Anti-cd39 humanized antibodies or antigen binding fragments thereof and uses thereof

Abstract

The application provides an anti-CD39 humanized antibody or an antigen binding fragment thereof and an application thereof, and relates to the technical field of biological medicines, and the antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, CDR regions of the heavy chain variable region comprise HCDR1, HCDR2 and HCDR3, and CDR regions of the light chain variable region comprise LCDR1, LCDR2 and LCDR3. The humanized antibody or the antigen binding fragment thereof reduces the risk of antibody immunogenicity, maintains the functional activity of the antibody, and can be widely used for preparing medicines for diseases, disorders or conditions related to CD39.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-CD39 humanized antibody or its antigen-binding fragment and its applications. Background Technology

[0002] CD39, or exonucleoside triphosphate diphosphate hydrolase 1 (ENTPDase 1), is a single-channel type II transmembrane protein that converts extracellular eATP or ADP into AMP. CD73 then converts AMP into adenosine, a metabolite that exerts immunosuppressive effects in the tumor microenvironment. When the produced adenosine binds to adenosine receptors on the surface of CD4 T cells, CD8 T cells, and natural killer (NK) cells, it inhibits T cell and NK cell responses, thereby suppressing the immune system and promoting tumor growth. Furthermore, adenosine binds to A2A or A2B receptors on macrophages and dendritic cells, thereby inhibiting phagocytosis and antigen presentation, and increasing the secretion of oncogenes (such as VEGF, TGF, and IL-6). Adenosine, on the other hand, promotes the immunosuppressive activity of Tregs. Within the tumor microenvironment (TME), the adenosine pathway refers to the conversion of eATP to adenosine by adenosine hydrolase and the signal transduction of adenosine through A2A / A2B adenosine receptors on immune cells. Under normal conditions, CD39 maintains a balance between the extracellular levels of immunosuppressive adenosine and immunostimulatory ATP. In healthy tissues, ATP is almost undetectable in the extracellular environment because it is rapidly broken down by CD39 to generate ADP or AMP, and then AMP is converted to adenosine by CD73. Under cellular stress conditions, including cancer, eATP levels are significantly elevated, but due to the successive hydrolysis by CD39 and CD73 to adenosine, the immune system's recognition and killing of tumors is hindered.

[0003] Therefore, the development and optimization of anti-CD39 antibodies are of great significance for the prevention or treatment of tumor-related diseases. Summary of the Invention

[0004] The purpose of this invention is to humanize antibodies that specifically bind to CD39 in order to reduce the risk of antibody immunogenicity while maintaining the functional activity of the antibody.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] In a first aspect, the antibody or its antigen-binding fragment comprises:

[0007] (a) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions shown in any one of SEQ ID NO. 1 to 8; and,

[0008] (b) Light chain variable region, which includes LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NO. 9 to 16;

[0009] In an optional implementation, the HCDR1, HCDR2, and HCDR3, as well as the LCDR1, LCDR2, and LCDR3, are determined according to the IMGT definition method, Kabat definition method, Chothia definition method, AbM definition method, or Contact definition method.

[0010] Secondly, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0011] (i) Polynucleotides, including nucleotide sequences encoding anti-CD39 humanized antibodies or antigen-binding fragments thereof in the first aspect.

[0012] (ii) A vector carrying the aforementioned (i) polynucleotide.

[0013] (iii) Cells carrying the polynucleotide in (i), or containing the carrier in (ii), or expressing the anti-CD39 humanized antibody or its antigen-binding fragment of the first aspect.

[0014] Thirdly, an immunoconjugate is also provided, which is formed by conjugating the anti-CD39 humanized antibody of the first aspect or its antigen-binding fragment with at least one diagnostic agent and / or therapeutic agent to form an immunoconjugate.

[0015] The diagnostic agent is selected from one or more of the following: radioactive contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photosensitizers.

[0016] The therapeutic agent is selected from one or more of the following: cytotoxic agents, drugs, radionuclides, boron atoms, immunomodulators, anti-apoptotic agents, photosensitizing therapeutic agents, immunoconjugates, and oligonucleotides.

[0017] Fourthly, the application of the anti-CD39 humanized antibody or its antigen-binding fragment from the first aspect, or the biological material from the second aspect, or the immunoconjugate from the third aspect, in any of the following:

[0018] (i) Detection of CD39 or CD39-expressing cells for non-diagnostic and non-therapeutic purposes;

[0019] (ii) Prepare products for detecting CD39 or cells expressing CD39;

[0020] (iii) Blocking the ATPase hydrolytic activity of CD39 on cells for non-diagnostic and therapeutic purposes;

[0021] (iv) Prepare an inhibitor for blocking the ATPase hydrolysis activity of CD39 on cells;

[0022] (v) To prepare medicines for the treatment, prevention or relief of diseases, symptoms or conditions associated with CD39.

