Anti-5T4 antibody-natural killer cell conjugates and uses thereof

Abstract

The present invention provides an anti-5T4 antibody-natural killer cell conjugate, a pharmaceutical composition containing the conjugate, and medical uses of the conjugate, as well as methods for producing the same.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 202211728539.X, filed on December 30, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present invention generally relates to the field of biopharmaceuticals, and specifically relates to an anti-5T4 antibody-natural killer cell conjugate, a pharmaceutical composition containing the conjugate, as well as pharmaceutical uses of the conjugate and methods for producing the same. [Background technology]

[0003] According to national cancer statistics released by the National Cancer Center of China in January 2019, there were approximately 3.929 million cases of malignant tumors and approximately 2.338 million deaths nationwide in 2015. Compared with past data, the cancer burden has been steadily increasing. In terms of case numbers, lung cancer, liver cancer, upper digestive system tumors, colorectal cancer, and female breast cancer remain the leading malignant tumors in China. According to national cancer statistics released by the National Cancer Center of China in February 2022, there were approximately 4.064 million cases of malignant tumors and approximately 2.4135 million deaths nationwide in 2016. Compared with 2015 data, new cases and deaths from malignant tumors continue to show an upward trend, with lung cancer consistently retaining the highest annual incidence rate in China.

[0004] Because early tumor symptoms are unclear, some tumors may recur later even if detected early, and the tumor itself is highly malignant, the proportion of patients with advanced-stage tumors is relatively high. Currently, common clinical treatments for solid tumors include surgery, radiation therapy, chemotherapy, traditional biological therapy, and traditional Chinese medicine. These treatments often involve certain toxicities and side effects. After surgical removal of the cancerous lesion, there may be varying degrees of complications, and there is a risk of postoperative recurrence. Radiation therapy and chemotherapy are the treatments with the strongest toxicities and side effects and may cause some damage to the patient's body. Traditional Chinese medicine treatments have fewer toxicities and side effects, but often only provide a supporting effect. Traditional biological therapy also has many unavoidable toxicities and side effects. However, the majority of patients with advanced solid tumors are no longer suitable for surgery, radiation therapy, or concurrent chemoradiotherapy, and have failed systematic standard treatments.

[0005] Therefore, there is an urgent need in this field to explore and develop new treatments for tumors. Summary of the Invention

[0006] According to a first aspect, the present invention provides an antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-5T4 antibody or antigen-binding fragment thereof is conjugated to the NK cell via a linker.

[0007] According to a second aspect, the present invention provides a cell population comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect.

[0008] According to a third aspect, the present invention provides a pharmaceutical composition comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, and a pharmaceutically acceptable carrier.

[0009] According to a fourth aspect, the present invention provides a method for treating a tumor in an individual, in which the tumor cells highly express 5T4 (5T4 + ) Use of an antibody-natural killer cell (NK cell) conjugate according to the first aspect or a cell population according to the second aspect in the manufacture of a medicament for the treatment of a tumor.

[0010] According to a fifth aspect, the present invention provides a method for treating a tumor in an individual, in which the tumor cells highly express 5T4 (5T4 + ) A method of treating a tumor, the method comprising administering to said individual an effective amount of the antibody-natural killer cell (NK cell) conjugate of the first aspect, or the cell population of the second aspect, or the pharmaceutical composition of the third aspect.

[0011] By way of non-limiting example, the present invention provides the following embodiments. [1] An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-5T4 antibody or antigen-binding fragment thereof is conjugated to the NK cell via a linker.

[0012] [2] The anti-5T4 antibody or antigen-binding fragment thereof HCDR1 as set forth in SEQ ID NO: 5 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 5; an HCDR2 as set forth in SEQ ID NO: 6 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 6; an HCDR3 as set forth in SEQ ID NO: 7 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 7; LCDR1 as set forth in SEQ ID NO: 8 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 8; An LCDR2 as set forth in SEQ ID NO: 9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 9, and comprising an LCDR3 as set forth in SEQ ID NO: 10 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 10; 2. The antibody-natural killer cell (NK cell) conjugate of embodiment 1, wherein the amino acid sequences of the HCDR and LCDR are as defined by Kabat.

[0013] [3] 3. The antibody-natural killer cell (NK cell) conjugate of embodiment 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-5T4 antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 3 or has at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 3.

[0014] [4] The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 3, wherein the anti-5T4 antibody comprises a heavy chain variable region shown in SEQ ID NO: 1, a heavy chain constant region shown in SEQ ID NO: 2, a light chain variable region shown in SEQ ID NO: 3, and a light chain constant region shown in SEQ ID NO: 4.

[0015] [5] the anti-5T4 antibody is a whole antibody, a single-chain antibody (scFv), or a bispecific antibody; and / or the antigen-binding fragment of the anti-5T4 antibody is Fab, Fab', Fv or F(ab')2, and / or the anti-5T4 antibody is a humanized or fully human antibody, and / or the anti-5T4 antibody is a monoclonal antibody, and / or the anti-5T4 antibody is of the IgG1, IgG2 or IgG4 isotype, and / or The anti-5T4 antibody comprises a light chain constant region of the κ or λ subtype. An antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 4.

[0016] [6] The NK cells are CD16 + and / or NKG2D + and preferably CD16 + NKG2D + The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 5, wherein:

[0017] [7] The conjugate CD16 + NKG2D + The proportion of NK cells is at least 90%, preferably CD56 + The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 6, wherein the proportion of NK cells is at least 95%.

[0018] [8] The NK cells are obtained from in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells, or The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood, or The NK cells are obtained from the in vitro culture and expansion of an NK cell line, or The NK cells are obtained from in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSCs / iPS cells) or mesenchymal stem cells (ESCs); An antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 7.

[0019] [9] 9. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 8, wherein the anti-5T4 antibody or antigen-binding fragment thereof and the NK cell are conjugated via a click chemistry reaction of a linker.

[0020]

[10] 10. The antibody-natural killer cell (NK cell) conjugate of embodiment 9, wherein the anti-5T4 antibody or antigen-binding fragment thereof and the NK cell are conjugated via a first linker and a second linker, wherein the first linker is conjugated to the anti-5T4 antibody or antigen-binding fragment thereof, the second linker is conjugated to the NK cell, and the first linker and the second linker are conjugated to each other to form the antibody-NK cell conjugate.

[0021]

[11] 11. The antibody-natural killer cell (NK cell) conjugate of embodiment 10, wherein the first linker is an active ester capable of forming conjugation to a lysine residue of the antibody via an ester bond to amide bond reaction, and the active ester is, for example, a pentafluorophenyl ester, for example, piperidine acid pentafluorophenyl ester.

[0022]

[12] The first linker further comprises a carbon-carbon triple bond structure capable of undergoing a cyclization reaction with the azide group to form a five-membered triazazole ring, for example, the carbon-carbon triple bond structure is an octyne group. 12. The antibody-natural killer cell (NK cell) conjugate of embodiment 11.

[0023]

[13] The first linker has the following structure: [ka] [In the formula, n is an integer of 0 to 8.] 13. The antibody-natural killer cell (NK cell) conjugate of embodiment 12, wherein the antibody-natural killer cell (NK cell) conjugate is dibenzoazacyclooctynyl-glutaryl-aminopolyethylene glycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0024]

[14] The first linker has the following structure: [ka] 14. The antibody-natural killer cell (NK cell) conjugate of embodiment 13, wherein the antibody-natural killer cell (NK cell) conjugate is dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0025]

[15] 11. The antibody-natural killer cell (NK cell) conjugate of embodiment 10, wherein said second linker is an azidoacetylcyclohexosamine, e.g., azidoacetylcyclogalactosamine, or azidoacetylglucosamine.

[0026]

[16] The second linker has the following structure: [ka] 16. The antibody-natural killer cell (NK cell) conjugate of embodiment 15, wherein the conjugate is 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose having the formula:

[0027]

[17] 17. The antibody-natural killer cell (NK cell) conjugate of embodiment 16, wherein the proportion of said second linkers in a single α or β configuration is at least 90%.

[0028]

[18] A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 17.

[0029]

[19] CD3 - CD56 + CD16 + and / or CD3 - CD56 + NKG2D + The number of cells accounts for at least 95%, preferably at least 98%, of the total number of cells in the cell population; 19. The cell population of embodiment 18.

[0030]

[20] CD3 + CD56 + the number of cells accounts for 5% or less of the total number of cells in said cell population; and / or CD3 - CD19 + the number of cells accounts for 2% or less of the total number of cells in said cell population; and / or CD3 + CD4 + and CD3 + CD8 + The number of cells accounts for 2% or less of the total number of cells in the cell population, 20. The cell population of embodiment 18 or 19.

[0031] 〔twenty one〕 21. The cell population of any one of embodiments 18 to 20, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of the total cells in the cell population.

[0032] 〔twenty two〕 A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 17 or the cell population of any one of embodiments 18 to 21, and a pharmaceutically acceptable carrier.

[0033] 〔twenty three〕 23. The pharmaceutical composition of embodiment 22, comprising sodium chloride and / or human serum albumin.

[0034] 〔twenty four〕 24. The pharmaceutical composition of embodiment 22 or 23, comprising trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0035] 〔twenty five〕 Tumors in individuals, especially tumor cells that are 5T4 (5T4 + 25. The pharmaceutical composition according to any one of embodiments 22 to 24, for use in treating a tumor highly expressing IL-16.

[0036]

[26] 26. The pharmaceutical composition of embodiment 25, wherein the tumor is a solid tumor.

[0037]

[27] 26. The pharmaceutical composition of embodiment 25, wherein the tumor is a malignant tumor.

[0038]

[28] The pharmaceutical composition of embodiment 25, wherein the tumor is cancer.

[0039]

[29] 29. The pharmaceutical composition of embodiment 28, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

[0040]

[30] The pharmaceutical composition of embodiment 28, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

[0041]

[31] The pharmaceutical composition of embodiment 28, wherein the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colorectal cancer.

[0042]

[32] Tumors in individuals, especially tumor cells that are 5T4 (5T4 + 22. Use of an antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 17 or a cell population according to any one of embodiments 18 to 21 in the manufacture of a medicament for the treatment of a tumor highly expressing NK cell.

[0043]

[33] The use of embodiment 32, wherein the tumor is a solid tumor.

[0044]

[34] The use of embodiment 32, wherein the tumor is a malignant tumor.

[0045]

[35] The use of embodiment 32, wherein the tumor is cancer.

[0046]

[36] The use of embodiment 35, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

[0047]

[37] The use of embodiment 35, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

[0048]

[38] The use of embodiment 35, wherein the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colorectal cancer.

[0049]

[39] Tumors in individuals, especially tumor cells that are 5T4 (5T4 + 31. A method for treating a tumor highly expressing a NK cell (anti-NK cell) gene, comprising administering to said individual an effective amount of the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1 to 17, the cell population of any one of embodiments 18 to 21, or the pharmaceutical composition of any one of embodiments 22 to 31.

[0050]

[40] 40. The method of embodiment 39, wherein the tumor is a solid tumor.

[0051]

[41] 40. The method of embodiment 39, wherein the tumor is a malignant tumor.

