereof (e.g., CDRH1-3, CDRL1-3, V...). L VH (One or more of light chains or heavy chains).

[0091] At least one of the nucleic acid molecules described in this application may be codon-optimized. For example, the methods for codon optimization include, but are not limited to: eliminating rare codons, adjusting GC content, increasing mRNA stability, adjusting mRNA secondary structure, rationally designing adapters, and adjusting the start codon environment.

[0092] The nucleic acid molecules described in this application can be isolated. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR), (ii) clonal recombination, (iii) purification, such as separation by enzyme digestion and gel electrophoresis, or (iv) synthesis, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0093] In this application, nucleic acids encoding the antibodies, their antigen-binding fragments, or variants thereof can be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlapping extension PCR using synthetic oligonucleotides, as detailed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0094] In another aspect, this application provides one or more vectors comprising one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed 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 regulating gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the species or cell type and function, but typically includes 5' non-transcriptional sequences and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may contain a promoter region, which may contain a promoter sequence for transcriptionally controlling functionally linked nucleic acids. The expression control sequence may also include enhancer sequences or upstream activator sequences. One or more nucleic acid molecules described in this application can be operatively linked to the expression control element.

[0095] The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the expression vector may be T-easy.

[0096] In another aspect, this application provides a host cell that may contain one or more nucleic acid molecules and / or one or more vectors as described in this application. In some embodiments, each or every host cell may contain one or more nucleic acid molecules or vectors as described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more (e.g., two or more) nucleic acid molecules or vectors as described in this application. For example, the vectors as described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. The vectors as described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the host cell may be CHO-K1.

[0097] In another aspect, this application provides a method for preparing the described antibody, its antigen-binding fragment, or variant. The method may include culturing the host cells described in this application under conditions that cause expression of the described antibody, its antigen-binding fragment, or variant. For example, this can be achieved by using suitable culture media, suitable temperatures, and culture times, methods known to those skilled in the art.

[0098] In some cases, the method may further include the steps of separating and / or purifying the antibody or its antigen-binding fragment. For example, affinity chromatography using protein G-agarose or protein A-agarose can be used, and the antibody or its antigen-binding fragment described in this application can also be purified and separated by gel electrophoresis and / or high-performance liquid chromatography. For example, Protein A affinity purification can also be used.

[0099] In another aspect, this application provides a pharmaceutical composition that may comprise the antibody described in this application, its antibody-binding fragment or variant, the nucleic acid molecule, the vector, the host cell, and optionally a pharmaceutically acceptable adjuvant.

[0100] Pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight peptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants, etc.

[0101] 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 reservoir.

[0102] The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition of this application can inhibit or delay the development or progression of a disease, reduce tumor size (or even essentially eliminate the tumor), and / or alleviate and / or stabilize a disease state.

[0103] The pharmaceutical composition described in this application may contain a therapeutically effective amount of the antibody or its antigen-binding fragment. The therapeutically effective amount is the dose required to prevent and / or treat (at least partially treat) a condition or disease (e.g., cancer) and / or any complications thereof in a subject who has or is at risk of developing such a condition.

[0104] On the other hand, this application provides the use of the said antibody, its antigen-binding fragment, or variant in the preparation of a medicament for the prevention or treatment of tumors.

[0105] On the other hand, this application provides the said antibody, its antigen-binding fragment, or variant for the prevention or treatment of tumors.

[0106] On the other hand, this application provides a method for preventing or treating tumors, comprising administering to a subject in need the antibody described in this application, its antigen-binding fragment or variant, the molecular nucleic acid, the vector, the host cell, and / or the pharmaceutical composition described herein.

[0107] The use of P2A1, or a fusion protein containing P2A1, in the preparation of reagents for determining the presence and / or amount of CD73 in a sample.

[0108] Pharmaceutical compositions comprising P2A1 of this application, a fusion protein comprising P2A1, one or more isolated nucleic acid molecules encoding P2A1, or one or more vectors comprising nucleic acid molecules encoding P2A1 and / or cells comprising the above-mentioned antibodies, fusion proteins, nucleic acid molecules, or vectors.

[0109] 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, leukemia, melanoma, glioma, thyroid cancer, prostate cancer, and breast cancer.

