CN117866090A

CN117866090A - Antibodies or antigen binding fragments thereof that bind human CD39, methods of making and uses thereof

Info

Publication number: CN117866090A
Application number: CN202211233611.1A
Authority: CN
Inventors: 黄浩旻; 张学赛; 徐菲; 邓岚
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2022-10-10
Filing date: 2022-10-10
Publication date: 2024-04-12
Also published as: TW202415680A; WO2024078381A1

Abstract

The invention belongs to the technical field of tumor treatment, and particularly relates to an antibody or an antigen binding fragment thereof for binding human CD39, a preparation method and application thereof. The antibody or antigen binding fragment thereof binding to human CD39 can effectively bind to human CD39, is applied to the preparation of medicines for treating diseases in which CD39 is overexpressed, and has good clinical prospect.

Description

Antibodies or antigen binding fragments thereof that bind human CD39, methods of making and uses thereof

Technical Field

The invention belongs to the field of tumor treatment, and relates to an antibody or antigen binding fragment thereof for binding human CD39, a preparation method and application thereof.

Background

Nucleotide hydrolase CD39 is the primary and rate-limiting enzyme in the tumor microenvironment that converts the immunostimulatory molecule ATP to immunosuppressive adenosine. In the tumor microenvironment, high levels of extracellular ATP will enhance the inflammatory activity of dendritic cells, T cells and other myelogenous cells, as well as activation of cells associated with adaptive immunity, while high levels of extracellular adenosine will enhance immunosuppressive activity of almost all immune cells in the microenvironment by binding to immune cell surface receptors; CD39 exhibits a high expression in various human tumors and tumor-infiltrating leukocytes, thereby helping tumor cells to achieve a certain degree of immune escape, the former including lymphomas, sarcomas, chronic lymphocytic leukemia, lung cancer, pancreatic cancer, ovarian cancer, renal cancer, thyroid cancer, testicular cancer, etc., and the latter including effector T cells, regulatory T cells (tregs), macrophages, etc. Currently, there are relatively few immunotherapeutic studies on CD39 antibodies, and clinical application needs are not met.

Disclosure of Invention

To solve the above problems, the inventors of the present invention conducted a number of experiments to screen for murine antibodies that specifically bind to human CD39 and further constructed and obtained chimeric antibodies as well as humanized antibodies on the basis of the results.

The present invention aims to provide a novel antibody or antigen binding fragment thereof that binds human CD39, a method of preparation and use thereof.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect of the invention there is provided an antibody or antigen-binding fragment thereof that binds human CD39, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises:

the amino acid sequences are shown in SEQ ID NO: 1. HCDR1, HCDR2 and HCDR3 shown in figures 2 and 3; or (b)

The amino acid sequences are shown in SEQ ID NO: 7. HCDR1, HCDR2 and HCDR3 shown in figures 8 and 9; or (b)

The amino acid sequences are shown in SEQ ID NO: 13. HCDR1, HCDR2 and HCDR3 shown in fig. 14 and 15; or (b)

The amino acid sequences are shown in SEQ ID NO: 19. HCDR1, HCDR2 and HCDR3 shown in 20 and 21;

the light chain variable region comprises:

the amino acid sequences are shown in SEQ ID NO: 4. LCDR1, LCDR2 and LCDR3 as shown in 5 and 6; or (b)

The amino acid sequences are shown in SEQ ID NO: 10. LCDR1, LCDR2 and LCDR3 shown in figures 11 and 12; or (b)

The amino acid sequences are shown in SEQ ID NO: 16. LCDR1, LCDR2 and LCDR3 as shown at 17 and 18; or (b)

The amino acid sequences are shown in SEQ ID NO: 22. 23 and 24, LCDR1, LCDR2 and LCDR3.

The antibodies of the invention consist of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) followed by a constant region at one end. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. The antibody of the present invention includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody) formed from at least two antibodies, and the like, and is preferably a murine antibody, a chimeric antibody, or a humanized antibody.

The antigen-binding fragments of the present invention refer to fragments of antibodies capable of specifically binding to human CD39, including Fab fragments, F (ab') ₂ Fragments, fv fragments, and the like.

Preferably, the antibody or antigen binding fragment thereof binding to human CD39 of the invention comprises: the heavy chain variable region comprises amino acid sequences as set forth in SEQ ID NOs: 1. 2 and 3, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 4. LCDR1, LCDR2 and LCDR3 as shown in 5 and 6; or the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 7. 8 and 9, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 10. LCDR1, LCDR2 and LCDR3 shown in figures 11 and 12; or the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 13. 14 and 15, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 16. LCDR1, LCDR2 and LCDR3 as shown at 17 and 18; or the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 19. 20 and 21, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 22. 23 and 24, LCDR1, LCDR2 and LCDR3.

Preferably, the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO: 25. 29, 33, 37, 43, 47, 50, 53 or 56. Preferably, the light chain variable region shown has the sequence set forth in SEQ ID NO: 27. 31, 35, 39, 59, 62, 65 or 68.

The antibodies or antigen-binding fragments of the invention that bind human CD39 comprise: a heavy chain variable region selected from any one of the following amino acid sequences: SEQ ID NO: 25. 29, 33, 37, 43, 47, 50, 53, 56; and/or a light chain variable region selected from any one of the following amino acid sequences: SEQ ID NO: 27. 31, 35, 39, 59, 62, 65, 68.

Preferably, the antibody or antigen binding fragment thereof that binds human CD39 of the invention comprises: the amino acid sequence is shown in SEQ ID NO:25 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:27, and a light chain variable region shown in seq id no; or the amino acid sequence is shown as SEQ ID NO:29 and the amino acid sequence of which is shown in SEQ ID NO:31, a light chain variable region shown in seq id no; or the amino acid sequence is shown as SEQ ID NO:33 and the amino acid sequence of which is shown in SEQ ID NO:35, a light chain variable region shown in seq id no; or the amino acid sequence is shown as SEQ ID NO:37 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:39, a light chain variable region shown in seq id no; or the amino acid sequence is shown as SEQ ID NO:43 and the amino acid sequence of which is shown in SEQ ID NO: 59; or the amino acid sequence is shown as SEQ ID NO:47 and the amino acid sequence of which is set forth in SEQ ID NO: 59; or the amino acid sequence is shown as SEQ ID NO:50 and the amino acid sequence of which is shown in SEQ ID NO: 62. Or the amino acid sequence is shown as SEQ ID NO:53 and the amino acid sequence of which is shown in SEQ ID NO:65, a light chain variable region; or the amino acid sequence is shown as SEQ ID NO:43 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:68, a light chain variable region shown at 68; or the amino acid sequence is shown as SEQ ID NO:56 and the amino acid sequence of which is shown in SEQ ID NO: 65.

The heavy chain of an antibody also includes a heavy chain constant region. Preferably, the heavy chain constant region is of human or murine origin. Further preferably, the heavy chain constant region is a human antibody heavy chain IgG4 (S228P) constant region.

The light chain of an antibody also includes a light chain constant region. Preferably, the light chain constant region is of human or murine origin. Further preferably, the light chain constant region is a human antibody light chain kappa constant region.

Preferably, the antibody or antigen binding fragment thereof that binds human CD39 of the invention comprises: selected from the group consisting of SEQ ID NOs: 44. 48, 51, 54, 57 or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:41 and a human IgG4 (S228P) constant region selected from the group consisting of SEQ ID NOs: 25. 29, 33, 37, and a heavy chain comprising a spliced heavy chain variable region comprising any one of the amino acid sequences shown in seq id no; selected from the group consisting of SEQ ID NOs: 60. 63, 66, 69 or a light chain comprising any one of the amino acid sequences set forth in SEQ ID NO:42 and a human kappa chain constant region as set forth in SEQ ID NO: 27. 31, 35, 39.