[0052]

[42] 40. The method of embodiment 39, wherein the tumor is cancer.

[0053]

[43] 43. The method of embodiment 42, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

[0054]

[44] The method of embodiment 42, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

[0055]

[45] 43. The method of embodiment 42, wherein the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colorectal cancer. [Brief explanation of the drawings]

[0056] [Figure 1] FIG. 1 shows the sequence and structural analysis of exemplary anti-5T4 antibodies of the invention. [Figure 2] FIG. 1 shows a structural schematic of an exemplary anti-5T4 antibody-natural killer cell conjugate of the present invention. [Figure 3] FIG. 1 shows flow cytometry analysis of IBR854 stock solution (Lot No. DSE20220711): CD56+, CD3-, CD16+, NKG2D+, CD19-. [Figure 4] FIG. 1 shows flow cytometry analysis results for IBR854 formulation (Lot No. FE20220717): CD56+, CD3-, CD16+, NKG2D+, CD19-. [Figure 5] FIG. 1 shows the results of flow cytometry analysis of the conjugation positivity rate and geometric mean value of UNK, IBR854 stock solution, and IBR854 formulations. [Figure 6] FIG. 1 shows a schematic diagram of the production process flow of an L2 linker. [Figure 7] FIG. 1 shows the structural formula of the L2 linker (Japanese name: (Z)-19-(1′-aza-2′-one-dibenzo[b,f]cyclo-7′-octynyl)-3,6,9,12-tetraoxa-15-aza-16,20-dioxodecanoylpiperidine-4-carboxylic acid pentafluorophenyl ester, English name: pentafluorophenyl (Z)-20-(1′-aza-2′-oxo-dibenzo[b,f]cyclo-7′-octynyl)-3,6,9,12-tetraoxa-15-aza-16,20-dioxodecanoyl piperidine-4-carboxylate). [Figure 8] FIG. 1 shows a schematic flow diagram of the manufacturing process of the N1 linker. [Figure 9] FIG. 1 shows the structural formula of the N1 linker (Japanese name: 1,3,4,6-tetra-O-acetyl-2-azidoacetylamido-2-deoxy-a,bD-mannopyranose, English name: 1,3,4,6-tetra-O-acetyl-2-azidoacetylamido-2-deoxy-a,bD-mannopyanose). [Figure 10] FIG. 1 shows a schematic diagram of the conjugation mechanism between the L2 linker and the anti-5T4 antibody. [Figure 11] FIG. 1 shows a schematic diagram of the binding mechanism between the N1 linker and NK cells. [Figure 12] FIG. 1 shows a schematic diagram of the structure of UNK connected with an N1 linker. [Figure 13] FIG. 1 shows a schematic diagram of the binding mechanism between UNK and an L2-conjugated antibody. [Figure 14] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on H1975 cells. [Figure 15] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on NCI-H292 cells. [Figure 16] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on NCI-H226 cells. [Figure 17] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on HCC827 cells. [Figure 18] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on Panc-1 cells. [Figure 19] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on BxPC3 cells. [Figure 20] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on MDA-MB-231 cells. [Figure 21] FIG. 1 shows a comparison of the cytotoxic effects of IBR854, UNK cells, and NK cells on MDA-MB-468 cells. [Figure 22] 1 shows the levels of TraiL following the action of IBR854 on H292 tumor cells, where panels A and B show the time course at an effector to target ratio of 10:1, and panel C shows the time course at different effector to target ratios. [Figure 23] Figure 1 shows the levels of FasL following the action of IBR854 on H292 tumor cells, where panels A and B show the time course at an effector to target ratio of 10:1, and panel C shows the time course at different effector to target ratios. [Figure 24]This figure shows the tumor volume curves of animals in each group during a study in which human lung cancer cells NCI-H292 M-NSG were transplanted into mice. Compared to the vehicle control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 25] This figure shows the tumor volume curves of animals in each group during a mouse tumor transplant study using human breast cancer cell MDA-MB-468. Compared to the vehicle control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 26] This figure shows a comparison of tumor weights in each group of mice on day 20 during a study in which human breast cancer cell MDA-MB-468 tumors were implanted in the mice. Compared to the vehicle control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 27] Figure 1 shows a comparison of the cytotoxic effects of anti-5T4 antibody-natural killer cell conjugates with different L linkers, UNK cells, and NK cells on H292 cells, where the bars for each effector-to-target ratio are, from left to right, L3, L2, NK, and UNK. [Figure 28] FIG. 1 shows the time course of the cytotoxic effects of IBR854, NK cells, and a mixture of UNK cells and an anti-5T4 conjugated antibody on LOVO cells. [Figure 29] FIG. 10 is a graph showing the cytotoxic effect of a mixture of IBR854, NK cells, and UNK cells plus an anti-5T4 conjugate antibody on LOVO cells after 55 hours of exposure. [Figure 30] FIG. 1 shows the time course of the cytotoxic effects of IBR854, NK cells, and a mixture of UNK cells and an anti-5T4 conjugated antibody on N87 cells. [Figure 31] FIG. 1 shows the cytotoxic effect of a mixture of IBR854, NK cells, and UNK cells plus an anti-5T4 conjugated antibody on N87 cells after 24 hours of exposure. DETAILED DESCRIPTION OF THE INVENTION

[0057] [Definition] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as understood by those skilled in the art. For definitions and terms in the art, those skilled in the art can refer, in particular, to "Current Protocols in Molecular Biology" (Ausubel). Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0058] Notwithstanding the numerical ranges and approximations of parameters set forth in the broad scope of the invention, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, all numerical values ​​inherently contain a certain degree of error due to the standard deviation that exists in each measurement. Furthermore, all ranges disclosed herein should be understood to cover all subranges contained within that range. For example, a recited range of "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (including the endpoints), i.e., all subranges beginning with a minimum value of 1 or greater, e.g., 1 to 6.1, and all subranges ending with a maximum value of 10 or less, e.g., 5.5 to 10. Furthermore, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0059] As used herein, the term "individual" or "subject" refers to a mammal such as a human, but may also refer to other animals such as wild, domestic, or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, round squirrels, etc.).

[0060] As used herein, the term "antigen" refers to a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, polypeptides, sugars, nucleic acids, lipids, haptens, or other natural or synthetic compounds.

[0061] In a broad sense, an "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses a complete antibody / full-length antibody, a single antibody chain, or any antigen-binding fragment of an antibody (also called an "antigen-binding portion"). When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" can be understood as being complete with respect to "antigen-binding fragment / antigen-binding portion," and both commonly correspond to the broad concept of an antibody.

[0062] The term "agonist antibody" refers to an antibody that elicits a response, e.g., an antibody that mimics at least one functional activity of a target polypeptide. Agonist antibodies include antibodies that are ligand mimetics, e.g., a ligand binds to a cell surface receptor, the binding of which induces cell signaling or activity through an intracellular cell signaling pathway, and an antibody induces similar cell signaling or activation.

[0063] A "full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together via disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises a CL domain. The VH and VL regions may be further subdivided into multiple regions of hypervariability called complementarity-determining regions (CDRs), interspersed with multiple more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains comprise the binding domains that interact with antigens. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A full-length antibody (intact antibody) can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), but antibodies need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the constant domain of their heavy chains. There are generally five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known. The antibodies of the present invention also include chimeric or humanized antibodies. Those skilled in the art know that the complementarity-determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest influence on the affinity and specificity of an antibody.There are many common definition methods for the CDR amino acid sequences of VH or VL, such as the Kabat definition, the IMGT definition, and the Chothia definition. For a given antibody variable region amino acid sequence, the CDR amino acid sequences in the VH and VL amino acid sequences can generally be determined by various definition methods. In an embodiment of the present invention, Kabat is used to define the CDR amino acid sequences. For a given antibody variable region amino acid sequence, the CDR amino acid sequences of the variable region amino acid sequence can be analyzed by various methods.

[0064] The term "humanized antibody" refers to an antibody obtained by grafting CDR sequences from other mammalian species, such as mice, onto human framework sequences. Some residues in the framework (referred to as FR) section can be modified to maintain binding affinity. The humanized antibody or fragment thereof according to the present invention can be produced by techniques well known to those skilled in the art.

[0065] The term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, e.g., whose variable region sequences are derived from a mouse antibody and whose constant region sequences are derived from a human antibody. The chimeric antibody or fragment thereof of the present invention can be produced using genetic engineering techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter, a sequence encoding the variable region of a non-human (especially mouse) monoclonal antibody described in the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene is, for example, a mouse-human chimera, and the antibody's specificity is determined by the variable region derived from the mouse DNA and its allotype is determined by the constant region derived from the human DNA.

[0066] The term "partially humanized antibody" refers to an antibody that comprises a constant region of human origin and a variable region (including CDRs) of non-human (such as murine) origin. The term "semi-humanized antibody" refers to a type of humanized antibody, in which one antibody chain contains a mouse variable region and the other antibody chain contains a humanized variable region, i.e., a semi-humanized antibody. The term "monoclonal antibody" refers to an antibody obtained from a nearly homogeneous antibody population, in which the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a few individuals.

[0067] As used herein, the terms "antigen-binding fragment" or "antigen-binding portion" or "antigen-binding region" are used interchangeably and refer to the portion of an antibody that contains the amino acid residues that interact with an antigen and confer specificity and affinity to the binder for the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, and Fab', or any fragment whose half-life can be extended by chemical modification or incorporation into liposomes. Such chemical modifications include, for example, the addition of poly(alkylene) glycols, such as polyethylene glycol ("pegylated, PEGylated") (referred to as "pegylated fragments" of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, and Fab'-PEG, where "PEG" is polyethylene glycol). Preferably, the functional fragment consists of or comprises a subsequence of the heavy or light variable chain of the antibody from which it is derived, the subsequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, and the functional fragment comprises at least 5 consecutive amino acids, preferably 10, 15, 25, 50, or 100 consecutive amino acids of the antibody sequence from which it is derived. Examples of antigen-binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain, (2) an F(ab')2 fragment, which may be a bivalent fragment having two Fab' fragments linked by a disulfide bridge in the hinge region (i.e., an Fab' dimer), and (3) an Fv fragment of the VL and VH domains comprising a single arm of an antibody.

[0068] The term "single-chain antibody (scFv)" refers to a single polypeptide chain in which a VH domain and a VL domain are linked via a peptide linker. (scFv)2 contains two VH domains linked via a peptide linker and two VL domains connected to the two VH domains via disulfide bridges.

[0069] The terms "Fc fragment," "Fc region," "Fc domain," "Fc portion," or similar terms refer to a portion of the antibody heavy chain constant region, including the hinge region, CH2 fragment, and CH3 fragment of the constant region. The Fc region of an antibody can be engineered or modified, including modifications related to effector function, for example, to reduce or eliminate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody.

