Anti-CD39 humanized antibody or antigen binding fragment thereof and application thereof

Abstract

The invention provides an anti-CD39 humanized antibody or an antigen binding fragment thereof and application thereof, and relates to the technical field of biological medicine, the antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and a CDR region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; the CDR region of the light chain variable region comprises 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 drugs for diseases, symptoms or conditions related to CD39.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-CD39 humanized antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] CD39, or ectonucleoside triphosphate diphosphohydrolase 1 (ENTPDase1), is a single-channel type II transmembrane protein that converts extracellular eATP or ADP into AMP, and then CD73 converts AMP into adenosine, a metabolite that exerts an immunosuppressive effect in the tumor microenvironment. When the generated adenosine binds to adenosine receptors on the surface of CD4 T cells, CD8 T cells, and natural killer (NK) cells, it can inhibit T cell and NK cell responses, thereby inhibiting the immune system and promoting tumor growth. In addition, adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, thereby inhibiting phagocytosis and antigen presentation, and increasing the secretion of tumor-promoting factors such as VEGF, TGF, and IL-6. Adenosine promotes the immunosuppressive activity of Tregs. In the TME, the adenosine pathway refers to the conversion of eATP to adenosine under the action of adenylate hydrolases, and the signaling 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 barely detectable in the extracellular environment because it is rapidly broken down by CD39 to generate ADP or AMP, which is then converted to adenosine by CD73. Under conditions of cellular stress, including cancer, eATP levels are significantly elevated, but are subsequently hydrolyzed by CD39 and CD73 to convert to adenosine, which hinders the immune system's recognition of tumors and their killing.

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

[0004] The purpose of the present invention is to humanize an antibody that specifically binds to CD39 to reduce the risk of antibody immunogenicity while maintaining the functional activity of the antibody.

[0005] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

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

[0007] (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs. 1 to 8; and,

[0008] (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NOs. 9 to 16;

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

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

[0011] (i) a polynucleotide comprising a nucleotide sequence encoding the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect.

[0012] (ii) a vector carrying the polynucleotide described in (i).

[0013] (iii) a cell, wherein the cell carries the polynucleotide described in (i), or contains the vector described in (ii), or expresses the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect.

[0014] In the third aspect, an immunoconjugate is also provided, wherein the immunoconjugate is formed by coupling the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect with at least one diagnostic agent and / or therapeutic agent.

[0015] The diagnostic agent is selected from one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent, and a photosensitizer;

[0016] The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photosensitive therapeutic agent, an immunoconjugate, and an oligonucleotide.

[0017] In a fourth aspect, there is also provided the use of the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect, or the biomaterial of the second aspect, or the immunoconjugate of the third aspect in any of the following:

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

[0019] (ii) preparing a product for detecting CD39 or cells expressing CD39;

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

[0021] (iv) preparing a blocking agent for blocking the ATPase hydrolysis activity of CD39 on cells;

[0022] (v) preparing a medicament for treating, preventing or ameliorating a disease, disorder or condition associated with CD39.

[0023] In a fifth aspect, a pharmaceutical composition is also provided, which contains the anti-CD39 humanized antibody or its antigen-binding fragment of the first aspect, or the biomaterial 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] The present invention reduces the risk of antibody immunogenicity and maintains the functional activity of the antibody by humanizing the mouse monoclonal antibody. Experimental verification shows that the anti-CD39 humanized antibody or its antigen-binding fragment provided by the present invention has binding activity with CD39 on cells and can block the ATPase hydrolysis activity of CD39 on the cell surface; administration to mice can improve the tumor condition of mice, indicating that the anti-CD39 humanized antibody or its antigen-binding fragment provided by the present invention can reduce adenosine in the tumor microenvironment, exert anti-tumor effects, and provide new possibilities for the treatment and / or prevention of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[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 humanized antibodies (R1808, R1809, R1810, R1839, R1840, R1841, and R1842) to Mino cells;

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

[0030] Figure 1d The binding activity of humanized 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 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 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 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 This is the inhibition curve of T cell proliferation mediated by CD39 hydrolysis of ATP by humanized antibodies (R1801, R1802, R1803 and R1804);