Further preferred, the antibody or antigen-binding fragment thereof binding to human CD39 of the present invention comprises: the amino acid sequence is shown in SEQ ID NO:44 and the amino acid sequence of which is shown in SEQ ID NO: 60. Or the amino acid sequence is shown as SEQ ID NO:48 and the amino acid sequence of the heavy chain is shown as SEQ ID NO: 60. Or the amino acid sequence is shown as SEQ ID NO:51 and the amino acid sequence of which is shown in SEQ ID NO: 63; or the amino acid sequence is shown as SEQ ID NO:54 and the amino acid sequence of which is shown in SEQ ID NO: 66. Or the amino acid sequence is shown as SEQ ID NO:44 and the amino acid sequence of which is shown in SEQ ID NO: 69; or the amino acid sequence is shown as SEQ ID NO:57 and the amino acid sequence of which is shown in SEQ ID NO: 66.

Further preferably, the antibody or antigen-binding fragment thereof of the present invention that binds human CD39 of the present invention comprises: (1) the amino acid sequence is shown in SEQ ID NO:25 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41 and a heavy chain formed by splicing constant regions of human IgG4 (S228P), and an amino acid sequence shown in SEQ ID NO:27 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:42, a light chain comprising a splice of human kappa chain constant regions; or (2) an amino acid sequence shown in SEQ ID NO:29 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41 and a heavy chain formed by splicing constant regions of human IgG4 (S228P), and an amino acid sequence shown in SEQ ID NO:31 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:42, a light chain comprising a splice of human kappa chain constant regions; or (3) the amino acid sequence is shown as SEQ ID NO:33 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41 and a heavy chain formed by splicing constant regions of human IgG4 (S228P), and an amino acid sequence shown in SEQ ID NO:35 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:42, a light chain comprising a splice of human kappa chain constant regions; or (4) the amino acid sequence is shown as SEQ ID NO:37 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41 and a heavy chain formed by splicing constant regions of human IgG4 (S228P), and an amino acid sequence shown in SEQ ID NO:39 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:42, and a light chain comprising a splice of human kappa chain constant regions.

In a second aspect, the present invention provides a nucleotide molecule, comprising:

a nucleotide molecule encoding the above antibody or antigen binding fragment thereof that binds human CD 39.

The nucleotide molecules of the invention include nucleotide molecules capable of encoding heavy chain variable regions such as SEQ ID NOs: 26. 30, 34, 38; and/or a nucleotide molecule capable of encoding a light chain variable region as set forth in SEQ ID NO: 28. 32, 36, 40.

The nucleotide molecules of the invention include nucleotide molecules capable of encoding heavy chains as shown in SEQ ID NO: 45. 46, 49, 52, 55; and/or a nucleotide molecule capable of encoding a light chain as set forth in SEQ ID NO: 58. 61, 64, 67.

The nucleotide molecules of the invention can be prepared by methods conventional in the art, such as by gene cloning techniques (PCR methods) or artificial sequence synthesis. It will be appreciated by those skilled in the art that nucleotide sequences encoding the amino acid sequences of the antibodies or antigen binding fragments thereof described above that bind human CD39 may be appropriately introduced into substitutions, deletions, alterations, insertions or additions to provide a homolog of a polynucleotide. Homologs of the polynucleotide may be made by substitution, deletion, or addition of one or more bases of the gene encoding the antibody or antigen binding fragment thereof that binds human CD39 within a range that retains antibody activity.

The third aspect of the invention provides an expression vector, which comprises the following specific schemes:

an expression vector comprising the nucleotide molecule described above.

The expression vectors of the present invention are expression vectors conventional in the art, such as expression vectors comprising appropriate regulatory sequences such as promoter sequences, terminator sequences, and the like. The expression vector may be a virus or a plasmid.

In a fourth aspect, the invention provides a host cell comprising:

a host cell comprising the expression vector described above.

The host cell of the present invention is a variety of host cells conventional in the art, such as prokaryotic expression cells and eukaryotic expression cells, as long as the above expression vector can stably self-replicate and the carried nucleotide can be efficiently expressed.

In a fifth aspect, the present invention provides a method of preparing an antibody or antigen binding fragment thereof as described above that binds human CD39, said method comprising the steps of:

a) Culturing a host cell as described above under expression conditions, thereby expressing said antibody or antigen-binding fragment thereof that binds human CD 39;

b) Isolating and purifying the antibody or antigen binding fragment thereof that binds human CD39 of a).

The method of culturing the host cell, and the method of isolating and purifying the antibody of the present invention are conventional methods in the art.

In a sixth aspect the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds human CD39 as described above and a pharmaceutically acceptable carrier.

The antibody or antigen binding fragment thereof binding to human CD39 provided by the invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition so as to exert curative effect more stably, and the preparations can ensure the conformational integrity of the antibody or antigen binding fragment thereof binding to human CD39 disclosed by the invention, and simultaneously protect the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation). The pharmaceutical composition is in the form of suspension, water injection, freeze-drying and other preparations commonly used in the pharmaceutical field.

The seventh aspect of the invention provides an application of the antibody or antigen binding fragment thereof or the pharmaceutical composition for binding human CD39 in preparing antitumor drugs. The tumor comprises lymphoma, sarcoma, chronic lymphocytic leukemia, lung cancer, pancreatic cancer, ovarian cancer, renal cancer, thyroid cancer, testicular cancer, etc.

The beneficial effects are that: the invention develops a specific targeted CD39 therapeutic antibody through mouse immunity, hybridoma cell preparation screening, antibody humanization, pharmacodynamics research and the like, and the antibody can inhibit the generation of adenosine by maintaining the ATP concentration in the tumor microenvironment so as to reduce immunosuppression, improve immune effect, play a synergistic anti-tumor role and have better application prospect.

Drawings

FIGS. 1 to 3 show the binding curves of different murine antibodies to the CD39-His protein in example 3;

FIGS. 4-6 are graphs showing the results of the test for the activity of the different murine antibodies and the control antibodies in example 4 against the 293E-CD39 cell surface CD39 protein;

FIG. 7 shows the results of the test for inhibition of exogenous CD39 protease activity by the chimeric antibodies and control antibodies of example 6;

FIG. 8 is a graph showing the results of the test for inhibition of SKMEL-28 cell surface CD39 protease activity by the chimeric antibody and the control antibody of example 7;

FIGS. 9-11 are graphs showing the binding of humanized antibodies and control antibodies to CD39 protein in example 9;

FIGS. 12 to 13 are the results of the test for the inhibition of exogenous CD39 protease activity by the humanized antibody of example 10;

FIG. 14 shows the results of an inhibition test of CD39 protease activity on the surface of tumor cells SKMEL-28 by the humanized antibody and the control antibody of example 11;

FIG. 15 shows the results of the test for the inhibition of CD39 protease activity on the surface of MOLP-8 of tumor cells by the humanized antibody and the control antibody of example 11;

FIG. 16 is a test result of the inhibition of CD39 protease activity on the surface of CD14+ monocytes by the humanized antibody and the control antibody of example 12;

FIG. 17 shows the results of an inhibition test of the B cell surface CD39 protease activity by the humanized antibody and the control antibody of example 13;

FIG. 18 shows the results of an inhibition test of CD39 protease activity on macrophage surface by the humanized antibody and the control antibody of example 14;

FIG. 19 is a graph showing the results of the test for the promotion of IL-1β secretion by macrophages by humanized antibodies and control antibodies in example 14;

FIG. 20 is a schematic diagram of the humanized antibody of example 15 promoting maturation of DC cells and promoting CD4 ⁺ Detection results of IFN-gamma secretion by T cells;

FIG. 21 is a schematic diagram of the humanized antibody of example 15 promoting maturation of DC cells and promoting CD8 ⁺ Detection results of IFN-gamma secretion by T cells;

FIGS. 22-23 show the results of cross-reaction experiments of humanized antibodies with different species of CD39 protein in example 17.

Detailed Description

The following examples are further illustrative of the invention and should not be construed as limiting the invention. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids or methods of introducing plasmids into host cells.