[0070] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0071] The term "bispecific antibody" refers to an antibody capable of simultaneously binding to two different antigenic epitopes. The two epitopes may be on different antigens or on the same antigen. Bispecific antibodies may have multiple structural configurations. For example, a bispecific antibody may be composed of two Fc fragments fused to two antigen-binding moieties (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding moieties may be in the form of a single-chain antibody (scfv) or Fab fragments. The two different binding moieties of a bispecific antibody are each attached to the N-terminus of a single Fc fragment, and the antigen-binding moieties of the two arms can be configured in four combinations: scfv + Fab fragment, Fab fragment + scfv, scfv + scfv, or Fab fragment + Fab fragment. The Fc fragment can contain mutations that ensure heavy chain heteromerization, and knob-in-hole (KIH) technology is a strategy to address heavy chain heteropolymerization. Generally, KIH technology refers to a technique that alters the amino acid sequence of the CH3 region to form a favorable structure for heterologous incomplete antibodies to pair with each other, thereby forming a bispecific antibody while maintaining the structure of a normal antibody as much as possible.

[0072] Generally, to prepare monoclonal antibodies or functional fragments thereof, in particular murine-derived monoclonal antibodies or functional fragments thereof, reference can be made in particular to the techniques described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988), or to the technique of preparation from hybridoma cells by Kohler and Milstein (Nature, 256: 495497, 1975).

[0073] The term "conservative variant" or "conservative amino acid substitution" refers to a substitution that does not substantially affect or reduce the affinity of a protein. For example, an antibody may contain about 1 or less, about 2 or less, about 5 or less, about 10 or less, or about 15 or less conservative substitutions and specifically bind to a target antigen. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, as long as the antibody specifically binds to a target antigen.

[0074] The term "isolated" biological component (e.g., nucleic acid, protein (including antibody), or organelle) refers to one that has been substantially separated or purified from other biological components (i.e., other chromosomal and additional chromosomal DNA and RNA, proteins and organelles) in the component's naturally occurring environment (e.g., a cell). Nucleic acids and proteins that have been "isolated" include those that have been purified by standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0075] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug and a pharmaceutically acceptable carrier or auxiliary substance that are combined together to achieve a specific purpose. In some embodiments, the pharmaceutical composition includes a combination of drugs separated in time and / or space, as long as they can act together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition (e.g., the antibody-cell conjugates of the present invention) may be administered to an individual all at once or separately. When the components contained in the pharmaceutical composition are administered to an individual separately, the components may be administered to the individual simultaneously or sequentially. The pharmaceutical composition of the present invention may contain conventional components for cell culture, particularly NK cell culture, to maintain the activity of NK cells in the conjugate. Pharmaceutically acceptable carriers may further include water, aqueous buffer solutions, isotonic salt solutions such as PBS (phosphate buffer), glucose, mannitol, dextroglucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol and triglycerides, etc. The pharmaceutical composition or formulation according to the present invention can be administered by any suitable route, such as intravenous administration, intradermal administration, subcutaneous administration, intramuscular injection, etc. The composition according to the present invention may contain additives such as wetting agents, emulsifying agents or buffer substances.

[0076] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show its benefit to the individual to whom it is administered. The actual amount administered, the rate and time course of administration depend on the condition and severity of the individual being treated. The prescription for treatment (e.g., determining the dosage) is ultimately the responsibility of specialists and other physicians, and is generally determined taking into account the disease to be treated, the individual patient's condition, delivery site, administration method, and other factors known to physicians.

[0077] EC 50The EC value primarily refers to the concentration of a corresponding drug, antibody, or toxin that can reach 50% of its maximum biological effect after a specific exposure time. In pharmacology, it is used to characterize the activation ability of agonists in in vitro experiments, and also to indicate the blood concentration required to reach half of the maximum biological effect in vivo. In some literature, the EC 50 is also used to characterize the potency (including agonism and antagonism) of compounds at a cellular level, and EC 50 The value can be measured by methods such as ELISA.

[0078] The term "identity / homology / matching" of an amino acid or nucleic acid sequence is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after sequence alignment and capping, if necessary, to reach the maximum percentage of identity. Methods and computer programs for alignment are known to those skilled in the art.

[0079] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors may be benign, precancerous, or malignant.

[0080] As used herein, the term "malignancy" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Exemplary malignancies include carcinomas, solid tumors, melanomas, sarcomas, hematologic tumors, germ cell tumors, and blastomas. More specific examples of malignant tumors include multiple myeloma, renal cancer, lung cancer (including non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), bladder cancer, breast cancer, cervical cancer, colon cancer, hepatic carcinoma, gastric cancer (including gastrointestinal cancer), prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer, liver cancer, urinary tract cancer, hepatocellular carcinoma, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., cutaneous squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and related metastases.

[0081] As used herein, the term "hematological tumor" refers to a tumor caused by the uncontrolled growth and proliferation of abnormal cells. Often, these abnormal cells originate from the bone marrow, which is the very place where blood cells are produced. Exemplary hematological tumors include various leukemias, multiple myeloma, and malignant lymphomas. More specific examples of hematological malignancies include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoblastic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia (Waldenstrom's lymphoma), and leukemia. macroglobulinemia (Waldenström's macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, B-cell chronic lymphocytic lymphoma, old These include classic Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Selgene syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorder, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma, unspecified type, and anaplastic large cell lymphoma.

[0082] As used herein, the term "solid tumor" refers to a tangible mass that can be detected (palpable) by radiography, CT scan, B-ultrasound, clinical examination, palpation, etc. Clinically diagnosed and treated solid tumors are divided into two types: malignant and benign. Malignant solid tumors include pediatric Hodgkin's lymphoma (lymphocyte-predominant, nodular sclerosis, mixed cell type, lymphopenic type), pediatric non-Hodgkin's lymphoma (prolymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt's lymphoma / non-Burkitt's lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.), pediatric kidney tumors (nephroblastoma (Wilm's tumor), renal clear cell carcinoma, renal rhabdomyosarcomatoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, etc.), pediatric neuroblastoma (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric extracranial germ cell tumors (mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma (spermatogonial tumor), dysblastoma (astrocytoma), choriocarcinoma (choriocarcinoma)). , embryonal carcinoma, etc.), osteosarcoma and chondrosarcoma, childhood rhabdomyosarcoma (embryonic, acinar, pleomorphic, etc.), childhood soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, alveolar soft part sarcoma, epithelioid sarcoma (epithelioid sarcoma), clear cell sarcoma, malignant melanoma, synovial sarcoma, desmoplastic small round cell tumor, etc.), Ewing's sarcoma family tumors (Ewing's sarcoma, primitive neuroectodermal tumor), childhood liver tumors (hepatoblastoma (embryonic, fetal, undifferentiated), hepatocellular carcinoma), retinoblastoma, other tumors (posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatoblastoma, islet cell tumor, ileocecal carcinoid, mesothelioma, etc.). Benign solid tumors include lymphangiomas, hemangiomas, and thyroglossal duct cysts.

[0083] Adoptive cellular immunotherapy for tumor treatment has become a research hotspot in China and worldwide, achieving better results in clinical trials for tumors. Adoptive cellular immunotherapy involves isolating autologous or allogeneic immune cells, activating or genetically modifying them in vitro, expanding them to a sufficient number of immune cells with antitumor activity, and then injecting them into tumor patients to enhance the patient's cellular immune function and improve antitumor efficacy. Currently, multiple types of cellular immunotherapy are being investigated, including chimeric antigen receptor-modified T cells (CAR-T), T cell receptor gene-modified T cells (TCR-T), dendritic cell (DC) vaccines, natural killer cells (NK), tumor-infiltrating lymphocytes (TIL), and cytokine-induced killer cells (CIK). Although CAR-T cell therapy has developed rapidly in recent years, many deficiencies and challenges remain in its clinical application, including varying degrees of neurotoxicity and the risk of cytokine storm, as well as very low efficacy in the treatment of solid tumors. Furthermore, most CAR-T cell immunotherapies require autologous adoptive cell transplantation because allogeneic T cells can cause graft-versus-host disease (GVHD) unless the HLA barrier is addressed. Furthermore, CAR-T cell immunotherapies can cause life-threatening side effects in patients, such as cytokine release syndrome.

[0084] In contrast, NK cell therapy has demonstrated excellent clinical efficacy in the treatment of hematologic malignancies and offers several advantages over CAR-T cell therapy: 1) NK cells do not induce graft-versus-host reaction (GVHD) like T cells, making them promising as a versatile cell therapy. 2) Mature NK cells have a relatively short lifespan (approximately 7–10 days), effectively killing tumor cells while reducing the likelihood of long-term adverse events. 3) NK cells are not restricted by antigen-specific major histocompatibility complex (MHC) binding, giving them a more potent cytotoxic potential. 4) NK cell therapy does not secrete inflammatory factors (e.g., IL-1 and IL-6), reducing the risk of cytokine storms and neurotoxic reactions. 5) The CD16 low-affinity molecule on the surface of NK cells can bind to IgG antibody conjugates on target cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and apoptosis via the Fas / FasL pathway. Furthermore, NK cells secrete low levels of programmed death receptor 1 (PD-1), which induces little immunosuppression, making them promising candidates for the treatment of solid tumors.

[0085] The inventors of the present application have conducted extensive research in the field of cellular immunotherapy and have developed anti-5T4 antibody-natural killer cell conjugates, particularly for tumor therapy. Figure 2 shows a structural schematic diagram of an exemplary anti-5T4 antibody-natural killer cell conjugate of the present invention.

[0086] Natural killer cells (NK cells) belong to the granular lymphocyte family and are a component of the human immune system. NK cells recognize target antigens without major histocompatibility complex (MHC) restriction, and allogeneic NK cells have an extremely low risk of causing graft-versus-host disease (GVHD). Therefore, allogeneic NK cell therapy is clinically feasible. Furthermore, the risk of NK cells causing cytokine release syndrome (CRS) is also extremely low. In 2020, Liu et al. published the results of a phase I / II clinical trial of CAR-NK cell therapy for lymphoma in the New England Journal of Medicine. Following CAR-NK cell infusion in 11 enrolled subjects, eight showed remission, seven of whom achieved complete remission, without any CRS, neurotoxicity, or GVHD. This indicates that CAR-NK cell therapy has a relatively high safety profile. Furthermore, NK cells can recognize target antigens without being restricted by MHC, allowing them to be manufactured into general-purpose products without being limited to autologous cells. Therefore, the NK cell sources used in this therapy can be selected from a wide range of sources, including allogeneic peripheral blood, umbilical cord blood, embryonic stem cells, human-induced pluripotent stem cells, and the NK-92 cell line.

[0087] The cytotoxic activity of NK cells is primarily mediated by: 1) Direct lysis of target cells: NK cells release cytotoxic particles such as perforin and granzymes via exocytosis, which activate the caspase pathway to induce necrosis or apoptosis of target cells. 2) Cytokine secretion: NK cells have cytokine-mediated damaging effects, and can synthesize and secrete various cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10, and G-CSF, which induce apoptosis of target cells. 3) Induction of apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, which induce apoptosis of CD95+ target cells and TRAIL receptor-positive target cells through a cascade of endogenous enzymes. 4)ADCC: Antibody-dependent cell-mediated cytotoxicity. 5) Immune checkpoint pathway: It exerts its effect by expressing programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), among others, and inhibiting immune checkpoints.

[0088] The multiple mechanisms of action described above, combined with the potential for commercial application and reliable safety profile, make NK cell therapy an attractive immunotherapy.