[0023] Fifthly, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the anti-CD39 humanized antibody of the first aspect or its antigen-binding fragment, or the biological material of the second aspect, or the immunoconjugate of the third aspect; and optionally a pharmaceutically acceptable carrier and / or excipient.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention humanizes murine monoclonal antibodies, reducing the risk of antibody immunogenicity while maintaining the antibody's functional activity. Experimental verification shows that the anti-CD39 humanized antibody or its antigen-binding fragment provided by this invention has binding activity to CD39 on cells, blocking the ATPase hydrolysis activity of CD39 on the cell surface. Application to mice improves tumor conditions, indicating that the anti-CD39 humanized antibody or its antigen-binding fragment provided by this invention can reduce adenosine in the tumor microenvironment, exerting an anti-tumor effect and providing new possibilities for cancer treatment and / or prevention. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1a The binding activity of humanized humanized antibodies (R1801, R1802, R1803, R1804, R1805, R1806 and R1807) to Mino cells;

[0028] Figure 1b The binding activity of humanized antibodies (R1808, R1809, R1810, R1839, R1840, R1841 and R1842) to Mino cells;

[0029] Figure 1c The binding activity of humanized antibodies (R1843, R1844, R1845, R1846, R1847, R1848 and R1849) to Mino cells;

[0030] Figure 1d The binding activity of humanized antibodies (R1850, R1851, R1852 and R1853) to Mino cells;

[0031] Figure 2a The blocking curves of humanized antibodies (R1801, R1802, R1803, R1804, R1805, R1806 and R1807) on the ATPase hydrolysis activity of CD39 on Mino cells;

[0032] Figure 2b The blocking curves of humanized antibodies (R1808, R1809, R1810, R1839, R1840, R1841 and R1842) on the ATPase hydrolysis activity of CD39 on Mino cells;

[0033] Figure 2c The blocking curves of humanized antibodies (R1843, R1844, R1845, R1846, R1847, R1848 and R1849) on the ATPase hydrolysis activity of CD39 on Mino cells.

[0034] Figure 2d The blocking curves of humanized antibodies (R1850, R1851, R1852 and R1853) on the ATPase hydrolysis activity of CD39 on Mino cells;

[0035] Figure 3a The blocking curves of humanized antibodies (R1801, R1802, R1803, R1804, R1805, R1806 and R1807) on the ATPase hydrolysis activity of CD39 on MOLP-8 cells.

[0036] Figure 3b The blocking curves of humanized antibodies (R1808, R1809, R1810, R1841, R1847, R1850 and R1853) on the ATPase hydrolysis activity of CD39 on MOLP-8 cells.

[0037] Figure 4a The inhibition curves of humanized antibodies (R1801, R1802, R1803 and R1804) on CD39-mediated ATP hydrolysis T cell proliferation;

[0038] Figure 4b The inhibition curves of humanized antibodies (R1805, R1806, R1807 and R1808) on CD39-mediated ATP hydrolysis T cell proliferation;

[0039] Figure 4cThe inhibition curves of humanized antibodies (R1809, R1810, R1841 and R1847) on CD39-mediated ATP hydrolysis T cell proliferation;

[0040] Figure 4d The inhibition curves of humanized antibodies (R1850 and R1853) on CD39-mediated ATP hydrolysis T cell proliferation;

[0041] Figure 5a The binding activity of humanized antibodies (R1803, R1804, R1806, R1807, R1841 and R1847) to CHO-K1-cynoCD39 cells;

[0042] Figure 5b The binding activity of the humanized antibody (R1853) to CHO-K1-cynoCD39 cells;

[0043] Figure 6 The binding activity of humanized antibodies (R2122 and R2123) to Mino cells;

[0044] Figure 7 The binding activity of humanized antibodies (R2122 and R2123) to MOLP-8 cells;

[0045] Figure 8 The blocking curves of humanized antibodies (R2122 and R2123) on the ATPase hydrolysis activity of CD39 on Mino cells;

[0046] Figure 9 The blocking curves of humanized antibodies (R2122 and R2123) on the ATPase hydrolysis activity of CD39 on MOLP-8 cells;

[0047] Figure 10 The inhibition curves of humanized antibodies (R2122 and R2123) on CD39-mediated ATP hydrolysis T cell proliferation;

[0048] Figure 11 Tumor volume change curves after mice were injected with MOLP-8 cells and given humanized antibodies (R2122 and R2123);

[0049] Figure 12 The curves showing the change in mouse body weight after injection of MOLP-8 cells and administration of humanized antibodies (R2122 and R2123). Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0051] definition:

[0052] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising" shall be understood to include the stated elements or components without excluding other elements or other components.

[0053] In this article, the term "CD39" is also referred to as ENTPD1 or ENTPD enzyme 1, which is a membrane protein that converts ATP into AMP. CD39 contains two transmembrane domains, a smaller cytoplasmic domain, and a larger extracellular hydrophobic domain.

[0054] In this article, "antibody or its antigen-binding fragment" refers to a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments containing a complementarity-determining region (CDR). Furthermore, "antibody or its antigen-binding fragment" also includes both naturally occurring antibodies and non-naturally occurring antibodies.

[0055] In this article, "antigen-binding fragment" refers to a substance containing an antibody CDR that lacks at least some amino acids present in the full-length chain but still specifically binds to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), and scFv dimers (bivalent bifunctional antibodies). The aforementioned antigen-binding fragments can bind to the same antigen as the parent antibody.

[0056] In this paper, the term "Fab" in antibody refers to a portion of an antibody composed of a single light chain (including variable and constant regions) and a single heavy chain whose variable and first constant regions are linked by disulfide bonds. A "Fab' fragment" refers to a Fab fragment containing a portion of the hinge region. "F(ab')2" refers to a Fab' dimer. An "Fv fragment" is composed of the variable regions of a single light chain and / or a single heavy chain. A "single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by the direct interconnection of light chain variable regions and heavy chain variable regions, or by linkage through peptide linker sequences. A "minimum recognition unit" refers to a structure containing only a single CDR within the variable region; although the minimum recognition unit has a small molecular weight and low affinity, it possesses the ability to bind to antigens.

