Anti-Trop2 antibody-natural killer cell conjugate and its use

Abstract

This invention provides an anti-Trop2 antibody-natural killer cell conjugate, a pharmaceutical composition containing the conjugate, pharmaceutical uses of the conjugate, and methods for producing the same.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to Chinese Patent Application No. 202310397151.4, filed on 10 April 2023, the entirety of which is incorporated herein by reference.

[0002] This invention generally relates to the field of biopharmaceuticals, and more specifically to anti-Trop2 antibody-natural killer cell conjugates, pharmaceutical compositions containing the conjugate, pharmaceutical uses of the conjugate, and methods for producing them. [Background technology]

[0003] According to national cancer statistics data released by the National Cancer Research Center of China in January 2019, there were approximately 3.929 million new cases of malignant tumors and approximately 2.338 million deaths nationwide in China in 2015. Compared to past data, the burden of cancer is on a sustained upward trend. In terms of the number of cases, lung cancer, liver cancer, upper gastrointestinal tumors, colorectal cancer, and female breast cancer remain the major malignant tumors in China. According to national cancer statistics data released by the National Cancer Research Center of China in February 2022, there were approximately 4.064 million new cases of malignant tumors and approximately 2.4135 million deaths nationwide in 2016. Compared to the 2015 data, the number of new cases and deaths from malignant tumors continues to show an increasing trend, with lung cancer consistently being the cancer with the highest annual incidence rate in China.

[0004] Because the early symptoms of tumors are not clear, some tumors may recur later even if detected early, and the proportion of patients with advanced-stage tumors is relatively high due to the high malignancy of the tumors themselves. Currently, common clinical treatments for solid tumors include surgery, radiotherapy, chemotherapy, conventional biological therapies, and traditional Chinese medicine. These treatments often involve a certain degree of toxicity and side effects. After surgical removal of the cancerous lesion, there is a possibility of varying degrees of complications, and there is a risk of recurrence after surgery. Radiotherapy and chemotherapy are the most toxic and have the strongest side effects, and can cause a certain degree of damage to the patient's body. Traditional Chinese medicine has fewer toxicity and side effects, but often only provides adjunctive effects. Conventional biological therapies also have many unavoidable toxicity and side effects. However, in patients with advanced-stage solid tumors, the majority are no longer suitable for surgery, radiotherapy, or concurrent chemoradiotherapy, and have failed systematic standard treatments.

[0005] Therefore, in this field, the search for and development of new treatments for tumors is urgently needed. [Overview of the project]

[0006] According to a first aspect, the present invention provides an antibody-natural killer cell (NK cell) conjugate in which the antibody is an anti-Trop2 antibody or its antigen-binding fragment, and the anti-Trop2 antibody or its antigen-binding fragment is conjugated to the NK cell via a linker.

[0007] According to a second aspect, the present invention provides a cell population comprising the antibody natural killer cell (NK cell) conjugate (complex) described in 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 as described in the first aspect, or a cell population as described in the second aspect, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition is provided in the form of a sterile fresh formulation or a sterile cryopreserved formulation.

[0009] 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 pharmaceutical for the treatment of tumors in an organism, particularly tumors in which tumor cells highly express Trop2 (Trop2+).

[0010] According to a fifth aspect, the present invention provides a method for treating a tumor in an individual, particularly a tumor in which tumor cells highly express Trop2 (Trop2+), comprising administering to the individual an effective amount of the antibody-natural killer cell (NK cell) conjugate according to the first aspect, the cell population according to the second aspect, or the pharmaceutical composition according to the third aspect.

[0011] As a non-limiting example, the present invention provides the following embodiments.

[0012] [1] An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or its antigen-binding fragment, and the anti-Trop2 antibody or its antigen-binding fragment is conjugated to the NK cell via a linker.

[0013] [2] The anti-Trop2 antibody or its antigen-binding fragment is HCDR1, which is shown in Sequence ID No. 5, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 5. HCDR2, which is shown in Sequence ID No. 6, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 6. HCDR3, which is shown in Sequence ID No. 7, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 7. LCDR1, which is shown in sequence number 8, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in sequence number 8. LCDR2, which is shown in sequence number 9, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in sequence number 9, and It includes an LCDR3 that is shown in SEQ ID NO: 10, or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO: 10, Here, the amino acid sequences of HCDR and LCDR are defined by Kabat. The antibody-natural killer cell (NK cell) conjugate described in Embodiment 1.

[0014] [3] The antibody-natural killer cell (NK cell) conjugate according to Embodiment 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or its antigen-binding fragment is shown in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3 or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO: 3.

[0015] [4] The anti-Trop2 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, as described in any of Embodiments 1 to 3.

[0016] [5] The anti-Trop2 antibody is a complete antibody, a single-chain antibody (scFv), or a bispecific antibody, and / or The antigen-binding fragment of the anti-Trop2 antibody is Fab, Fab', Fv or F(ab')2, and / or The anti-Trop2 antibody is a humanized antibody or a fully human antibody, and / or The aforementioned anti-Trop2 antibody is a monoclonal antibody, and / or, The anti-Trop2 antibody is of the IgG1, IgG2, or IgG4 isotype and / or The anti-Trop2 antibody comprises a constant light chain region of the κ subtype or λ subtype, and / or The aforementioned anti-Trop2 antibody specifically binds to Trop2 in primates (e.g., humans or rhesus monkeys), but does not bind to Trop2 in rodents (e.g., rats or mice). An antibody-natural killer cell (NK cell) conjugate according to any one of Embodiments 1 to 4.

[0017] [6] The aforementioned 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.

[0018] [7] CD16 of the aforementioned conjugate + 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 Embodiments 1 to 6, wherein the proportion of NK cells is at least 95%.

[0019] [8] The aforementioned NK cells were obtained from in vitro culture and proliferation of NK cells derived from peripheral blood mononuclear cells (PBMCs), or The NK cells mentioned above are obtained from in vitro culture and proliferation of NK cells derived from umbilical cord blood, or The aforementioned NK cells are obtained from in vitro culture and proliferation of NK cell lines, or The aforementioned NK cells are obtained from in vitro induction, culture, and proliferation of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs). An antibody-natural killer cell (NK cell) conjugate according to any one of Embodiments 1 to 7.

[0020] [9] The antibody-natural killer cell (NK cell) conjugate according to any one of Embodiments 1 to 8, wherein the anti-Trop2 antibody or its antigen-binding fragment and the NK cells are conjugated via a click chemistry reaction of a linker.

[0021]

[10] The antibody-natural killer cell (NK cell) conjugate according to Embodiment 9, wherein the anti-Trop2 antibody or its antigen-binding fragment and the NK cells are conjugated via a first linker and a second linker, the first linker being conjugated to the anti-Trop2 antibody or its antigen-binding fragment, the second linker being conjugated to the NK cells, and the first linker and the second linker being conjugated to each other to form an antibody-NK cell conjugate.

[0022]

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

[0023]

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

[0024]

[13] The first linker described above has the following structure: [ka] [In the formula, n is an integer between 0 and 8.] The antibody-natural killer cell (NK cell) conjugate according to Embodiment 12, which is dibenzoazacyclooctinyl-glutaryl-aminopolyethylene glycol-acetylpiperidine pentafluorophenyl ester having the above.

[0025]

[14] The first linker described above has the following structure: [ka] The antibody-natural killer cell (NK cell) conjugate according to Embodiment 13, which is dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester having the above.

[0026]

[15] The antibody-natural killer cell (NK cell) conjugate according to Embodiment 10, wherein the second linker is azidoacetylated cyclohexosamine, for example, azidoacetylated cyclogalactosamine, or azidoacetylated glucosamine.

[0027]

[16] The second linker described above has the following structure: [ka] The antibody-natural killer cell (NK cell) conjugate according to embodiment 15, which is 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-α,β-D-galactose.

[0028] 〔17〕 The antibody-natural killer cell (NK cell) conjugate according to embodiment 16, wherein the ratio of the single α or β configuration of the second linker is at least 90%.

[0029] 〔18〕 A cell population comprising the antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 17.

[0030] 〔19〕 CD3 - CD56 + CD16 + The number of cells accounts for at least 95%, preferably at least 98% of the total number of cells in the cell population, and / or CD3 - CD56 + NKG2D + The cell population according to embodiment 18, wherein the number of cells accounts for at least 95%, preferably at least 98% of the total number of cells in the cell population.

[0031] 〔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 + The number of cells accounts for 2% or less of the total number of cells in the cell population. The cell population according to embodiment 18 or 19.

[0032] 〔21〕<0The cell population according to any one of Embodiments 18 to 20, wherein the antibody-natural killer cell (NK cell) conjugate constitutes 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.

[0033] 〔twenty two〕 A pharmaceutical composition comprising 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, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition is provided in the form of a sterile fresh formulation or a sterile cryopreserved formulation.

[0034] 〔twenty three〕 The pharmaceutical composition according to Embodiment 22, comprising sodium chloride and / or human serum albumin.

[0035] 〔twenty four〕 A pharmaceutical composition according to Embodiment 22 or 23, comprising trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0036] 〔twenty five〕 A pharmaceutical composition according to any one of embodiments 22 to 24 for use in treating tumors in an organism, particularly tumors in which tumor cells highly express Trop2 (Trop2+).

[0037]

[26] The pharmaceutical composition according to Embodiment 25, wherein the tumor is a solid tumor.

[0038]

[27] The pharmaceutical composition according to Embodiment 25, wherein the tumor is a malignant tumor.

[0039]

[28] The pharmaceutical composition according to Embodiment 25, wherein the tumor is cancer.

[0040]

[29] The pharmaceutical composition according to 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, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

[0041]

[30] The pharmaceutical composition according to 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, cervical cancer, and endometrial cancer or uterine cancer.

[0042]

[31] 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 pharmaceutical for the treatment of tumors in an organism, particularly tumors in which tumor cells highly express Trop2 (Trop2+).

[0043]

[32] The use according to embodiment 31, wherein the tumor is a solid tumor.