[0089] The antigen target selected in this invention is trophoblast glycoprotein 5T4 (5T4 oncofetal trophoblast glycoprotein, referred to as "5T4" in this invention). 5T4 is an N-glycosylated transmembrane protein with a molecular weight of 72 kDa that is highly expressed on the surface of trophoblast cells (trophoblast cells) and various solid tumors (non-small cell lung cancer, breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, etc.) and tumor stem cells, but is expressed at low or no levels in normal adult tissues. The extracellular domain of the 5T4 protein contains multiple leucine repeats involved in protein binding, promoting cell-matrix, i.e., cell-cell, interactions.

[0090] According to a first aspect, the present invention provides an antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-5T4 antibody or antigen-binding fragment thereof is conjugated to the NK cell via a linker.

[0091] In some embodiments, the anti-5T4 antibody or antigen-binding fragment thereof is HCDR1 as set forth in SEQ ID NO: 5 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 5; an HCDR2 as set forth in SEQ ID NO: 6 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 6; an HCDR3 as set forth in SEQ ID NO: 7 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 7; LCDR1 as set forth in SEQ ID NO: 8 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 8; An LCDR2 as set forth in SEQ ID NO: 9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 9, and comprising an LCDR3 as set forth in SEQ ID NO: 10 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 10; Here, the amino acid sequences of the HCDR and LCDR are defined by Kabat.

[0092] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-5T4 antibody or antigen-binding fragment thereof is set forth in SEQ ID NO:1 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:3 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:3.

[0093] In some embodiments, the amino acid sequence of the heavy chain variable region of an anti-5T4 antibody or antigen-binding fragment thereof differs from the amino acid sequence set forth in SEQ ID NO: 1 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 1 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids and still maintain similar function as the heavy chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 1 and the resulting amino acid sequence still maintains similar function as the heavy chain variable region of the antibody. In some embodiments, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1, as long as the modified amino acid sequence maintains substantially similar functions to the heavy chain variable region of the antibody.

[0094] In some embodiments, the amino acid sequence of the light chain variable region of an anti-5T4 antibody or antigen-binding fragment thereof differs from the amino acid sequence set forth in SEQ ID NO: 3 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 3 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids and still maintain similar function to the light chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids may be further added to the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 3 and still maintain similar function to the light chain variable region of the antibody. In some embodiments, the modified amino acid sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 3, as long as the modified amino acid sequence maintains substantially similar function to the light chain variable region of the antibody.

[0095] In some embodiments, the anti-5T4 antibody has a heavy chain variable region set forth in SEQ ID NO: 1, a heavy chain constant region set forth in SEQ ID NO: 2, a light chain variable region set forth in SEQ ID NO: 3, and a light chain constant region set forth in SEQ ID NO: 4.

[0096] FIG. 1 shows the sequence and structural analysis of an exemplary anti-5T4 antibody. In some embodiments, the anti-5T4 antibody is a whole antibody, a single chain antibody (scFv), or a bispecific antibody. In some embodiments, the antigen-binding fragment of the anti-5T4 antibody is Fab, Fab', Fv, or F(ab')2. In some embodiments, the anti-5T4 antibody is a humanized or fully human antibody.

[0097] In some embodiments, the anti-5T4 antibody is a monoclonal antibody. In some embodiments, the anti-5T4 antibody is an IgG1, IgG2, or IgG4 isotype. In some embodiments, the anti-5T4 antibody is an IgG1 isotype. In some embodiments, the anti-5T4 antibody comprises a light chain constant region of the kappa or lambda subtype. Once the structure / sequence of an antibody has been identified, techniques for producing the corresponding antibody are within the skill of the art.

[0098] As a non-limiting example, the sequence of an anti-5T4 monoclonal antibody can be derived from screening a phage library, and then confirmed and selected by screening for binding activity to 5T4 and 5T4-expressing cells and affinity studies to obtain an anti-5T4 monoclonal antibody, wherein the anti-5T4 monoclonal antibody comprises a heavy chain variable region shown in SEQ ID NO: 1 (HCDRs 1-3 are SEQ ID NOs: 5, 6, and 7, respectively), a heavy chain constant region shown in SEQ ID NO: 2, a light chain variable region shown in SEQ ID NO: 3 (LCDRs 1-3 are SEQ ID NOs: 8, 9, and 10, respectively), and a light chain constant region shown in SEQ ID NO: 4. The DNA sequence of the anti-5T4 monoclonal antibody is determined, and a recombinant plasmid (IB12) expressing the anti-5T4 monoclonal antibody is constructed. The construction of the monoclonal cell line was based on known techniques. The IB12 plasmid was transfected into CHO cells by electroporation (electrotransfection). Minipools were screened once, followed by two rounds of screening after plating. Initial stable cultures were confirmed, resulting in a cell line capable of stably expressing anti-5T4 monoclonal antibody. Anti-5T4 monoclonal antibody was produced using known production protocols in the field, including cell culture and protein purification. Working cells were harvested, resuscitated, and grown for 3–5 rounds at medium scale. The unprocessed cell suspension (UPB) was then collected and clarified and filtered to obtain the cell culture supernatant. The anti-5T4 monoclonal antibody was then purified using a three-step protein A affinity chromatography, anion exchange, and cation exchange chromatography.

[0099] In some embodiments, the NK cells are CD16 + and / or NKG2D + In some embodiments, the NK cells are CD16 + NKG2D + is.

[0100] In some embodiments, the CD16 + NKG2D +In some embodiments, the percentage of NK cells is at least 90%, e.g., at least 95%, at least 98%, or at least 99%. + NKG2D + The proportion of NK cells is at least 90% and CD56 + The proportion of NK cells is at least 95% (eg, at least 96%, at least 97%, at least 98%, or at least 99%).

[0101] There are a wide variety of techniques for culturing, expanding and obtaining NK cells in vitro, the general principles and methodologies of which are known to those skilled in the art.

[0102] In some embodiments, NK cells are obtained by in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells, which is also an exemplary method in the Examples of this application. PBMCs are one of the major sources of NK cells, and have the advantages of being relatively easy to collect, easy to expand in vitro, and without toxicity or side effects. However, the percentage of NK cells in PBMCs is only 10% to 15%. Methods for expanding PBMC-derived NK cells include stimulating in vitro proliferation of NK cells using a combination of cytokines, feeder cells, or membrane particles; these different expansion systems exhibit different levels of NK cell expansion efficiency. In some embodiments, PBMCs are screened for HLA and KIR. In some embodiments, PBMCs are screened for CD16a variants, i.e., 176V and 176F. In some embodiments, one or more cytokines are used to maintain or activate the activity of natural killer cells in culture. In some embodiments, one or more immunoglobulins or fusion proteins are used to suppress the proliferation of B cells, macrophages, and other immune cells.

[0103] PBMCs may be derived from peripheral blood lymphocyte collection from allogeneic healthy donors. After separation of T cells and red blood cells, cells are transferred to primary cell cryopreservation solution, resulting in a total of 6.0 x 10 viable cells per vial. 7 The PBMCs are then aliquoted into 100 or more aliquots to obtain PBMCs. PBMCs are stored long-term in liquid nitrogen at temperatures below -175°C. PBMCs cryopreserved in liquid nitrogen are removed, resuscitated in an adapted medium, and expanded in culture (i.e., by increasing the medium volume). NK cell-associated cytokines, including but not limited to IL2 and IL15, are added to maintain their proliferation capacity and activity. For an exemplary production method, see Example 1 of the present application.

[0104] In some embodiments, NK cells are obtained by in vitro culture and expansion of NK cells derived from umbilical cord blood. There are generally two different methods for obtaining large amounts of NK cells from umbilical cord blood. One method is to expand NK cells in umbilical cord blood, and the other method is to extract CD34 + The goal is to induce hematopoietic stem / progenitor cells to differentiate into NK cells and then proliferate.

[0105] In some embodiments, the NK cells are obtained by in vitro culture and expansion of an NK cell line. As an example, NK-92, a homogeneous immortalized NK lymphoma cell line, is the first NK cell-based immunotherapy approved by the FDA for clinical trials.

[0106] In some embodiments, NK cells are obtained by in vitro derivation, culture, and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs). In some embodiments, the anti-5T4 antibody or antigen-binding fragment thereof and the NK cell are conjugated via a click chemistry reaction of a linker.

[0107] "Click chemistry," also known as "link chemistry" or "speed-matching combined chemistry," is a synthetic concept introduced by chemist Barry Sharpless in 2001. Its main purpose is to rapidly and reliably complete the chemical synthesis of various molecules by linking small units. There are four main types of click chemistry reactions: cycloaddition reactions, nucleophilic ring-opening reactions, non-aldol carbonyl chemical reactions, and carbon-carbon multiple bond addition reactions.

[0108] In some embodiments, the anti-5T4 antibody or antigen-binding fragment thereof and the NK cell are conjugated via a first linker and a second linker, wherein the first linker is conjugated to the anti-5T4 antibody or antigen-binding fragment thereof, the second linker is conjugated to the NK cell, and the first linker and the second linker are conjugated to each other to form an antibody-NK cell conjugate.

[0109] In some embodiments, the first linker is an activated ester that can form conjugation to a lysine residue of an antibody through an ester bond-to-amide bond reaction, and the activated ester is, for example, a pentafluorophenyl ester, such as piperidine acid pentafluorophenyl ester. The activated ester may be a molecule that is stable in an aqueous phase and can specifically covalently bond to a lysine residue of an antibody. In some embodiments, the activated ester has a tetraethylene glycol chain structure and can be gradually hydrolyzed in water. In some embodiments, the activated ester reacts with an amino group at the hydrophilic interface of a protein to convert the ester bond to an amide bond, thereby establishing a binding mechanism (conjugation mechanism).

[0110] In some embodiments, the first linker further comprises a carbon-carbon triple bond structure that can undergo a cyclization reaction with the azide group to form a five-membered triazazole ring, for example, the carbon-carbon triple bond structure is an octyne group.

[0111] In some embodiments, the first linker has the following structure: [ka] [In the formula, n is an integer of 0 to 8.] Dibenzoazacyclooctynyl-glutaryl-aminopolyethylene glycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0112] In some embodiments, the first linker has the following structure: [ka] Dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0113] Dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester is also referred to as L2 linker in the present invention. The synthesis pathway of L2 linker is shown in Figure 6, and its structural formula is shown in Figure 7. It includes three main synthesis steps: amide condensation, hydrolysis reaction, and synthesis of activated ester.

[0114] <Step 1> Amide condensation Dibenzoazacyclooctyne glutaric acid (L2-1) and aminotetraethyleneglycol acetylpiperidine methyl ester (L2-2) were obtained commercially and then subjected to a chemical condensation reaction to obtain the intermediate dibenzoazacyclooctynylglutaryl aminotetraethyleneglycol acetylpiperidine methyl ester (L2-3), which is a stable compound.

[0115] Specifically, compounds L2-1 and L2-2 (1:1.1, L2-2 is in excess) were dissolved in dichloromethane (DCM), and hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide (EDCI) were added, followed by triethanolamine (TEA). The mixture was stirred at room temperature for 4-12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain compound L2-3 as a yellow oil.