[0057] In this article, the "variable region" or "variable domain" of an antibody or its antigen-binding fragment refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing the antigen. The heavy chain variable region can be referred to as "VH," and the light chain variable region can be referred to as "VL." Variable domains contain antigen-binding sites. The variable regions of both the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). Typically, the VL / VH variable regions of the heavy and light chains are obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0058] The CDR boundaries of antibodies or their antigen-binding fragments in this article can be defined or identified according to the definitions of IMGT, Kabat, Chothia, AbM, and Contact. CDRs defined in other ways acceptable in the art are also within the scope of protection of this invention (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005);

[0059] RMMacCallum et al., Antibody–antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996);

[0060] Martin, ACRProtein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001);

[0061] Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulinand T cell receptor variable domains and Ig superfamily V-like domains,Developmental and Comparative Immunology 27(2003)55–77).

[0062] In this article, "humanized antibody" refers to an antibody that retains the reactivity of a non-human antibody while exhibiting low immunogenicity in humans. For example, it can be constructed by retaining the CDR region of a non-human antibody and replacing the rest of the antibody with a human antibody counterpart (i.e., the framework portion of the constant region and the variable region).

[0063] The terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of a binding protein to an epitope on a pre-defined antigen. Typically, binding proteins bind at a rate of approximately less than 10... -5 M, for example, approximately less than 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Value binding. The K value of the antibody can be determined using methods well-established in the art. D Values. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry.

[0064] The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Polynucleotides contain a portion encoding the aforementioned antibody or its antigen-binding fragment, optionally encoding either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0065] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0066] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0067] The terms “cell,” “cell line,” and “cell culture” used in this article are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, for example, in morphological and / or genomic DNA differences.

[0068] In this article, the term "Mino cell" refers to a human non-Hodgkin lymphoma cell line that naturally expresses the human CD39 protein on its cell surface.

[0069] In this paper, the term "MOLP-8 cell" refers to a human multiple myeloma cell line that naturally expresses human CD39 protein on its cell surface. Multiple myeloma models established by inoculating these cells into NOD-SCID mice can be used to detect the in vivo anti-tumor effect of anti-CD39 antibodies.

[0070] In this document, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition is administered. In some specific embodiments, the antibody contained in or expressed in the pharmaceutical composition is capable of specifically targeting and binding to CD39, blocking the hydrolysis of ATP by CD39.

[0071] In a first aspect, an anti-CD39 humanized antibody or an antigen-binding fragment thereof is provided, the anti-CD39 humanized antibody or the antigen-binding fragment thereof comprising (a) and (b):

[0072] (a) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions shown in any one of SEQ ID NO. 1 to 8; and,

[0073] (b) Light chain variable region, which includes LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NO. 9 to 16.

[0074] In an optional implementation, the HCDR1, HCDR2, and HCDR3, as well as the LCDR1, LCDR2, and LCDR3, are determined according to the IMGT definition method, Kabat definition method, Chothia definition method, AbM definition method, or Contact definition method.

[0075] Table 1 shows the CDR amino acid sequences of exemplary antibodies 1847 (the amino acid sequence of the heavy chain variable region H6 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region L8 is shown in SEQ ID NO.9) and 1853 (the amino acid sequence of the heavy chain variable region H8 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region L8 is shown in SEQ ID NO.9) defined according to different definitions.

[0076] Table 1. CDR amino acid sequences of exemplary monoclonal antibodies

[0077]

[0078]

[0079] In an optional embodiment, the HCDR1, HCDR2, and HCDR3 of the anti-CD39 humanized antibody or its antigen-binding fragment, as well as LCDR1, LCDR2, and LCDR3, are determined according to the IMGT definition:

[0080] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.17; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.22; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.27; the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.30; the LCDR2 comprises the amino acid sequence shown in YT; and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.36.

[0081] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD39 humanized antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO. 1 to 8.

[0082] In an optional embodiment, an antibody or its antigen-binding fragment having a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO. 1 to 8, and an amino acid sequence of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO. 9 to 16.

[0083] In an optional embodiment, the amino acid sequence of the light chain variable region of the anti-CD39 humanized antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO. 9 to 16.

[0084] In an optional embodiment, an antibody or its antigen-binding fragment having an amino acid sequence as shown in any one of SEQ ID NO. 9 to 16, and an amino acid sequence of the heavy chain variable region as shown in any one of SEQ ID NO. 1 to 18.

[0085] In an optional embodiment, the heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment are selected from any combination of the following, as shown in Table 2:

[0086] Table 2

[0087]

[0088]

[0089] In an optional embodiment, the antigen-binding fragment includes one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibodies, and antibody minimum recognition units.

[0090] In an optional embodiment, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region;

[0091] The heavy chain constant region contains a sequence of part or all of the constant region of any one of IgG1, IgG1.8, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD;

[0092] The light chain constant region is a κ or λ chain;

[0093] In an optional implementation, the constant region source species include one or more combinations of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, and humans.

[0094] In an optional embodiment, the heavy chain constant region is selected from IgG1; the amino acid sequence of the heavy chain constant region is preferably as shown in SEQ ID NO.39.

[0095] In an optional embodiment, the heavy chain constant region is selected from IgG1.8; the amino acid sequence of the heavy chain constant region is preferably as shown in SEQ ID NO.40;

[0096] In an optional embodiment, the light chain constant region is a κ chain; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.41.

[0097] Secondly, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0098] (i) Polynucleotides, including nucleotide sequences encoding anti-CD39 humanized antibodies or antigen-binding fragments thereof in the first aspect.

[0099] (ii) A vector carrying the aforementioned (i) polynucleotide.

[0100] (iii) Cells carrying the polynucleotide in (i), or containing the carrier in (ii), or expressing the anti-CD39 humanized antibody or its antigen-binding fragment of the first aspect.

