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

Abstract

The present application provides an anti-Trop2 antibody-natural killer cell conjugate, a pharmaceutical composition comprising the conjugate, and the medical use and preparation method of the conjugate.

Description

Technical Field

[0001] The present application generally relates to the field of biopharmaceuticals, and specifically, to anti-Trop2 antibody-natural killer cell conjugates, pharmaceutical compositions comprising the conjugates, and pharmaceutical uses and preparation methods of the conjugates. Background Art

[0002] The national cancer statistics released by the National Cancer Center in January 2019 show that in 2015, there were about 3.929 million cases of malignant tumors and about 2.338 million deaths nationwide. Compared with historical data, the cancer burden continues to rise. In terms of the number of cases, lung cancer, liver cancer, upper digestive system tumors, colorectal cancer and female breast cancer are still the main malignant tumors in my country. The national cancer statistics released by the National Cancer Center in February 2022 show that in 2016, there were about 4.064 million cases of malignant tumors and about 2.4135 million deaths nationwide. Compared with the data in 2015, the number of new cases and deaths from malignant tumors is still on the rise, among which lung cancer has always been the cancer with the highest incidence in my country.

[0003] The number of patients with advanced tumors is relatively high due to the fact that the early symptoms of tumors are not obvious, some tumors are discovered early but recur later, and the tumor itself is highly malignant. At present, the commonly used clinical treatments for solid tumors include surgery, radiotherapy, chemotherapy, conventional biological therapy, and traditional Chinese medicine treatment. These treatments often have certain toxic side effects. After surgical resection of the cancerous body, there may be complications of varying degrees, and there is a risk of recurrence after surgery. Radiotherapy and chemotherapy are the treatments with the greatest toxic side effects, which will cause a certain degree of damage to the patient's body. Although traditional Chinese medicine treatment has few toxic side effects, it often only plays an auxiliary role. Conventional biological treatments also have many unavoidable toxic side effects. However, for patients with advanced solid tumors, most are no longer suitable for surgery, radiotherapy or synchronous radiotherapy and chemotherapy, and have failed systemic standard treatments.

[0004] Therefore, there is an urgent need to explore new treatment methods for tumors in this field. SUMMARY OF THE INVENTION

[0005] In a first aspect, the present application provides an antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or the antigen-binding fragment thereof is coupled to the NK cell via a linker.

[0006] In a second aspect, the present application provides a cell population comprising the antibody-natural killer cell (NK cell) conjugate described in the first aspect.

[0007] In a third aspect, the present application provides a pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate described in the first aspect or the cell population described in the second aspect, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition is provided in the form of a sterile fresh preparation or a sterile frozen preparation.

[0008] In a fourth aspect, the present application provides the antibody-natural killer cell (NK cell) conjugate described in the first aspect or the cell population described in the second aspect for use in the preparation of a method for treating an individual tumor, particularly a tumor cell that highly expresses Trop2 (Trop2 + ) in medicine for treating tumors.

[0009] In a fifth aspect, the present application provides a method for treating a tumor in an individual, particularly a tumor cell that highly expresses Trop2 (Trop2 + ) tumor, the method comprising administering to the individual an effective amount of the antibody-natural killer cell (NK cell) conjugate described in the first aspect, the cell population described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0010] As non-limiting examples, the present application provides the following embodiments:

[0011] 1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or the antigen-binding fragment thereof is coupled to the NK cell via a linker.

[0012] 2. The antibody-natural killer cell (NK cell) conjugate according to embodiment 1, wherein the anti-Trop2 antibody or antigen-binding fragment thereof comprises:

[0013] A HCDR1 as shown in SEQ ID NO:5 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as shown in SEQ ID NO:5,

[0014] a HCDR2 as set forth in SEQ ID NO:6 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:6,

[0015] a HCDR3 as set forth in SEQ ID NO:7 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:7,

[0016] A LCDR1 as set forth in SEQ ID NO:8 or having at least 80%, 85%, 90%, 95% or 99% identity thereto,

[0017] A LCDR2 as set forth in SEQ ID NO:9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:9, and

[0018] LCDR3 as set forth in SEQ ID NO: 10 or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 10;

[0019] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0020] 3. The antibody-natural killer cell (NK cell) conjugate of embodiment 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof is as 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 as 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.