[0116] <Step 2> Hydrolysis reaction The intermediate L2-3 obtained in Step 1 was hydrolyzed to give the intermediate dibenzoazacyclooctynyl glutaryl aminotetraethylene glycol acetylpiperidine acid (L2-4). The hydrolysis reaction is usually quantitative, and the product is used directly in the next synthesis step without further purification.

[0117] Specifically, compound L2-3 was dissolved in a mixture of methanol and water, cooled to 0°C, and then 1 mol / L aqueous lithium hydroxide (LiOH) solution was added. The mixture was stirred at 0°C to room temperature for 4 to 12 hours, acidified to pH 2 to 3 with 1 mol / L hydrochloric acid, extracted three times with ethyl acetate (EA), and dried to obtain the intermediate dibenzoazacyclooctynyl glutarylaminotetraethylene glycol acetylpiperidine acid (L2-4) as a yellow oil.

[0118] <Step 3> Synthesis of activated ester The intermediate L2-4 (unpurified) obtained in Step 2 was condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to give L2 (i.e., dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester).

[0119] Specifically, compound L2-4 was dissolved in tetrahydrofuran (THF) and cooled to 0 °C. Then, pentafluorophenol, hydroxybenzotriazole (HOBt), and dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred at 0 °C to room temperature for 4 to 12 hours, extracted three times with ethyl acetate (EA), dried, and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester (L2) as a yellow oil (see Figure 7 for the structural formula).

[0120] The L2 linker and anti-5T4 monoclonal antibody can then undergo a site-directed coupling reaction in a PBS system at pH 7.2 to 7.4 (preferably HEPES at pH 7.2) (see Figure 10 for a schematic diagram). Typically, one anti-5T4 monoclonal antibody can be bound to one to four (preferably one to two) L2 linkers. The reaction can be stopped by adjusting the pH to approximately 5.0, yielding an L2-conjugated antibody.

[0121] In some embodiments, the second linker is an azidoacetylated cyclohexosamine, such as azidoacetylated cyclogalactosamine or azidoacetylated glucosamine. In some embodiments, the second linker is a molecule stable in an aqueous phase that can specifically and covalently bind to a membrane protein modified with sialic acid. In some embodiments, the second linker is transferred to the membrane protein modified with sialic acid through the cell's own metabolic pathway during cell culture. In some embodiments, the azidoacetyl group of the second linker can undergo a cyclization reaction with the carbon-carbon triple bond of the first linker to form a stable five-membered triazole ring.

[0122] In some embodiments, the second linker has the following structure: [ka] and 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose having the formula:

[0123] In some embodiments, the proportion of a single α or β configuration of 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-α,bD-galactose is at least 90%, for example, at least 95%, at least 98%, or at least 99%.

[0124] 1,3,4,6-Oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose is also referred to as the N1 linker in this invention. The synthesis pathway of the N1 linker is shown in Figure 8, and its structural formula is shown in Figure 9. The N1 linker involves two major synthesis steps: aminoazidoacetylation and hydroxyacetylation.

[0125] <Step 1> Aminoazide acetylation 1.2 times the amount of α-azidoacetic acid (compound 1), 2 times the amount of hydroxybenzotriazole (HOBt), and triethylamine (EtN) were added to a solution of D-galactosamine hydrochloride (compound 2) in N,N-dimethylformamide (DMF). A small amount of methanol (MeOH) was added to aid dissolution. The mixture was allowed to react at room temperature for 12 hours. The reaction mixture was then poured into dichloromethane (DCM) / methanol (MeOH), shaken to mix well, and ether was added to precipitate the oily product (compound 3). The ether layer was poured off and this process was repeated twice. The mixture was then dried under vacuum. The product was then used directly for the next step without further purification.

[0126] <Step 2> Hydroxyacetyl esterification Compound 3 obtained above was added to anhydrous pyridine (Pyr) and acetic anhydride (AcO) and 4-dimethylaminopyridine (DMAP) catalyst at room temperature. The reaction was allowed to proceed for 12 hours. The reaction was detected to be nearly complete by HPLC and then concentrated to yield a racemic mixture of tetraacetyl-N-azidoacetyl-a,b-D-galactosamine. The solid was precipitated in ethyl acetate / petroleum ether, which revealed the product to be predominantly in the b-configuration (β-configuration) with a purity of over 85%. Further purification using a silica gel column (dichloromethane:methanol = 20:1) yielded the N1 product with a single enantiomer purity of over 90%. The two a,b-isomers can isomerize with each other intracellularly, and through multiple metabolic and synthetic steps, they are converted to N-azidoacetylsialic acid, which is ultimately expressed on the surface of NK cells. The structural formula of the N1 linker is shown in Figure 9.

[0127] After culturing the NK cells for 15 to 16 days, N1 linker is added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (also referred to as "UNK" in the examples of the present application) (see Figures 11 and 12 for the binding mechanism between the N1 linker and NK cells and the structural schematic of UNK).

[0128] Finally, to obtain an antibody-NK cell conjugate, UNK and the L2 conjugated antibody are subjected to a coupling reaction in the medium (see FIG. 13 for the reaction mechanism), and the antibody-NK cell conjugate can be obtained.

[0129] Subsequent processes further include producing an antibody-NK cell conjugate formulation to effectively extend the stability of the antibody-NK cell conjugate. The formulation may contain components that maintain isotonicity, such as sodium chloride and human serum albumin, and may also contain components that maintain the low-temperature resistance of cells and the activity of proteins and enzymes, such as trehalose, sucrose, dextran, and DMSO.

[0130] According to a second aspect, the present invention provides a cell population comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect. In some embodiments, CD3 - CD56 + CD16 + In some embodiments, the CD3 - CD56 + CD16 + In some embodiments, the CD3 - CD56 + NKG2D + In some embodiments, the CD3 - CD56 + NKG2D + The number of cells represents at least 98% of the total number of cells in said cell population.

[0131] In some embodiments, CD3 + CD56 + The number of cells accounts for 5% or less of the total number of cells in the cell population. In some embodiments, CD3 - CD19 + The number of cells accounts for 2% or less of the total number of cells in the cell population. In some embodiments, CD3 + CD4 + and CD3 + The number of CD8 cells accounts for 2% or less of the total number of cells in the cell population.

[0132] In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total cells in the cell population. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 95% of the total cells in the cell population. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 98% of the total cells in the cell population. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 99% of the total cells in the cell population.

[0133] According to a third aspect, the present invention provides a pharmaceutical composition comprising an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, and a pharmaceutically acceptable carrier.

[0134] In some embodiments, the pharmaceutical composition comprises sodium chloride and / or human serum albumin. In some embodiments, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof. In some embodiments, the pharmaceutical composition is used to treat a tumor in an individual.

[0135] In some embodiments, the tumor is 5T4 (5T4 + In some embodiments, the tumor is highly expressing 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 +A tumor that highly expresses 5T4 refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that is highly 5T4-expressing refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express 5T4.

[0136] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a hematological tumor. In some embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is a cancer.

[0137] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell cancer (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

[0138] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colon cancer, and ovarian cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colon cancer.

[0139] According to a fourth aspect, the present invention provides the use of an antibody-natural killer cell (NK cell) conjugate according to the first aspect or a cell population according to the second aspect in the manufacture of a medicament for the treatment of a tumour in an individual.

[0140] In some embodiments, the tumor is 5T4 (5T4 + In some embodiments, the tumor is highly expressing 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that is highly 5T4-expressing refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express 5T4.

[0141] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a hematological tumor. In some embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is a cancer.

[0142] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell cancer (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

[0143] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colon cancer, and ovarian cancer.

[0144] In some embodiments, the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colon cancer.

[0145] According to a fifth aspect, the present invention provides a method of treating a tumour in an individual, the method comprising administering to said individual an effective amount of an antibody-natural killer cell (NK cell) conjugate according to the first aspect, or a cell population according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0146] In some embodiments, the tumor is 5T4 (5T4 + In some embodiments, the tumor is highly expressing 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 +A tumor that highly expresses 5T4 refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that highly expresses 5T4 refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express 5T4. In some embodiments, 5T4 (5T4 + A tumor that is highly 5T4-expressing refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express 5T4.

[0147] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a hematological tumor. In some embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is a cancer.

[0148] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell cancer (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

[0149] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colon cancer, and ovarian cancer.

[0150] In some embodiments, the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colon cancer.

[0151] It should be understood that the above detailed description is intended only to enable those skilled in the art to more clearly understand the present invention, but is not intended to limit any aspect thereof. Those skilled in the art may make various modifications and changes to the above embodiments.

[0152] The present invention will now be further described with reference to specific examples, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. [Example]

[0153] Example 1: Preparation of anti-5T4 antibody-NK cell conjugate The production of the antibody-NK cell conjugate in this example generally involves: (1) Production steps of anti-5T4 monoclonal antibody; (2) NK cell manufacturing steps; (3) Preparation of the antibody linker (also referred to as the L2 linker in this example); (4) Preparation of the NK cell linker (referred to as N1 linker in this example); (5) applying an antibody linker (L2 linker) to the antibody; (6) applying an NK cell linker (N1 linker) to the NK cells; and (7) Conjugating each antibody having a linker to an NK cell. It can be divided into:

[0154] (1) Production of anti-5T4 monoclonal antibodies, Briefly, the sequence of the anti-5T4 monoclonal antibody was derived from phage library screening, and then confirmed and selected by screening for binding activity to 5T4 and 5T4-expressing cells and affinity studies to obtain an anti-5T4 monoclonal antibody. The anti-5T4 monoclonal antibody contains a heavy chain variable region (HCDRs 1-3) represented by SEQ ID NO: 1 (SEQ ID NOs: 5, 6, and 7, respectively), a heavy chain constant region (SEQ ID NO: 2), a light chain variable region (LCDRs 1-3) represented by SEQ ID NO: 3 (SEQ ID NOs: 8, 9, and 10, respectively), and a light chain constant region (SEQ ID NO: 4). The DNA sequence of the anti-5T4 monoclonal antibody was determined, and a recombinant plasmid (IB12) expressing the anti-5T4 monoclonal antibody was constructed.

[0155] Construction of monoclonal cell lines is based on known techniques, in which the IB12 plasmid is transfected into CHO cells by electroporation (electrotransfection), followed by one round of screening of minipools and two rounds of screening after plating of monoclones, and confirmation by early passage stable culture to obtain cell lines capable of stably expressing anti-5T4 monoclonal antibody.

[0156] Anti-5T4 monoclonal antibodies are produced using well-known protocols, including cell culture and protein purification. Working cells are harvested, resuscitated, and grown for 3-5 rounds at medium scale. The unprocessed cell suspension (UPB) is then collected and clarified and filtered to obtain the cell culture supernatant. Anti-5T4 monoclonal antibodies are then purified using a three-step protein A affinity chromatography, anion exchange, and cation exchange chromatography.

[0157] (2) NK cell production PBMCs are derived from peripheral blood lymphocyte collection from allogeneic healthy donors. After separation of T cells and red blood cells, cells are transferred to primary cell cryopreservation solution, resulting in a total of 6.0 × 10 viable cells per vial. 7 The cells are then aliquoted to give PBMCs, which are then stored for long periods in liquid nitrogen at temperatures below -175°C.