[0101] Thirdly, an immunoconjugate is also provided, which is formed by conjugating the anti-CD39 humanized antibody of the first aspect or its antigen-binding fragment with at least one diagnostic agent and / or therapeutic agent to form an immunoconjugate.

[0102] The diagnostic agent is selected from one or more of the following: radioactive contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photosensitizers.

[0103] The therapeutic agent is selected from one or more of the following: cytotoxic agents, drugs, radionuclides, boron atoms, immunomodulators, anti-apoptotic agents, photosensitizing therapeutic agents, immunoconjugates, and oligonucleotides.

[0104] Radioactive nuclides include, but are not limited to, 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 One or more of Sr.

[0105] Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0106] Fluorescent markers include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C. Phycocyanin R. Thiamine, Phycoerythrin, Phycoerythrin R. REG. Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (Tetramethylrhodamine isothiol), Tetramethylrhodamine, and Texas Red.

[0107] Oligonucleotides include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

[0108] Drugs include, but are not limited to, methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vincristine, paclitaxel, cephalotaxine alkaloids, and L-asparaginase.

[0109] Immunomodulators include, but are not limited to, one or more of the following: cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factors (CSF), interferons, erythropoietin, thrombopoietin, tumor necrosis factor (TNF), interleukins (IL), granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and stem cell growth factors.

[0110] Radioactive nuclides include, but are not limited to, 111 In、 111 At、 177 Lu、 211 Bi、 212 Bi、 213 Bi、 211 At、 62 Cu、 67 Cu、 90 Y、 125 I, 131 I, 133 I, 32 P, 33 P, 47 Sc、 111 Ag、 67 Ga、 153 Sm、 161 Tb, 152 Dy、 166 Dy、 161 Ho、 166 Ho、 186 Re、 188 Re、 189 Re、 211 Pb, 212 Pb, 223 Ra、 225 Ac、 77 As、 89 Sr、 99 Mo、 105 Rh、 149 Pm, 169 Er、 194 Ir、 58 Co、 80 mBr、 99 mTc, 103 mRh、 109 Pt, 119 Sb、 189 mOs, 192 Ir、 219 Rn、 215 Po、 221 Fr、 255 Fm、 11 C 13 N、 15 O、 75 Br、 198 Au、 199 Au、 224 Ac、 77 Br、 113 mIn, 95 Ru、 97 Ru、103 Ru、 105 Ru、 107 Hg, 203 Hg, 121 mTe, 122 mTe, 125 mTe, 165 Tm、 167 Tm、 168 Tm、 197 Pt, 109 Pd, 142 Pr、 143 Pr、 161 Tb, 57 Co、 58 Co、 51 Cr 59 Fe、 75 Se、 201 Tl、 76 Br and 169 One or more of Yb.

[0111] Fourthly, the application of the anti-CD39 humanized antibody or its antigen-binding fragment from the first aspect, or the biological material from the second aspect, or the immunoconjugate from the third aspect, in any of the following:

[0112] (i) Detection of CD39 or CD39-expressing cells for non-diagnostic and non-therapeutic purposes;

[0113] (ii) Prepare products for detecting CD39 or cells expressing CD39;

[0114] (iii) Blocking the ATPase hydrolytic activity of CD39 on cells for non-diagnostic and therapeutic purposes;

[0115] (iv) Prepare an inhibitor for blocking the ATPase hydrolysis activity of CD39 on cells;

[0116] (v) To prepare medicines for the treatment, prevention or relief of diseases, symptoms or conditions associated with CD39.

[0117] In an optional implementation, the application of aspect (i) above can utilize the ability of anti-CD39 antibodies or their antigen-binding fragments to specifically target and bind to CD39, enabling the detection of CD39 or CD39-expressing cells based on immunoassay techniques. For example, when the antibody or its antigen-binding fragment chain is an immunoconjugate, such as one linked to a fluorescent group, a fluorescence detection device can be used to locate or detect CD39 in real time. This can be used in techniques such as immunoblotting, immunoprecipitation, or flow cytometry, which involve utilizing the specific binding properties of CD39 antigen and antibody to detect CD39 or CD39-expressing cells. Correspondingly, regarding aspect (ii) above, those skilled in the art can select the reagent composition in the kit according to the actual detection method, including but not limited to antagonists, anti-CD39 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc.

[0118] In an optional embodiment, the application of step (iii) above can allow the cells whose ATPase hydrolysis activity is to be blocked to be in full contact with the anti-CD39 antibody or its antigen-binding fragment, for example, but not limited to, placing the cells whose ATPase hydrolysis activity is to be blocked in a culture medium containing the anti-CD39 antibody or its antigen-binding fragment for incubation.

[0119] In an optional embodiment, the inhibitor used to block the ATPase hydrolysis activity of CD39 on cells also contains excipients acceptable in the art, including but not limited to culture media, preservatives, and buffer components.

[0120] Fifthly, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the anti-CD39 humanized antibody of the first aspect or its antigen-binding fragment, or the biological material of the second aspect, or the immunoconjugate of the third aspect; and optionally a pharmaceutically acceptable carrier and / or excipient.

[0121] The term "optionally" indicates that the pharmaceutical composition may or may not contain a pharmaceutically acceptable carrier and / or excipient. In an optional embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipient may be any carrier and / or excipient known in the art and conventionally available. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, used to extend the shelf life or potency of antibodies; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, solubilizers, and emulsifiers.