The experimental materials used in the following examples are all commercially available conventional products, and specific sources are described below:

(1) Mouse myeloma cells SP2/0: purchased from ATCC under accession number CRL-1581; (2) Balb/c mice: purchased from Shanghai Ling Biotechnology Co., ltd; (3) SKMEL-28 cells: purchased from ATCC under accession number HTB-72; (4) MOLP-8 cells: purchased from the southern Beijing department of herborist, cat No. CBP60562; (5) CD39 ⁺ 293 cells are laboratory constructs of the antibody protein engineering department; (6) HRP-goat anti-human IgG Fc secondary antibody: purchased from Sigma, cat# A0170-1ML; (7) FITC-labeled goat anti-human IgG-Fc secondary antibody: purchased from Abcam, cat number 97224; (8) TMB: purchased from KPL company under the number 52-00-03; (9) Bovine Serum Albumin (BSA): purchased from a manufacturer, cat No. a600332-0100; (10) RPMI 1640Medium: purchased from Gibco company under the accession number 61870127; (11) Penicillin-streptomycin (Penicillin-streptomycin): purchased from Gibco company under the accession number 15140122; (12) Fetal Bovine Serum (FBS): purchased from Gibco company under the accession number 10091-148; (13) polyethylene glycol solution is available from sigma company under the product number P7181; (14) Hybridoma-SFM was purchased from life technologies, cat No. 12045-076; (15) HAT: purchased from Gibco, cat No. 21060017; (16) pcDNA 3.4: purchased from thermo fisher, cat No. a14697; (17) HEK-293F: purchased from Thermo Fisher, cat No. a14527; (18) Streptavidin HRP available from BD Pharmingen, cat# 554066 (19) EZ-Link NHS-Biotin Reagent: purchased from Thermo Fisher, cat No. 20217; (20) Cell Titer-Glo: purchased from Promega, cat No. G7570; (21) ATP: purchased from Sigma, cat No. a7655; (22) HBS-EP pH7.4 buffer: purchased from GE Healthcare under the accession number BR-1006-69; (23) Protein A/G chip: purchased from GE Healthcare under the accession number BR-1005-30; (24) CD19 microblades, human: available from Miltenyi Biotec, cat# 130-050-301; (25) CD14 microblades, human: available from Miltenyi Biotec, cat# 130-050-201; (26) CD4 microblades, human: available from miltenyibiocec under the designation 130-045-101; (27) CD8 microblades, human: available from miltenyibiocec under the accession number 130-097-057; (28) LS Columns plus tubes: available from Miltenyi Biotec,130-122-7291.

EXAMPLE 1 preparation of control antibodies

The amino acid sequences of the heavy chain and light chain variable regions of the reference antibody TTX-030 are from patent WO 2019027935A1, the heavy chain variable regions are connected with a human IgG4 (S228P) constant region (the amino acid sequence is shown as SEQ ID NO 41) by using a conventional gene synthesis and molecular cloning method, the light chain variable regions are connected with a human kappa chain (the amino acid sequence is shown as SEQ ID NO 42) constant region, the N end of the light chain variable regions is added with a signal peptide sequence to construct into a pcDNA 3.4 expression vector, a heavy chain plasmid TTX-030-HC and a light chain plasmid TTX-030-LC for expressing TTX-030 are respectively obtained, HEK-293F cells are co-transfected by the heavy chain plasmid and the light chain plasmid, after 5d expression, cell culture supernatants are collected and purified by a Protein A affinity chromatography column to obtain the TTX-030 Protein. The heavy chain amino acid sequence of the control antibody TTX-030 is shown as SEQ ID NO 70, and the light chain amino acid sequence is shown as SEQ ID NO 71.

EXAMPLE 2 preparation and screening of antigen-immunized animals and hybridomas

(1) Expression of antigens

The extracellular region gene of CD39 (the sequence is from UniProt database, accession number P49961, and the extracellular region amino acid sequence T38-V478 of CD39 is shown as SEQ ID NO 72) was constructed into pcDNA 3.4 expression vector by conventional gene synthesis and molecular cloning method, and signal peptide sequence was added at its N-terminal, 6 XHis tag was added at its C-terminal, HEK-293F cell was transfected, after 5d expression, cell culture supernatant was collected and purified by Ni+ affinity chromatography column to obtain CD39-His protein.

(2) Antigen immunized mice

Balb/c mice were routinely immunized with human CD39-His protein prepared as described above (purity > 95%). Balb/c mice were subcutaneously multi-injected (100. Mu.g of human CD 39-His/mouse/0.5 mL) after emulsification of soluble human CD39-His protein with Freund's complete adjuvant, balb/c mice were subcutaneously injected (50. Mu.g of human CD 39-His/mouse/0.5 mL) after emulsification of soluble human CD39-His protein with Freund's incomplete adjuvant, and mice spleens were taken for fusion experiments after 3-4 days after three weeks.

(3) Hybridoma cell preparation and screening

The spleen cells of the mice were PEG-fused with myeloma cells SP2/0 of the mice 3-4 days after the last immunization of the mice using conventional hybridoma protocols. The fused cells were suspended uniformly in complete medium composed of RPMI1640-GLUMAX added with 1% penicillin-streptomycin, 20% fbs (fetal bovine serum), and 1×hat. The fused cells were cultured at a rate of 3X 10 ⁴ Cells/200. Mu.L/well were plated in 60 96 well plates. After 7-12 days, the supernatant was harvested and hybridoma wells positive for human CD39 binding activity were screened by ELISA.

Wherein, the ELISA method is used for screening hybridoma holes positive for human CD39 binding activity, and the method is as follows: CD39-His protein was diluted to 2. Mu.g/mL with PBS buffer, 100. Mu.L/well was added to the plate and incubated overnight at 4 ℃; throwing off the supernatant the next day, adding 5% skimmed milk powder, sealing at 37 ℃ for 2 hours, and washing the PBST plate for 3 times for later use; sequentially adding collected hybridoma supernatants into the closed plate, and standing at 37deg.C for 1 hr at 100 μl/well; PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30min; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu L of TMB is added into each hole, and the plate is placed for 5min at room temperature and in a dark place; 50 mu L of 2M H are added to each well ₂ SO ₄ The stopping solution stops the substrate reaction, the OD value is read at 450nm of the enzyme-labeled instrument, and the binding capacity of the antibody to be detected and the target antigen CD39 is analyzed. Amplifying and screening the Hybridoma cell strain in the complete culture medium containing serum, centrifuging and changing the liquid to a serum-free culture medium hybrid oma-SFM culture medium to ensure that the cell density is 1-2 multiplied by 10 ⁷ Per mL, at 8% CO ₂ Culturing for 1 week at 37 ℃, centrifuging to obtain a culture supernatant, and purifying by Protein G affinity chromatography to obtain various mouse source antibodies of the anti-human CD39 Protein. A total of 28 hybridoma cell lines were obtained in each round of screening.

EXAMPLE 3 test of the binding Capacity of murine antibody to human CD39-his protein

Each of the murine antibodies obtained in example 2 was named and its binding ability to human CD39-His protein was measured by indirect enzyme-linked immunosorbent assay (ELISA). The specific measurement method is as follows: the CD39-His protein was coated with a coating solution (50 mM carbonate coating buffer,pH 9.6) was diluted to 2. Mu.g/mL and ELISA plates were coated overnight at 4 ℃; sealing with 5% skimmed milk powder, and incubating at 37deg.C for 2 hr; after washing the plates 3 times with PBST, the murine antibody against human CD39 protein was diluted in a gradient with 1% BSA buffer and added to the plates pre-coated with CD39-His protein at 100. Mu.L/well and incubated for one hour at 37 ℃; PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30min; after PBST washing the plate 3 times, the residual liquid drop is beaten as much as possible on the absorbent paper, 100 mu L of TMB is added into each hole, the plate is placed for 5min at room temperature and protected from light, and 50 mu L of 2M H is added into each hole ₂ SO ₄ The stopping solution stops the substrate reaction, the OD value is read at 450nm of the enzyme-labeled instrument, and the binding capacity of each murine antibody to be detected and the antigen human CD39-His is analyzed.

The binding curves of each murine antibody and CD39-His protein are shown in FIGS. 1-3, EC ₅₀ And binding activity high plateau values (Top) are shown in tables 1 to 3. As can be seen from fig. 1 to 3, murine antibodies 302B4H10, 317B1A5, 321D9E5, 339E8A9, 343E7G6, 344E2C6, 346A1B12, 355F9D6, 406a10E3, 439G11A9, 435H6H8, 453D9A3, 426F10E2, 403A1B3, 431H1F7, 438C1B6, 52a12H7F1 have relatively excellent binding activity to the target antigen CD 39-His.