[0021] 4. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-3, 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.

[0022] 5. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-4, wherein:

[0023] The anti-Trop2 antibody is a whole antibody, a single-chain antibody (scFv) or a bispecific antibody; and / or

[0024] The antigen-binding fragment of the anti-Trop2 antibody is Fab, Fab', Fv or F(ab') 2 ; and / or

[0025] The anti-Trop2 antibody is a humanized antibody or a fully human antibody; and / or

[0026] The anti-Trop2 antibody is a monoclonal antibody; and / or

[0027] The anti-Trop2 antibody is of IgG1, IgG2 or IgG4 isotype; and / or

[0028] The anti-Trop2 antibody comprises a light chain constant region of a κ subtype or a λ subtype; and / or

[0029] The anti-Trop2 antibody specifically binds to primate (eg, human or rhesus monkey) Trop2, for example, and does not bind to rodent (eg, rat or mouse) Trop2.

[0030] 6. The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 5, wherein the NK cell is CD16 + and / or NKG2D + , preferably CD16 + NKG2D + .

[0031] 7. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-6, wherein the CD16 + NKG2D + The proportion of NK cells is at least 90%, preferably, CD56 + The NK cell ratio is at least 95%.

[0032] 8. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-7, wherein

[0033] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC); or

[0034] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood; or

[0035] The NK cells are obtained from the in vitro culture and expansion of NK cell lines; or

[0036] The NK cells are obtained from in vitro induction, culture and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells (MSC).

[0037] 9. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-8, wherein the anti-Trop2 antibody or antigen-binding fragment thereof and the NK cell are coupled together by a click chemistry reaction of a linker.

[0038] 10. The antibody-natural killer cell (NK cell) conjugate as described in embodiment 9, wherein the anti-Trop2 antibody or its antigen-binding fragment and the NK cell are coupled together by a first linker and a second linker, the first linker is coupled to the anti-Trop2 antibody or its antigen-binding fragment, the second linker is coupled to the NK cell, and the first linker and the second linker are coupled to form the antibody-NK cell conjugate.

[0039] 11. The antibody-natural killer cell (NK cell) conjugate as described in embodiment 10, wherein the first linker is an active ester, which can form a coupling with a lysine residue of the antibody by reacting an ester bond to an amide bond, for example, the active ester is a pentafluorophenyl ester, such as pentafluorophenyl pipecolic acid.

[0040] 12. An antibody-natural killer cell (NK cell) conjugate as described in embodiment 11, wherein the first linker also comprises a carbon-carbon triple bond structure that can undergo a cyclization reaction with an azide group to form a triazole five-membered ring, for example, the carbon-carbon triple bond structure is an octyne group.

[0041] 13. The antibody-natural killer cell (NK cell) conjugate according to embodiment 12, wherein the first linker is dibenzazepine octyne-glutaryl-amino ethylene glycol acetal-acetyl pipecolic acid pentafluorophenyl ester, the structure of which is as follows:

[0042]

[0043] Wherein n is an integer from 0 to 8.

[0044] 14. The antibody-natural killer cell (NK cell) conjugate according to embodiment 13, wherein the first linker is dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester, and the structure is as follows:

[0045]

[0046] 15. The antibody-natural killer cell (NK cell) conjugate of embodiment 10, wherein the second linker is an azidoacetylated cyclohexosamine, for example, azidoacetylated galactosamine or azidoacetylated glucosamine.

[0047] 16. The antibody-natural killer cell (NK cell) conjugate according to embodiment 15, wherein the second linker is 1,3,4,6-oxy-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose, and the structure is as follows:

[0048]

[0049] 17. The antibody-natural killer cell (NK cell) conjugate of embodiment 16, wherein the proportion of the α or β single configuration of the second linker is at least 90%.

[0050] 18. A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-17.

[0051] 19. The cell population as described in embodiment 18, wherein 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.

[0052] 20. The cell population of embodiment 18 or 19, wherein:

[0053] CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population; and / or

[0054] CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population; and / or

[0055] CD3 + CD4 + and CD3 + CD8 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0056] 21. A cell population as described in any of embodiments 18-20, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total cell number in the cell population, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

[0057] 22. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate described in any one of embodiments 1-17 or the cell population described in any one of embodiments 18-21, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is optionally provided in the form of a sterile fresh preparation or a sterile frozen preparation.