[0122] In optional embodiments, the pharmaceutical composition may further include one or more active pharmaceutical ingredients with other therapeutic effects, including but not limited to one or more of the following: chemotherapeutic agents, anticancer drugs, radiotherapy agents, immunotherapy agents, antiangiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0123] In an optional embodiment, the pharmaceutical composition is a medicine for treating, preventing, or alleviating diseases, symptoms, or conditions related to CD39.

[0124] The CD39-related diseases, conditions, or circumstances mentioned in the fourth and fifth aspects above refer to any disease or circumstance caused, aggravated, or associated with an increase or decrease in CD39 expression or activity. In some embodiments, CD39-related diseases, conditions, or circumstances are circumstances related to excessive cell proliferation, such as cancer. In some embodiments, CD39-related diseases or circumstances are characterized by the expression or overexpression of CD39 and / or CD39-related genes, such as ENTPD1, ​​2, 3, 4, 5, 6, 7, or 8 genes.

[0125] In alternative implementations, "CD39-related" diseases, conditions, or circumstances include, but are not limited to, cancer, autoimmune diseases, and infections.

[0126] In an alternative implementation, "CD39-related" cancers include cancers in which CD39 is expressed in cancer cells or immune cells or immunosuppressive cells infiltrating the tumor, and in which the level of CD39 expression in cancer cells or immune cells or immunosuppressive cells infiltrating the tumor is significantly higher than the level expected in normal cells.

[0127] In optional implementations, examples of "CD39-related" cancers include, but are not limited to, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, and gastrointestinal cancer. Cancer including esophageal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T- or B-cell lymphoma, gastrointestinal stromal tumors, soft tissue tumors, hepatocellular carcinoma, and adenocarcinoma. In some embodiments, the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer.

[0128] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0130] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The invention is further illustrated below by specific examples; however, it should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention in any way. The references cited are: *Reaction* (Mullis et al., ed., 1994); and *Current Protocols in Immunology* (JEColigan et al., ed., 1991), each of which is expressly incorporated herein by reference.

[0131] Example 1: Humanization of a monoclonal antibody against CD39

[0132] Recombinant human CD39 fusion protein and constructed CHOK1-hCD39 were used as immunogens to generate anti-human CD39 antibodies. Further screening yielded #110 mouse monoclonal antibody, which exhibited superior binding affinity for CHO-hCD39, Mino, Molp-8, and cynoCD39. The antibody effectively blocked CD39 enzyme activity on Mino and Molp-8 cells. Furthermore, functional experiments showed that the antibody reduced ADO production by blocking CD39 enzyme activity on T cells, thereby promoting T cell proliferation (the weight-variable region and light chain variable region of #110 mouse monoclonal antibody are shown in Table 3). Therefore, this embodiment humanized the above-mentioned #110 mouse monoclonal antibody using the following specific method:

[0133] Humanization was performed using complementarity-determining region (CDR) grafting. First, antibody homology modeling of the Fv region of the #110 mouse monoclonal antibody was conducted using MOE (Molecular Operating Environment) software. Based on the model structure, key amino acid residues affecting the conformational stability of the antigen-binding region were analyzed. Subsequently, a human immunoglobulin database was searched, and human IGVH and IGVK sequences with high homology to #110 were used as templates for humanization. #110 was compared with matching human IGVH and IGVK sequences, focusing on analyzing sites on the original mouse monoclonal antibody where key amino acid residues affecting the conformational stability of the antigen-binding region were inconsistent with the human IGVH and IGVK sequences. The possibility of humanization replacement was observed within the model. Depending on the degree of humanization replacement, multiple humanized sequences could be generated simultaneously from a single parent sequence.

[0134] For molecule #110, the humanized heavy chain sequence is shown in Table 3 below:

[0135] Table 3

[0136]

[0137] For molecule #110, the humanized light chain sequence is shown in Table 4 below:

[0138] Table 4

[0139]

[0140]

[0141] Combining different humanized light and heavy chains can produce different humanized antibody molecules. The main consideration in different combinations is balancing the degree of humanization with the maintenance of antibody activity after humanization. Simply put, pairing the light and heavy chains with the highest degree of humanization yields the antibody molecule with the highest degree of humanization. This implies potentially lower immunogenicity, but a high degree of humanization may lead to reduced activity of the modified molecule (key amino acid residues affecting the activity of the parent antibody are replaced with humanized amino acids, causing changes in the antigen-binding region of the modified antibody). Conversely, pairing the light and heavy chains with the lowest degree of humanization yields functional activity more consistent with the parent antibody (because most key amino acid residues are retained), but the degree of humanization is also lower, potentially leading to a higher risk of immunogenicity. This is a balancing act; the preparation of humanized antibodies requires expression of various light and heavy chain combinations, followed by validation through in vivo and in vitro functional experiments, ultimately screening for suitable humanized antibody molecules.

[0142] For molecule #110, the humanized antibody sequence is shown in Table 5 below:

[0143] Table 5

[0144]

[0145] The constant region sequences can be used to construct complete antibodies, as shown in Table 6 below:

[0146] Table 6

[0147]

[0148]

[0149] Example 2. Expression and purification of humanized antibodies

[0150] Antibody preparation was performed using standard methods, and the expression supernatant was purified by ProA affinity chromatography. The procedure was as follows: using an AKTA Avant 150 chromatography system, the column (e.g., MabSelect SuRe LX, GE) was equilibrated with at least 5 CV equilibration buffer (10 mM PBS). The sample was loaded onto the column, allowing the target protein to adsorb onto the column while other impurities permeated and separated. After loading, the column was washed again with at least 5 CV equilibration buffer (10 mM PBS), followed by elution with elution buffer (20 mM NaAc, pH 3.4). Neutralization buffer (1 M Tris, pH 8.0) was pre-added to the collection tube; the volume of neutralization buffer added depended on the estimated concentration of the eluted sample, generally 10% of the elution volume.