[0110] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating how the products, methods or systems of this application work, and are not intended to limit the scope of the invention.

[0111] Example

[0112] Example 1: Generation of anti-CD73 antibody P2A1

[0113] Various immunization strategies (DNA immunization, protein immunization, and cell immunization) were used to immunize mice of different species, including Balb / c and SJL breeds. Beacon was used to screen mouse B cells that were positive for binding to human CD73, and the candidate molecule P2A1, which specifically recognizes human CD73, was isolated through sequence extraction and sequencing.

[0114] The amino acid sequence of the antibody variable region was analyzed using the Kabat sequence determination method, as shown below.

[0115] In this embodiment, the complementarity-determining region (CDRH1) sequence of the heavy chain variable region of anti-CD73 antibody P2A1 is: ASYMH, the CDRH2 sequence is: LVYPYSGGTNYNQKFKG, and the CDRH3 sequence is: VQDYGYGLDY. In this embodiment, the CDRL1 sequence of the complementarity-determining region (CDRL3) of the light chain variable region of anti-CD73 antibody P2A1 is: RASQDISNYLN, the CDRL2 sequence is: YTSRLHS, and the CDRL3 sequence is: QQGSTLPRT.

[0116] Furthermore, in this application, the heavy chain variable region V of the anti-CD73 antibody P2A1... H The sequence is:

[0117] EVQLQQSGPVLVKPGPSVKISCKASGFTFTASYMHWVKQSHGKSLEWIGLVYPYSGGTNYNQKFKGKATLTVDTSSSTAYMELNSLTSEDSAVYYCARVQDYGYGLDYWGQGTTLTVSS;

[0118] Light chain variable region V L The sequence is:

[0119] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTNYSLTISNLEQEDIATYFCQQGSTLPRTFGGGTKLEIK.

[0120] Based on the Kabat sequence determination method, the full-length amino acid sequence of the antibody was analyzed, and the constant regions of its heavy and light chains are shown below:

[0121] The heavy chain constant region sequence of anti-CD73 antibody P2A1 is as follows:

[0122] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0123] The light chain constant region sequence of anti-CD73 antibody P2A1 is as follows:

[0124] AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0125] Example 2: Species cross-reactivity of P2A1

[0126] The species cross-reactivity of P2A1 was evaluated by detecting its binding to CHOK1 overexpressing CD73 in different species using FACS.

[0127] Following the standard operating procedure (SOP) for cell culture, culture and process the corresponding stable cell lines expressing human, monkey, and mouse CD73 antigens, including CHOK1-huCD73, CHOK1-cynoCD73, and CHOK1-mCD73. After digestion, harvest the cells and centrifuge at 300g for 5 minutes at room temperature. Discard the supernatant and wash twice with FACS buffer (PBS + 2% FBS). Place (2-5) × 10⁶ cells into 96-well plates. 5 Cells in each well were centrifuged and the supernatant discarded. The cells were then resuspended in diluted antibody solution (100 μL / well), mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded. Cells were washed twice with FACS buffer, and 100 μL / well of the appropriate secondary antibody solution was added to each well. Cells were incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant and antibody solution were discarded. Cells were washed twice with FACS buffer. Finally, the cells were resuspended in PBS, and signal values ​​were detected using a BD flow cytometer.

[0128] Figure 1 The species cross-reactivity of P2A1 according to one embodiment of this application is shown. The results are as follows... Figure 1 As shown, P2A1 can recognize and bind to CD73 in primates and cynomolgus monkeys, but cannot recognize CD73 in rodent mice.

[0129] Example 3: Binding activity of P2A1

[0130] The binding activity of P2A1 to huCD73 on the cell surface was evaluated by measuring the EC50 at different concentrations using FACS.

[0131] As described above, the CHOK1-huCD73 cell line, which expresses human CD73 antigen, was cultured and processed using flow cytometry. After digestion, the harvested cells were centrifuged at 300g for 5 minutes at room temperature. The supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). (2-5) × 10⁶ cells were then placed in 96-well plates. 5Cells in each well were centrifuged and the supernatant discarded. The cells were then resuspended in a gradient of diluted antibody solutions (100 μL / well), mixed, and incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded. Cells were washed twice with FACS buffer, and the corresponding secondary antibody solution (100 μL / well) was added to each well. Cells were incubated at 4°C for 1 hour. After centrifugation at 300 g for 5 minutes at 4°C, the supernatant antibody solution was discarded. Cells were washed twice with FACS buffer. Finally, the cells were resuspended in PBS, and signal values ​​were detected using a BD flow cytometer.