[0158] PBMCs cryopreserved in liquid nitrogen are removed, resuscitated in adapted medium, and culture scale expanded (i.e., expanded with increased medium), with the addition of NK cell-associated cytokines, including, but not limited to, IL2 and IL15, to maintain proliferation capacity and activity. Exemplary NK cell culture steps, process parameters, and process control indicators are shown in Table 1 below.

[0159] [Table 1]

[0160] Quality control for final cell purity of NK cells meets the requirements in Table 2 below (other requirements such as cell density and biosafety comply with industry standards).

[0161] [Table 2]

[0162] Figures 3 and 4 show the flow cytometry detection results of the above immunological markers in the final antibody-NK cell conjugate stock solution and formulation.

[0163] (3) Preparation of antibody linker (also referred to as L2 linker in this example) The synthetic pathway diagram of the L2 linker is shown in Figure 6 and the structural formula is shown in Figure 7, which includes three main synthetic steps: amide condensation, hydrolysis reaction, and synthesis of the activated ester.

[0164] <Step 1> Amide condensation Dibenzoazacyclooctynyl glutaric acid (L2-1) and aminotetraethylene glycol acetylpiperidine methyl ester (L2-2) were obtained commercially and then subjected to a chemical condensation reaction to obtain the intermediate dibenzoazacyclooctynyl glutaryl aminotetraethylene glycol acetylpiperidine methyl ester (L2-3), which is a stable compound.

[0165] Specifically, compounds L2-1 and L2-2 (1:1.1, L2-2 is in excess) were dissolved in dichloromethane (DCM), and hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide (EDCI) were added, followed by triethanolamine (TEA). The mixture was stirred at room temperature for 4-12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain compound L2-3 as a yellow oil.

[0166] <Step 2> Hydrolysis reaction The intermediate L2-3 obtained in Step 1 was hydrolyzed to give the intermediate dibenzoazacyclooctynyl glutaryl aminotetraethylene glycol acetylpiperidine acid (L2-4). The hydrolysis reaction is usually quantitative, and the product is used directly in the next synthesis step without further purification.

[0167] Specifically, compound L2-3 was dissolved in a mixture of methanol and water, cooled to 0°C, and then 1 mol / L aqueous lithium hydroxide (LiOH) solution was added. The mixture was stirred at 0°C to room temperature for 4 to 12 hours, acidified to pH 2 to 3 with 1 mol / L hydrochloric acid, extracted three times with ethyl acetate (EA), and dried to obtain the intermediate dibenzoazacyclooctynyl glutarylaminotetraethylene glycol acetylpiperidine acid (L2-4) as a yellow oil.

[0168] <Step 3> Synthesis of activated ester The intermediate L2-4 (unpurified) obtained in Step 2 was condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to give L2 (i.e., dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester).

[0169] Specifically, compound L2-4 was dissolved in tetrahydrofuran (THF) and cooled to 0 °C. Then, pentafluorophenol, hydroxybenzotriazole (HOBt), and dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred at 0 °C to room temperature for 4 to 12 hours, extracted three times with ethyl acetate (EA), dried, and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester (L2) as a yellow oil (see Figure 7 for the structural formula).

[0170] (4) Production of NK cell linker (referred to as N1 linker in this example) The synthetic pathway of the N1 linker is shown in Figure 8, and the structural formula is shown in Figure 9. The N1 linker includes two major synthetic steps: aminoazidoacetylation and hydroxyacetylation.

[0171] <Step 1> Aminoazide acetylation 1.2 times the amount of α-azidoacetic acid (compound 1), 2 times the amount of hydroxybenzotriazole (HOBt), and triethylamine (EtN) were added to a solution of D-galactosamine hydrochloride (compound 2) in N,N-dimethylformamide (DMF). A small amount of methanol (MeOH) was added to aid dissolution. The mixture was allowed to react at room temperature for 12 hours. The reaction mixture was then poured into dichloromethane (DCM) / methanol (MeOH), shaken to mix well, and ether was added to precipitate the oily product (compound 3). The ether layer was poured off and this process was repeated twice. The mixture was then dried under vacuum. The product was then used directly for the next step without further purification.

[0172] <Step 2> Hydroxyacetyl esterification Compound 3 obtained above was added to anhydrous pyridine (Pyr) and acetic anhydride (AcO) and 4-dimethylaminopyridine (DMAP) catalyst at room temperature. The reaction was allowed to proceed for 12 hours. The reaction was detected to be nearly complete by HPLC and then concentrated to yield a racemic mixture of tetraacetyl-N-azidoacetyl-a,b-D-galactosamine. The solid was precipitated in ethyl acetate / petroleum ether, which revealed the product to be predominantly in the b-configuration (β-configuration) with a purity of over 85%. Further purification using a silica gel column (dichloromethane:methanol = 20:1) yielded the N1 product with a single enantiomer purity of over 90%. The two a,b-isomers can isomerize with each other intracellularly, and through multiple metabolic and synthetic steps, they are converted to N-azidoacetylsialic acid, which is ultimately expressed on the surface of NK cells. The structural formula of the N1 linker is shown in Figure 9.

[0173] (5) Applying an antibody linker (L2 linker) to the antibody The L2 linker and anti-5T4 monoclonal antibody undergo a site-specific coupling reaction in a PBS system at pH 7.2 to 7.4 (preferably HEPES pH 7.2) (see Figure 10 for a schematic diagram). Typically, one anti-5T4 monoclonal antibody can be bound to one to four (preferably one to two) L2 linkers. The reaction is terminated by adjusting the pH to approximately 5.0, and an L2-conjugated antibody can be obtained.

[0174] (6) Applying NK cell linker (N1 linker) to NK cells After culturing the NK cells for 15 to 16 days, N1 linker is added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (also referred to as "UNK" in the examples of the present application) (see Figures 11 and 12 for the binding mechanism between the N1 linker and NK cells and the structural schematic of UNK).

[0175] (7) Conjugating antibodies with linkers to NK cells The UNK and L2 conjugated antibodies were subjected to a coupling reaction in the culture medium (see Figure 13 for the reaction mechanism) to obtain a stock solution (DS) of the antibody-NK cell conjugate. The anti-5T4 monoclonal antibody-NK cell conjugate was named "IBR854," and this name will be used in subsequent examples to represent the anti-5T4 antibody-NK cell conjugate.

[0176] Subsequent processes further include producing an antibody-NK cell conjugate formulation to effectively extend the stability of the antibody-NK cell conjugate. The formulation may contain components that maintain isotonicity, such as sodium chloride and human serum albumin, and may also contain components that maintain the low-temperature resistance of cells and the activity of proteins and enzymes, such as trehalose, sucrose, dextran, and DMSO.

[0177] Flow cytometry was used to detect the NK cell purity in the antibody-NK cell conjugate stock solution (Figure 3) and formulation (Figure 4), and the results were over 98%, exceeding the standard requirement of 95%. Furthermore, flow cytometry was used to detect the binding positivity rate of the antibody-NK cell conjugate stock solution and formulation using UNK as a control. The results showed that the binding positivity rate of both the stock solution and formulation exceeded 98% (Figure 5), compared to the standard requirement of 90%.

[0178] Example 2: Comparison of basic antibody properties between conjugate IBR854 and anti-5T4 monoclonal antibody In this example, several basic antibody properties of the conjugate IBR854 prepared in Example 1 were compared with those of the anti-5T4 monoclonal antibody prepared in step (1) of Example 1. The results are shown in Table 3 below. The methodology for each test item follows conventional testing methods in the art.

[0179] [Table 3]

[0180] As can be seen from the results in Table 3, conjugation of anti-5T4 monoclonal antibody with NK cells does not significantly affect the properties of the antibody itself, and the desirable properties of the antibody are maintained.

[0181] Example 3: Evaluation of the in vitro cytotoxic activity of conjugate IBR854 against lung cancer, breast cancer, and pancreatic cancer cells For the evaluation of cytotoxic activity in this example, eight tumor cell lines expressing 5T4 were selected, including lung cancer cell lines (H1975, NCI-H292, NCI-H226, and HCC827), breast cancer cell lines (MDA-MB-231 and MDA-MB-468), and pancreatic cancer cell lines (Panc-1 and BxPC3), and their previously measured 5T4 expression levels (flow cytometry measurement) are shown in Table 4 below.

[0182] [Table 4]

[0183] First, we used real-time, label-free cellular assay technology to detect the cytotoxic activity of conjugated IBR854 against cells from eight tumor cell lines, as well as NK cells (obtained in step (2) of Example 1) and UNK cells (obtained in step (6) of Example 1) as controls. A control group containing only tumor cells was also included in the experiment. NK cells, UNK cells, and conjugated IBR854 were each administered at three concentrations, with each concentration administered in three replicate wells. Tumor cells were seeded onto a detection plate, and NK cells, UNK cells, and IBR854 were added. The RTCA instrument monitored and recorded the cell index (CI) values ​​every 15 minutes. The cytotoxicity rate was calculated based on changes in the cell index. The results are shown in Figures 14 to 21. The results showed that IBR854 had a cytotoxicity rate of over 85% against a total of eight cell lines, including lung cancer, breast cancer, and pancreatic cancer cells, and its cytotoxic activity was higher than that of NK cells and UNK cells.

[0184] Next, we used calcein AM release assay to detect the dose-effect relationship of IBR854 in cytotoxicity against eight tumor cell lines. Eight concentrations of IBR854 were administered, with six replicate wells per concentration. Changes in fluorescence intensity were detected using a microplate reader, and the cytotoxicity rate and EC 50 The calculated values ​​are shown in Table 5 below.

[0185] As a result, IBR854 had a significant cytotoxic effect on all eight tumor cell lines, and EC 50 The effector-to-target ratio (Effector-to-target ratio) values ​​were in the range of 0.86 to 5.06, indicating that the maximum inhibition rate reached 75% or more.

[0186] [Table 5]

[0187] Example 4: Evaluation of the factor-releasing function of conjugate IBR854 In this example, we functionally investigated the effect of conjugate IBR854 on cytokine secretion. The cytokines studied included IL-2 (interleukin-2), IL-6 (interleukin-6), IL-15 (interleukin-15), IL-1β (interleukin-1β), TNFα (tumor necrosis factor α), IFNγ (interferon-γ), IL-8 (interleukin-8), CCL2 (chemokine 2), CCL3 (chemokine 3), and CCL5 (chemokine 5). Cytokines were measured using commercially available ELISA reagent kits in various cell culture supernatants according to the manufacturer's instructions.

[0188] (1) Conjugate IBR854 was co-incubated with three tumor effector cells (H1975, BxPC-3, and BxPC-3) at different effector-to-target ratios (1:1, 3:1, 10:1, and 30:1), and then the secretion levels of IL-2, IL-6, IL-15, and IL-1β in the cell culture supernatant were measured by ELISA. The results are shown in Tables 6 to 9.