[0038] Figure 4b This is the inhibition curve of T cell proliferation mediated by CD39 hydrolysis of ATP by humanized antibodies (R1805, R1806, R1807 and R1808);

[0039] Figure 4cThis is the inhibition curve of T cell proliferation mediated by CD39 hydrolysis of ATP by humanized antibodies (R1809, R1810, R1841 and R1847);

[0040] Figure 4d This is the inhibition curve of T cell proliferation mediated by CD39 hydrolysis of ATP by humanized antibodies (R1850 and R1853);

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

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

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

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

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

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

[0047] Fig.10 This is the inhibition curve of T cell proliferation mediated by CD39 hydrolysis of ATP by humanized antibodies (R2122 and R2123);

[0048] Fig.11 The curve of tumor volume changes after mice were injected with MOLP-8 cells and given humanized antibodies (R2122 and R2123);

[0049] Fig.12 The weight change curve of mice after being injected with MOLP-8 cells and then given humanized antibodies (R2122 and R2123). DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] definition:

[0052] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising” will be understood to include the stated elements or components but not to exclude other elements or components.

[0053] As used herein, 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, which generally refers to all proteins and protein fragments containing a complementary determining region (CDR region). In addition, "antibody or its antigen-binding fragment" also includes naturally occurring antibodies and non-naturally occurring antibodies.

[0055] In this article, "antigen binding fragment" is a substance containing antibody CDRs, which lacks at least some of the amino acids present in the full-length chain but is still able to specifically bind to the 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), scFv dimers (bivalent bifunctional antibodies). The above-mentioned antigen binding fragments are able to bind to the same antigen as the parent antibody.

[0056] In this article, the term "Fab" of an antibody refers to a part of an antibody composed of a single light chain (including a variable region and a constant region) and the variable region and the first constant region of a single heavy chain bound together by a disulfide bond. "Fab' fragment" refers to a Fab fragment that includes part of the hinge region. "F(ab')2" refers to a dimer of Fab'. "Fv fragment" is composed of the variable region of a single light chain and / or the variable region of a single heavy chain. "Single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by the light chain variable region and the heavy chain variable region directly connected to each other or connected by a peptide linker sequence. "Antibody minimum recognition unit" refers to a single CDR structure containing only a variable region. Although the minimum recognition unit has a small molecular weight and low affinity, it has the ability to bind to the antigen.

[0057] As used herein, the "variable region" or "variable domain" of an antibody or its antigen-binding fragment refers to the domain of the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen, and the composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". The variable domain contains an antigen-binding site. The variable regions of 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 variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0058] The CDR boundaries of the antibody or antigen-binding fragment thereof herein can be defined or identified according to the IMGT, Kabat, Chothia, AbM, Contact definitions, and CDRs defined in other acceptable ways in the art also fall within the scope of protection of the present 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] Herein, "humanized antibody" is an antibody that retains the reactivity of non-human antibodies while having lower immunogenicity in humans. For example, it can be constructed by retaining non-human antibody CDR regions and replacing the rest of the antibody with human antibody counterparts (i.e., constant regions and variable region framework portions).

[0063] The terms "specific recognition", "selective binding", "selectively binds" and "specifically binds" or similar expressions refer to the binding of a binding protein to an epitope on a predetermined antigen. Typically, the binding protein is present at a rate of less than about 10 -5 M, for example, less than about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D The K value of an antibody can be determined using methods well established in the art. D Other standard assays for evaluating the binding ability of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis.

[0064] The term "polynucleotide" 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 natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide comprises a portion encoding the above-mentioned antibody or its antigen-binding fragment, and the encoding is optionally encoding a sense strand or an antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0065] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.

[0066] The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0067] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Progeny may not necessarily be completely identical to the original cell, e.g., differ in morphology and / or in genomic DNA from the original cell due to natural, accidental or deliberate mutation.

[0068] As used herein, the term "Mino cell" refers to a human non-Hodgkin's lymphoma cell line that naturally expresses human CD39 protein on its cell surface.