[0132] Figure 2 The binding curves of P2A1 and the reference antibody MEDI-9447 to CHOK1-huCD73 cells according to one embodiment of this application are shown. Table 1 shows the binding parameters of P2A1 and the reference antibody MEDI-9447 to CHOK1-huCD73 cells according to one embodiment of this application. The results are as follows: Figure 2 As shown in Table 1, P2A1 exhibits strong binding activity to human CD73 on the cell surface. This indicates that P2A1 can effectively bind to the surface of CHOK1-huCD73 cells overexpressing human CD73, and this binding activity is dose-dependent, with superior binding ability compared to the reference antibody MEDI-9447.

[0133] Table 1: Binding parameters of P2A1 to reference antibody MEDI-9447 in CHOK1-huCD73 cells

[0134] Top 209234 198582 4597 EC50(nM) 0.2713 1.98 NA <![CDATA[Goodness of fit (R 2 )]]> 0.9932 0.9992 0.9998

[0135] Example 4: Protein-level enzyme activity inhibition function of P2A1

[0136] The biological function of P2A1 was evaluated by detecting its inhibitory function at the protein level.

[0137] Dilute 5× antigen protein solution (final concentration 300 ng / ml) in TM buffer and add 50 μL / well of diluent to a 96-well plate. Dilute a certain proportion of 5× antibody diluent in TM buffer and add 50 μL / well of diluent to a 96-well plate. Mix well and incubate at 37°C for 30 minutes. Dilute 10× AMP (final concentration 200 μM) in TM buffer and add 25 μL / well of diluent to a 96-well plate. Incubate at 37°C for 30 minutes. Transfer 50 μL of supernatant to a new 96-well plate, add 2× ATP solution diluted in TM buffer (final concentration 65 μM), add 50 μL / well to the 50 μL supernatant, and then add 100 μL / well of a CellTiter Glo Reagent fluorescent cell activity detection system. Mix well at room temperature and incubate for 10 minutes. Read the luminescence signal.

[0138] Figure 3 The protein-level enzyme activity inhibition of P2A1 with reference antibody MEDI-9447 according to one embodiment of this application is shown. Results are as follows... Figure 3 As shown, P2A1, similar to the reference antibody MEDI-9447, exhibits reduced protein-level enzyme activity inhibition at high antibody concentrations ("hook effect"), which may be related to the binding epitope. Compared to the reference antibody MEDI-9447, P2A1 shows... Figure 3 As shown, the concentration that produces the hook effect is higher, and it still has enzyme activity inhibition function at a concentration of 17 nM, while the reference antibody MEDI-9447 has no function at this concentration. P2A1 can better exert its enzyme activity inhibition function and has better protein level enzyme activity inhibition activity.

[0139] Example 5: Cellular-level enzyme activity inhibition function of P2A1

[0140] The biological function of P2A1 was evaluated by detecting its inhibitory function at the cellular level.

[0141] Dilute 5× cell suspension (3200 CHOK1-hCD73 cells / well) in TM buffer and add 50 μL / well of diluent to a 96-well plate. Dilute a certain proportion of 5× antibody diluent in TM buffer and add 50 μL / well of diluent to a 96-well plate. Mix well and incubate at 37°C for 30 minutes. Dilute 10× AMP (final concentration 200 μM) in TM buffer and add 25 μL / well of diluent to a 96-well plate. Incubate at 37°C for 30 minutes. Transfer 50 μL of supernatant to a new 96-well plate, add 2× ATP solution (final concentration 65 μM) diluted in TM buffer, add 50 μL / well to the 50 μL supernatant, and then add 100 μL / well of a CellTiter Glo Reagent fluorescent cell activity assay system. Mix well at room temperature and incubate for 10 minutes. Read the luminescence signal.