[0044]

[33] The use according to Embodiment 31, wherein the tumor is a malignant tumor.

[0045]

[34] The use according to embodiment 31, wherein the tumor is cancer.

[0046]

[35] The use according to Embodiment 34, 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, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

[0047]

[36] The use according to Embodiment 34, 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, cervical cancer, and endometrial cancer or uterine cancer.

[0048]

[37] A method for treating a tumor in an individual, particularly a tumor in which tumor cells highly express Trop2 (Trop2+), comprising administering to the individual an effective amount of an antibody-natural killer cell (NK cell) conjugate according to any one of Embodiments 1 to 17, a cell population according to any one of Embodiments 18 to 21, or a pharmaceutical composition according to any one of Embodiments 22 to 31.

[0049]

[38] The method according to embodiment 37, wherein the tumor is a solid tumor.

[0050]

[39] The method according to embodiment 37, wherein the tumor is a malignant tumor.

[0051]

[40] The method according to embodiment 37, wherein the tumor is cancerous.

[0052]

[41] The method according to Embodiment 40, 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, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

[0053]

[42] The method according to Embodiment 40, 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, cervical cancer, and endometrial cancer or uterine cancer. [Brief explanation of the drawing]

[0054] [Figure 1] This is a schematic diagram illustrating the structure of an exemplary anti-Trop2 antibody-natural killer cell conjugate of the present invention. [Figure 2] This figure shows the flow cytometry analysis results of the IBR822 formulation (lot number FE20230105): CD56+, CD3-, CD16+, NKG2D+, CD19-. [Figure 3] This figure shows the flow cytometry analysis results regarding the conjugation positivity rate and geometric mean of UNK and the IBR822 formulation (lot number FE20230105). [Figure 4] This is a schematic diagram showing the manufacturing process flow of the L2 linker. [Figure 5]This figure 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-carboxylate, 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).

[0055] [Figure 6] This is a schematic diagram showing the flow of the N1 linker manufacturing process. [Figure 7] This figure 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-mannopyranose). [Figure 8] This is a schematic diagram showing the conjugation mechanism between the L2 linker and the anti-Trop2 antibody. [Figure 9] This is a schematic diagram illustrating the binding mechanism between the N1 linker and NK cells. [Figure 10] This is a schematic diagram showing the structure of UNK with the N1 linker connected.

[0056] [Figure 11] This is a schematic diagram showing the binding mechanism between UNK and the L2 conjugate antibody. [Figure 12] This figure shows the tumor volume curves for each group in the animal experiment during the human breast cancer cell MDA-MB-468 transplanted tumor mouse study. Compared to the solvent control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 13]This figure shows a comparison of tumor weight at day 20 for each group in the human breast cancer cell MDA-MB-468 transplanted tumor mouse study compared to the animal experiment. Compared to the solvent control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 14] This figure shows the effect of IBR822 cell injection on tumor volume in a mouse tumor model of human prostate cancer (DU145) cells. [Figure 15] This figure shows the effect of IBR822 cell injection on the relative tumor volume of a mouse tumor model transplanted with human prostate cancer (DU145) cells. [Modes for carrying out the invention]

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

[0058] Regardless of the numerical ranges and parameter approximations shown in the broader scope of this invention, the numerical values ​​shown in the specific examples are described as accurately as possible. However, all numerical values ​​inherently contain a certain degree of error due to the standard deviation present in each measurement. Furthermore, all ranges disclosed herein should be understood as covering all subranges included within that range. For example, the described range "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 starting from a minimum value of 1 or greater, e.g., 1 to 6.1, and all subranges ending at a maximum value of 10 or less, e.g., 5.5 to 10. Also, any references referred to as "incorporated herein" should be understood as incorporated as a whole.

[0059] As used herein, the terms “individual” or “subject” mean a mammal such as a human, but may also mean other animals such as wild animals, domesticated animals, or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, tarbagans, ground 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, "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 therefore encompasses complete antibodies / full-length antibodies, single-chain antibodies, or any antigen-binding fragment of an antibody (also called the "antigen-binding moiety"). When "antibody" and "antigen-binding fragment / antigen-binding moiety" appear in the same context, "antibody" can be understood as the complete form of "antigen-binding fragment / antigen-binding moiety," and both commonly correspond to the broad concept of antibody.

[0062] The term "agonist antibody" refers to an antibody that induces a response, such as an antibody that mimics at least one functional activity of a target polypeptide. Agonist antibodies include ligand mimics, for example, a ligand that binds to a cell surface receptor, and that binding induces cell signaling or activity via an intracellular signaling pathway, while the antibody induces similar cell signaling or activation.

[0063] A "full-length antibody" refers to a protein containing at least two heavy chains (H) and two light chains (L) linked to each other via disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains domain CL. The VH and VL regions may be further subdivided into multiple hypervariable regions called complementarity-determining regions (CDRs), with more conservative regions called framework regions (FRs) scattered between them. Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. These variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant domain of an antibody can mediate the binding of immunoglobulins to various cells of the immune system (such as effector cells) and to host tissues or factors, including the first component (Clq) of the classical complement system. Full-length antibodies (intact antibodies) may be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or any of the aforementioned subclasses), but the antibody does not need to belong to any particular class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence of the constant domain of the heavy chain. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further classified 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 structures of different classes of immunoglobulins are known. Chimeric antibodies or humanized antibodies are also included in the antibodies according to the present invention. Those skilled in the art know that the complementarity-determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions within the variable region that have the greatest influence on the affinity and specificity of the antibody.There are many common definition methods for the VH or VL CDR amino acid sequence, such as the Kabat definition, IMGT definition, and Chothia definition. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can usually be determined by various definition methods. In embodiments of the present invention, Kabat is used to define the CDR amino acid sequence. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence of the variable region amino acid sequence can be analyzed in various ways.

[0064] The term "humanized antibody" refers to an antibody obtained by transplanting a CDR sequence derived from another mammalian species, such as a mouse, onto a human framework sequence. Several residues in the backbone (called FR) section can be modified to maintain binding affinity. The humanized antibody or fragment thereof according to the present invention can be manufactured by techniques well known to those skilled in the art.

[0065] The term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody. The chimeric antibody or fragment thereof according to the present invention can be produced using genetic recombination technology. 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 (particularly mouse) monoclonal antibody described in the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody according to the present invention encoded by such a recombinant gene is, for example, a mouse-human chimera, and the specificity of the antibody is determined by the variable region derived from mouse DNA, and its isotype is determined by the constant region derived from human DNA.

[0066] The term "partially humanized antibody" refers to an antibody that contains a constant region derived from humans and a variable region (including the CDR) derived from non-human organisms (such as mice).

[0067] The term "semi-humanized antibody" refers to a type of humanized antibody in which one antibody chain contains the mouse variable region and the other antibody chain contains the humanized variable region; in other words, a semi-humanized antibody.

[0068] The term "monoclonal antibody" refers to an antibody obtained from a nearly identical antibody population (where the individual antibodies constituting the population are identical, except for the possibility of spontaneous mutations occurring in a small number of individuals).

[0069] As used herein, the terms “antigen-binding fragment,” “antigen-binding portion,” or “antigen-binding region” refer to a portion of an antibody containing amino acid residues that are interchangeable, interact with an antigen, and confer specificity and affinity to the antigen to the binder, and more specifically, to antibody fragments, such as Fv, Fab, F(ab')2, or Fab', or any fragment whose half-life can be extended by chemical modification or introduction into liposomes. Examples of such chemical modifications include the addition of poly(alkylene) glycol, such as polyethylene glycol ("polyethylene glycolation, PEGation") (referred to as "polyethylene glycolated fragments" such as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol). Preferably, the functional fragment consists of or includes a sub-sequence of the variable chain of the heavy or light chain of the derived antibody, the sub-sequence being sufficient to maintain the same binding specificity and sufficient affinity as the derived antibody, and the functional fragment includes at least five amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of the derived antibody sequence. 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) a 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., a dimer of Fab'), and (3) an Fv fragment with a single arm of the antibody having a VL domain and a VH domain.

[0070] The term "single-chain antibody (scFv)" refers to a single polypeptide chain in which VH domains and VL domains are linked via a peptide linker. (scFv)2 contains two VH domains linked via a peptide linker and two VL domains linked to these two VH domains via disulfide crosslinks.

[0071] The terms “Fc fragment,” “Fc region,” “Fc domain,” “Fc portion,” or similar terms refer to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment, and the 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 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.

[0072] 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.

[0073] The term "bispecific antibody" refers to an antibody that has the ability to simultaneously bind to two different antigenic epitopes. The two antigenic epitopes may be located on different antigens or on the same antigen. Bispecific antibodies may have multiple structural configurations. For example, a bispecific antibody may consist of two Fc fragments and two antigen-binding moieties fused to each of them (similar to a native antibody except that the two arms bind to different antigenic targets or epitopes), and the antigen-binding moieties may be in the form of a single-chain antibody (scfv) or a Fab fragment. The two different binding moieties of a bispecific antibody are each bound 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, and Fab fragment + Fab fragment. The Fc fragments may contain mutations that ensure heavy chain heteromerization, and KIH (knob-in-hole) technology is a strategy to address heavy chain heteropolymerization. Typically, KIH technology refers to a technique that modifies the amino acid sequence of the CH3 region to create a favorable structure for pairing heterologous incomplete antibodies, thereby maintaining the structure of a normal antibody as much as possible while constructing a bispecific antibody.

[0074] Typically, to produce monoclonal antibodies or their functional fragments, particularly mouse-derived monoclonal antibodies or their functional fragments, one can refer 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 the technique by Kohler and Milstein for production from hybridoma cells (Nature, 256:495497, 1975).

[0075] The terms “conservative variant” or “conservative amino acid substitution” refer to substitutions that do not substantially affect or reduce the affinity of a protein. For example, an antibody may contain approximately one or fewer, two or fewer, five or fewer, ten or fewer, or fifteen or fewer conservative substitutions and still bind specifically to the target antigen. The term “conservative variant” also includes using a substituted amino acid instead of an unsubstituted parent amino acid, as long as the antibody binds specifically to the target antigen.