TABLE 1 EC of each murine antibody binding to CD39-His protein ₅₀

Sample of	302B4H10	317B1A5	318F8A9	321D9E5	326D6A6	336A9E2	339E8A9
								EC ₅₀ (nM)	0.195	0.146	1.071	0.094	17683.000	28.280	0.118
Top	2.174	2.284	0.939	1.754	1.193	1.781	1.899
								Sample of	343E7G6	344E2C6	346A1B12	346B11G3	355F9D6	406A10E3	419H10A4
EC ₅₀ (nM)	0.135	0.174	0.183	0.248	0.168	0.171	0.399
								Top	2.114	2.012	2.180	2.236	2.043	2.279	1.846

Table 2: EC of murine antibody binding to CD39-His protein ₅₀

Sample of	405G5H7	439G11A9	447F2H11	441G2A5	435H6H8	453D9A3	426F10E2
								EC ₅₀ (nM)	1.280E-29	0.111	0.106	0.070	0.115	0.067	0.144
Top	1.298	1.424	1.294	1.025	1.387	1.481	1.763
								Sample of	403A1B3	431H1F7	438C1B6	460A4D6	417B7C7	415A12B5	447F2H11
EC ₅₀ (nM)	0.122	0.065	0.045	39.220	219.000	118.600	2.521
								Top	1.974	1.535	1.409	1.296	1.243	1.817	1.789

Table 3: EC of murine antibody binding to CD39-His protein ₅₀

Sample of	1C6B4D6	2D2G2F2	21B5H7A3	52A12H7F1
					EC ₅₀ (nM)	0.097	0.126	0.226	0.037
Top	1.428	1.999	1.103	2.039

Example 4 detection of the enzymatic Activity of murine antibodies against cell surface CD39 protein

The inhibition of CD39 enzyme activity by each murine antibody and control antibody TTX-030 was determined by the method of detecting ATP, and the specific method is as follows: HEK293E cells (293E-CD 39) which over-express CD39 were collected, and 300g of the cell culture broth was removed by centrifugation and washed 1 time with PBS buffer; count and use Tris buffer (25mM Tris,5mM CaCl) ₂ PH 7.5) diluted to 1X 10 ⁴ Well, spreading cells into 96-well cell culture plates, centrifuging and discarding cell culture supernatant; diluting each murine antibody to be tested and a reference antibody TTX-030 to 10 mug/mL by using Tris buffer, carrying out 3-time gradient dilution, adding 11 holes in 100 uL/hole, adding into a culture plate, and incubating for 60min in a culture box at 37 ℃; centrifuging to remove the supernatant, adding ATP (adenosine triphosphate) with final concentration of 50 mu M200 mu L/hole into a Cell culture plate, incubating for 60min at 37 ℃, centrifuging to obtain 50 mu L of culture supernatant, placing the culture supernatant into a 96-hole white opaque detection plate, adding 50 mu L of Cell Titer-Glo detection reagent, incubating for 5min at room temperature, and reading fluorescence intensity in a multifunctional enzyme-labeled instrument and analyzing.

The inhibitory enzyme activity curves of the murine antibodies are shown in FIGS. 4 to 6, corresponding to the enzyme activity IC ₅₀ And the maximum inhibitory activity (Top) results are shown in tables 4 to 6. From FIGS. 4 to 6, it is clear that murine antibodies 419H10A4, 439G11A9, 52A12H7F1, 2D2G2F2 significantly inhibited the activity of CD39 enzyme on the surface of 293E-CD39 cells, and that 439G11A9, 52A12H7F1 were significantly better than the control antibody TTX-030.

Table 4: murine antibody inhibits the enzyme activity IC of 293E-CD39 cell surface CD39 protein ₅₀

NA: data unavailable

Table 5: murine antibody inhibits the enzyme activity IC of 293E-CD39 cell surface CD39 protein ₅₀

Table 6: murine antibody inhibits the enzyme activity IC of 293E-CD39 cell surface CD39 protein ₅₀

Sample of	TTX-030	1C6B4D6	2D2G2F2	21B5H7A3	52A12H7F1
						IC ₅₀ (nM)	1.450	NA	0.182	NA	0.143
Top	117885	43261	82249	37488	112931

EXAMPLE 5 variable region Gene acquisition of candidate antibody and preparation of chimeric antibody

(1) Acquisition of candidate antibody variable region genes

Four candidate murine antibodies 419H10A4, 439G11A9, 52A12H7F1, 2D2G2F2 with good effects were obtained by related methods of molecular biology, and the specific schemes are as follows.

RNA from each hybridoma was extracted by Trizol, mRNA was reverse transcribed using Oligo-dT primers to obtain cDNA, and then PCR was performed using cDNA as a template, using heavy and light chain degenerate primers of murine antibodies (Antibody Engineering, volume1, edited by Roland Kontermann and Stefan D, and the sequence of the combined primers was from page 323), and the obtained PCR product was sequenced and analyzed by kabat database to determine that the obtained sequence was the variable region sequence of murine antibodies. Specific information for each murine antibody is:

419H10A4: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1. 2, 3 (the full length is 369bp, and codes for 123 amino acid residues) of the heavy chain variable region coding nucleotide sequence is shown in SEQ ID NO: 26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27, the amino acid sequences of LCDR1, LCDR2, LCDR3 are respectively as shown in SEQ ID NO: 4. 5, 6, the light chain variable region coding nucleotide sequence (the total length is 321bp, and the coding sequence is 107 amino acid residues) is shown in SEQ ID NO: 28.

439G11A9: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:29, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 7. 8 and 9, the heavy chain variable region coding nucleotide sequence (the full length is 366bp, and the coding number of the amino acid residues is 122) is shown as SEQ ID NO: shown at 30; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:31, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 10. 11, 12, the light chain variable region coding nucleotide sequence (the full length is 333bp, and codes for 111 amino acid residues) is shown in SEQ ID NO: shown at 32.

52a12H7F1: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:33, the amino acid sequences of HCDR1, HCDR2, HCDR3 are respectively shown in SEQ ID NOs: 13. 14, 15 (full length of 360bp, encoding 120 amino acid residues) as set forth in SEQ ID NO: shown at 34; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35, the amino acid sequences of LCDR1, LCDR2, LCDR3 are respectively shown in SEQ ID NOs: 16. 17, 18, the light chain variable region coding nucleotide sequence (full length is 336bp, 112 amino acid residues are coded) is shown in SEQ ID NO: shown at 36.

2D2G2F2: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:37, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NOs: 19. 20, 21 (351 bp in full length, encoding 117 amino acid residues) as set forth in SEQ ID NO: shown at 38; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:39, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 22. 23, 24, the light chain variable region coding nucleotide sequence (full length is 318bp, codes for 106 amino acid residues) is shown in SEQ ID NO: 40.

(2) Preparation of chimeric antibodies

The heavy chain variable region sequence of four candidate mouse source antibodies is spliced with a human IgG4 (S228P) constant region (the amino acid sequence is shown as SEQ ID NO: 41), the light chain variable region sequence is spliced with a human kappa chain constant region (the amino acid sequence is shown as SEQ ID NO: 42), heavy chain and light chain-to-pcDNA3.4 expression vectors of the chimeric antibodies are respectively constructed, HEK-293F cells are subjected to cotransfection and purification to obtain the chimeric antibodies, and the chimeric antibodies corresponding to the mouse source antibodies 419H10A4, 439G11A9, 52A12H7F1, 2D2G2F2 are respectively named 419H-ch, 439G-ch, 52A12-ch and 2D2-ch.