[0189] The secretion levels of IL-2, IL-6, IL-15, and IL-1β were all lower than the average levels in healthy individuals. IL-2 levels were all less than 2.0 pg / mL, showing a dose-dependent increase, and were lower than the IL-2 serum level of 9.40 ± 2.31 ng / mL in healthy individuals. IL-6 levels were all less than 0.3 pg / mL, lower than the IL-6 serum level of 60.32 ± 3.24 pg / mL in healthy individuals. IL-15 levels were all less than 0.5 pg / mL, lower than the IL-15 serum level of 13.38 ± 4.41 pg / mL in healthy individuals. IL-1β secretion levels were all less than 0.5 pg / mL.

[0190] [Table 6]

[0191] [Table 7]

[0192] [Table 8]

[0193] [Table 9]

[0194] (2) The IBR854 conjugate was co-incubated with NCI-H292 and MDA-MB-231 tumor cells at effector-to-target ratios of 10:1, 3:1, and 1:1 (simultaneously, a control containing the IBR854 conjugate at the same effector-to-target ratio but without tumor cells was set up), and the secretion levels of TNFα and IFNγ in the cell culture supernatant were measured by ELISA. As can be seen from the results in Tables 10 and 11, after co-incubation of IBR854 with NCI-H292 and MDA-MB-231 tumor cells, the secretion levels of TNFα and IFNγ were positively correlated with the dose, and the TNFα level was lower than the average level of healthy subjects, 22.75±6.28 μg / mL. After co-incubation with tumor cells, the secretion levels of TNFα and IFNγ significantly increased, indicating a certain correlation with the tumor-damaging effect.

[0195] [Table 10]

[0196] [Table 11]

[0197] (3) The IBR854 conjugate was co-incubated with tumor cells H1975, BXPC-3, and Panc-1 at different effector-to-target ratios (1:1, 3:1, 10:1, and 30:1), and the secretion levels of CCL2, CCL3, CCL5, and IL-8 (CXCL8) in the cell culture supernatant were measured by ELISA. As shown in Tables 12 to 14, the levels of CCL3, CCL5, and IL-8 (CXCL8) were positively correlated with the IBR854 exposure dose. CCL3, CCL5, and IL-8 (CXCL8) may contribute to the cytotoxic effect of the IBR854 conjugate on tumor cells, which is achieved by chemotaxis of related immune cells such as macrophages and leukocytes. The factor secretion level of CCL2 was negatively correlated with the exposure dose of IBR854, which may be because tumor cells could achieve migration and invasion by secreting CCL2, while NK cells suppressed the chemotactic ability of tumor cells, as evidenced by the decrease in CCL2 concentration with an increase in the effector-to-target ratio of NK cells.

[0198] [Table 12]

[0199] [Table 13]

[0200] [Table 14]

[0201] (4) The conjugate IBR854 was co-incubated with NCI-H292 and MDA-MB-231 tumor cells at different effector-to-target ratios of 10:1, 3:1, and 1:1 (a control containing the conjugate IBR854 at the same effector-to-target ratio but without tumor cells was also established). The cell culture supernatant was collected and the secretion levels of granzyme B and perforin were detected by ELISA. As shown in Tables 15 and 16, IBR854 alone secreted a certain amount of granzyme B and perforin in a dose-dependent manner. When IBR854 was co-incubated with NCI-H292 and MDA-MB-231 tumor cells, the expression level of granzyme B increased before and after tumor stimulation, but perforin secretion did not change significantly.

[0202] [Table 15]

[0203] [Table 16]

[0204] (5) Flow cytometry was used to detect changes in FasL / TRAIL secretion levels after co-incubation of IBR854 and H292 tumor cells. After co-incubation of IBR854 and H292 tumor cells for 1, 2, 3, and 4 hours, respectively, the cells were stained with TRAIL and FasL flow cytometry antibodies, respectively, and then the expression of TRAIL and FasL on the IBR854 cell surface was analyzed using flow cytometry. When IBR854 cytotoxicated H292 tumor cells, the expression of FasL and TRAIL on the NK cell surface changed over time, initially increasing and then decreasing, but no significant correlation with dose was observed. The results are shown in Figures 22 and 23.

[0205] Example 5: Efficacy study of conjugate IBR854 on NCI-H292 tumor-bearing mouse model Seventy M-NSG mice were inoculated with 5.0 × 10 6 NCI-H292 cells were inoculated, and the average tumor volume was approximately 50 mm 3 When the tumor volume reached 100 mg / kg, the animals were randomly divided into seven groups according to tumor volume and administered the treatment. Group 1: Vehicle control (buffer solution for formulation) group Group 2: antibody control group (anti-5T4 antibody in Example 1, 0.75 mg / kg) Group 3: IL-15 group, Group 4: IL-15+IBR854 low dose (2.5×10 8 cells / kg) group, Group 5: IL-15 + IBR854 medium dose (5.0×10 8 cells / kg) group, Group 6: IL-15+IBR854 high dose (1.0×10 9 cells / kg) group, Group 7: IBR854 medium dose (5.0×10 8 cells / kg) group.

[0206] In vivo efficacy studies, exogenous IL-15 was administered to extend the duration of IBR854 in animals. IL-15 (90 μg / kg) was administered intraperitoneally once a week for a total of three doses. Other groups received IL-15 via tail vein injection twice a week, one day apart, for a total of six doses. Body weight and tumor volume were measured twice a week. M-NSG mice were euthanized on day 28 and tumor masses were weighed. Table 17 shows the experimental design for efficacy in a mouse model of tumors transplanted with human lung cancer cells NCI-H292.

[0207] [Table 17]

[0208] The results are shown below: 1) At the end of the study, no mice died in any group. There was no significant decrease in body weight of mice in each IBR854 dose group compared to the vehicle control group, demonstrating the safety and tolerability of IBR854. 2) From day 8, the tumor volumes of mice in each IL-15+IBR854 dose group were reduced to varying degrees compared to the vehicle control group, and on day 28, the tumor volume, relative tumor volume (RTV), and relative tumor growth rate (T / C) of the high-dose IL-15+IBR854 group were all statistically significantly reduced compared to the vehicle control group. See Figure 24 and Table 18 for the results.

[0209] [Table 18]

[0210] Compared with the vehicle control group, *: p<0.05, **: p<0.01, ***: p<0.001; compared with the IL-15 group, ##: p<0.01. Tumor volume V = 1 / 2 × a × b 2 , a and b represent the long and short diameters of the tumor, respectively. Relative tumor volume (RTV) = Vt / V1, where V1 is the tumor volume measured at the time of group administration (i.e., D1), and Vt is the tumor volume at each measurement. T / C (%) = TRTV / CRTV x 100%, where TRTV is the RTV of the treatment group, and CRTV is the RTV of the vehicle control group. IR (%) = (1 - TWt / TWc) x 100%, where TWt is the tumor weight in the treatment group, and TWc is the tumor weight in the vehicle control group.

[0211] Therefore, under the experimental conditions of this example, IB854 was added at 2.5 × 10 8 ~10.0×10 8 High-dose tail vein administration of IL-15 and IBR854 (twice weekly for a total of six doses) significantly suppressed tumor growth in mice bearing human lung cancer cells NCI-H292.

[0212] Example 6: Efficacy study of conjugate IBR854 on MDA-MB-468 tumor-bearing mouse model 50 NCG mice, each with 4 × 10 6MDA-MB-468 cells were seeded, and the tumor volume was approximately 30–80 mm 3 When the tumor volume reached 100 mg / kg, the animals were randomly divided into five groups according to tumor volume and administered the treatment. Group 1: Vehicle control (buffer solution for formulation) group Group 2: IL-15 group, Group 3: IL-15+IBR854 low dose (5.0×10 8 individual cells / kg) group, Group 4: IL-15 + IBR854 medium dose (1.0×10 9 individual cells / kg) group, Group 5: IL-15+IBR854 high dose (1.5×10 9 individual cells / kg) group.

[0213] In vivo efficacy studies, exogenous IL-15 was administered to extend the duration of IBR854 in animals. IL-15 (0.18 mg / kg) was administered intraperitoneally once a week for a total of three doses. Other groups received IL-15 via tail vein injection twice a week for a total of six doses. Body weight and tumor volume were measured twice a week. On day 20, M-NCG mice were euthanized and tumor masses were weighed. Table 19 shows the experimental design for efficacy in a mouse model of tumors transplanted with human breast cancer cells MDA-MB-468.

[0214] [Table 19]

[0215] The results are shown below: 1) Weight and clinical observations: In this example, no significant weight loss or abnormal behavior was observed in any of the mice, indicating that the mice tolerated the IBR854 injection well under the experimental conditions.

[0216] 2) Tumor volume: On the 20th day of administration, the mean tumor volume in Group 1 (vehicle control group) was 161.44 ± 6.46 mm 3Compared with the vehicle control group, the mean tumor volumes of Group 2 (IL-15 group), Group 3 (IL-15 + IBR854 low dose group), Group 4 (IL-15 + IBR854 medium dose group), and Group 5 (IL-15 + IBR854 high dose group) were 160.18 ± 11.36 mm, respectively. 3 , 134.94±5.11mm 3 (p<0.05), 124.02±6.91mm 3 (p<0.001), and 114.19±3.50mm 3 (p<0.001), and the tumor volume gradually decreased in the IL-15+IBR854 low-dose group, medium-dose group, and high-dose group, indicating that there was a significant dose-dependent tumor suppression effect on tumor volume.

[0217] 3) Tumor growth inhibition rate (TGI): Compared with the vehicle control group, the tumor growth inhibition rates (TGI) of the IL-15 group, IL-15 + IBR854 low-dose group, IL-15 + IBR854 medium-dose group, and IL-15 + IBR854 high-dose group were 0.74%, 31.49%, 44.11%, and 56.66%, respectively.

[0218] 4) Tumor weight: The tumor weight reduction rates were -6.00%, 16.85%, 22.97%, and 32.97%, respectively. Compared with the vehicle control group, tumor volume and weight were significantly reduced in each IBR854 dose group (p<0.05), demonstrating a highly significant dose-dependent tumor suppression effect. See Figures 25 and 26 and Table 20 for specific results.

[0219] [Table 20]

[0220] Tumor volume V = 0.5 × a × b 2 , a and b represent the long and wide diameters of the tumor, respectively. Tumor growth inhibition rate TGI (%) = (1-T / C) × 100%. T / C% is the relative growth rate of the tumor, and T / C% = (T i -T0) / (V i-V0) × 100%, i.e., at a specific time point, T i is the mean tumor volume after the start of administration in the treatment group, T0 is the mean tumor volume at the first administration in the treatment group, V0 is the mean tumor volume at the first administration in the vehicle control group, and V i is the average tumor volume after the start of administration in the vehicle control group. Also, T / C = T RTV / C RTV ×100%, where T RTV is the RTV of the treatment group, C RTV is the RTV of the vehicle control group.

[0221] Therefore, under the experimental conditions of this example, IB854 was added at 5.0 × 10 8 ~1.5×10 9 Animals receiving doses in the range of cells / kg were well tolerated. Tail vein administration of IL-15 and IBR854 (twice weekly, a total of six doses) significantly inhibited tumor growth in a dose-dependent manner in mice bearing human breast cancer cells, MDA-MB-468.