[0076] The term "isolated" refers to a biological component (e.g., nucleic acid, protein (including antibodies), or organelle) that has been substantially isolated or purified from other biological components (i.e., other chromosomes and additional chromosomal DNA or RNA, proteins, and organelles) in its naturally occurring environment (e.g., cells). Already "isolated" nucleic acids and proteins include those purified using standard purification methods. This term also includes nucleic acids and proteins produced by recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0077] As used herein, the term “pharmaceutical composition” refers to a combination of at least one drug and a pharmaceutically usable carrier or auxiliary substance, which are combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition includes combinations that are temporally and / or spatially separated, insofar 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 conjugate according to the present invention) may be administered to an individual all at once or divided into individual doses. When the components contained in the pharmaceutical composition are administered to an individual divided into individual doses, the components may be administered to the individual simultaneously or sequentially. The pharmaceutical composition according to the present invention may include conventional components of cell culture, particularly NK cell culture, to maintain the activity of NK cells in the conjugate. Pharmaceutically acceptable carriers may further include water, buffered aqueous solutions, isotonic salt solutions such as PBS (phosphate buffer), glucose, mannitol, dextrorotatory glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, and polyalkylene glycols such as polypropylene glycol and triglycerides. The pharmaceutical composition or pharmaceutical preparation according to the present invention can be administered by any suitable route, such as intravenous administration, intradermal administration, subcutaneous administration, or intramuscular injection. The composition according to the present invention may contain a wetting agent, an emulsifier, or a buffering substance as an additive.

[0078] As used herein, the terms “therapeutic effective dose” or “effective dose” refer to a dose sufficient to demonstrate its benefit to the individual being administered. The actual amount administered, the rate of administration, and the course of time will depend on the condition and severity of the individual being treated. The prescription for treatment (e.g., dosage determination) is ultimately the responsibility of the specialist and other physicians and is generally determined by considering the disease being treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to the physician.

[0079] EC 50The 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 activating ability of agonists in in vitro experiments, as well as to indicate the blood concentration required to reach half of the maximum biological effect in vivo. In some literature, the EC 50 It is also used to characterize the potency of a compound at a certain cellular level (including agonism and antagonistism), and EC 50 The value can be measured by methods such as ELISA.

[0080] The term "identity / homology / consistency" of an amino acid or nucleic acid sequence is defined as the proportion of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and capping, to the maximum proportion of identity as required. Methods and computer programs for alignment are known to those skilled in the art.

[0081] In this specification, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell proliferation. Tumors may be benign, precancerous, or malignant.

[0082] In this specification, the term “malignant tumor” refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. Exemplary malignant tumors include cancer, solid tumors, melanoma, sarcoma, hematological malignancies, germ cell tumors, and blastomas. More specific examples of malignant tumors include multiple myeloma, kidney cancer (also known as 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, liver cancer (hepatic carcinoma), stomach 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 associated metastases.

[0083] In this specification, the term “hematological malignancy” refers to tumors caused by the uncontrolled growth and proliferation of abnormal cells. Often, these abnormal cells originate in the bone marrow, which is also where blood cells are produced. Exemplary hematological malignancies include various leukemias, multiple myeloma, and malignant lymphoma. More specific examples of hematological malignancies include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), pilocytic cell leukemia (HCL), pre-T-cell lymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, pre-B-cell lymphocytic leukemia, Burkitt's leukemia and adult T-cell leukemia, non-Hodgkin lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoblastic lymphoma, lymphoplasmacytic lymphoma, and primary macroglobulinemia (Waldenstrom). macroglobulinemia (Waldensström 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 exudative lymphoma, Burkitt lymphoma, B-cell chronic lymphocytic lymphoma, ancient Examples include typical 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, Celgene syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorder, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, nonspecific peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0084] In this specification, the term "solid tumor" refers to a tangible mass that can be detected (palpated) by clinical examinations such as radiography, CT scans, B-ultrasound, and palpation. Clinically diagnosed and treated solid tumors are classified into two types: malignant and benign. Malignant solid tumors include pediatric Hodgkin lymphoma (lymphocyte-dominant type, nodular sclerosis type, mixed cell type, lymphopenic type), pediatric non-Hodgkin lymphoma (prolymphoblastic lymphoma, small non-incisional nuclear cell lymphoma (Burkitt lymphoma / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.), pediatric renal tumors (nephroblastoma (Wilms' tumor), clear cell carcinoma, rhabdomyosarcomatoid tumor, clear cell sarcoma, renal primitive neuroectoderm tumor, etc.), pediatric neuroblastoma (neuroblastoma, ganglioneuroblastoma), pediatric extracranial germ cell tumors (mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminomas (spermatogonia), dyserocarcinoma (asexual cell tumor), choriocarcinoma (choriocarcinoma), embryogenetic These include osteosarcoma and chondrosarcoma, pediatric rhabdomyosarcoma (embryonic, acinar, pleomorphic, etc.), pediatric soft tissue sarcoma (fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, fibroplastic round cell tumor, etc.), Ewing sarcoma family tumors (Ewing sarcoma, primitive neuroectodermal tumor), pediatric liver tumors (hepatoblastoma (embryonic, fetal, undifferentiated), hepatocellular carcinoma), retinoblastoma, and other tumors (posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoid, mesothelioma, etc.). Benign solid tumors include lymphangiomas, hemangiomas, and thyroglossal duct cysts.

[0085] For the treatment of tumors, adoptive cell immunotherapy has become a hot spot for research in China and globally, achieving better results in clinical trials for tumors. Adoptive cell immunotherapy is a treatment method that involves isolating autoimmune cells or allogeneic (allogeneic) immune cells, activating or genetically modifying them in vitro, proliferating them to a sufficient quantity of immune cells with antitumor activity, and then injecting them into tumor patients to enhance the patient's cellular immune function and improve the antitumor effect. Currently, several types of cell immunotherapies are being studied, 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 (TILs), and cytokine-induced killer cells (CIK). Although CAR-T cell therapy has developed rapidly in recent years, many shortcomings and challenges still exist in clinical application, such as varying degrees of neurotoxicity, the risk of cytokine storms, and very low efficacy in treating 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 treated. In addition, CAR-T cell immunotherapy can cause life-threatening side effects in patients, such as cytokine release syndrome.

[0086] In contrast, NK cell therapy has shown superior clinical efficacy in the treatment of hematological malignancies and offers several advantages over CAR-T cell therapy, including: 1) NK cells do not cause graft-versus-host (GVHD) reactions like T cells, making them promising as a general-purpose cell therapy. 2) Mature NK cells have a relatively short lifespan (approximately 7-10 days), allowing them to effectively damage tumor cells while reducing the probability of long-term adverse events. 3) NK cells are not limited by antigen-specific major histocompatibility complexes (MHC) and possess more potent damaging capabilities. 4) NK cell therapy does not secrete inflammatory factors (IL-1, IL-6, etc.), resulting in a lower risk of cytokine storms and neurotoxic reactions. 5) The low-affinity CD16 molecule on the surface of NK cells can bind to IgG antibody conjugates on the surface of target cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and can also mediate apoptosis via the Fas / FasL pathway. Furthermore, because NK cells secrete low levels of programmed death receptor 1 (PD-1) and induce little immunosuppression, NK cells are expected to have excellent applications in the treatment of solid tumors.

[0087] The inventors of this application have conducted detailed research in the field of cellular immunotherapy, and have developed anti-Trop2 antibody-natural killer cell conjugates, particularly for tumor treatment. Figure 1 shows a schematic structural diagram of an exemplary anti-Trop2 antibody-natural killer cell conjugate of the present invention.

[0088] Natural killer cells (NK cells) belong to the granular lymphocyte class and are components of the human immune system. NK cells recognize target antigens without major histocompatibility conjugate (MHC) restriction, and allogeneic (allogeneic) NK cells have a very 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 very low. In 2020, Liu E 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, showing that after 11 enrolled subjects received CAR-NK cell infusion, 8 subjects showed remission of their disease, 7 of whom achieved complete remission, and no cases of CRS, neurotoxicity, or GvHD occurred. This indicates that CAR-NK cell therapy is relatively safe. Furthermore, based on the characteristic that NK cells recognize target antigens without being restricted by MHC, they can be manufactured into general-purpose products without being limited to autologous cells. For this reason, a wide range of NK cell sources can be selected for use in this therapy, including allogeneic peripheral blood, umbilical cord blood, embryonic stem cells, human-induced pluripotent stem cells, and NK-92 cell lines.

[0089] The toxic activity of NK cells is primarily mediated as follows: 1) Direct lysis of target cells: NK cells release cytotoxic particles such as perforin and granzymes via exocytosis, activating the caspase pathway to induce necrosis or apoptosis of target cells. 2) Cytokine secretion: There is a cytokine-mediated toxic effect, and NK cells can synthesize and secrete various cytokines such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10, and G-CSF, thereby inducing apoptosis in target cells. 3) Induction of apoptosis: Activated NK cells express Fas(CD95) ligand and tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) molecules, inducing apoptosis in CD95+ target cells and TRAIL receptor-positive target cells via a cascade of endogenous enzymes. 4)ADCC: Antibody-dependent cell-mediated cytotoxicity. 5) Immune checkpoint pathway: This pathway exerts its effects by inhibiting immune checkpoints through the expression of programmed death receptor 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4), among others.

[0090] Due to its multiple mechanisms of action, potential for general application as a product, and reliable safety, NK cell therapy is an attractive immunotherapy.