[0069] In this article, the term "MOLP-8 cells" refers to a human multiple myeloma cell line that naturally expresses human CD39 protein on its cell surface. The multiple myeloma model 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] As used herein, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. In some specific embodiments, the antibody contained in the above-mentioned pharmaceutical composition or the expressed antibody can specifically target and bind to CD39, blocking CD39 from hydrolyzing ATP.

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

[0072] (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs. 1 to 8; and,

[0073] (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NOs. 9 to 16.

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

[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 definition methods.

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

[0077]

[0078]

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

[0080] The HCDR1 includes the amino acid sequence shown as SEQ ID NO.17; the HCDR2 includes the amino acid sequence shown as SEQ ID NO.22; the HCDR3 includes the amino acid sequence shown as SEQ ID NO.27; the LCDR1 includes the amino acid sequence shown as SEQ ID NO.30; the LCDR2 includes the amino acid sequence shown as YT; the LCDR3 includes the amino acid sequence shown as 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 antigen-binding fragment thereof is shown in any one of SEQ ID NOs. 1 to 8.

[0082] In an optional embodiment, the antibody or antigen-binding fragment thereof having a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 8, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NOs. 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 antigen-binding fragment thereof is shown in any one of SEQ ID NOs. 9 to 16.

[0084] In an optional embodiment, the antibody or antigen-binding fragment thereof having a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs.9 to 16, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs.1 to 18.

[0085] In an optional embodiment, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof are selected from any of the following combinations, see 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 antibody and antibody minimum recognition unit.

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

[0091] The heavy chain constant region contains a sequence of a partial or complete 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 embodiment, the constant region is derived from species including one or more 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] In a second aspect, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0098] (i) a polynucleotide comprising a nucleotide sequence encoding the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect.

[0099] (ii) a vector carrying the polynucleotide described in (i).

[0100] (iii) a cell, wherein the cell carries the polynucleotide described in (i), or contains the vector described in (ii), or expresses the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect.

[0101] In the third aspect, an immunoconjugate is also provided, wherein the immunoconjugate is formed by coupling the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect with at least one diagnostic agent and / or therapeutic agent.

[0102] The diagnostic agent is selected from one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent, and a photosensitizer;

[0103] The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photosensitive therapeutic agent, an immunoconjugate, and an oligonucleotide.

[0104] Radionuclides 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), iron (II), 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 labels 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, 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 fast purple, cresol blue purple, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, trisbipyridyl diamine europium, europium cryptates or chelates, diamines, bicyanines, La Jolla blue dye, allophycocyanin, allococyanin B, one or more of 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, vinblastine, paclitaxel, cephalotaxel alkaloids, and L-asparaginase.

[0109] Immunomodulators include, but are not limited to, one or more of 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] Radionuclides 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 2. 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 Such as 97 Such as103 Such as 105 Such as 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] In a fourth aspect, there is also provided the use of the anti-CD39 humanized antibody or antigen-binding fragment thereof of the first aspect, or the biomaterial of the second aspect, or the immunoconjugate of the third aspect in any of the following:

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

[0113] (ii) preparing a product for detecting CD39 or cells expressing CD39;

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

[0115] (iv) preparing a blocking agent for blocking the ATPase hydrolysis activity of CD39 on cells;

[0116] (v) preparing a medicament for treating, preventing or ameliorating a disease, disorder or condition associated with CD39.

[0117] In an optional embodiment, the application of the above-mentioned aspect (i) can utilize anti-CD39 antibodies or their antigen-binding fragments to specifically target and bind to CD39, and realize the detection of CD39 or cells expressing CD39 based on immunodetection technology. For example, when the antibody or its antigen-binding fragment chain is an immunoconjugate, for example, it is connected with a fluorescent group, and a fluorescent detection device can be used to realize the positioning or real-time detection of CD39. For example, it can be used for immunoblotting, immunoprecipitation or flow cytometry, etc., which involve the use of the specific binding properties of CD39 antigen and antibody to detect CD39 or cells expressing CD39. Correspondingly, in the above-mentioned aspect (ii), those skilled in the art can select the reagent composition in the kit according to the actual detection means, 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 the above-mentioned (iii) can allow the cells whose ATPase hydrolysis activity is to be blocked to be fully contacted 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 and incubating them.