[0222] Example 7: Evaluation of in vitro cytotoxic activity of anti-5T4 antibody-natural killer cell conjugates with different L linkers An example of a linker conjugated to the antibody portion of the anti-5T4 antibody-natural killer cell conjugate of the present invention has the following structure: [ka] [In the formula, n is an integer of 0 to 8.] Dibenzoazacyclooctynyl-glutaryl-aminopolyethylene glycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

[0223] In this example, the in vitro cytotoxic activity of conjugates having two antibody moiety linkers, L2 (n=3) and L3 (n=1), was tested.

[0224] The L2 linker is the linker of IBR854 prepared in Example 1. The preparation process of the conjugate with the L3 linker is similar to that of IBR854 in Example 1, except that a different L2-2 substrate was selected when preparing the L3 linker, and the corresponding substrate is also commercially available.

[0225] The in vitro cytotoxicity test method was similar to that in Example 3, the test cell line was H292, and NK cells (obtained in step (2) of Example 1) and UNK cells (obtained in step (6) of Example 1) were similarly selected as controls.

[0226] As is clear from the results in Figure 27, the conjugates having the L2 and L3 linkers had higher cytotoxic activity against H292 cells than against NK and UNK cells, respectively, and the conjugates containing the L2 (i.e., IBR854) and L3 linkers showed equivalent cytotoxic activity.

[0227] Example 8: Evaluation of the in vitro inhibitory activity of conjugate IBR854 against colon cancer and gastric cancer cells LOVO cells are a type of colon cancer cell line. LOVO cells were cultured in 96-well plates at 10,000 cells / well and allowed to adhere. Cytostatic activity studies were performed using IBR854 (effector-to-target ratio 2:1). The xCELLigence system was used to monitor tumor cell proliferation and the time course of the inhibitory effects of effector cells (IBR854, NK cells as a control, and a mixture of UNK cells and antibodies) (Figure 28). After 55 hours of treatment, the NK cell inhibition rate was 43.6%, the UNK plus anti-5T4 conjugated antibody (10 μg / mL) inhibition rate was 64.3%, and the IBR854 cell inhibition rate was 86.8%. The control group did not contain effector cells (Figure 29).

[0228] N87 cells are a type of gastric adenocarcinoma cell line. N87 cells were cultured in 8-well plates at 80,000 cells / well and allowed to adhere to the plate. Cytostatic activity studies were performed using IBR854 (effector-to-target ratio 2:1, 24-hour treatment period). The xCELLigence system was used to monitor the time course of tumor cell proliferation and the inhibitory effects of effector cells (IBR854, NK cells as a control, and UNK cells + antibody mixture) (Figure 30). After 24 hours of treatment, the NK cell inhibition rate was 30.5%, the UNK + anti-5T4 conjugate antibody (10 μg / mL) inhibition rate was 56.9%, and the IBR854 cell inhibition rate was 63.5% (Figure 31). The control group contained no effector cells.

[0229] The use of any embodiments or exemplary language (e.g., "such as," "such as," etc.) provided herein is intended to facilitate a better understanding of the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element required to practice the invention.

[0230] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, any theory, mechanism, demonstration, or discovery described herein is intended to further enhance the understanding of the present invention, and is not intended to limit the present invention in any way to such theory, mechanism, demonstration, or discovery. While the present invention has been shown and described in detail in the accompanying drawings and the foregoing description, it should be considered illustrative and not limiting of the present invention.

Claims

1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-5T4 antibody or antigen-binding fragment thereof is conjugated to the NK cell via a linker.

2. the anti-5T4 antibody or antigen-binding fragment thereof HCDR1 as set forth in SEQ ID NO: 5 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 5; HCDR2 as set forth in SEQ ID NO:6 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:6; HCDR3 as set forth in SEQ ID NO:7 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:7; LCDR1 as set forth in SEQ ID NO: 8 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 8; An LCDR2 set forth in SEQ ID NO: 9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 9, and comprising an LCDR3 as set forth in SEQ ID NO: 10 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 10; wherein the amino acid sequences of HCDR and LCDR are as defined by Kabat: The antibody-natural killer cell (NK cell) conjugate of claim 1.

3. 3. The antibody-natural killer cell (NK cell) conjugate of claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-5T4 antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95%, or 99% identity to the sequence set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 3 or has at least 80%, 85%, 90%, 95%, or 99% identity to the sequence set forth in SEQ ID NO:

3.

4. The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 3, wherein the anti-5T4 antibody comprises a heavy chain variable region shown in SEQ ID NO: 1, a heavy chain constant region shown in SEQ ID NO: 2, a light chain variable region shown in SEQ ID NO: 3, and a light chain constant region shown in SEQ ID NO:

4.

5. the anti-5T4 antibody is a whole antibody, a single-chain antibody (scFv), or a bispecific antibody; and / or The antigen-binding fragment of the anti-5T4 antibody is Fab, Fab', Fv or F(ab'). ２ and / or the anti-5T4 antibody is a humanized or fully human antibody, and / or the anti-5T4 antibody is a monoclonal antibody, and / or the anti-5T4 antibody is of the IgG1, IgG2 or IgG4 isotype, and / or the anti-5T4 antibody comprises a light chain constant region of the κ or λ subtype; The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 4.

6. The NK cells are CD16 ＋ and / or NKG2D ＋ and preferably CD16 ＋ NKG2D ＋ The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 5, wherein

7. The conjugate CD16 ＋ NKG2D ＋ The proportion of NK cells is at least 90%, preferably CD56 ＋ The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 6, wherein the proportion of NK cells is at least 95%.

8. The NK cells are obtained from in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells, or The NK cells are obtained from the in vitro culture and expansion of umbilical cord blood-derived NK cells, or The NK cells are obtained from the in vitro culture and expansion of an NK cell line, or The NK cells are obtained from in vitro derivation, culture, and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs); The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 7.

9. The antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 8, wherein the anti-5T4 antibody or antigen-binding fragment thereof and the NK cell are conjugated via a click chemistry reaction of a linker.

10. 10. The antibody-natural killer cell (NK cell) conjugate of claim 9, wherein the anti-5T4 antibody or antigen-binding fragment thereof and the NK cell are conjugated via a first linker and a second linker, wherein the first linker is conjugated to the anti-5T4 antibody or antigen-binding fragment thereof, the second linker is conjugated to the NK cell, and the first linker and the second linker are conjugated to each other to form the antibody-NK cell conjugate.

11. 11. The antibody-natural killer cell (NK cell) conjugate of claim 10, wherein the first linker is an active ester capable of forming conjugation to a lysine residue of an antibody via an ester bond to amide bond reaction, the active ester being, for example, a pentafluorophenyl ester, for example, piperidine acid pentafluorophenyl ester.

12. The antibody-natural killer cell (NK cell) conjugate of claim 11, wherein the first linker further comprises a carbon-carbon triple bond structure capable of undergoing a cyclization reaction with the azide group to form a five-membered triazazole ring, for example, the carbon-carbon triple bond structure is an octyne group.

13. The first linker has the following structure: 【Chemistry 1】 [In the formula, n is an integer of 0 to 8.] 13. The antibody-natural killer cell (NK cell) conjugate of claim 12, which is dibenzoazacyclooctynyl-glutaryl-aminopolyethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

14. The first linker has the following structure: 【Chemistry 2】 14. The antibody-natural killer cell (NK cell) conjugate of claim 13, which is dibenzoazacyclooctynyl-glutaryl-aminotetraethyleneglycol-acetylpiperidine acid pentafluorophenyl ester having the formula:

15. The antibody-natural killer cell (NK cell) conjugate of claim 10, wherein the second linker is an azidoacetylated cyclohexosamine, e.g., azidoacetylated cyclogalactosamine, or azidoacetylated glucosamine.

16. The second linker has the following structure: 【Transformation 3】 16. The antibody-natural killer cell (NK cell) conjugate of claim 15, wherein the conjugate is 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,b-D-galactose having the formula:

17. 17. The antibody-natural killer cell (NK cell) conjugate of claim 16, wherein the proportion of the second linker in a single α or β configuration is at least 90%.

18. A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 17.

19. CD3 － CD56 ＋ CD16 ＋ and / or CD3 － CD56 ＋ NKG2D ＋ 19. The cell population of claim 18, wherein the number of cells represents at least 95%, preferably at least 98%, of the total number of cells in the cell population.

20. CD3 ＋ CD56 ＋ the number of cells accounts for 5% or less of the total number of cells in the cell population; and / or CD3 － CD19 ＋ the number of cells accounts for 2% or less of the total number of cells in the cell population; and / or CD3 ＋ CD4 ＋ and CD3 ＋ CD8 ＋ 20. The cell population of claim 18 or 19, wherein the number of cells accounts for 2% or less of the total number of cells in the cell population.

21. 21. The cell population of any one of claims 18 to 20, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of all cells in the cell population.

22. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 17 or the cell population of any one of claims 18 to 21, and a pharmaceutically acceptable carrier.

23. 23. The pharmaceutical composition of claim 22, comprising sodium chloride and / or human serum albumin.

24. 24. The pharmaceutical composition of claim 22 or 23, comprising trehalose, sucrose, dextran, DMSO, or any combination thereof.

25. Tumors in individuals, particularly tumor cells that are 5T4 (5T4 ＋ The pharmaceutical composition according to any one of claims 22 to 24, for use in treating a tumor that highly expresses the IL-1 receptor agonist (IL-1).

26. 26. The pharmaceutical composition of claim 25, wherein the tumor is a solid tumor.

27. 26. The pharmaceutical composition of claim 25, wherein the tumor is a malignant tumor.

28. 26. The pharmaceutical composition of claim 25, wherein the tumor is cancer.

29. 29. The pharmaceutical composition of claim 28, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell cancer (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

30. 29. The pharmaceutical composition of claim 28, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

31. 29. The pharmaceutical composition of claim 28, wherein the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colorectal cancer.

32. Tumors in individuals, particularly tumor cells, highly express 5T4 (5T4 ＋ 22.) Use of an antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 17 or a cell population according to any one of claims 18 to 21 in the manufacture of a medicament for the treatment of a tumor.

33. 33. The use according to claim 32, wherein the tumor is a solid tumor.

34. 33. The use according to claim 32, wherein the tumor is a malignant tumor.

35. 33. The use of claim 32, wherein the tumor is cancer.

36. 36. The use of claim 35, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell cancer (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

37. 36. The use of claim 35, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

38. 36. The use of claim 35, wherein the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colorectal cancer.

39. Tumors in individuals, particularly tumor cells, highly express 5T4 (5T4 ＋ 32. A method for treating a tumor, comprising administering to said individual an effective amount of the antibody-natural killer cell (NK cell) conjugate of any one of claims 1 to 17, or the cell population of any one of claims 18 to 21, or the pharmaceutical composition of any one of claims 22 to 31.

40. 40. The method of claim 39, wherein the tumor is a solid tumor.

41. 40. The method of claim 39, wherein the tumor is a malignant tumor.

42. 40. The method of claim 39, wherein the tumor is cancer.

43. 43. The method of claim 42, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell cancer (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, and metastases of these cancers.

44. 43. The method of claim 42, wherein the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer.

45. 43. The method of claim 42, wherein the cancer is selected from the group consisting of lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, and colorectal cancer.

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