EXAMPLE 6 inhibition of exogenous CD39 protease Activity by chimeric antibodies

In this example, the inhibition of the activity of exogenous free CD39 protease by each chimeric antibody and control antibody was determined by the method of detecting ATP, as follows: the CD39-His protein prepared and stored by the method is resuspended in 0.01%BSA 25mM Tris,5mM CaCl ₂ ，pH 7.5(Tris buBuffer), 25 μl per well was added to a white opaque 96 well plate at a concentration of 160 nM; diluting the antibody to be detected by Tris buffer according to 3 times of gradient, continuously diluting each group for 12 gradients, wherein the highest working concentration of each antibody is 1 mu M, adding 25 mu L of diluted antibody to be detected into the 96-well plate, and the final volume of each well is 50 mu L; incubating the 96-well plate at room temperature for 2 hours; after the incubation, dilute ATP to 1000. Mu.M with Tris buffer, add to 96 well plate, 50. Mu.L/well, mix well, incubate at 37℃for 60min; the Cell Titer-Glo reagent was then added directly to the 96-well plate at a 1:1 volume ratio, and the fluorescence intensity was read in a multifunctional microplate reader and the data analyzed.

The results of the inhibition test of exogenous CD39 protease activity by each chimeric antibody are shown in FIG. 7, corresponding to enzyme activity IC ₅₀ And the maximum inhibitory activity (Top) results are shown in Table 7.

TABLE 7 IC of chimeric antibodies inhibiting exogenous CD39 protease Activity ₅₀

Example 7 detection of chimeric antibody inhibiting tumor cell surface CD39 enzymatic Activity

Collecting malignant melanoma cells SKMEL-28 in logarithmic growth phase, centrifuging to remove cell culture solution, and washing the cells with PBS for 1 time; count and re-suspend with complete medium (1640+10%FBS+1%Pen Strep+1%Sodium Pyruvate+1%GlutaMAX) at 3.5X10) ⁴ Inoculating the individual cells/well into a round bottom 96 cell culture well plate, culturing at 100 mu L/well in a cell culture box at 37 ℃ overnight, discarding cell culture supernatant, diluting the chimeric antibody to be detected and the control antibody to 1000nM (SKMEL-28) by using Tris buffer, diluting with 3-fold gradient, adding into the cell culture plate according to 100 mu L/well, and incubating at 37 ℃ for 60min; then adding ATP with final concentration of 50 μm, incubating at 37deg.C for 60min, collecting 50 μl culture supernatant, and making 96-well white and impermeableTo the photo detection plate, 50. Mu.L of Cell Titer-Glo detection reagent was added, incubated at room temperature for 5min, and fluorescence intensity was read and analyzed in a multifunctional microplate reader.

The inhibition test curves of the chimeric antibody and the control antibody on the activity of the SKMEL-28 cell surface CD39 protease are shown in FIG. 8, and the corresponding enzyme activity IC ₅₀ And the maximum inhibitory activity (Top) results are shown in Table 8.

Table 8: IC of each chimeric antibody for inhibiting CD39 protease activity on SKMEL-28 cell surface ₅₀

Sample of	TTX-030	2D2-ch	419H-ch	52A12-ch	439G-ch
						IC ₅₀ (nM)	0.029	NA	0.020	0.005	0.348
Top	366766	274694	347808	215229	753306

EXAMPLE 8 construction and preparation of humanized antibodies

The amino acid sequences of the murine antibody 439G11A9 light chain variable region (amino acid sequence shown in SEQ ID NO: 31) and heavy chain variable region (amino acid sequence shown in SEQ ID NO: 29) were analyzed to determine the heavy chain, light chain antigen binding Complementarity Determining Regions (CDRs) and Framework Regions (FRs) of 439G11A9 according to the Kabat rules. The amino acid sequences of complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region amino acids are shown in SEQ ID NO: 7. 8, 9, the amino acid sequences of the complementarity determining regions LCDR1, LCDR2, LCDR3 of the light chain variable region are respectively set forth in SEQ ID NOs: 10. 11, 12.

Selecting a human IgG Germline sequence template which is matched with a heavy chain variable region non-CDR sequence of the 439G11A9 antibody in a Germline database, wherein the heavy chain variable region selects IGHV14-3 x 02 as a humanized template, the light chain variable region selects IGKV3-2 x 01 as the humanized template, then transplanting the heavy chain variable region and the CDR region of the light chain of the 439G11A9 antibody to the selected human template respectively to replace the CDR region in the humanized template, and simultaneously carrying out back mutation on embedded residues, residues with direct interaction with the CDR region and residues with important influence on the VL and VH conformation of the antibody based on the three-dimensional structure of the antibody to finally obtain the heavy chain variable region and the light chain variable region of the multi-humanized antibody; each humanized heavy chain variable region was recombined with a human IgG4 (S228P) constant region, each humanized light chain variable region was recombined with a human kappa chain constant region, and constructed into pcdna3.4 expression vectors, expressed on self-pairing co-transfected HEK-293F cells and purified to obtain each humanized antibody. 439G11A9, the weight, light chain designations, nucleotide and amino acid sequences of the respective humanized antibodies were as follows:

Heavy chain:

(1) 439G-HC-Hu: the amino acid sequence is shown in SEQ ID NO:44, the variable region amino acid sequence of which is shown in SEQ ID NO:43, the coding nucleotide sequence is shown as SEQ ID NO: 45.

(2) 439G-HC-G: the amino acid sequence is shown in SEQ ID NO:48, the amino acid sequence of the variable region is shown as SEQ ID NO:47, the coding nucleotide sequence is shown as SEQ ID NO: 46.

(3) 439G-HC-HuME: the amino acid sequence is shown in SEQ ID NO:51, the amino acid sequence of the variable region is shown as SEQ ID NO:50, and the coding nucleotide sequence is shown as SEQ ID NO: shown at 49.

(4) 439G-HC-HuMK: the amino acid sequence is shown in SEQ ID NO:54, the amino acid sequence of the variable region is shown as SEQ ID NO:53, the coding nucleotide sequence is shown as SEQ ID NO: 52.

(5) 439G-HC-HuML: the amino acid sequence is shown in SEQ ID NO:57, the amino acid sequence of the variable region is shown as SEQ ID NO:56, the coding nucleotide sequence is shown as SEQ ID NO: shown at 55.

Light chain:

(1) 439G-LC-G: the amino acid sequence is shown in SEQ ID NO:60, the amino acid sequence of the variable region is shown as SEQ ID NO:59, and the coding nucleotide sequence is shown as SEQ ID NO: shown at 58.

(2) 439G-LC-HuM100Y: the amino acid sequence is shown in SEQ ID NO:63, the amino acid sequence of the variable region is shown as SEQ ID NO:62, the coding nucleotide sequence is shown as SEQ ID NO: shown at 61.

(3) 439G-LC-HuMut3: the amino acid sequence is shown in SEQ ID NO:66, the amino acid sequence of the variable region is shown as SEQ ID NO:65, the coding nucleotide sequence is shown as SEQ ID NO: shown at 64.

(4) 439G-LC-HuM85E: the amino acid sequence is shown in SEQ ID NO:69, the amino acid sequence of the variable region is shown as SEQ ID NO:68, and the coding nucleotide sequence is shown as SEQ ID NO: 67.

The nomenclature of the antibodies obtained by pairing expression of each humanized antibody heavy and light chain combination is shown in Table 9.

TABLE 9 antibodies obtained by the combined paired expression of the heavy and light chains of each humanized antibody

EXAMPLE 9 binding Activity of humanized antibodies to human CD39-His protein

The binding capacity of each humanized antibody to human CD39-His protein was determined herein using an indirect enzyme-linked immunosorbent assay (ELISA). The specific method comprises the following steps:

the CD39-his protein was diluted to 0.5. Mu.g/mL with coating solution (50 mM carbonate coating buffer, pH 9.6), ELISA plates were coated, 100. Mu.L/well, placed in a wet box, coated at 4℃for 16h; washing the ELISA plate three times by PBST, removing unbound antigen, drying the ELISA plate on absorbent paper, removing redundant liquid, and then blocking with 2% BSA prepared by PBS, 200 mu L/hole for 2h at room temperature; washing with PBST once, washing out excessive blocking solution, drying ELISA plate, removing excessive liquid, diluting each antibody with 1% BSA (antibody diluent) prepared by PBST according to 3-time gradient, diluting 12 gradients with the highest concentration of 20nM, adding ELISA hole, 100 μl/hole, and incubating at room temperature for 1h; washing out unbound or non-specifically bound primary antibodies, diluting HRP-labeled Anti-Human Fc (1:5000) (sigma, A0170) secondary antibodies to a suitable concentration with antibody diluent, adding ELISA plate, 100 μl/well, and incubating at room temperature for 1h; washing with PBST for five times, drying ELISA plate on absorbent paper, removing excessive liquid, adding TMB color development liquid, developing to appropriate depth at 100 μl/hole, adding 2M H ₂ SO ₄ 50. Mu.L/well to terminate the development, OD was read at 450nm in a multifunctional microplate reader, data were collected and assayed for binding of the test antibody to the target antigen human CD 39-His.