[0119] In an optional embodiment, the blocker for blocking the ATPase hydrolysis activity of CD39 on cells further contains excipients acceptable in the art, including but not limited to culture medium, preservatives and buffer components.

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

[0121] Wherein "optionally" means that the pharmaceutical composition contains or does 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 can be selected from any conventional carrier and / or excipient known in the art. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent and delayed absorption agent, etc., which are used to extend the shelf life or efficacy of the antibody; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, cosolvents and emulsifiers, etc.

[0122] In an optional embodiment, the pharmaceutical composition further comprises one or more active pharmaceutical ingredients having other therapeutic effects, including but not limited to one or more combinations of chemotherapeutic agents, anticancer drugs, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0123] In an optional embodiment, the pharmaceutical composition is used as a drug for treating, preventing or alleviating a disease, disorder or condition associated with CD39.

[0124] The diseases, disorders or conditions associated with CD39 in the fourth and fifth aspects above refer to any disease or condition caused, aggravated or associated with an increase or decrease in the expression or activity of CD39. In some embodiments, the disease, disorder or condition associated with CD39 is a disorder associated with excessive cell proliferation, such as cancer. In some embodiments, the disease or condition associated with CD39 is characterized by 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 embodiments, "CD39-related" diseases, disorders or conditions include, but are not limited to, cancer, autoimmune diseases and infections.

[0126] In an optional embodiment, "CD39-associated" cancer includes cancers in which CD39 is expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells, and the level of CD39 expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells is significantly higher than the expected level in normal cells.

[0127] In an optional embodiment, examples of "CD39-associated" 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 cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra 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, gastrointestinal cancer, 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] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If no specific conditions are specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments used is not specified, they 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 those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0130] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., 1994); and Current Protocols in Immunology (JE Coligan et al., 1991), each of which is expressly incorporated herein by reference. The present invention is further described below by specific examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the present invention in any form.

[0131] Example 1 Humanization of monoclonal antibodies against CD39

[0132] Recombinant human CD39 fusion protein and constructed CHOK1-hCD39 were used as immunogens to generate anti-human CD39 antibodies. After further screening, the obtained #110 mouse monoclonal antibody had a better binding affinity to CHO-hCD39, Mino, Molp-8 and cynoCD39. It can be detected on Mino and Molp-8 that the antibody can well block the enzymatic activity of CD39, and in the functional experiment part, it can also be seen that the antibody reduces the production of ADO by blocking the enzymatic activity of CD39 on T cells, thereby promoting the proliferation of T cells (the weight variable region and light chain variable region of the #110 mouse monoclonal antibody are shown in Table 3); Therefore, in this example, the above #110 mouse monoclonal antibody is humanized, and the specific method is as follows:

[0133] Humanization was performed by the complementary determining region transplantation (CDR-grafting) method. First, the MOE (Molecular Operating Environment) software was used to perform antibody homology modeling on the Fv region of the #110 mouse monoclonal antibody. According to the model structure, the key amino acid residues that can affect the conformational stability of the antigen binding region were analyzed. Subsequently, the human immunoglobulin database was searched to search for human IGVH and IGVK sequences with high homology to #110 in the human germline antibody library as humanization templates. #110 was compared with the matching human IGVH and IGVK sequences respectively, focusing on analyzing the key amino acid residues on the original mouse monoclonal antibody that can affect the conformational stability of the antigen binding region and the sites that are inconsistent with the human IGVH and IGVK sequences, and observing whether humanized replacements can be performed in the model. According to different degrees of humanized replacement, multiple humanized sequences can be generated simultaneously based on a parent sequence.

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

[0135] Table 3

[0136]

[0137] For the #110 molecule, 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. Different combinations mainly consider the balance between the degree of humanization and the maintenance of antibody activity after humanization. Simply put, pairing the light and heavy chains with the highest degree of humanization can obtain the antibody molecule with the highest degree of humanization, which means a possible lower immunogenicity, but a high degree of humanization may lead to a decrease in the activity of the modified molecule (the key amino acid residues that affect the activity of the parent antibody are replaced by humanized amino acids, resulting in changes in the antigen binding region of the modified antibody). Conversely, pairing the light and heavy chains with the lowest degree of humanization can obtain functional activity that is more consistent with the parent antibody (because most of the key amino acid residues are retained), but the degree of humanization is also low, and there may be a risk of higher immunogenicity. This is a balancing process. In the process of preparing humanized antibodies, it is necessary to express and prepare a variety of light and heavy chain combinations, and further verify them through in vivo and in vitro functional tests, and finally screen suitable humanized antibody molecules.