[0091] The antigen target selected in this application is Trop2, whose formal name is Human Trophoblast Cell-Surface Antigen 2. Trop2 is a cell surface glycoprotein belonging to the TACSTD family, encoded and expressed by the TACSTD2 gene, and is also known as tumor-associated calcium signaling factor 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). Trop2 is overexpressed in various malignant tumors and is an oncogene involved in the development, invasion, and metastasis of malignant tumors, as well as a signaling molecule that can regulate tumor cell proliferation. The Trop2 gene is located on the short arm of chromosome 1, specifically localized to 1p32.1. The full length of this gene is 9072 bp, it has no introns, and it has only one exon. The primary structure of the Trop2 protein is a 36 kDa polypeptide composed of 323 amino acids, and it is a single-pass transmembrane surface glycoprotein. Trop-2 plays a role in regulating tumor cell proliferation and may represent a novel cell surface receptor that functions as a regulator in cell self-renewal, proliferation, and metastasis.

[0092] According to a first aspect, the present invention provides an antibody-natural killer cell (NK cell) conjugate in which the antibody is an anti-Trop2 antibody or its antigen-binding fragment, and the anti-Trop2 antibody or its antigen-binding fragment is conjugated to the NK cell via a linker.

[0093] In some embodiments, the anti-Trop2 antibody or its antigen-binding fragment is HCDR1, which is shown in Sequence ID No. 5, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 5. HCDR2, which is shown in Sequence ID No. 6, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 6. HCDR3, which is shown in Sequence ID No. 7, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 7. LCDR1, which is shown in sequence number 8, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in sequence number 8. LCDR2, which is shown in sequence number 9, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in sequence number 9, and It includes an LCDR3 that is shown in SEQ ID NO: 10, or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO: 10, Here, the amino acid sequences of HCDR and LCDR are defined by Kabat.

[0094] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or its antigen-binding fragment is shown 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 with the sequence shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown 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 with the sequence shown in SEQ ID NO: 3.

[0095] In some embodiments, the amino acid sequence of the heavy chain variable region of an anti-Trop2 antibody or its antigen-binding fragment differs from the amino acid sequence shown 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 shown 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 the function similar to that of the heavy chain variable region of the antibody is still maintained. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 1, and the resulting amino acid sequence still maintains the function similar to that of 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 substantially maintains a function similar to that of the heavy chain variable region of the antibody.

[0096] In some embodiments, the amino acid sequence of the light chain variable region of an anti-Trop2 antibody or its antigen-binding fragment differs from the amino acid sequence shown 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 shown 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 the function similar to that of the light chain variable region of the antibody is still maintained. 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 shown in SEQ ID NO: 3, and the resulting amino acid sequence still maintains a function similar to that of 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 substantially maintains a function similar to that of the light chain variable region of the antibody.

[0097] In some embodiments, the anti-Trop2 antibody has a heavy chain variable region indicated by SEQ ID NO: 1, a heavy chain constant region indicated by SEQ ID NO: 2, a light chain variable region indicated by SEQ ID NO: 3, and a light chain constant region indicated by SEQ ID NO: 4.

[0098] In some embodiments, the anti-Trop2 antibody specifically binds to Trop2 in primates (e.g., humans or rhesus monkeys) but does not bind to Trop2 in rodents (e.g., rats or mice).

[0099] In some embodiments, the anti-Trop2 antibody is a complete antibody, a single-chain antibody (scFv), or a bispecific antibody. In some embodiments, the antigen-binding fragment of the anti-Trop2 antibody is Fab, Fab', Fv, or F(ab')2.

[0100] In some embodiments, the anti-Trop2 antibody is a humanized antibody or a fully human antibody. In some embodiments, the anti-Trop2 antibody is a monoclonal antibody.

[0101] In some embodiments, the anti-Trop2 antibody is IgG1, IgG2, or IgG4 isotype. In some embodiments, the anti-Trop2 antibody is IgG1 isotype. In some embodiments, the anti-Trop2 antibody includes a constant light chain region of either the κ subtype or the λ subtype.

[0102] Once the structure / sequence of an antibody is identified, the techniques for producing the corresponding antibody are acquired by those skilled in the art.

[0103] As a non-limiting example, the sequences of anti-Trop2 monoclonal antibodies can be derived from phage library screening, confirmed and selected through screening of binding activity of Trop2 and Trop2-expressing cells, interspecies cross-comparison studies, and affinity studies to obtain anti-Trop2 monoclonal antibodies. Here, the anti-Trop2 monoclonal antibody includes the heavy chain variable region shown in SEQ ID NO: 1 (HCDR1-3 are SEQ ID NOs. 5, 6, and 7, respectively), the heavy chain constant region shown in SEQ ID NO: 2, the light chain variable region shown in SEQ ID NO: 3 (LCDR1-3 are SEQ ID NOs. 8, 9, and 10, respectively), and the light chain constant region shown in SEQ ID NO: 4. The DNA sequence of the anti-Trop2 monoclonal antibody is determined, and a recombinant plasmid (IB12) expressing the anti-Trop2 monoclonal antibody is constructed. The construction of the monoclonal cell line is based on known techniques. The IB12 plasmid is transfected into CHO cells by electroporation, followed by one round of screening in a minipool and two rounds of screening after plate seeding of monoclonal cells. Confirmation is then achieved through stable culture during initial passaging, resulting in a cell line capable of stably expressing anti-Trop2 monoclonal antibody. The anti-Trop2 monoclonal antibody is manufactured based on known production protocols in this field, including cell culture and protein purification. Working cells are collected, resuscitated, grown for 3-5 rounds, cultured on a medium scale, and the untreated cell suspension (UPB) is collected. Cell culture supernatant is obtained after clarification and filtration. Subsequently, the anti-Trop2 monoclonal antibody is obtained using a protein purification process via three-step chromatography: protein A affinity, anion exchange, and cation exchange.

[0104] In some embodiments, NK cells are CD16 + and / or NKG2D + In some embodiments, NK cells are CD16 + NKG2D + That is the case.

[0105] In some embodiments, the conjugate CD16 + NKG2D +The percentage of NK cells is at least 90%, for example, at least 95%, at least 98%, or at least 99%. In some embodiments, the conjugate CD16 + NKG2D + The proportion of NK cells is at least 90%, and CD56 + The percentage of NK cells is at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%).

[0106] There are many different techniques for culturing, growing, and obtaining NK cells in vitro, and their general principles and methodologies are known to those skilled in the art.

[0107] In some embodiments, NK cells are obtained from in vitro culture and proliferation of NK cells derived from peripheral blood mononuclear cells (PBMCs), which is an exemplary method in the embodiments of this application. PBMCs are one of the primary sources of NK cells, offering the advantages of being relatively easy to collect, readily proliferating in vitro, and free from toxicity and side effects. However, the proportion of NK cells in PBMCs is only 10% to 15%, and methods for proliferating PBMC-derived NK cells include stimulating in vitro proliferation of NK cells using a combination of cytokines, feeder cells, or membrane particles, and these different proliferation systems exhibit different levels of NK cell proliferation efficiency. In some embodiments, PBMCs are screened for HLA, KIR. In some embodiments, PBMCs are screened for CD16a variants, i.e., 176V, 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.

[0108] PBMCs may be derived from peripheral blood lymphocytes isolated from allogeneic healthy donors. After separating T cells and red blood cells, the cells are transferred to primary cell cryopreservation solution, with a total number of viable cells per vial of 6.0 × 10⁶. 7 The cells are dispensed into portions of 1 or more to obtain PBMCs. The PBMCs are stored long-term in liquid nitrogen at -175°C or below. The PBMCs, which have been cryopreserved in liquid nitrogen, are taken out, revived in an appropriate medium, and the culture scale is expanded (i.e., the medium is increased for expanded culture), and NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their proliferative capacity and activity. For an exemplary manufacturing method, refer to Example 1 of this application.

[0109] In some embodiments, NK cells are obtained from in vitro culture and proliferation of NK cells derived from umbilical cord blood. There are generally two different methods for obtaining a large number of NK cells from umbilical cord blood. One method is to proliferate NK cells in umbilical cord blood, and the other method is to extract CD34 cells from umbilical cord blood. + This involves inducing hematopoietic stem cells / progenitor cells, differentiating them into NK cells, and then proliferating them.

[0110] In some embodiments, NK cells are obtained from in vitro culture and proliferation of NK cell lines. As an example, NK-92 as homogeneous immortalized NK lymphoma cells is the first NK cell-based immunotherapy approved by the FDA for clinical trials.

[0111] In some embodiments, NK cells are obtained by in vitro induction, culture, and proliferation of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs).

[0112] In some embodiments, an anti-Trop2 antibody or its antigen-binding fragment and NK cells are conjugated via a linker click chemistry reaction.

[0113] Click chemistry, also known as link chemistry or speed-matching / combined chemistry, is a synthetic concept introduced in 2001 by chemist Barry Sharpless. 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: ring addition reactions, nucleophilic ring-opening reactions, non-aldol carbonyl chemical reactions, and carbon-carbon multiple bond addition reactions.

[0114] In some embodiments, an anti-Trop2 antibody or its antigen-binding fragment and NK cells are conjugated via a first linker and a second linker, the first linker being conjugated to the anti-Trop2 antibody or its antigen-binding fragment, the second linker being conjugated to the NK cells, and the first and second linkers being conjugated to each other to form an antibody-NK cell conjugate.

[0115] In some embodiments, the first linker is an active ester capable of forming a conjugation to a lysine residue of an antibody via a reaction from an ester bond to an amide bond, the active ester being, for example, a pentafluorophenyl ester, such as pentafluorophenyl piperidine. The active ester may be an aqueous-phase stable molecule that can specifically covalently bond to a lysine residue of an antibody. In some embodiments, the active ester has a tetraethylene glycol chain structure and can be gradually hydrolyzed in water. In some embodiments, the active ester reacts with an amino group at the hydrophilic interface of the protein to convert the ester bond to an amide bond, thereby establishing the binding mechanism (conjugation mechanism).

[0116] In some embodiments, the first linker further comprises a carbon-carbon triple bond structure that can undergo a cyclization reaction with an azide group to form a five-membered ring of triazazole, for example, the carbon-carbon triple bond structure being an octin group.