The experimental results are shown in FIGS. 9 to 11 and tables 10 to 12. As can be seen from FIG. 9 and Table 10, the affinity of 439G-HuG in the humanized antibodies was significantly stronger than that of 439G-GG and the control antibody TTX-030. As can be seen from FIGS. 10 to 11 and tables 11 to 12, the affinity of 439G-Hu-HMELW100Y and 439G-Hu-HMKLmut3 in the humanized antibodies for CD39 protein was optimal and significantly stronger than that of the other humanized antibodies and the control antibody TTX-030.

Table 10: EC of humanized antibodies to CD39 protein binding ₅₀

Sample of	EC ₅₀ (nM)	Top
			TTX-030	0.099	0.774
439G-HuG	0.201	0.991
			439G-GG	0.414	0.617

Table 11: EC of humanized antibodies to CD39 protein binding ₅₀

Sample of	EC ₅₀ (nM)	Top
			TTX-030	0.157	1.083
439G-HuG	0.734	1.602
			439G-HuG-LN85E	0.412	1.691
439G-Hu-HMELW100Y	0.244	1.821

Table 12: EC of humanized antibodies to CD39 protein binding ₅₀

Sample of	EC ₅₀ (nM)	Top
			TTX-030	0.069	0.637
439G-HuG	0.304	0.909
			439G-HuG-LN85E	0.448	0.911
439G-Hu-HMLLmut3	0.184	0.910
			439G-Hu-HMKLmut3	0.143	1.201

EXAMPLE 10 inhibition of exogenous CD39 protease Activity by humanized antibodies

The inhibitory capacity of each humanized antibody against exogenous CD39 protease activity was determined with reference to example 6.

As shown in FIGS. 12-13 and tables 13-14, the humanized antibodies 439G-HuG, 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3, 439G-Hu-HMLLmut3 had equivalent inhibitory effects on the ATP degrading activity of the exogenous free CD39 protein, which were significantly better than that of the control antibody TTX-030.

Table 13: IC of humanized antibody inhibiting exogenous CD39 proteinase activity ₅₀

Sample of	IC ₅₀ (nM)	Top
			TTX-030	1.168	329268
439G-HuG	2.56	1842143
			439G-Hu-HMELW100Y	3.311	1830883
439G-HuG-LN85E	3.109	1829711

Table 14: IC of humanized antibody inhibiting exogenous CD39 proteinase activity ₅₀

Sample of	IC ₅₀ (nM)	Top
			TTX-030	7.479	728382
439G-HuG	4.242	1807346
			439G-HuG-LN85E	5.290	1842214
439G-Hu-HMLLmut3	4.845	1919578
			439G-Hu-HMKLmut3	3.810	1760895

EXAMPLE 11 detection of humanized antibody inhibition of tumor cell surface CD39 protease Activity

Each humanized antibody was assayed for its ability to inhibit CD39 enzymatic activity on the cell surface of malignant melanoma SKMEL-28 and multiple myeloma MOLP-8 as described in reference example 7. The test results are shown in FIGS. 14 to 15 and tables 15 to 16, respectively.

Table 15: IC of humanized antibody for inhibiting CD39 proteinase activity on surface of tumor cell SKMEL-28 ₅₀

Sample of	IC ₅₀ (nM)	Top
			TTX-030	1.091	738472
439G-HuG	1.037	1398523
			439G-HuG-LN85E	0.934	1412268
439G-Hu-HMELW100Y	0.768	1385164
			439G-Hu-HMKLmut3	0.847	1372273
439G-Hu-HMLLmut3	0.904	1403902

Table 16: IC of humanized antibody for inhibiting tumor cell MOLP-8 surface CD39 proteinase activity ₅₀

Sample of	IC ₅₀ (nM)	Top
			TTX-030	0.413	1070214
439G-HuG	2.089	1839684
			439G-HuG-LN85E	1.553	1824891
439G-Hu-HMELW100Y	1.367	1777186
			439G-Hu-HMKLmut3	1.259	1811259
439G-Hu-HMLLmut3	1.699	1857517

As can be seen from FIG. 14 and Table 15, the humanized antibodies 439G-HuG, 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3, 439G-Hu-HMLLmut3 were equivalent in activity in inhibiting the degradation of ATP by the CD39 protein on the cell surface of SKMEL-28, and significantly superior to the control antibody TTX-030.

From FIG. 15 and Table 16, it can be seen that the humanized antibodies 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3, 439G-Hu-HMLLmut3 were equivalent in activity of inhibiting degradation of ATP by CD39 protein on the surface of MOLP-8, slightly superior to 439G-HuG, and significantly superior to control antibody TTX-030.

EXAMPLE 12 humanized antibodies inhibit CD14 ⁺ Detection of monocyte surface CD39 protease Activity

This example measures CD14 in PBMC from antibodies by detecting ATP ⁺ Inhibition of monocyte surface CD39 protease activity. The specific method comprises the following steps:

CD14 was obtained by sorting from human PBMC (from Shanghai bamboo hat Biotechnology Co., ltd.) according to the CD14 microblades specification ⁺ Mononuclear cells were centrifuged to remove cell culture, cells were washed 1 time with PBS buffer, counted and resuspended in complete medium (1640+10%FBS+1%Pen Strep+1%Sodium Pyruvate+1%GlutaMAX) at 1.0X10 ⁵ Individual cells/well were seeded into round bottom 96 wells100. Mu.L/well in plates, incubated overnight at 37℃in a cell incubator; the next day, the cell culture supernatant was discarded, the antibody to be tested was diluted to 100nM with Tris buffer, 3-fold gradient dilution, then added to the above cell culture plate at 100. Mu.L/well, and incubated at 37℃for 60min; then, ATP with a final concentration of 20. Mu.M was added, 50. Mu.L/well was incubated at 37℃for 60min, 50. Mu.L of the culture supernatant was placed in a 96-well white opaque assay plate, 50. Mu.L of Cell Titer-Glo assay reagent was added, incubated at room temperature for 5min, and fluorescence intensity was read and analyzed in a multifunctional microplate reader.

As shown in FIG. 16 and Table 17, the humanized antibodies 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3 inhibited CD14 ⁺ The activity of the CD39 protein on the surface of the mononuclear cells for degrading ATP is equivalent, is slightly better than 439G-HuG and 439G-Hu-HMLLmut3, and is obviously better than that of a control antibody TTX-030.

Table 17: humanized antibody pair CD14 ⁺ IC for inhibition of monocyte surface CD39 protease activity ₅₀

EXAMPLE 13 detection of humanized antibodies inhibiting B cell surface CD39 protease Activity

B cells were isolated from human PBMC (from Shanghai bamboo hat Biotechnology Co., ltd.) according to the CD19 MicroBeads instructions, the cell culture broth was removed by centrifugation, and the cells were washed 1 pass with PBS buffer; cells were counted and resuspended in complete medium (1640+10%FBS+1%Pen Strep+1%Sodium Pyruvate+1%GlutaMAX) at 5.0X10 cells ⁴ Individual cells/well were seeded into round bottom 96 well plates, 100 μl/well, and incubated overnight in a cell incubator at 37 ℃; the next day, the cell culture supernatant was discarded, the antibody to be tested was diluted to 100nM with Tris buffer, 3-fold gradient dilution, then added to the above cell culture plate at 100. Mu.L/well, and incubated at 37℃for 60min; then adding ATP with a final concentration of 50 mu M, 50 mu L/well and 37 DEG CIncubation was performed for 60min, 50 μl of culture supernatant was taken in a 96-well white opaque assay plate, 50 μl of Cell Titer-Glo assay reagent was added, incubated at room temperature for 5min, and fluorescence intensity was read and analyzed in a multifunctional microplate reader.