[0058] 23. The pharmaceutical composition of embodiment 22, comprising sodium chloride and / or human serum albumin.

[0059] 24. The pharmaceutical composition of embodiment 22 or 23, comprising trehalose, sucrose, dextran, DMSO or any combination thereof.

[0060] 25. A pharmaceutical composition as described in any one of embodiments 22-24, which is used to treat a tumor in an individual, especially a tumor cell that highly expresses Trop2 (Trop2 + ) tumors.

[0061] 26. A pharmaceutical composition as described in embodiment 25, wherein the tumor is a solid tumor.

[0062] 27. A pharmaceutical composition as described in embodiment 25, wherein the tumor is a malignant tumor.

[0063] 28. A pharmaceutical composition as described in embodiment 25, wherein the tumor is cancer.

[0064] 29. A pharmaceutical composition as described in embodiment 28, wherein the cancer is selected from: 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 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 and head and neck cancer, and metastases of the above cancers.

[0065] 30. A pharmaceutical composition as described in embodiment 28, wherein the cancer is selected from: 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 or uterine cancer.

[0066] 31. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-17 or the cell population of any one of embodiments 18-21 is used in the preparation of a method for treating a tumor in an individual, especially a tumor cell that highly expresses Trop2 (Trop2 + ) in medicine for treating tumors.

[0067] 32. The use according to embodiment 31, wherein the tumor is a solid tumor.

[0068] 33. The use according to embodiment 31, wherein the tumor is a malignant tumor.

[0069] 34. The use according to embodiment 31, wherein the tumor is cancer.

[0070] 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 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, and head and neck cancer, and metastases of the above cancers.

[0071] 36. The use according to embodiment 34, wherein the cancer is selected from: 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 or uterine cancer.

[0072] 37. Treating tumors in individuals, especially those whose tumor cells highly express Trop2 (Trop2 + ) of a tumor, the method comprising administering to the individual an effective amount of an antibody-natural killer cell (NK cell) conjugate as described in any one of embodiments 1-17, or a cell population as described in any one of embodiments 18-21, or a pharmaceutical composition as described in any one of embodiments 22-31.

[0073] 38. The method of embodiment 37, wherein the tumor is a solid tumor.

[0074] 39. The method of embodiment 37, wherein the tumor is a malignant tumor.

[0075] 40. The method of embodiment 37, wherein the tumor is cancer.

[0076] 41. A method as described in embodiment 40, wherein the cancer is selected from: 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 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 and head and neck cancer, and metastases of the above cancers.

[0077] 42. The method of embodiment 40, wherein the cancer is selected from: 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 or uterine cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 A schematic diagram of the structure of an exemplary anti-Trop2 antibody-natural killer cell conjugate of the present application is shown.

[0079] Figure 2 Flow cytometry results of IBR822 preparation (lot number FE20230105) are shown: CD56 + 、CD3 - 、CD16 + NKG2D + 、CD19 - .

[0080] Figure 3 Flow cytometric analysis results for UNK and IBR822 preparations (lot number FE20230105) are shown for coupling positivity and coupling geometric mean.

[0081] Figure 4 A schematic diagram of the process flow for preparing the L2 linker is shown.

[0082] Figure 5 The structural formula of the L2 linker is shown, and the Chinese name is: (Z)-19-(1'-aza-2'-keto-dibenzo[b,f]cyclo-7'-octynyl)-3,6,9,12-tetraoxa-15-aza-16,20-dione-eicosylpiperidine-4-acid pentafluorophenyl ester; the English name is: 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.

[0083] Figure 6 A schematic diagram of the process flow for preparing the N1 linker is shown.

[0084] Figure 7 The structural formula of the N1 linker is shown, and the Chinese name is 1,3,4,6-oxy-tetraacetyl-2-azidoacetylamido-2-deoxy-a,bD-galactose; the English name is 1,3,4,6-tetra-O-acetyl-2-azidoacetylamido-2-deoxy-a,bD-mannopyanose.

[0085] Figure 8 A schematic diagram of the coupling mechanism of the L2 linker to the anti-Trop2 antibody is shown.

[0086] Fig. 9A schematic diagram of the coupling mechanism of N1 linker and NK cells is shown.