[0117] In some embodiments, the first linker has the following structure: [ka] [In the formula, n is an integer between 0 and 8.] It is a dibenzoazacyclooctinyl-glutaryl-aminopolyethylene glycol-acetylpiperidine pentafluorophenyl ester having [a specific characteristic].

[0118] In some embodiments, the first linker has the following structure: [ka] It is a dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester having [a specific characteristic].

[0119] Dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester, also known as the L2 linker in this invention, has a synthetic pathway diagram for the L2 linker shown in Figure 4 and a structural formula shown in Figure 5, and includes three main synthetic steps: amide condensation, hydrolysis reaction, and synthesis of the active ester.

[0120] <Step 1> Amide condensation Dibenzoazacyclooctinyl glutaric acid (L2-1) and aminotetraethylene glycol acetylpiperidine methyl ester (L2-2) were obtained commercially, and the intermediate dibenzoazacyclooctinyl glutarylaminotetraethylene glycol acetylpiperidine methyl ester (L2-3) was obtained by chemical condensation reaction. L2-3 is a stable compound.

[0121] Specifically, compounds L2-1 and L2-2 (1:1.1, with L2-2 in excess) were dissolved in dichloromethane (DCM) solution, and hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide (EDCI) were added to each, followed by the addition of triethanolamine (TEA). The mixture was stirred at room temperature for 4 to 12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified by silica gel column (dichloromethane:methanol = 20:1) to obtain yellow oily compound L2-3.

[0122] <Step 2> Hydrolysis reaction The intermediate L2-3 obtained in Step 1 was subjected to a hydrolysis reaction to obtain the intermediate dibenzoazacyclooctinylglutarylaminotetraethylene glycol acetylpiperidine acid (L2-4). Hydrolysis is usually a quantitative reaction and is used directly in the synthesis of the next step without further purification.

[0123] Specifically, compound L2-3 was dissolved in a methanol and water mixture, cooled to 0°C, and then a 1 mol / L lithium hydroxide (LiOH) aqueous solution was added. The mixture was stirred from 0°C to room temperature for 4 to 12 hours, the pH was acidified to 2 to 3 with 1 mol / L hydrochloric acid, extracted three times with ethyl acetate (EA), and dried to obtain the intermediate dibenzoazacyclooctinylglutarylaminotetraethylene glycol acetylpiperidine acid (L2-4) as a yellow oily substance.

[0124] <Step 3> Synthesis of activated esters The intermediate L2-4 obtained in Step 2 (without purification) was condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2 (i.e., dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester).

[0125] Specifically, compounds L2-4 were dissolved in tetrahydrofuran (THF), cooled to 0°C, and 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 by silica gel column (dichloromethane:methanol = 20:1) to obtain dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester (L2) (see Figure 5 for structural formula) as a yellow oily substance.

[0126] Subsequently, the L2 linker and the anti-Trop2 monoclonal antibody can undergo a site-directed coupling reaction in a PBS system with a pH of 7.2-7.4 (preferably HEPES with a pH of 7.2) (see Figure 8 for a schematic diagram). Typically, one anti-Trop2 monoclonal antibody can be conjugated to 1-4 (preferably 1-2) L2 linkers. The reaction can be stopped by adjusting the pH to approximately 5.0 to obtain an L2 conjugate antibody.

[0127] In some embodiments, the second linker is azidoacetylated cyclohexosamine, e.g., azidoacetylated cyclogalactosamine, or azidoacetylated glucosamine. In some embodiments, the second linker is an aqueous-phase stable molecule that can specifically covalently bond to the sialic acid-modified membrane protein. In some embodiments, the second linker translocates to the sialic acid-modified membrane protein via the cell's own metabolic pathway during the cell culture stage. 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 triazole five-membered ring.

[0128] In some embodiments, the second linker has the following structure: [ka] It is 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose containing [a specific compound].

[0129] In some embodiments, the proportion of a single α or β configuration of 1,3,4,6-oxo-tetraacetyl-2-azidoacetamido-2-deoxy-a,bD-galactose is at least 90%, for example, at least 95%, at least 98%, or at least 99%.

[0130] 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 synthetic pathway diagram of the N1 linker is shown in Figure 6, and its structural formula is shown in Figure 7. It includes two main synthetic steps: aminoazidoacetylation and hydroxyacetylation.

[0131] <Step 1> Aminoazidoacetylation Add 1.2 times α-azidoacetic acid (compound 1), 2 times hydroxybenzotriazole (HOBt), and triethylamine (Et3N) to a solution of D-galactosamine hydrochloride (compound 2) in N,N-dimethylformamide (DMF), add a small amount of methanol (MeOH) to aid dissolution, and react at room temperature for 12 hours. Pour the reaction mixture into dichloromethane (DCM) / methanol (MeOH), shake well to mix, add ether to precipitate an oily product (compound 3), pour out the ether layer, and repeat this process twice, drying under vacuum. Without further purification of the product, proceed directly to the next step of the reaction.

[0132] <Step 2> Hydroxyacetylation Compound 3 obtained above was added to pyridine anhydride (Pyr.) and acetic anhydride (Ac2O), and a 4-dimethylaminopyridine (DMAP) catalyst was added at room temperature. The reaction was allowed to proceed for 12 hours, and HPLC was used to detect that the reaction was nearly complete. The mixture was then concentrated to obtain a racemic mixture of tetraacetyl-N-azidoacetyl-α,bD-galactosamine. The solid was precipitated in ethyl acetate / petroleum ether, and the product was mainly the β-configuration, with a purity exceeding 85%. Further purification by silica gel column (dichloromethane:methanol = 20:1) yielded the N1 product with a single optical isomer purity exceeding 90%. The two α,b isomers can isomerize with each other intracellularly and are converted to N-azidoacetylsialic acid through multiple metabolic and synthetic steps, ultimately being expressed on glycoproteins on the surface of NK cells. The structural formula of the N1 linker is shown in Figure 7.

[0133] When NK cells are cultured for 15-16 days, an N1 linker is added to the culture medium and incubated for 12-18 hours to obtain NK cells modified with the N1 linker (also referred to as "UNK" in the embodiments of this application) (see Figures 9 and 10 for the binding mechanism between the N1 linker and NK cells, and a schematic diagram of the UNK structure).

[0134] Finally, to obtain the antibody-NK cell conjugate, the UNK cell and the L2 conjugate antibody are coupled in culture medium (see Figure 11 for the reaction mechanism), thereby obtaining the antibody-NK cell conjugate.

[0135] The subsequent process further involves the production of antibody-NK cell conjugate formulations to effectively extend the stability of the antibody-NK cell conjugates. The formulations may contain isotonic components such as sodium chloride and human serum albumin, and may also contain components that maintain the cell's cold tolerance and the activity of proteins and enzymes, such as trehalose, sucrose, dextran, and DMSO.

[0136] According to a second aspect, the present invention provides a cell population comprising the antibody natural killer cell (NK cell) conjugate (complex) described in the first aspect.

[0137] In some embodiments, CD3 - CD56 + CD16 + The number of cells accounts for at least 95% of the total number of cells in the cell population. In some embodiments, CD3 - CD56 + CD16 + The number of cells accounts for at least 98% of the total number of cells in the cell population. In some embodiments, CD3 - CD56 + NKG2D + The number of cells accounts for at least 95% of the total number of cells in the cell population. In some embodiments, CD3 - CD56 + NKG2D + The number of cells accounts for at least 98% of the total number of cells in the cell population.

[0138] In some embodiments, CD3 + CD56 + The number of cells accounts for 5% or less of the total number of cells in the aforementioned cell population. In some embodiments, CD3 - CD19 + The number of cells accounts for 2% or less of the total number of cells in the aforementioned cell population. In some embodiments, CD3 + CD4 + and CD3 + CD8 cells account for 2% or less of the total number of cells in the aforementioned cell population.

[0139] 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.

[0140] According to a third aspect, the present invention provides a pharmaceutical composition comprising an antibody-natural killer cell (NK cell) conjugate as described in the first aspect, or a cell population as described in the second aspect, and a pharmaceutically acceptable carrier.

[0141] Where conditions permit, the pharmaceutical composition may be provided directly to the patient in the form of a sterile, fresh formulation. If long-term storage (e.g., for multiple doses) or transportation is required, the pharmaceutical composition may be provided in the form of a sterile, cryopreserved formulation that is thawed and regenerated before use.

[0142] 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 tumors in an individual.

[0143] In some embodiments, the tumor is a Trop2+ tumor. In some embodiments, a Trop2+ tumor means that at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2+) means that at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2+) means that at least 99% of the tumor cells in the tumor cell population express Trop2.

[0144] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a hematological malignancy. In some embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is cancer.

[0145] In some manner, cancers are 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, stomach cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

[0146] In some embodiments, 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 endometrial cancer or uterine cancer.

[0147] 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 pharmaceutical for the treatment of tumors in an individual.

[0148] In some embodiments, the tumor is a Trop2+ tumor. In some embodiments, a Trop2+ tumor means that at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2+) means that at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2+) means that at least 99% of the tumor cells in the tumor cell population express Trop2.

[0149] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a hematological malignancy. In some embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is cancer.

[0150] In some manner, cancers are 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, stomach cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

[0151] In some embodiments, 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 endometrial cancer or uterine cancer.

[0152] According to a fifth aspect, the present invention provides a method for treating a tumor in an individual, comprising administering to the individual an effective amount of an antibody-natural killer cell (NK cell) conjugate according to the first aspect, a cell population according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0153] In some embodiments, the tumor is a Trop2+ tumor. In some embodiments, a Trop2+ tumor means that at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a Trop2+ tumor means that at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2+) means that at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2+) means that at least 99% of the tumor cells in the tumor cell population express Trop2.

[0154] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a hematological malignancy. In some embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is cancer.

[0155] In some manner, cancers are 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, stomach cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

[0156] In some embodiments, 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 endometrial cancer or uterine cancer.