[0151] Sample concentration was determined using the Biotek-Epoch-Take-3 assay. Antibody concentration was detected using the A280 method, with an extinction coefficient EC = 1.37 and a path length of 0.05 mm (slight differences in path length may occur between wells in the Take-3 plate, which is automatically corrected). The absorbance of the sample was measured using the instrument, and the concentration of the antibody was calculated according to the Lambert-Beer law. If the sample concentration was too low, ultrafiltration concentration was required using an ultrafiltration concentrator (…). Using the Ultra-15 Centrifugal Filter Devices (30kD), concentrate the sample concentration to >0.5mg / ml according to the general operating procedure provided in the instruction manual; collect the concentrated sample, sterilize it with a 0.22μm sterile needle filter (Cobbat, PES, 0.22μm, 13mm diameter), and then aliquot and freeze for later use.

[0152] The preparation data of the #110 humanized antibody are shown in Table 7 below.

[0153] Table 7

[0154]

[0155]

[0156] Note: In the purity column of the table, the leftmost value is the percentage of aggregates, the rightmost value is the percentage of fragments, and the middle value is the percentage of antibody monomers. "-" indicates a value of 0%. For example, "8.98% / 91.02% / -" indicates that the percentage of aggregates in the antibody solution is 8.98%, the percentage of antibody monomer purity is 91.02%, and the percentage of fragments is 0%.

[0157] Example 3. Binding activity of humanized antibodies to Mino cells

[0158] Mino cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and seeded into 96-well V plates at a ratio of 2E5 cells / well. CD39 monoclonal antibody and control antibody were added to each well, and the plates were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PE Goat anti-mouse IgG Fc (1:500 dilution) per well. The cells were resuspended and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PBS per well to resuspend the cells. The binding differences of CD39 antibody to Mino cells were assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figures 1a to 1dAs shown, R1839, R1840, R1842, R1843, R1844, R1845, R1846, R1848, R1849, R1851, and R1852 showed no or weak binding activity with Mino cells. The affinity of these 11 humanized antibodies was significantly lower than that of the chimeric antibody R1796 (#110-hIgG1) and the positive control antibody R1800 (ES002-hIgG1), while the remaining antibodies were superior to or on par with the positive antibody.

[0159] The amino acid sequence of the heavy chain variable region of the chimeric antibody R1796 (#110-hIgG1) is shown in SEQ ID NO.37, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.39, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.38, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.41.

[0160] The amino acid sequence of the heavy chain variable region of the positive control antibody R1800 (ES002-hIgG1) is shown in SEQ ID NO.42, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.39, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.43, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.41.

[0161] Example 4. Detection of ATPase hydrolysis activity of CD39 on Mino cells blocked by humanized antibody.

[0162] After adjusting the density of Mino cells to 4E6 cells / mL, transfer 50 μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend in fresh culture medium). Prepare antibody at a starting concentration of 60 μg / mL using culture medium. Perform 3-fold dilutions to obtain 10 concentrations. Add 100 μL of antibody to each well of the cells, mix well, and incubate at 37°C for 60 min. Prepare ATP at 200 μM using culture medium, add 50 μL per well to the cells, mix well, and incubate at 37°C for 45 min. Centrifuge the cells at 350G for 5 min, collect 80 μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, and incubate at room temperature in the dark for 10 min before analysis. The blocking curve is shown below. Figures 2a to 2d As shown, similar to the binding activity results detected on Mino cells, R1839, R1840, R1842, R1843, R1844, R1845, R1846, R1848, R1849, R1851, and R1852 showed no or weak blocking activity against CD39 enzyme activity on Mino cells. These 11 humanized antibodies had significantly lower affinity and blocking activity than the chimeric antibody R1796, and were therefore excluded. These 11 humanized antibodies will not be tested in subsequent tests.

[0163] Example 5. Detection of ATPase hydrolysis activity of CD39 in MOLP-8 cells blocked by humanized antibody.

[0164] After adjusting the density of MOLP-8 cells to 3E6 cells / ml, transfer 50 μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend in fresh culture medium). Prepare antibody at a starting concentration of 60 μg / ml. Perform 3-fold dilutions to obtain 10 concentrations. Add 100 μL of antibody to each well, mix well, and incubate at 37°C for 60 min. Prepare ATP at 200 μM, add 50 μL per well to the cells, mix well, and incubate at 37°C for 35 min. Centrifuge the cells at 350G for 5 min, collect 80 μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, and incubate at room temperature in the dark for 10 min before analysis. The antibody blocking activity curve is shown below. Figure 3a and Figure 3b As shown, R1803, R1804, R1805, R1806, R1807, R1841, R1847, R1850, and R1853 exhibited blocking activities against CD39 hydrolase on MOLP-8 cells that were similar to or superior to those of the chimeric antibody R1796, while the remaining antibodies R1801, R1802, R1808, R1809, and R1810 showed relatively poor blocking activities. Further functional experiments will be conducted to evaluate these 14 antibodies.

[0165] Example 6. Analysis of T cell proliferation inhibition mediated by CD39 hydrolysis ATP mediated by humanized antibodies.