[0087] Fig.10 A schematic diagram of the UNK structure with an N1 linker attached is shown.

[0088] Fig.11 A schematic diagram of the coupling mechanism of UNK and L2-conjugated antibodies is shown.

[0089] Fig.12 The figure shows the tumor volume curves of each group of animals in the test of human breast cancer cell MDA-MB-468 transplanted tumor mice. Compared with the vehicle control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0090] Fig.13 The figure shows the comparison of tumor weight of each group of animals on day 20 in the test of human breast cancer cell MDA-MB-468 transplanted tumor mice. Compared with the vehicle control group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0091] definition

[0092] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0093] Although the numerical ranges and parameter approximations shown in the broad scope of the present application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0094] As used herein, the term "individual" or "subject" refers to a mammal, such as a human, but may also be other animals, such as wild animals, livestock or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0095] The term "antigen" as used herein is 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 naturally occurring or synthetic compounds.

[0096] In a broad sense, "antibody" may refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses a complete antibody / full-length antibody, an antibody single chain, or any antigen-binding fragment (also referred to as an "antigen-binding portion") of an antibody. When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" may be understood as a complete body relative to the "antigen-binding fragment / antigen-binding portion", and the two together correspond to the broad concept of an antibody.

[0097] The term "agonist antibody" is an antibody that elicits a response, e.g., an antibody that mimics at least one functional activity of a target polypeptide. Agonist antibodies include antibodies that are ligand mimetics, e.g., where the ligand binds to a cell surface receptor, the binding induces cell signaling or activity through an intracellular cell signaling pathway, and where the antibody induces similar cell signaling or activation.

[0098] "Full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into multiple regions with high variability, called complementary determining regions (CDRs), interspersed with multiple more conservative regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or the subclasses described above), but the antibody does not need to belong to any particular class. According to the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be designated as different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional structure of immunoglobulins of different classes are well known. Chimeric or humanized antibodies are also encompassed in antibodies according to the present application. It is well known to those skilled in the art that the complementary determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are multiple common definitions of the CDR amino acid sequence of VH or VL, such as the Kabat definition, the IMGT definition, the Chothia definition, etc. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence in the VH and VL amino acid sequence can usually be determined according to different definitions. In an embodiment of the present application, the CDR amino acid sequence is defined using Kabat. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in a variety of ways.

[0099] The term "humanized antibody" means an antibody obtained by grafting CDR sequences derived from another mammalian species, such as a mouse germline, onto human framework sequences. In order to retain binding affinity, some residues of the framework (called FR) segment may be modified. Humanized antibodies or fragments thereof according to the present application may be prepared by techniques known to those skilled in the art;

[0100] The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, such as an antibody in which the variable region sequence is from a mouse antibody and the constant region sequence is from a human antibody. The chimeric antibody or its fragment according to the present application can be prepared by using genetic recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA, which comprises a promoter and a sequence encoding the variable region of a non-human, especially mouse monoclonal antibody according to the present application, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present application encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of the antibody being determined by the variable region derived from the mouse DNA, and its isotype being determined by the constant region derived from the human DNA.

[0101] The term "partially humanized antibody" refers to an antibody that contains a constant region from human and a variable region (including CDR) from non-human (eg, mouse).

[0102] The term "semi-humanized antibody" is a type of humanized antibody, and refers to an antibody in which one antibody chain comprises a murine variable region and the other antibody chain comprises a humanized variable region, i.e., a semi-humanized antibody.

[0103] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may occur in minor individuals.

[0104] As used herein, the terms "antigen binding fragment" or "antigen binding portion" or "antigen binding region" are used interchangeably and refer to the portion of an antibody that contains the amino acid residues that interact with an antigen and confer upon the binding agent its specificity and affinity for the antigen, particularly antibody fragments such as Fv, Fab, F(ab') 2 or Fab', or any fragment that should be able to increase half-life by chemical modification, such as the addition of poly(alkylene) glycols such as polyethylene glycol ("PEGylation") (referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab') or by incorporation into liposomes. 2-PEG or Fab'-PEG PEGylated fragment) ("PEG" is polyethylene glycol). Preferably, the functional fragment will be composed of or contain a partial sequence of the heavy chain or light chain variable chain of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. Such a functional fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived. Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab') 2 A fragment which may be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (ie, a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody.