The results of the experiment are shown in FIG. 17 and Table 18, and each humanized antibody significantly inhibited the degradation of ATP by the B cell surface CD39 protein, which was slightly better than that of control antibody TTX-030, which was shown to be 439G-Hu-HMKLmut 3.

Table 18: IC of humanized antibody for inhibiting B cell surface CD39 proteinase activity ₅₀

Sample of	IC ₅₀ (nM)	Top
			TTX-030	0.231	1610394
439G-HuG	0.162	1673126
			439G-HuG-LN85E	0.081	1684727
439G-Hu-HMELW100Y	0.100	1689281
			439G-Hu-HMKLmut3	0.064	1675509
439G-Hu-HMLLmut3	0.083	1632797

EXAMPLE 14 detection of humanized antibody inhibiting macrophage surface CD39 protease Activity

CD14 was selected from human PBMC (available from Shanghai bamboo hat Biotechnology Co., ltd.) according to the CD14 microblades specification ⁺ Is a monocyte of (a). CD14 to be sorted out ⁺ Monocytes were resuspended in RPMI-1640 complete medium containing 400ng/mL GM-CSF (RPMI-1640 medium containing 10% FBS), and differentiation was induced by adding 20mL of cell suspension to a T75 cell culture flask at a cell density of 1.5X10 ⁵ individual/mL; on day seven, half-changing was performed with fresh complete medium containing 400ng/mL GM-CSF; on the tenth day, the cell culture supernatant was removed, the cells were washed once with serum-free medium RPMI-1640, the cells were digested by adding 3mL of TrypLE Select, and after the cells shed, the digestion was stopped with complete medium, centrifuged, and the cells were washed twice with PBS to obtain macrophages. Resuspension of macrophages with complete medium to adjust cell density to 5×10 ⁵ Per mL, 100. Mu.L per well, plated in round bottom 96 well plates; the antibody to be detected is diluted in a gradient way by using a complete culture medium, the highest concentration of the antibody is 1000nM, and 12 gradients are diluted according to 3 times of gradients; centrifuging the 96-well plate paved with macrophages, completely discarding cell supernatant, adding the antibody to be tested with gradient dilution, adding 50 mu L of the antibody to be tested into each hole, and placing the mixture in a cell incubator for incubation for 1h; preparing LPS solution with the final concentration of 10ng/mL by using a complete culture medium, adding 50 mu L of each hole into the 96-well plate, and placing the mixture into a cell culture box for culturing for 3 hours; preparing an ATP solution with a final concentration of 1000 mu M by using a complete culture medium, adding 100 mu L of the ATP solution into a 96-well plate in each well, and placing the mixture into a cell culture box for culturing for 2 hours; after the completion of the culture, cell supernatants were collected for detection of IL-1β and takenmu.L of the supernatant was added with 50. Mu.L of Cell Titer-Glo detection reagent, incubated at room temperature for 5min, and fluorescence intensity was read in a multifunctional microplate reader for analysis and determination of ATP levels in the supernatant.

As shown in FIG. 18, humanized antibodies 439G-HuG, 439G-Hu-HMKLmut3, 439G-HuG-LN85E were effective in inhibiting macrophage surface CD39 protease activity to maintain extracellular ATP concentration.

As shown in fig. 19 and table 19, each humanized antibody was effective in stimulating the secretion of IL-1 β by macrophages by inhibiting ATP retained by the surface CD39 protease activity of macrophages in the presence of LPS.

Table 19: IC of humanized antibody for promoting macrophage to secrete IL-1 beta ₅₀

Sample of	IC ₅₀ (nM)	Top
			TTX-030	52.030	407.200
439G-HuG	50.910	109.800
			439G-HuG-LN85E	0.433	75.720
439G-Hu-HMKLmut3	0.375	98.640

EXAMPLE 15 humanized antibodies to T cell activation

CD14 was obtained by sorting from human PBMC (from Shanghai bamboo hat Biotechnology Co., ltd.) according to the CD14 microblades specification ⁺ Monocytes were washed 1 time with PBS buffer and resuspended to 30mL 1640 complete medium containing 400ng/mLGM-CSF and 20ng/mL IL-4 and added to T75 cell flasks to induce differentiation at a cell density of 1X 10 ⁵ individual/mL; on day seven, half of the cell culture supernatant was discarded and replaced with fresh 1640 complete medium containing 400ng/mL GM-CSF and 20ng/mL IL-4; on day ten, suspension cells were harvested and washed once with serum-free medium 1640; cell numbers and viability were counted. DC cells were grown according to 1X 10 ⁴ Spreading individual cells/hole in 96-hole round-bottom cell culture plate, diluting antibody to be tested with 10% FBS 1640 culture medium in gradient, adding into the 96-hole plate, 100 mu L/hole, and incubating the 96-hole plate in cell culture box for 1 hr; the final concentration of 1000. Mu.M ATP solution was prepared in 10% FBS 1640 medium, 50. Mu.L of each well was added to a 96-well plate, the 96-well plate was placed in a cell culture incubator to culture for 48 hours, after the completion of the culture, the cell supernatant was removed by centrifugation, and DC cells were washed 2 times with PBS.

CD4 was obtained by sorting from another batch of human PBMC (from Shanghai bamboo hat Biotechnology Co., ltd.) according to the CD4 MicroBeads, CD MicroBeads Specification ⁺ And CD8 ⁺ T cells. CD4 ⁺ T and CD8 ⁺ T cells were resuspended in 1640 complete medium, respectively, according to DC: t=1: 10 is added to the above-mentioned mixture containing 1X 10 ⁴ In a 96-well plate of each DC cell/well, 100. Mu.L/well, and 100IU/mL of IL-2 were added to all wells at the same time, and the cell plates were placed in a 37℃cell incubator for 5 days, and the cell supernatants were collected for IFN- γ detection.

As shown in FIG. 20, the results of experiments show that the cells 439G-HuG, 439G-Hu-HMKLmut3 and 439G-HuG-LN85E can significantly promote the maturation of DC cells and promote CD4 in a dose-dependent manner ⁺ T cells secrete IFN-gamma.

As shown in FIG. 21, 439G-HuG, 439G-Hu-HMKLmut3, 439G-HuG-LN85E also significantly promoted maturation of DC cells and dose-dependent promotion of CD8 ⁺ T cells secrete IFN-gamma.

EXAMPLE 16 detection of binding kinetics of humanized antibodies to CD39 protein

Antibodies were captured using a chip (commercially available from GE Healthcare, cat. No. 29139131-AA) covalently coupled to Protein A, and the relevant operating parameters were as follows: the concentration of the antibody is 1 mug/mL, the contact time is 60s, the flow rate is 10 mug/min, and the regeneration contact time is 30s; diluting the CD39 antigen by using HBS-EP+pH7.4 buffer solution, wherein the highest concentration is 80nM, diluting to 0.8192nM and 0 concentration point according to 2.5 times, adopting 6M guanidine hydrochloride solution as regeneration buffer solution, injecting sample on Biacore 8K according to the following parameters, wherein the combination time is 180s, the dissociation time is 720s, the flow rate is 30L/min, and the regeneration contact time is 30s; data were collected and analyzed using Biacore 8K Evaluation Software.

As shown in Table 20, the binding kinetics of each humanized antibody to CD39 protein was significantly better than that of the control antibody TTX-030, and was mainly characterized by slower dissociation rates, with the particular advantage of 439G-Hu-HMKLmut3, which was fast and slow.