[0142] Figure 4 Table 2 shows the cellular-level enzyme activity inhibition of P2A1 with reference antibodies TJD5 and MEDI-9447 according to one embodiment of this application. The results are as follows: Figure 4 As shown in Table 2, both P2A1 and the reference antibody MEDI-9447 can inhibit the enzyme activity of CD73 overexpressed in cells, and this inhibition is dose-dependent. However, P2A1 has a smaller IC50 and exhibits superior enzyme activity inhibition.

[0143] Table 2: Cellular-level enzyme activity inhibition of P2A1 compared to reference antibodies TJD5 and MEDI-9447

[0144] P2A1 93.586 0.230 MEDI9447 83.135 0.417 TJD5 99.332 0.669

[0145] Example 6: Affinity detection of P2A1

[0146] The affinity of P2A1 was detected using the protein-protein interaction analysis platform (Biacore).

[0147] The CM5 chip uses an amino-coupled method to capture a certain amount of anti-human IgG antibody, and then captures the antibody using a capture method. Different concentrations of human CD73-his are analyzed by passing the flow through the chip to detect its affinity.

[0148] Figure 5 The binding dissociation constants of P2A1 and human CD73 according to one embodiment of this application are shown in Table 3. The results are as follows: Figure 5 As shown in Table 3, antibody P2A1 has a high affinity for the CD73 target protein, with a KD value of 1.11 × 10⁻⁶. -10 M.

[0149] Table 3: Binding dissociation constant parameters of P2A1 and human CD73

[0150]

[0151] Example 7: Detection of P2A1 endocytic activity

[0152] The endocytic activity of P2A1 and the reference antibody was detected by FACS.

[0153] Add 100 μL of a 2×10⁻⁶ concentration to a 96-well plate. 6 CHOK1-huCD73 cell suspension was centrifuged at 300g for 5 minutes at 4°C, and the supernatant was discarded. 100 μL of CD73 Tab antibody was added to each well to bring the final antibody concentration to 20 μg / mL, and the cells were incubated at 4°C for 30 minutes. Cells were washed twice with FACS buffer to remove unbound excess antibody. After resuspending the cells in 100 μL of FACS buffer, endocytosis was performed at 4°C and 37°C for 1, 2, and 4 hours, respectively. Cells at different time points and temperatures were fixed on ice with 2% paraformaldehyde for 15 minutes, the supernatant was washed away, and 100 μL of the corresponding secondary antibody solution was added to each well, and the cells were incubated at 4°C for 1 hour. The cells were centrifuged at 300g for 5 minutes at 4°C, the supernatant antibody solution was discarded, and the cells were washed twice with FACS buffer. Finally, the cells were resuspended in PBS, and the signal values ​​were detected using a BD flow cytometer.

[0154] Table 4: Detection of endocytic activity of P2A1 with reference antibodies TJD5 and MEDI-9447

[0155]

[0156] Figure 6 The table shows the endocytic activity detection parameters of P2A1 with reference antibodies TJD5 and MEDI-9447 according to one embodiment of this application. Table 4 shows the endocytic activity detection results of P2A1 with reference antibodies TJD5 and MEDI-9447 according to one embodiment of this application. Figure 6 As shown, P2A1 exhibits significantly superior endocytic activity compared to the reference antibodies MEDI-9447 and TJD5, and is better able to reduce CD73 on the cell surface, thus demonstrating better biological activity.

[0157] Example 8: Detection of P2A1 by Mixed Lymphatic Reaction (MLR)

[0158] The in vitro immune activation function of P2A1 was evaluated by MLR detection.

[0159] Adjust the DC (stimulator) cell concentration to 1×10⁻⁶. 6 Add 10 μg / ml mitomycin C to inhibit DC proliferation, and incubate at 37°C for 45 minutes. Centrifuge at 500g for 5 minutes and wash twice with 10 mL PBS. Follow the kit instructions (EasySep). TM CD4+ T cells (Responders) were isolated and purified from PBMCs using a Human CD4+ T Cell Isolation Kit. The CD4+ T cell density was adjusted to 2 × 10⁶ cells / cells using X-VIVO15 medium. 6 / ml, adjust the density of DC cells to 4×10⁶ 5 CD4+ T cells and DC cells were mixed in equal volumes (1:1, DC:CD4+ T = 1:20) at a ratio of 1:1. 100 μL / well of the mixed cell suspension was added to a 96-well plate, followed by 50 μL / well of 5× anti-PD1 (final concentration 10 nM), 50 μL / well of 5× AMP (final concentration 40 μM), and 50 μL / well of serially diluted antibody. The plate was incubated at 37°C for 120 hours. After centrifugation at 500 g for 5 min, the supernatant was collected, and the IFN-γ content in the supernatant was determined according to the instructions of the IFN-γ HTRF kit (Cisbio, CAT#62HIFNGPEG).