[0105] The term "single-chain antibody (scFv)" refers to a single polypeptide chain consisting of a VH domain and a VL domain connected by a peptide linker. (scFv) 2 It comprises two VH domains connected by a peptide linker and two VL domains, wherein the two VL domains are combined with the two VH domains via disulfide bridges.

[0106] The term "Fc fragment", "Fc region", "Fc domain", "Fc part" or similar terms refers to a portion of the constant region of the heavy chain of an antibody, 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 effector function-related modifications, such as to reduce or eliminate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions / mutations in the Fc region of the antibody.

[0107] 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.

[0108] The term "bispecific antibody" refers to an antibody that has the ability to bind to two antigen epitopes at the same time. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two antigen binding parts fused thereto respectively (similar to natural antibodies, the difference is that the two arms bind to different antigen targets or epitopes), and the antigen binding part can be in the form of a single-chain antibody (scFv) or a Fab fragment. The two different binding parts of the bispecific antibody each bind to the N-terminus of an Fc fragment, and the antigen binding part configuration of the two arms can have four combinations: scFv+Fab fragment, Fab fragment+scFv, scFv+scFv, Fab fragment+Fab fragment. The Fc fragment can contain mutations that can ensure heavy chain heteropolymerization, and KIH technology (knob-in-hole, KIH) is a strategy to solve heavy chain heteropolymerization. Generally, KIH technology refers to the formation of a structure that is conducive to the mutual pairing of heterogeneous half antibodies by modifying the amino acid sequence of the CH3 region, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible.

[0109] Generally, in order to prepare monoclonal antibodies or functional fragments thereof, especially murine monoclonal antibodies or functional fragments thereof, reference can be made to the techniques described in the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the techniques for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0110] The term "conservative variant" or "conservative amino acid substitution" is a substitution that does not substantially affect or reduce the affinity of the protein. For example, an antibody may include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions and specifically bind to the target antigen. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parent amino acids, as long as the antibody specifically binds to the target antigen.

[0111] The term "isolated" biological components (e.g., nucleic acids, proteins (including antibodies), or organelles) have been substantially separated or purified from other biological components (i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., cells) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids.

[0112] The term "pharmaceutical composition" used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that is combined together to achieve a certain specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present application. For example, the ingredients contained in the pharmaceutical composition (for example, the antibody-cell conjugate according to the present application) can be applied to an individual as a whole, or separately to an individual. When the ingredients contained in the pharmaceutical composition are applied separately to an individual, the ingredients can be applied to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application can include conventional components of cell culture, especially NK cell culture, to maintain the activity of NK cells in the conjugate. Pharmaceutically acceptable carriers can also include water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any suitable route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0113] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the individual to whom it is administered. The actual amount administered, as well as the rate and time course of administration will depend on the individual condition and severity of the condition being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by general practitioners and other physicians, typically taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0114] EC 50 EC value mainly refers to the concentration of drugs, antibodies or toxins that can achieve 50% of the maximum biological effect after a specific exposure time. In pharmacy, in addition to being used to characterize the activation ability of agonists in in vitro experiments, it can also be used to indicate the blood drug concentration required to achieve half of the maximum biological effect in vivo. In some literature, EC 50It is also used to characterize the efficacy of a compound at the cellular level (including agonism and antagonism). EC can be determined by ELISA and other methods. 50 value.

[0115] The term "identity / homology / consistency" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for alignment are well known in the art.

[0116] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant or malignant.

[0117] As used herein, the term "malignant tumor" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary malignant tumors include: carcinomas, solid tumors, melanoma sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignant tumors include: multiple myeloma, renal cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, liver cancer (hepatic carcinoma), gastric cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer, liver cancer (liver cancer), urinary tract cancer, hepatoma, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma and B-cell lymphoma, brain cancer and head and neck cancer and related metastatic lesions.