[0157] The above detailed description is intended only to make the present invention clearly understandable to those skilled in the art, and should be understood not to limit any aspect. Those skilled in the art can make various modifications and changes to the above embodiments. [Examples]

[0158] The present invention will be further described below with reference to specific examples. Please understand that these examples are merely for illustrative purposes and do not limit the scope of the present invention.

[0159] [Example 1: Production of anti-Trop2 antibody-NK cell conjugates] The production of the antibody-NK cell conjugate in this example generally involves the following steps: (1) Steps for manufacturing an anti-Trop2 monoclonal antibody, (2) Steps for manufacturing NK cells, (3) Manufacturing steps for the antibody linker (also called the L2 linker in this example), (4) Steps for manufacturing the NK cell linker (referred to as the N1 linker in this embodiment), (5) Step of applying the antibody linker (L2 linker) to the antibody, (6) The step of applying an NK cell linker (N1 linker) to NK cells, (7) The process can be divided into steps of conjugating NK cells with antibodies that each have a linker.

[0160] (1) Production of anti-Trop2 monoclonal antibody Simply put, the anti-Trop2 monoclonal antibody sequence was derived from a phage library screening, confirmed and selected through screening of binding activity to Trop2 and Trop2-expressing cells, interspecies cross-reactivity studies (human, monkey, and mouse), and affinity studies, resulting in the acquisition of the anti-Trop2 monoclonal antibody. Here, the anti-Trop2 monoclonal antibody includes the heavy chain variable region shown in SEQ ID NO: 1 (HCDR1-3 correspond to SEQ ID NOs. 5, 6, and 7, respectively), the heavy chain constant region shown in SEQ ID NO: 2, the light chain variable region shown in SEQ ID NO: 3 (LCDR1-3 correspond to SEQ ID NOs. 8, 9, and 10, respectively), and the light chain constant region shown in SEQ ID NO: 4. The DNA sequence of the anti-Trop2 monoclonal antibody was determined, and a recombinant plasmid (IB12) expressing the anti-Trop2 monoclonal antibody was constructed.

[0161] The construction of the monoclonal cell line is based on known techniques. The IB12 plasmid is transfected into CHO cells by electropermeation, followed by one round of screening in a minipool and two rounds of screening after plate seeding of monoclonal cells. Confirmation is then made through stable culture during the initial passaging, resulting in a cell line capable of stably expressing the anti-Trop2 monoclonal antibody.

[0162] Anti-Trop2 monoclonal antibodies are manufactured based on known production protocols in this field, such as cell culture and protein purification. Working cells are collected, resuscitated, grown for 3-5 rounds, cultured on a medium scale, and the untreated cell suspension (UPB) is collected. The cell culture supernatant is obtained after clarification and filtration. Subsequently, the anti-Trop2 monoclonal antibody is obtained by a three-step chromatography process using protein A affinity, anion exchange, and cation exchange, employing a protein purification process.

[0163] (2) Production of NK cells PBMCs are derived from peripheral blood lymphocytes isolated from allogeneic healthy donors. After separating T cells and red blood cells, the cells are transferred to primary cell cryopreservation solution, with a total number of viable cells per vial being 6.0 × 10⁶. 7 Dispense the mixture into portions of at least one to obtain PBMCs. Store the PBMCs long-term in liquid nitrogen at -175°C or below.

[0164] PBMCs cryopreserved in liquid nitrogen are removed, revived in appropriate culture medium, and the culture scale is expanded (i.e., the medium is increased for expanded culture) and NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their proliferative capacity and activity. Exemplary NK cell culture steps, process parameters, and process control indicators are shown in Table 1 below.

[0165] [Table 1]

[0166] Quality control for the 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).

[0167] [Table 2]

[0168] Figure 2 shows the flow cytometry detection results of the above immunological markers for the final antibody-NK cell conjugate formulation.

[0169] (3) Manufacturing of antibody linker (also called L2 linker in this example) The synthesis pathway diagram for the L2 linker is shown in Figure 4, and its structural formula is shown in Figure 5. It includes three main synthesis steps: amide condensation, hydrolysis, and synthesis of the active ester.

[0170] <Step 1> Amide condensation Dibenzoazacyclooctinyl glutaric acid (L2-1) and aminotetraethylene glycol acetylpiperidine methyl ester (L2-2) were obtained commercially, and the intermediate dibenzoazacyclooctinyl glutarylaminotetraethylene glycol acetylpiperidine methyl ester (L2-3) was obtained by chemical condensation reaction. L2-3 is a stable compound.

[0171] Specifically, compounds L2-1 and L2-2 (1:1.1, with L2-2 in excess) were dissolved in dichloromethane (DCM) solution, and hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide (EDCI) were added to each, followed by the addition of triethanolamine (TEA). The mixture was stirred at room temperature for 4 to 12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified by silica gel column (dichloromethane:methanol = 20:1) to obtain yellow oily compound L2-3.

[0172] <Step 2> Hydrolysis reaction The intermediate L2-3 obtained in Step 1 was subjected to a hydrolysis reaction to obtain the intermediate dibenzoazacyclooctinylglutarylaminotetraethylene glycol acetylpiperidine acid (L2-4). Hydrolysis is usually a quantitative reaction and is used directly in the synthesis of the next step without further purification.

[0173] Specifically, compound L2-3 was dissolved in a methanol and water mixture, cooled to 0°C, and then a 1 mol / L lithium hydroxide (LiOH) aqueous solution was added. The mixture was stirred from 0°C to room temperature for 4 to 12 hours, the pH was acidified to 2 to 3 with 1 mol / L hydrochloric acid, extracted three times with ethyl acetate (EA), and dried to obtain the intermediate dibenzoazacyclooctinylglutarylaminotetraethylene glycol acetylpiperidine acid (L2-4) as a yellow oily substance.

[0174] <Step 3> Synthesis of activated esters The intermediate L2-4 obtained in Step 2 (without purification) was condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2 (i.e., dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester).

[0175] Specifically, compounds L2-4 were dissolved in tetrahydrofuran (THF), cooled to 0°C, and 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 by silica gel column (dichloromethane:methanol = 20:1) to obtain dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester (L2) (see Figure 5 for structural formula) as a yellow oily substance.

[0176] (4) Production of NK cell linker (referred to as N1 linker in this example) The synthesis pathway diagram of the N1 linker is shown in Figure 6, and its structural formula is shown in Figure 7. It includes two main synthesis steps: aminoazidoacetylation and hydroxyacetylation.

[0177] <Step 1> Aminoazidoacetylation Add 1.2 times α-azidoacetic acid (compound 1), 2 times hydroxybenzotriazole (HOBt), and triethylamine (Et3N) to a solution of D-galactosamine hydrochloride (compound 2) in N,N-dimethylformamide (DMF), add a small amount of methanol (MeOH) to aid dissolution, and react at room temperature for 12 hours. Pour the reaction mixture into dichloromethane (DCM) / methanol (MeOH), shake well to mix, add ether to precipitate the oily product (compound 3), pour out the ether layer, and repeat this process twice, drying under vacuum. Without further purification of the product, proceed directly to the next step of the reaction.

[0178] <Step 2> Hydroxyacetylation Compound 3 obtained above was added to pyridine anhydride (Pyr.) and acetic anhydride (Ac2O), and a 4-dimethylaminopyridine (DMAP) catalyst was added at room temperature. The reaction was allowed to proceed for 12 hours, and HPLC was used to detect that the reaction was nearly complete. The mixture was then concentrated to obtain a racemic mixture of tetraacetyl-N-azidoacetyl-α,bD-galactosamine. The solid was precipitated in ethyl acetate / petroleum ether, and the product was mainly the β-configuration, with a purity exceeding 85%. Further purification by silica gel column (dichloromethane:methanol = 20:1) yielded the N1 product with a single optical isomer purity exceeding 90%. The two α,b isomers can isomerize with each other intracellularly and are converted to N-azidoacetylsialic acid through multiple metabolic and synthetic steps, ultimately being expressed on glycoproteins on the surface of NK cells. The structural formula of the N1 linker is shown in Figure 7.

[0179] (5) Apply the antibody linker (L2 linker) to the antibody. L2 linkers and anti-Trop2 monoclonal antibodies can undergo a site-directed coupling reaction in a PBS system at pH 7.2-7.4 (preferably HEPES at pH 7.2) (see Figure 8 for a schematic diagram). Typically, one anti-Trop2 monoclonal antibody can conjugate to 1-4 (preferably 1-2) L2 linkers. The reaction can be stopped by adjusting the pH to approximately 5.0 to obtain an L2 conjugate antibody.

[0180] (6) Apply the NK cell linker (N1 linker) to NK cells. When NK cells are cultured for 15-16 days, an N1 linker is added to the culture medium and incubated for 12-18 hours to obtain NK cells modified with the N1 linker (also referred to as "UNK" in the embodiments of this application) (see Figures 9 and 10 for the binding mechanism between the N1 linker and NK cells, and a schematic diagram of the UNK structure).

[0181] (7) Conjugate NK cells with antibodies that each have a linker. The UNK cell and the L2 conjugate antibody are coupled in culture medium (see Figure 11 for the reaction mechanism) to obtain a stock solution (DS) of the antibody-NK cell conjugate. This anti-Trop2 monoclonal antibody-NK cell conjugate is named "IBR822," and this name will be used in subsequent examples to refer to the anti-Trop2 antibody-NK cell conjugate.

[0182] The subsequent process further involves the production of antibody-NK cell conjugate formulations to effectively extend the stability of the antibody-NK cell conjugates. The formulations may contain isotonic components such as sodium chloride and human serum albumin, and may also contain components that maintain the cell's cold tolerance and the activity of proteins and enzymes, such as trehalose, sucrose, dextran, and DMSO.

[0183] Using flow cytometry, the NK cell purity of the antibody-NK cell conjugate formulation was detected to be over 98%, exceeding the standard requirement of 95% (Figure 2). Furthermore, flow cytometry was used to detect the binding positivity of the antibody-NK cell conjugate stock solution and the formulation, using UNK as a control. The results showed that the binding positivity of the formulation exceeded 98% in all cases (Figure 3), while the usual specification requirement is 90%.