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

[0143] Table 5

[0144]

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

[0146] Table 6

[0147]

[0148]

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

[0150] The antibody was prepared by conventional methods, and the expression supernatant was purified by ProA affinity chromatography. The process is as follows: using an AKTA avant 150 chromatography device, the chromatography column (such as MabSelectSuRe LX, GE) was equilibrated with at least 5CV equilibration buffer (10mM PBS), and the sample was loaded onto the chromatography column, so that the target protein was adsorbed on the chromatography column and other impurities penetrated and separated. After the sample is loaded, the chromatography column is rinsed again with at least 5CV equilibration buffer (10mM PBS), and then the target protein is eluted with elution buffer (20mM NaAc, pH=3.4). Neutralization buffer (1M Tris, pH8.0) was pre-added to the collection tube, and the volume of neutralization buffer added was determined according to the estimated content of the eluted sample, and generally 10% of the elution volume was added.

[0151] The sample was measured using Biotek-Epoch-Take-3, and the antibody concentration was detected using the A280 method, i.e., the extinction coefficient EC = 1.37, the optical path = 0.05 mm (the optical path of different holes in the Take-3 plate is slightly different, which will be automatically corrected), the sample absorbance value was detected by the equipment, and the concentration of the antibody to be tested was calculated according to the Lambert-Beer law. If the sample concentration is too low, ultrafiltration concentration is required, using an ultrafiltration concentration tube ( Ultra-15 Centrifugal Filter Devices, 30kD) was used to concentrate the sample to a concentration of >0.5 mg / ml according to the general operating method provided in the instructions; the sample at the concentrated end was collected, sterilized and filtered through a 0.22 μm sterile syringe filter (Cobetter, PES, 0.22 μm, diameter 13 mm), and then aliquoted and frozen for later use.

[0152] The preparation data of humanized antibody #110 is 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, the middle value is the percentage of antibody monomers, and "-" indicates a value of 0%, for example, "8.98% / 91.02% / -", which indicates that in the antibody solution, the percentage of aggregates 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 plated on V-shaped 96-well plates at 2E5 / well. CD39 monoclonal antibody and control antibody were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200μL of PBS was added to each well to wash the cells, and then 100μl / well of PE Goat anti mouse IgG Fc (1:500 dilution) was added. After resuspending the cells, the cells were incubated at 4°C for 30 minutes. After centrifugation, 200μL of PBS was added to each well to wash the cells, and then 100μL of PBS was added to each well to resuspend the cells, and the binding difference of CD39 antibody to Mino cells was evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 1a to Figure 1dAs shown, R1839, R1840, R1842, R1843, R1844, R1845, R1846, R1848, R1849, R1851 and R1852 had no binding activity or weak binding activity to 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), and the remaining antibodies were better than or equal to the positive antibodies.

[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, and 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, and 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 antibodies

[0162] Adjust the density of Mino cells to 4E6 cells / mL and transfer 50μl per well to a 96-well U-bottom cell culture plate (be sure to resuspend with fresh culture medium); prepare the antibody culture medium at a starting value of 60μg / ml. Dilution was made 3 times, with a total of 10 concentrations. Add 100μL of the antibody to the cells per well, mix well, and incubate at 37°C for 60min. Prepare ATP culture medium to 200μM and add 50μL / well to the cells. Mix well and incubate at 37°C for 45min. Centrifuge the cells at 350G for 5min, take 80μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, incubate at room temperature in the dark for 10min, and detect on the machine. The blocking curve is as follows Figure 2a to Figure 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 had no blocking activity or weak blocking activity on the enzymatic activity of CD39 on Mino cells. The affinity and blocking activity of these 11 humanized antibodies were significantly lower than that of the chimeric antibody R1796, so they were excluded and will no longer be tested in subsequent tests.