[0166] First, the T cell density was adjusted to 5E6 / mL. T cells were then labeled with CFSE (10mM stock solution) using DPBS as buffer to a final concentration of 0.5μM. Labeling was performed in a 37°C water bath for 20 min in the dark. Then, 12mL of complete culture medium (10%) was added. FBS (pre-cooled at 4℃) was used to terminate the CFSE reaction; the antibody was diluted with cell culture medium to prepare a concentration of 400 nM, serially diluted 10×, with 5 spots; the labeled cells were centrifuged at 300 G for 5 min, the supernatant was removed, the cells were resuspended in complete culture medium and the cell count was adjusted to 2E6 / ml (total cells are counted here, not live cells), and 50 μL / well was added to a 96-well U-shaped plate, i.e., the final cell count was 1E5 / well; the prepared antibody was added to the cells at 50 μL / well; the beads were diluted according to the number of cells to ensure that the final cell:bead ratio was 20:1, and 50 μL / well was added to a 96-well U-shaped plate; after three days (72 h), the culture medium was diluted with ATP to 1200 μM and added to a 96-well plate at 50 μL / well, i.e., the final volume was 200 μL. Cells were incubated statically in an incubator for two days (48 hours). After centrifugation, the supernatant was discarded. A flow cytometry antibody (anti-human CD8T, 400× dilution; FITC fluorescent antibody cannot be used) was prepared using BSA and incubated at 4°C for 30 minutes. T cell proliferation was then detected by flow cytometry (10,000 cells were collected, ensuring a smooth peak shape). The analysis was performed according to the "CytoFLEX Flow Cytometer Standard Operating Procedure". The fitted curve is shown below. Figures 4a to 4d As shown, R1847 and R1853 are superior to the chimeric antibody R1796.

[0167] Example 7. Binding activity of humanized antibody to CHO-K1-cynoCD39 cells

[0168] CHO-K1-cynoCD39 cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and seeded into 96-well V plates at a ratio of 2E5 cells / well. Antibody was prepared in culture medium at a starting concentration of 60 μg / mL. Ten concentrations were obtained by 3-fold dilution, with CD39 humanized antibody and chimeric antibody added to each well, and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PE Goat anti-mouse IgG Fc (1:500 dilution) per well. Cells were resuspended and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PBS per well to resuspend the cells. The binding differences of CD39 antibody to CHO-K1-cynoCD39 cells were assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figure 5a and Figure 5bAs shown, the humanized and chimeric antibodies against CD39 exhibit similar binding activity to CHO-K1-cynoCD39 cells.

[0169] Based on the above results, we screened out antibodies R1847 and R1853, which performed better in binding blocking and functional experiments. Therefore, we selected these two antibodies for subsequent antibody subtype modification.

[0170] Example 8. Fc modification of candidate humanized antibodies

[0171] To prevent the antibody from producing strong ADCC and ADCP effects in the body, we mutated the hIgG1-Fc of the candidate protein. The correspondence between the molecular numbers before and after the modification is shown in Table 8 below:

[0172] Table 8

[0173] R1847 R2122 R1853 R2123 Positive control antibody R1800 R1289

[0174] Example 9. Binding activity of humanized antibodies with different subtypes to Mino cells

[0175] Mino cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and seeded into 96-well V plates at a ratio of 2E5 cells / well. CD39 monoclonal antibody and control antibody were added to each well, and the plates were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PE Goat anti-mouse IgG Fc (1:500 dilution) per well. The cells were resuspended and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PBS per well to resuspend the cells. The binding differences of CD39 antibody to Mino cells were assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figure 6 As shown, R2122 and R2123 exhibit similar binding activity to Mino cells, and their plateau values ​​are superior to those of the positive control antibody R1289.

[0176] Example 10. Binding activity of humanized antibody with different subtypes to MOLP-8 cells

[0177] MOLP-8 cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and seeded into 96-well V plates at a ratio of 2E5 cells / well. CD39 monoclonal antibody and control antibody were added to each well, and the plates were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PE Goat anti-mouse IgG Fc (1:500 dilution) per well. The cells were resuspended and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, followed by 100 μL of PBS per well to resuspend the cells. The binding difference of CD39 antibody to Mino cells was assessed using a Cytoflex flow cytometer (Beckman Countler). The binding curves are shown below. Figure 7 As shown, R2122 and R2123 exhibit similar binding activity to MOLP-8 cells, and their plateau values ​​are superior to those of the positive control antibody R1289.

[0178] Example 11. Detection of ATPase hydrolysis activity of CD39 on Mino cells blocked by humanized antibody.

[0179] After adjusting the density of Mino cells to 4E6 cells / ml, transfer 50 μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend in fresh culture medium). Prepare antibody at a starting concentration of 60 μg / mL using culture medium. Perform 3-fold dilutions to obtain 10 concentrations. Add 100 μL of antibody and hybridoma supernatant to each well of the cell culture plate, mix well, and incubate at 37°C for 60 min. Prepare ATP at 200 μM using culture medium, add 50 μL per well to the cell culture plate, mix well, and incubate at 37°C for 45 min. Centrifuge the cells at 350G for 5 min, collect 80 μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, and incubate at room temperature in the dark for 10 min before analysis. The blocking curve is shown below. Figure 8 As shown, R2122 and R2123 exhibited superior blocking activity in Mino cells.

[0180] Example 12. Detection of ATPase hydrolysis activity of CD39 in MOLP-8 cells blocked by humanized antibody.