[0160] Figure 7 The MLR detection of P2A1 with reference antibodies TJD5 and MEDI-9447 according to one embodiment of this application is shown. Results are as follows... Figure 7As shown, P2A1 can antagonize AMP-mediated immunosuppression by promoting interferon release from CD4+ T cells. The addition of a certain concentration of P2A1 significantly promoted the release of interferon from CD4+ T cells in a dose-dependent manner. Its ability to promote interferon release was significantly superior to the reference antibodies TJD5 and MEDI-9447, demonstrating the biological activity of P2A1.

[0161] Example 9: Detection of P2A1 reversing AMP-mediated T cell proliferation

[0162] Use EasySep TM The human CD4+ T cell extraction kit (Stemcell, Cat#17952) was used to extract CD4+ T cells from PBMCs. The extracted CD4+ T cells were mixed with 30 mL of PBS, centrifuged at 500 g for 5 minutes, and then resuspended in 0.1% BSA / PBS to adjust the cell density to 2 × 10⁻⁶ cells / mL. 6 / ml. Mix T cells with an equal volume of CFSE (4uM) and incubate at 37°C for 10 minutes. Add 40% volume of FBS to stop staining and incubate at 37°C for 10 minutes. Wash twice with T cell culture medium (10% FBS in RPMI 1640), 500g, 5 minutes. Adjust T cell density to 1×10⁶ cells / ml. 6 Add Anti-CD2 / CD3 / CD28 beads (1 bead: 2 cells) to each well. Transfer T cell suspension to a 96-well cell culture plate (Corning #3799), 100 μL / well. Add 50 μL of serially diluted 4× antibody and 50 μL of 4× AMP (final concentration 800 μM or 1000 μM) to each well. Incubate at 37°C, 5% CO2 for 96 hours, centrifuge at 400g for 5 minutes, discard the supernatant, resuspend in 100 μL PBS, and detect CFSE signal using a BD flow cytometer.

[0163] Figure 8 The table shows the detection of P2A1 reversing AMP-mediated T cell proliferation according to one embodiment of this application. Table 5 shows the detection of P1G11 reversing AMP-mediated T cell proliferation according to one embodiment of this application. Figure 8 As shown in Table 5, P2A1 can reverse AMP-mediated T cell proliferation. The addition of a certain concentration of P2A1 significantly promotes T cell proliferation in a dose-dependent manner, and its ability to promote T cell proliferation is superior to that of the reference antibody MEDI-9447.

[0164] Table 5: Detection of P2A1 reversing AMP-mediated T cell proliferation

[0165]

[0166] Example 10: Classification of P2A1 antigenic epitopes

[0167] To further clarify the epitope of P2A1 binding to CD73, the antigen-binding region was determined by competitive FACS method against reference antibodies MEDI9447, BMS986179 and TJD5, which are known to bind to different regions of CD73 epitopes.

[0168] Following the standard operating procedure (SOP) for cell culture, culture and process stable cell lines expressing the corresponding antigens. After digestion, harvested cells were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with FACS buffer (PBS + 2% FBS). (2-5)×E+05 cells / well were placed in a 96-well plate, centrifuged, and the supernatant was discarded. The cells were resuspended in 40ug / ml unlabeled antibody solution (100µL / well), mixed, and incubated at 4°C for 30 minutes. Without washing, the cells were resuspended in fluorescently labeled antibody solution (100µL / well), mixed, and incubated at 4°C for 45 minutes. After centrifugation at 300g for 5 minutes at 4°C, the supernatant antibody solution was discarded, and the cells were washed three times with FACS buffer. Finally, the cells were resuspended in PBS, and the signal values ​​were detected using a BD flow cytometer.