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

[0164] Adjust the density of MOLP-8 cells to 3E6 cells / ml and transfer 50μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend with fresh culture medium); prepare the antibody in culture medium at a starting value of 60μg / ml. Dilution was made 3 times, with a total of 10 concentrations. Add 100μL of the antibody to the cells in each well, mix well, and incubate at 37°C for 60min. Prepare ATP in culture medium to 200μM and add 50μL / well to the cells. Mix well and incubate at 37°C for 35min. Centrifuge the cells at 350G for 5min, take 80μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, incubate at room temperature in the dark for 10min, and detect on a machine. The blocking activity curve of the antibody is shown in the figure below. Figure 3a and Figure 3b As shown, the blocking activities of R1803, R1804, R1805, R1806, R1807, R1841, R1847, R1850 and R1853 on CD39 hydrolase on MOLP-8 cells are similar to or better than those of chimeric antibody R1796, while the blocking activities of the remaining R1801, R1802, R1808, R1809 and R1810 are relatively poor. These 14 antibodies will be tested in functional experiments in the future.

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

[0166] First, the T cell density was adjusted to 5E6 / mL, and CFSE (10mM stock solution) was used as the buffer to label the T cells, with a final concentration of 0.5μM, and the cells were labeled in a 37°C water bath in the dark for 20 minutes; 12mL of complete culture medium (10% FBS, precooled at 4°C) was used to terminate the CFSE reaction; the antibody was diluted with cell culture medium to a concentration of 400nM, 10× gradient dilution, and 5 points were set; the labeled cells were centrifuged at 300G for 5 minutes to remove the supernatant, resuspended in complete culture medium and counted, and the density was adjusted to 2E6 / ml (total cells are counted here, not living cells), and 50μL / well was added to a 96-well U-shaped plate, that is, the final cell was 1E5 / well; the prepared antibody was added to the cells at 50μL / well; the beads were diluted according to the number of cells to ensure that the cell:bead ratio in the final system was 20:1, and added to the 96-well U-shaped plate at 50μL / well; three days (72h) later, the culture medium diluted ATP to 1200μM and added to a 96-well plate at 50μL / well, that is, the final system was 200μL. The cells were placed in an incubator and cultured for two days (48 hours). The cell plate was centrifuged and the supernatant was discarded. BSA was prepared with flow cytometry antibodies (anti-human CD8T, 400× dilution, FITC fluorescent antibodies cannot be used), incubated at 4°C for 30 minutes, and T cell proliferation was detected by flow cytometry (10,000 cells were collected to ensure smooth peaks). The test was performed on the machine according to the "CytoFLEX Flow Cytometer Standard Operating Procedure". The fitting curve is shown in the figure below. Figure 4a to Figure 4d As shown, R1847 and R1853 were superior to the chimeric antibody R1796.

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

[0168] CHO-K1-cynoCD39 cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and plated into V-shaped 96-well plates at 2E5 / well. The antibody culture medium was configured to start at 60μg / mL. Three-fold dilutions were made, with a total of 10 concentrations. CD39 humanized antibodies and chimeric antibodies were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200μL of PBS was added to each well to wash the cells, and then 100μL / well of PE Goat anti mouse IgG Fc (1:500 dilution) was added. After resuspending the cells, the cells were incubated at 4°C for 30 minutes. After centrifugation, 200μL of PBS was added to each well to wash the cells, and then 100μL of PBS was added to each well to resuspend the cells, and the binding differences of CD39 antibodies to CHO-K1-cynoCD39 cells were evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 5a and Figure 5bAs shown, the binding activity of the humanized antibody and chimeric antibody against CD39 on CHO-K1-cynoCD39 cells was similar.

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

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

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

[0172] Table 8

[0173] hIgG1 subtype hIgG1.8 subtype R1847 R2122 R1853 R2123 Positive control antibody R1800 R1289

[0174] Example 9. Binding activity of humanized antibodies after subtype change to Mino cells

[0175] Mino cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and plated into V-shaped 96-well plates at 2E5 / well. CD39 monoclonal antibody and control antibody were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL / well of PE Goat anti mouse IgG Fc (1:500 dilution) was added. After resuspending the cells, the cells were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL of PBS was added to each well to resuspend the cells, and the binding difference of CD39 antibody to Mino cells was evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 6 As shown, R2122 and R2123 had similar binding activities to Mino cells, and the platform values ​​were better than that of the positive control antibody R1289.