[0184] [Example 2: Affinity of L2-conjugated antibodies against Trop2 and interspecies cross-detection] In this example, the affinity of the conjugate antibodies prepared in step (5) of Example 1 for Trop2 from different species was detected using an OctetR8 instrument (BLI method). Specifically, various Trop2 proteins were loaded onto His-K probes and conjugated to free L2 conjugate antibodies in solution (diluted with PBS (pH 7.4)). Binding was determined based on the maximum response value. The dissociation constant KD value was calculated based on the binding and dissociation rates. The results are as follows.

[0185] [Table 3]

[0186] As can be seen from the results in Table 3, the antibodies obtained in this application show specificity to human or primate Trop2 such as rhesus monkeys, but do not bind to rodent Trop2 such as rats or mice.

[0187] [Example 3: Evaluation of in vitro toxic activity of conjugated IBR822 against different tumor cells] In evaluating cytotoxic activity in this embodiment, 11 tumor cell lines expressing Trop2 were selected. The cell line names, cancer types, and pre-measured Trop2 expression levels (measured by flow cytometry) are shown in Table 4 below.

[0188] [Table 4]

[0189] Using the calcein AM fluorescence labeling method, the cytotoxic activities of NK cells (obtained in step (2) of Example 1), UNK cells (obtained in step (6) of Example 1), and IBR822 against multiple tumor cells with high Trop2 expression, such as non-small cell lung cancer cells (NCI-H292, HCC827), breast cancer cells (MDA-MB-231), esophageal cancer cells (KYSE140, OE33), prostate cancer cells (DU-145), ovarian cancer cells (SKOV-3), and cervical cancer cells (SiHa), were measured.

[0190] After staining the tumor cells with calcein AM for 30 minutes, they were washed three times, and the tumor cell concentration was adjusted to 1.5×10 5 cells / mL and seeded into a 96-well cell culture plate at 100 μL / well. NK cells, UNK cells, and IBR822 were adjusted to concentrations of 1.5×10 6 cells / mL, 5×10 5 cells / mL, and 1.7×10 5 cells / mL, and six replicate wells were created for each concentration gradient and added to the 96-well plate at 100 μL / well, and the final effector-to-target ratio was adjusted to be approximately 10:1, 3:1, and 1:1. Then, corresponding control groups were set up and cultured at 37°C under 5% CO2 conditions for 4 hours. 120 μL / well of the supernatant was transferred in parallel to a black 96-well microplate, and the fluorescence intensity was measured by selecting an excitation wavelength of 490 nm and an emission wavelength of 535 nm.

[0191] In Table 5 below, NK cells, UNK cells, and IBR822 all showed cytotoxic effects on the said cells. It was shown that the cytotoxic activity of IBR822 against the said cells was higher than that of NK cells and UNK cells.

[0192]

Table 5

[0193] Next, the calcein AM release assay was used to detect the dose-effect relationship of three batches of conjugate IBR822 in damaging tumor cells. After the tumor cells were stained with calcein AM for 30 minutes, they were washed three times, and the tumor cell concentration was adjusted to 1.5×10 5 cells / mL and seeded into a 96-well cell culture plate at 100 μL / well. IBR822 was adjusted to concentrations of 6.0×10 6 cells / mL, 3.0×10 6 cells / mL, 1.5×10 6 cells / mL, 5×10 5 cells / mL, 1.7×10 5 cells / mL, 5.6×10 4 cells / mL, 1.9×10 4 cells / mL, 6.2×10 3 cells / mL. Six replicate wells were created for each concentration gradient and added to the 96-well plate at 100 μL / well, and the final effector-to-target ratio was adjusted to be 40:1, 20:1, 10:1, 3:1, 1:1, 1:3, 1:9, 1:27. The cells were cultured at 37 °C and 5% CO2 for 6 hours.

[0194] 120 μL / well of the supernatant was transferred in parallel to a fully black 96-well microplate, and the fluorescence intensity was measured by selecting an excitation wavelength of 490 nm and an emission wavelength of 535 nm.

[0195] With the final effector-to-target ratio of the test product on the x-axis and the average value of the cell lysis rate on the y-axis, a regression model of a four-parameter equation was selected to create an "S" curve, and the software automatically generated the "C" value, that is, the half-maximal effective concentration (EC 50 50). The results are shown in Table 6 below.

[0196]

Table 6

[0197] The results indicate that IBR822 has significant damaging activity against the above-mentioned tumor cells, with an EC50 value of 0.15 - 4.00, a maximum damage rate exceeding 65%, and the damaging effect showing a dose-effect relationship.

[0198] [Example 4: Evaluation of factor release function of conjugate IBR822] In this study, the functional effects of conjugate IBR822 on cytokine secretion were investigated. The cytokines studied included IL-2 (interleukin-2), IL-6 (interleukin-6), IL-8 (interleukin-8), TNFα (tumor necrosis factor α), IFNγ (interferon-γ), CCL2 (chemokine 2), CCL3 (chemokine 3), and CCL5 (chemokine 5). A commercially available ELISA reagent kit was used to measure cytokine levels in various cell culture supernatants according to the manufacturer's instructions.

[0199] NCI-H292 was selected as the representative tumor cell line. Conjugated IBR822 and NCI-H292 cells (8,000 cells / well) were co-incubated at different effector-to-target ratios (1:1, 3:1, 10:1, with corresponding control groups excluding tumor cells). CCL2, CCL3, CCL5, IL-2, IL-6, IL-8, IFN-γ, and TNF-α secretion levels in the cell culture supernatant were then measured by ELISA. The results are shown in Table 7.

[0200] [Table 7]

[0201] The results showed that the secretion levels of each factor were lower than the average levels in healthy individuals.

[0202] [Example 5: Efficacy study of conjugate IBR822 in an MDA-MB-468 tumor-bearing mouse model] 4 x 10 per NCG mouse 6 Individual MDA-MB-468 cells were seeded, and the tumor volume was approximately 30-60 mm. 3 When the tumor volume reached a certain point, the animals were randomly divided into groups according to their tumor volume and administered the drug. The experiment consisted of 10 animals per group, divided into the following five groups: Group 1: Solvent control (pharmaceutical buffer) group, Group 2: IL-15 group, Group 3: IL-15+IBR822 low dose (5.0×10 8 cells / kg) group, Group 4: IL-15 + IBR822 medium dose (1.0×10 9 cells / kg) group, Group 5: IL-15+IBR822 high dose (1.5×10 9 The cells were divided into groups (cells / kg).

[0203] In an in vivo efficacy study, exogenous IL-15 levels were increased, extending the duration of IBR822 in animals. IL-15 (0.18 mg / kg) was administered intraperitoneally once a week for a total of three times. Other groups received tail vein injections twice a week for a total of six times. Body weight and tumor volume were measured twice a week, and on day 20, M-NCG mice were euthanized and the tumor mass was weighed.

[0204] Table 8 shows the design of the drug efficacy experiment in a mouse model of human breast cancer cells MDA-MB-468 transplanted tumor.

[0205] [Table 8]

[0206] The results were as follows: 1) Body weight and clinical observations: In this example, no significant decrease in body weight or behavioral abnormalities were observed in any of the mice, indicating that the mice had good tolerance to IBR822 injection under these experimental conditions.

[0207] 2) Tumor volume: On day 20 of administration, the mean tumor volume of Group 1 (solvent control group) was 161.44 ± 6.46 mm². 3 Compared to the solvent control group, the mean tumor volume for group 2 (IL-15 group), group 3 (IL-15 + IBR822 low-dose group), group 4 (IL-15 + IBR822 medium-dose group), and group 5 (IL-15 + IBR822 high-dose group) was 160.18 ± 11.36 mm², respectively. 3 , 118.03±3.49mm 3, 107.83 ± 5.29 mm 3 , and 105.43 ± 4.27 mm 3 and the tumor growth inhibition rates (TGI) are 0.74%, 51.64%, 63.63%, and 66.95% respectively.

[0208] 3) Tumor weight: The average tumor weight of the solvent control group was 0.146 ± 0.009 g, and the average tumor weights of the IL-15 group, IL-15 + IBR822 low-dose group, IL-15 + IBR822 medium-dose group, and IL-15 + IBR822 high-dose group were 0.155 ± 0.011 g, 0.118 ± 0.005 g, 0.109 ± 0.007 g, and 0.102 ± 0.005 g respectively. Compared with the solvent control group, the tumor weight reduction rates were -6.16%, 19.18%, 25.34%, and 30.14% respectively.

[0209] Overall, in the IL-15 + IBR822 low-dose group, IL-15 + IBR822 medium-dose group, and IL-15 + IBR822 high-dose group, the tumor volume and weight were significantly reduced compared with the solvent control group (p < 0.01), showing a significant tumor suppression effect, and a dose-dependent relationship was observed. See Figures 12, 13 and Table 9 for specific results.

[0210]

Table 9

[0211] Compared with the solvent control group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Tumor volume V = 0.5 × a × b 2 , where a and b represent the major axis and minor axis of the tumor respectively.

[0212] 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% is calculated, that is, at a specific time point, T iis the average tumor volume after the start of administration in the administration group, T0 is the average tumor volume at the first administration in the administration group, V0 is the average tumor volume at the first administration in the solvent control group, and V i is the average tumor volume after the start of administration in the solvent control group. Also, T / C = T RTV / C RTV × 100%, where T RTV is the RTV of the administration group, and C RTV is the RTV of the solvent control group.

[0213] Therefore, under the experimental conditions of this example, animals administered IB822 in the range of 5.0 × 10 8 ~1.5 × 10 9 cells / kg showed good tolerance. Intravenous administration of IL-15 + IBR822 (twice a week for a total of 6 administrations) significantly inhibited tumor growth in a dose-dependent manner in human breast cancer cell MDA-MB-468 transplanted mice.