[0169] Table 6: Classification of antigenic epitopes of P2A1

[0170]

[0171] Table 6 shows the antigenic epitope classification of P2A1 according to one embodiment of this application. As shown in Table 6, the binding epitopes of P2A1 overlap with those of BMS986179 and MEDI9447, proving that the epitope of P2A1 binding to the CD73 antigen is the N-terminal domain.

[0172] Example 11: In vivo efficacy of P2A1

[0173] To evaluate the in vivo efficacy of P2A1 and the similar reference antibody MEDI-9447 in an A375 cell xenograft tumor model.

[0174] Will contain 4×10 6 0.2 mL of A375 cell suspension (200 μL DMEM basal medium) was subcutaneously injected into the right posterior dorsal region of each BALB / c nude mouse. On day 21 post-inoculation, the average tumor volume reached 99.16 mm². 3 The administration was started in groups of 8 mice. Each group was given 10 mg / kg three times a week. If the weight loss exceeded 15%, the administration was stopped immediately until the weight recovered to more than 98% of the initial weight, at which point normal administration was resumed. If the weight loss exceeded 20%, the animals were euthanized.

[0175] Figure 9 The relative changes in tumor volume from A375 cell xenografts according to one embodiment of this application are shown in Table 7. Figure 9 As shown in Table 7, P2A1 showed a significantly better tumor inhibition rate than MEDI9447.

[0176] Table 7: Relative changes in tumor volume after A375 cell xenografting

[0177]

[0178] In summary, this invention provides CD73 antibodies that, in both soluble and cell membrane surface forms, exhibit high binding affinity for human and cynomolgus monkey CD73 proteins; and possess potent inhibitory activity against CD73 enzyme activity at both the soluble protein and cellular levels. Furthermore, the binding of these antibodies induces CD73 internalization in tumor cells, leading to a further reduction in CD73 activity on the cell surface. Compared to Medimmune's MEDI9447, a known antibody with similar properties (binding to the N-terminal domain), the antibodies of this invention demonstrate superior in vitro and in vivo antitumor activity, making them excellent candidate drug molecules for therapeutic and diagnostic applications.

[0179] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. An antibody that specifically binds to CD73 or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes: 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 includes: CDRL1, whose sequence is shown in SEQ ID NO.4; CDRL2, the sequence of which is shown in SEQ ID NO.5; and CDRL3, whose sequence is shown in SEQ ID NO.

6.

2. The antibody or its antigen-binding fragment according to claim 1, wherein: The sequence of the heavy chain variable region is shown in SEQ ID NO. 7; The sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain constant region and a light chain constant region, wherein: The constant region of the antibody heavy chain is selected from one or more of IgG, IgM, IgA, IgE or IgD; the constant region of the light chain is selected from the κ or λ chain.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the IgG series antibody is selected from one or more of IgG1, IgG2 and IgG4.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from whole antibodies.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof is selected from monoclonal antibodies.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof is selected from humanized antibodies.

8. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of chimeric antibodies and fully human antibodies.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment and ScFv.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD73 is selected from primate CD73.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the primate is selected from humans and cynomolgus monkeys.

12. One or more isolated nucleic acid molecules encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-11.

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 comprising a CAR-T or CAR-NK cell containing one or more isolated nucleic acid molecules of claim 12 or one or more vectors of claim 13.

16. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1-11, comprising culturing cells according to claim 12 or 13 under conditions that enable the expression of an antibody or antigen-binding fragment thereof according to any one of claims 1-11.

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

18. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1-11, one or more isolated nucleic acid molecules according to claim 12, one or more carriers according to claim 13, and / or cells according to claim 14 or 15 in the preparation of a medicament for treating cancer or tumor; wherein the cancer or tumor is a CD73-positive cancer or tumor; wherein the cancer or tumor is selected from colorectal cancer, pancreatic cancer, non-small cell lung cancer, ovarian cancer, melanoma, prostate cancer, and breast cancer.

19. The use according to claim 18, wherein, The drug is a cell therapy drug; The cancer or tumor is a CD73-positive cancer or tumor; wherein the cancer or tumor is selected from colorectal cancer, pancreatic cancer, non-small cell lung cancer, ovarian cancer, melanoma, prostate cancer, and breast cancer.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-11 in the preparation of a reagent for determining the presence and / or amount of CD73 in a sample.

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

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