[0176] Example 10. Binding activity of humanized antibodies after subtype change to MOLP-8 cells

[0177] MOLP-8 cells were collected, centrifuged at 350G for 5 minutes, resuspended in PBS, and plated into V-shaped 96-well plates at 2E5 / well. CD39 monoclonal antibody and control antibody were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL / well of PE Goat anti mouse IgG Fc (1:500 dilution) was added. After resuspending the cells, the cells were incubated at 4°C for 30 minutes. After centrifugation, 200 μL of PBS was added to each well to wash the cells, and then 100 μL of PBS was added to each well to resuspend the cells, and the binding difference of CD39 antibody to Mino cells was evaluated using a Cytoflex flow cytometer (Beckman Countler). The binding curve is shown in the figure. Figure 7 As shown, R2122 and R2123 had similar binding activities to MOLP-8 cells, and the platform value was better than that of the positive control antibody R1289.

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

[0179] Adjust the density of Mino cells to 4E6 cells / ml and transfer 50μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend with fresh culture medium); prepare the antibody culture medium at a starting value of 60μg / mL. Dilution was made 3 times, with a total of 10 concentrations. Add 100μL of antibody and hybridoma supernatant to the cells at each well, mix well, and incubate at 37°C for 60min. Prepare ATP culture medium at 200μM and add 50μL / well to the cells. Mix well and incubate at 37°C for 45min. Centrifuge the cells at 350G for 5min, take 80μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, incubate at room temperature in the dark for 10min, and then detect on a machine. The blocking curve is shown below. Figure 8 As shown, R2122 and R2123 exhibited superior blocking activity on Mino cells.

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

[0181] Adjust the density of MOLP-8 cells to 3E6 cells / ml and transfer 50μL per well to a 96-well U-bottom cell culture plate (be sure to resuspend with fresh culture medium); prepare the antibody culture medium at a starting value of 60μg / mL. Make 3-fold dilutions, a total of 10 concentrations, add 100μL of the antibody and hybridoma supernatant to the cells per well, mix well, and incubate at 37°C for 60min; prepare ATP culture medium to 200μM, add 50μL / well to the cells, mix well, and incubate at 37°C for 35min; centrifuge the cells at 350G for 5min, take 80μL of the supernatant, transfer to a white plate, add an equal volume of Cell Titer-Glo, incubate at room temperature in the dark for 10min, and detect on the machine. The blocking activity curve of the antibody is shown in the figure below. Fig. 9 As shown, the blocking activity IC50 of R2123 in MOLP-8 cells was similar to that of R2122, and the platform value was better than that of R2122 and positive antibody R1289.

[0182] Example 13. Analysis of T cell proliferation inhibition mediated by humanized antibodies blocking CD39 hydrolysis of ATP

[0183] First, the T cell density was adjusted to 5E6 / ml, and CFSE (10mM stock solution) was used as the buffer to label the T cells, with a final concentration of 0.5μM, and the cells were labeled in a 37°C water bath in the dark for 20 minutes; 12mL of complete culture medium (10% FBS, precooled at 4°C) was used to terminate the CFSE reaction; the antibody was diluted with cell culture medium to a concentration of 400nM, 10× gradient dilution, and 5 points were set; the labeled cells were centrifuged at 300G for 5 minutes to remove the supernatant, resuspended in complete culture medium and counted, and the density was adjusted to 2E6 (total cells are counted here, not living cells), and 50μL / well was added to a 96-well U-shaped plate, that is, the final cell was 1E5 / well; the prepared antibody was added to the cells at 50μL / well; the beads were diluted according to the number of cells to ensure that the cell:bead ratio in the final system was 20:1, and added to a 96-well U-shaped plate at 50μL / well; three days (72h) later, the culture medium diluted ATP to 1200μM and added to a 96-well plate at 50μL / well, that is, the final system was 200μL. The cells were placed in an incubator and cultured for two days (48 hours). The cell plate was centrifuged and the supernatant was discarded. BSA was prepared with flow cytometry antibodies (anti-human CD8T, 400× dilution, FITC fluorescent antibodies cannot be used), incubated at 4°C for 30 minutes, and T cell proliferation was detected by flow cytometry (10,000 cells were collected to ensure smooth peaks). The test was performed on the machine according to the "CytoFLEX Flow Cytometer Standard Operating Procedure". The fitting curve is shown in the figure below. Fig.10As shown, R2122 has better blocking activity against T cell proliferation inhibition than R2123 in this experimental system. Since there are only two candidate molecules, and each has advantages in blocking activity and functional experiments, both candidate antibodies are retained for subsequent functional experimental testing.