[0214] [Example 6: Pharmacodynamic study of conjugate IBR822 on a mouse model bearing human prostate cancer cells DU145] Human prostate cancer DU145 cells in the logarithmic growth phase were collected and inoculated at 5 × 10 6 cells / mouse under the left rib of NCG mice. Wait until the average tumor volume grows to about 30 - 60 mm 3 . Thirty-two NCG mice were randomly divided into 4 groups based on tumor size: (1) Negative control group (frozen preparation buffer), (2) IBR822 cell low-dose group (5.0 × 10 8 cells / kg), (3) IBR822 cell medium-dose group (1.0 × 10 9 cells / kg), (4) IBR822 cell high-dose group (1.5 × 10 9 cells / kg). There were 8 females in each group. Each group was administered twice a week for a total of 4 weeks according to the animal's body weight. The body weight was weighed, observed, and the tumor volume was measured twice every week. On D29, the body weight was weighed, the tumor volume was measured, the mice were euthanized, the tumor mass was weighed, and photographs were taken.

[0215] Table 10 shows the experimental design of the drug efficacy in a mouse model of transplanted tumors of human prostate cancer cell DU145.

[0216] [Table 10]

[0217] The results were as follows. Body weight: At the end of the experiment, no deaths were observed in the mice of each administration group, and there was no significant change in body weight compared with the negative control group.

[0218] Tumor volume (TV): The tumor volume of the mice in the negative control group (frozen preparation buffer) was 300 ± 27 mm 3 Compared with this, the tumor volumes of the low, medium, and high dose groups of IBR822 cells were 231 ± 21 mm 3 (P < 0.05), 233 ± 11 mm 3 (P < 0.05), 211 ± 17 mm 3 (P < 0.01). (For details, see Table 11 and Figure 14).

[0219] Relative tumor volume (RTV) and T / C: Compared with the relative tumor volume of the negative control group (frozen preparation buffer) of 6.35 ± \alpha, the relative tumor volumes of the low, medium, and high dose groups of IBR822 cells were 4.92 ± 0.47 (P < 0.05), 5.05 ± 0.37 (P < 0.05), and 4.50 ± 0.32 (P < 0.01), respectively. The T / Cs were 77.51%, 79.62%, and 70.85%, respectively. (For details, see Table 11 and Figure 15).

[0220] Under the conditions of this experiment, the IBR822 cell injection solution inhibited tumor growth in a mouse transplanted tumor model of human prostate cancer (DU145) cells, and no significant cytotoxicity was observed. For specific results, see Figure 14, Figure 15, and Table 11.

[0221] [Table 11]

[0222] Compared to the cryopreservation preparation buffer group, * p < 0.05 and ** p < 0.01. Tumor volume V = 0.5 × a × b 2 a and b represent the longest diameter and widest diameter of the tumor, respectively.

[0223] T / C% is the relative growth rate of the tumor, and T / C% = (T i -T0) / (V i It is calculated as -V0) × 100%, that is, at a specific point in time, T i V0 is the mean tumor volume after the start of administration in the treatment group, T0 is the mean tumor volume at the first dose in the treatment group, V0 is the mean tumor volume at the first dose in the solvent control group, and V i This represents the mean tumor volume after the start of administration in the solvent control group. Also, T / C = T RTV / C RTV ×100%, here T RTV This is the RTV of the treatment group, and C RTV This is the RTV of the solvent control group.

[0224] Any embodiments or exemplary language provided herein (e.g., the use of "etc." or "like" is intended to better illustrate the invention and, unless otherwise claimed, does not limit the scope of the invention. The language herein should not be construed as indicating that any non-claimed element is necessary for the implementation of the invention.

[0225] All publications and patent applications cited herein are incorporated herein by reference and by means of such reference as each individual publication or patent application is specifically and individually indicated. Furthermore, any theories, mechanisms, demonstrations, or discoveries described herein are intended to further enhance the understanding of the present invention and are not intended to limit the invention in any way to such theories, mechanisms, demonstrations, or discoveries. The present invention has been shown and described in detail in the accompanying drawings and the foregoing, but should be considered illustrative and not limiting to the invention.

Claims

1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or its antigen-binding fragment, and the anti-Trop2 antibody or its antigen-binding fragment is conjugated to the NK cell via a linker.

2. The anti-Trop2 antibody or its antigen-binding fragment HCDR1, which is shown in Sequence ID No. 5, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No.

5. HCDR2, which is shown in Sequence ID No. 6, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 6, HCDR3, which is shown in Sequence ID No. 7, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 7, LCDR1, which is shown in Sequence ID No. 8, or which has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No.

8. LCDR2 shown in Sequence ID No. 9, or having at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No. 9, and The LCDR3 comprises the sequence shown in Sequence ID No. 10, or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in Sequence ID No.

10. Here, the amino acid sequences of HCDR and LCDR are defined by Kabat. The antibody-natural killer cell (NK cell) conjugate according to claim 1.

3. The antibody-natural killer cell (NK cell) conjugate according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or its antigen-binding fragment is shown in SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3 or has at least 80%, 85%, 90%, 95%, or 99% identity with the sequence shown 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-Trop2 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-Trop2 antibody is a complete antibody, a single-chain antibody (scFv), or a bispecific antibody, and / or The antigen-binding fragment of the anti-Trop2 antibody is Fab, Fab', Fv, or F(ab'). ２ and / or, The anti-Trop2 antibody is a humanized antibody or a fully human antibody, and / or The anti-Trop2 antibody is a monoclonal antibody and / or The anti-Trop2 antibody is an IgG1, IgG2, or IgG4 isotype, and / or The anti-Trop2 antibody comprises a constant light chain region of the κ subtype or λ subtype, and / or The aforementioned anti-Trop2 antibody specifically binds to Top2 in primates (e.g., humans or rhesus monkeys), but does not bind to Top2 in rodents (e.g., rats or mice). The antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 4.

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

7. CD16 of the aforementioned conjugate ＋ 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 mentioned above are obtained from in vitro culture and proliferation of NK cells derived from peripheral blood mononuclear cells (PBMCs), or The NK cells mentioned above are obtained from in vitro culture and proliferation of NK cells derived from umbilical cord blood, or The NK cells mentioned above are obtained from in vitro culture and proliferation of NK cell lines, or The NK cells are obtained from in vitro induction, culture, and proliferation 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 according to any one of claims 1 to 8, wherein the anti-Trop2 antibody or its antigen-binding fragment and the NK cells are conjugated via a click chemistry reaction of a linker.

10. The antibody-natural killer cell (NK cell) conjugate according to claim 9, wherein the anti-Trop2 antibody or its antigen-binding fragment and the NK cells are conjugated via a first linker and a second linker, the first linker being conjugated to the anti-Trop2 antibody or its antigen-binding fragment, the second linker being conjugated to the NK cells, and the first linker and the second linker being conjugated to each other to form an antibody-NK cell conjugate.

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

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

13. The first linker has the following structure: 【Chemistry 1】 [In the formula, n is an integer between 0 and 8.] The antibody-natural killer cell (NK cell) conjugate according to claim 12, wherein the antibody is dibenzoazacyclooctinyl-glutaryl-aminopolyethylene glycol-acetylpiperidine pentafluorophenyl ester having the above properties.

14. The first linker has the following structure: 【Chemistry 2】 The antibody-natural killer cell (NK cell) conjugate according to claim 13, wherein the antibody is dibenzoazacyclooctinyl-glutaryl-aminotetraethylene glycol-acetylpiperidine pentafluorophenyl ester having the above properties.

15. The antibody-natural killer cell (NK cell) conjugate according to claim 10, wherein the second linker is azidoacetylated cyclohexosamine, for example, azidoacetylated cyclogalactosamine, or azidoacetylated glucosamine.

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

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

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

19. CD3 － CD56 ＋ CD16 ＋ The number of cells accounts for at least 95%, preferably at least 98% of the total number of cells in the cell population, 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, The cell population according to claim 18.

20. CD3 ＋ CD56 ＋ The number of cells accounts for 5% or less of the total number of cells in the aforementioned cell population, and / or CD3 ＋ CD19 ＋ The number of cells accounts for 2% or less of the total number of cells in the aforementioned 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 aforementioned cell population. The cell population according to claim 18 or 19.

21. The antibody-natural killer cell (NK cell) conjugate constitutes 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. A cell population according to any one of claims 18 to 20.

22. A pharmaceutical composition comprising 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, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may be provided in the form of a sterile fresh preparation or a sterile cryopreserved preparation.

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

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

25. A pharmaceutical composition according to any one of claims 22 to 24, for use in treating tumors in an organism, particularly tumors in which tumor cells highly express Trop2 (Trop2+).

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

27. The pharmaceutical composition according to claim 25, wherein the tumor is a malignant tumor.

28. The pharmaceutical composition according to claim 25, wherein the tumor is cancer.

29. 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, stomach cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers. The pharmaceutical composition according to claim 28.

30. The pharmaceutical composition according to 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, cervical cancer, and endometrial cancer or uterine cancer.

31. 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 pharmaceutical for the treatment of tumors in an individual, particularly tumors in which tumor cells highly express Top2 (Top2+).

32. The use according to claim 31, wherein the tumor is a solid tumor.

33. The use according to claim 31, wherein the tumor is a malignant tumor.

34. The use according to claim 31, wherein the tumor is cancerous.

35. The use according to claim 34, 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, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

36. The use according to claim 34, 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, cervical cancer, and endometrial cancer or uterine cancer.

37. A method for treating a tumor in an individual, particularly a tumor in which tumor cells highly express Top2 (Trop2+), comprising administering to the individual an effective amount of an antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 17, a cell population according to any one of claims 18 to 21, or a pharmaceutical composition according to any one of claims 22 to 31.

38. The method according to claim 37, wherein the tumor is a solid tumor.

39. The method according to claim 37, wherein the tumor is a malignant tumor.

40. The method according to claim 37, wherein the tumor is cancerous.

41. The method according to claim 40, 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, cervical cancer, endometrial cancer or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal 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 metastatic lesions of these cancers.

42. The method according to claim 40, 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, cervical cancer, and endometrial cancer or uterine cancer.

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