[0181] After adjusting the density of MOLP-8 cells to 3E6 cells / ml, transfer 50 μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend in fresh culture medium). Prepare antibody at a starting concentration of 60 μg / mL using culture medium. Perform 3-fold dilutions to obtain 10 concentrations. Add 100 μL of antibody and hybridoma supernatant to each well of the cell culture plate, mix well, and incubate at 37°C for 60 min. Prepare 200 μM ATP in culture medium and add 50 μL / well to the cell culture plate, mix well, and incubate at 37°C for 35 min. Centrifuge the cells at 350G for 5 min, collect 80 μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, and incubate at room temperature in the dark for 10 min before analysis. The antibody blocking activity curve is shown below. Figure 9 As shown, the IC50 of the blocking activity of R2123 on MOLP-8 cells was similar to that of R2122, and the plateau value was superior to that of R2122 and the positive antibody R1289.

[0182] Example 13. Analysis of humanized antibody blocking CD39-mediated ATP hydrolysis-induced T cell proliferation inhibition

[0183] First, the T cell density was adjusted to 5E6 / ml. T cells were then labeled with CFSE (10mM stock solution) using DPBS as buffer to a final concentration of 0.5μM. Labeling was performed in a 37°C water bath for 20 min in the dark. Then, 12mL of complete culture medium (10%) was added. FBS (pre-cooled at 4℃) was used to terminate the CFSE reaction; the antibody was diluted with cell culture medium to prepare a concentration of 400 nM, serially diluted 10×, with 5 spots; the labeled cells were centrifuged at 300 G for 5 min, the supernatant was removed, the cells were resuspended in complete culture medium and the cells were counted, and the density was adjusted to 2E6 (total cells are counted here, not live cells), and 50 μL / well was added to a 96-well U-shaped plate, i.e., the final cells were 1E5 / well; the prepared antibody was added to the cells at 50 μL / well; the beads were diluted according to the number of cells to ensure that the final system of cells:beads was 20:1, and 50 μL / well was added to a 96-well U-shaped plate; after three days (72 h), the culture medium was diluted with ATP to 1200 μM and added to a 96-well plate at 50 μL / well, i.e., the final system was 200 μL. Cells were incubated statically in an incubator for two days (48 hours). After centrifugation, the supernatant was discarded. A flow cytometry antibody (anti-human CD8T, 400× dilution; FITC fluorescent antibody cannot be used) was prepared using BSA and incubated at 4°C for 30 minutes. T cell proliferation was then detected by flow cytometry (10,000 cells were collected, ensuring a smooth peak shape). The analysis was performed according to the "CytoFLEX Flow Cytometer Standard Operating Procedure". The fitted curve is shown below. Figure 10As shown, R2122 exhibited superior T-cell proliferation inhibition activity compared to R2123 in this experimental system. Since only two candidate molecules were available, and each had its own advantages in blocking activity and functional experiments, both candidate antibodies were retained for subsequent functional assays.

[0184] Example 14. Detection of the role of humanized antibodies in MOLP-8 xenografted mice

[0185] Female NOD SCID mice aged 6-8 weeks were subcutaneously injected with 5×10⁻⁶ mg / L on day 0. 6 MOLP-8 cells were administered CD39 antibody at a dose of 30 mg / kg on days 1, 5, 8 and 12, respectively. Figure 11 The curve depicts the average tumor volume, the error bars represent SEM, the red triangles on the X-axis indicate the time points of compression, the X-axis shows the number of days after implantation, and the Y-axis represents the tumor volume. Figure 12 The curves depict the average mouse body weight, the error bars represent SEM, the arrows on the X-axis indicate the compression time points, the X-axis shows the number of days after implantation, and the Y-axis represents the mouse body weight. The results showed that all CD39 humanized antibodies had antitumor effects compared to the blank control, with R2123 showing better antitumor effects than R2122. Furthermore, there was no significant difference in mouse body weight between groups during the administration process.

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-CD39 humanized antibody or its antigen-binding fragment, characterized in that, The heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment are selected from any combination of the following: 。 2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The CDR regions of the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the CDR regions of the light chain variable region include LCDR1, LCDR2 and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.17; the amino acid sequence of HCDR2 is shown in SEQ ID NO.22; the amino acid sequence of HCDR3 is shown in SEQ ID NO.27; the amino acid sequence of LCDR1 is shown in SEQ ID NO.30; the amino acid sequence of LCDR2 is shown in YT; and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

36.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment includes one or more of F(ab')2, Fab', Fab, Fv, scFv, and dsFv.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; The heavy chain constant region contains a sequence of part or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; The constant region of the light chain is a κ or λ chain.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The constant region source species include one or more combinations of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, and humans.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The heavy chain constant region is selected from IgG1; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

39.

7. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

40.

8. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The light chain constant region is a κ chain; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

41.

9. A biomaterial, characterized in that, The biomaterial is selected from any one of (i) to (iii): (i) Polynucleotides, including nucleotide sequences encoding the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 8; (ii) A vector carrying the aforementioned (i) polynucleotide; (iii) A cell carrying the polynucleotide of (i), or containing the carrier of (ii), or expressing the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the biological material according to claim 9, in any one of the following: (i) Detection of CD39 or CD39-expressing cells for non-diagnostic and non-therapeutic purposes; (ii) Prepare products for detecting CD39 or cells expressing CD39; (iii) Blocking the ATPase hydrolysis activity of CD39 on cells for non-diagnostic and non-therapeutic purposes; (iv) Prepare an inhibitor for blocking the ATPase hydrolysis activity of CD39 on cells; (v) To prepare medicines for the treatment, prevention or relief of diseases, symptoms or conditions related to CD39; in, Diseases, conditions or conditions associated with CD39 are selected from: human multiple myeloma, human lymphocytic leukemia.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, or the biological material as described in claim 9; and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, or the biological material as described in claim 9; and a pharmaceutically acceptable excipient.

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