Table 20: binding kinetics of humanized antibodies to CD39 protein

Sample of	ka(1/Ms)	kd(1/s)	KD(M)
				TTX-030	1.20E+05	2.78E-03	2.32E-08
439G-HuG	1.67E+05	3.33E-04	2.00E-09
				439G-HuG-LN85E	1.98E+05	3.96E-04	2.01E-09
439G-Hu-HMKLmut3	7.89E+05	1.61E-04	2.04E-10

EXAMPLE 17 humanized antibody species Cross-reaction

Human (Human), macaque (Maca, SEQ ID NO: XP-015311944 from NCBI database, accession number) Mouse (Mouse, SEQ ID NO: P55772 from UniProt database, SEQ ID NO: XP-032747831.1) Rat (Rat, SEQ ID NO: XP-032747831.1 from NCBI database, SEQ ID NO: Q9MYU 4) CD39-His protein was diluted to 2. Mu.g/mL with ELISA coating solution, 100. Mu.L/well coated ELISA plates, placed in a wet box, coated at 4℃for 16h; washing the ELISA plate three times with PBST, removing unbound antigen, and drying the ELISA plate on absorbent paper to remove excess liquid, then blocking with PBS-formulated 2% BSA,200 μl/well for 2h at room temperature; washing with PBST once, washing out excessive blocking solution, drying ELISA plate, removing excessive liquid, measuring antibody with 1% BSA prepared by PBST according to 3 times gradient, the highest concentration is 20nM, diluting 11 gradients 3 times, incubating for 1h according to 100 mu L/Kong Jiaru ELISA hole at room temperature, and making 2 duplicate holes in each sample; washing the unbound or non-specifically bound primary antibody, HRP-labeled Anti-Human Fc secondary antibody was diluted 1:5000 with antibody dilution, 100. Mu.L/Kong Jiaru ELISA wells incubated for 1h at room temperature; washing with PBST three times, drying ELISA plate on absorbent paper, removing excessive liquid, adding TMB color development liquid, developing to appropriate depth at 100 μl/hole, adding 2M H ₂ SO ₄ 50. Mu.L/well to terminate the color development and its absorbance was measured at a wavelength of 450nm in a multifunctional microplate reader, and the data was analyzed.

As shown in FIGS. 22 and 23, humanized antibody 439G-Hu-HMKLmut3 cross-reacted with cynomolgus CD39 protein (Maca CD 39), EC ₅₀ EC binding to Human CD39 protein (Human) at 0.145nM ₅₀ 0.172nM.

Claims

1. An antibody or antigen-binding fragment thereof that binds human CD39, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising:

The light chain variable region comprises

2. The antibody or antigen-binding fragment thereof that binds human CD39 of claim 1, comprising:

the heavy chain variable region comprises amino acid sequences as set forth in SEQ ID NOs: 1. 2 and 3, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 4. LCDR1, LCDR2 and LCDR3 as shown in 5 and 6; or (b)

The heavy chain variable region comprises amino acid sequences as set forth in SEQ ID NOs: 7. 8 and 9, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 10. LCDR1, LCDR2 and LCDR3 shown in figures 11 and 12;

or (b)

The heavy chain variable region comprises amino acid sequences as set forth in SEQ ID NOs: 13. 14 and 15, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 16. LCDR1, LCDR2 and LCDR3 as shown at 17 and 18;

Or (b)

The heavy chain variable region comprises amino acid sequences as set forth in SEQ ID NOs: 19. 20 and 21, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NOs: 22. 23 and 24, LCDR1, LCDR2 and LCDR3.

3. The antibody or antigen-binding fragment thereof that binds human CD39 of claim 1 or 2, comprising: a heavy chain variable region selected from any one of the following amino acid sequences: SEQ ID NO: 25. 29, 33, 37, 43, 47, 50, 53, 56; and/or

A light chain variable region selected from any one of the following amino acid sequences: SEQ ID NO: 27. 31, 35, 39, 59, 62, 65, 68; preferably, it comprises an amino acid sequence as set forth in SEQ ID NO:25 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:27, and a light chain variable region shown in seq id no; or (b)

The amino acid sequence is shown in SEQ ID NO:29 and the amino acid sequence of which is shown in SEQ ID NO:31, a light chain variable region shown in seq id no; or (b)

The amino acid sequence is shown in SEQ ID NO:33 and the amino acid sequence of which is shown in SEQ ID NO:35, a light chain variable region shown in seq id no; or (b)

The amino acid sequence is shown in SEQ ID NO:37 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:39, a light chain variable region shown in seq id no; or (b)

The amino acid sequence is shown in SEQ ID NO:43 and the amino acid sequence of which is shown in SEQ ID NO: 59; or (b)

The amino acid sequence is shown in SEQ ID NO:47 and the amino acid sequence of which is shown in SEQ ID NO: 59; or (b)

The amino acid sequence is shown in SEQ ID NO:50 and the amino acid sequence of which is shown in SEQ ID NO: 62. Or (b)

The amino acid sequence is shown in SEQ ID NO:53 and the amino acid sequence of which is shown in SEQ ID NO:65, a light chain variable region; or (b)

The amino acid sequence is shown in SEQ ID NO:43 and the amino acid sequence of which is shown in SEQ ID NO:68, a light chain variable region shown at 68; or (b)

The amino acid sequence is shown in SEQ ID NO:56 and the amino acid sequence of which is shown in SEQ ID NO: 65.

4. The antibody or antigen-binding fragment thereof that binds human CD39 of claim 1 or 2, comprising: selected from the group consisting of SEQ ID NOs: 44. 48, 51, 54, 57 or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:41 and a human IgG4 constant region selected from the group consisting of SEQ ID NOs: 25. 29, 33, 37, and a heavy chain comprising a spliced heavy chain variable region comprising any one of the amino acid sequences shown in seq id no;

Selected from the group consisting of SEQ ID NOs: 60. 63, 66, 69 or a light chain comprising any one of the amino acid sequences set forth in SEQ ID NO:42 and a human kappa chain constant region as set forth in SEQ ID NO: 27. 31, 35, 39.

Preferably, the method comprises the steps of:

the amino acid sequence is shown in SEQ ID NO:44 and the amino acid sequence of which is shown in SEQ ID NO: 60. Or (b)

The amino acid sequence is shown in SEQ ID NO:48 and the amino acid sequence of the heavy chain is shown as SEQ ID NO: 60. Or (b)

The amino acid sequence is shown in SEQ ID NO:51 and the amino acid sequence of which is shown in SEQ ID NO: 63; or (b)

The amino acid sequence is shown in SEQ ID NO:54 and the amino acid sequence of which is shown in SEQ ID NO: 66. Or (b)

The amino acid sequence is shown in SEQ ID NO:44 and the amino acid sequence of which is shown in SEQ ID NO: 69; or (b)

The amino acid sequence is shown in SEQ ID NO:57 and the amino acid sequence of which is shown in SEQ ID NO: 66. Or (b)

The amino acid sequence is shown as SEQ ID NO:25 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41, and a heavy chain formed by splicing human IgG4 constant regions and consisting of a polypeptide having an amino acid sequence as set forth in SEQ ID NO:27 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:42, a light chain comprising a splice of human kappa chain constant regions; or (b)

The amino acid sequence is shown as SEQ ID NO:29 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41, and a heavy chain formed by splicing human IgG4 constant regions and consisting of a polypeptide having an amino acid sequence as set forth in SEQ ID NO:31 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:42, a light chain comprising a splice of human kappa chain constant regions; or (b)

The amino acid sequence is shown as SEQ ID NO:33 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41, and a heavy chain formed by splicing human IgG4 constant regions and consisting of a polypeptide having an amino acid sequence as set forth in SEQ ID NO:35 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:42, a light chain comprising a splice of human kappa chain constant regions; or (b)

The amino acid sequence is shown as SEQ ID NO:37 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:41, and a heavy chain formed by splicing human IgG4 constant regions and consisting of a polypeptide having an amino acid sequence as set forth in SEQ ID NO:39 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:42, and a light chain comprising a splice of human kappa chain constant regions.

5. A nucleotide molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 that binds human CD 39.

6. An expression vector comprising the nucleotide molecule of claim 5.

7. A host cell comprising the expression vector of claim 6.

8. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-4 that binds human CD39, the method comprising the steps of:

a) Culturing the host cell of claim 7 under expression conditions, thereby expressing said antibody or antigen-binding fragment thereof that binds human CD 39;

9. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 that binds human CD39 and a pharmaceutically acceptable carrier.

10. Use of an antibody or antigen-binding fragment thereof that binds human CD39 according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 9 for the preparation of an anti-tumor medicament.