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

[0185] On day 0, 6-8 week old female NOD SCID mice were injected subcutaneously with 5 × 10 6 MOLP-8 cells were cultured and CD39 antibody was administered at a dose of 30 mg / kg on days 1, 5, 8, and 12. Fig.11 The curve depicts the mean tumor volume, the error bars represent the SEM, the red triangles on the X-axis mark the time points of compression, the X-axis shows the days after implantation, and the Y-axis represents the volume of the tumor; Fig.12 The curve depicts the average mouse weight, the error bar represents SEM, the arrow on the X-axis marks the time point of compression, the X-axis shows the number of days after implantation, and the Y-axis represents the mouse weight. The results show that compared with the blank control, the CD39 humanized antibodies all have tumor inhibition effects, among which R2123 has a better tumor inhibition effect than R2122, and there is no significant difference in the weight of mice 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs. 1 to 8; and, (b) a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NOs. 9 to 16; Preferably, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are determined according to the IMGT definition, the Kabat definition, the Chothia definition, the AbM definition or the Contact definition.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The CDR region of the heavy chain variable region includes HCDR1, HCDR2 and HCDR3; the CDR region of the light chain variable region includes LCDR1, LCDR2 and LCDR3; The HCDR1 includes the amino acid sequence shown as SEQ ID NO.17; the HCDR2 includes the amino acid sequence shown as SEQ ID NO.22; the HCDR3 includes the amino acid sequence shown as SEQ ID NO.27; the LCDR1 includes the amino acid sequence shown as SEQ ID NO.30; the LCDR2 includes the amino acid sequence shown as YT; the LCDR3 includes the amino acid sequence shown as SEQ ID NO.

36.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs. 1 to 8.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs. 9 to 16.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs. 9 to 16.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs. 1 to 8.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof are selected from any combination of:

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antigen binding fragment includes one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody and antibody minimum recognition unit.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; The heavy chain constant region contains a sequence of a partial or complete constant region of any one of IgG1, IgG1.8, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; The light chain constant region is a κ or λ chain; Preferably, the constant region is derived from species including one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans; Preferably, 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; Preferably, 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; Preferably, 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.

10. Biomaterial, characterized in that The biological material is selected from any one of (i) to (iii): (i) a polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; (ii) a vector carrying the polynucleotide (i) described above; (iii) a cell, wherein the cell carries the polynucleotide described in (i), or contains the vector described in (ii), or expresses the antibody or antigen-binding fragment thereof described in any one of claims 1 to 9.

11. An immunoconjugate, characterized in that The anti-CD39 humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 is coupled with at least one diagnostic agent and / or therapeutic agent to form an immunoconjugate; The diagnostic agent is selected from one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent, and a photosensitizer; The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photosensitive therapeutic agent, an immunoconjugate, and an oligonucleotide.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the biomaterial according to claim 10, or the immunoconjugate according to claim 11 in any of the following: (i) Detection of CD39 or cells expressing CD39 for non-diagnostic and non-therapeutic purposes; (ii) preparing a product 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) preparing a blocking agent for blocking the ATPase hydrolysis activity of CD39 on cells; (v) preparing a medicament for treating, preventing or ameliorating a disease, disorder or condition associated with CD39.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the biomaterial according to claim 10, or the immunoconjugate according to claim 11; and optionally a pharmaceutically acceptable carrier and / or excipient.

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