[0118] As used herein, the term "hematologic malignancy" refers to malignant malignancies caused by the uncontrolled growth and proliferation of abnormal cells. In most cases, the origin of these abnormal cells is the bone marrow, which is where blood cells are produced. Exemplary hematologic malignancies include various types of leukemia, multiple myeloma, and malignant lymphoma. More specific examples of hematologic malignancies include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, Nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Say-Schermer syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disease, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, not otherwise specified peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0119] The term "solid tumor" as used herein refers to a tangible mass that can be felt through clinical examinations such as X-rays, CT scans, B-ultrasound or palpation. Solid tumors that have been clinically diagnosed and treated are divided into malignant and benign tumors. Malignant solid tumors include: Childhood Hodgkin's lymphoma: lymphocyte-predominant type, nodular sclerosis type, mixed cell type, lymphocyte-depleted type; Childhood non-Hodgkin's lymphoma: prolymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.; Childhood kidney tumors: Wilms tumor (Wilms tumor), renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, etc.; Childhood neuroblastoma: neuroblastoma, ganglioneuroblastoma, ganglioneuroma; Childhood extracranial germ cell tumor: mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma, dysgerminoma, choriocarcinoma, embryonal carcinoma etc.; osteosarcoma and chondrosarcoma; childhood rhabdomyosarcoma: embryonic, alveolar, pleomorphic, etc.; childhood 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, desmoplastic small round cell tumor, etc.; Ewing family sarcoma: Ewing sarcoma, primitive neuroectodermal tumor; childhood liver tumor: hepatoblastoma (embryonal, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; 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: lymphangioma, hemangioma, thyroglossal duct cyst, etc.

[0120] For the treatment of tumors, adoptive cell immunotherapy has become a hot topic of research at home and abroad, and has achieved good results in clinical trials of tumors. Adoptive cell immunotherapy is a treatment method that separates autologous or allogeneic immune cells, amplifies a sufficient amount of immune cells with anti-tumor activity after in vitro activation or gene modification, and infuses them into tumor patients to amplify the cellular immune function in the patient's body to improve the anti-tumor effect. Currently, there are many types of cell immunotherapy under study, including chimeric antigen receptor modified T cells (CAR-T), T cell receptor gene modified T cells (TCR-T), dendritic cell (DC) vaccines, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), cytokine-induced killer cells (CIK), etc. Although CAR-T cell therapy has developed rapidly in recent years, there are still many shortcomings and challenges in clinical application, such as varying degrees of neurotoxicity and cytokine storm risks, and low efficacy in the treatment of solid tumors. In addition, most CAR-T cell immunotherapy requires autologous adoptive cell transplantation because allogeneic T cells may cause graft-versus-host disease (GVHD) unless the HLA barrier is addressed; in addition, CAR-T cell immunotherapy may produce some side effects that endanger the patient's life, such as cytokine release syndrome.

[0121] In contrast, NK cell therapy has shown good clinical effects in the treatment of hematological tumors, and has more advantages than CAR-T cell therapy: 1) NK cells do not produce graft-versus-host reactions (GVHD) like T cells, and are expected to become a universal cell therapy; 2) The life span of mature NK cells is relatively short (about 7 to 10 days), which can effectively kill tumor cells while reducing the probability of long-term adverse events; 3) NK cells are not restricted by antigen-specific major histocompatibility complex (MHC) and have stronger killing ability; 4) NK cell therapy does not secrete inflammatory factors (IL-1, IL-6, etc.), and the risk of cytokine storm and neurotoxic reactions is low; 5) The low-affinity CD16 molecule on the surface of NK cells can bind to the IgG antibody complex on the surface of target cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), and can also mediate cell apoptosis through the Fas / FasL pathway. In addition, the level of programmed death receptor 1 (PD-1) secreted by NK cells is low, and it hardly induces immunosuppression, so NK cells have good application prospects in the treatment of solid tumors.

[0122] The inventors of the present application have conducted in-depth research in the field of cell immunotherapy, and in particular have developed an anti-Trop2 antibody-natural killer cell conjugate for tumor treatment. Figure 1 A schematic diagram of the structure of an exemplary anti-Trop2 antibody-natural killer cell conjugate of the present application is shown.

[0123] Natural Killer cells (NK cells) are granular lymphocytes and are part of the human immune system. NK cells recognize target antigens without major histocompatibility complex (MHC) restriction, and the risk of allogeneic NK cells causing graft-versus-host disease (GvHD) is extremely low. Therefore, allogeneic NK cell therapy can be performed clinically. In addition, the risk of NK cells causing cytokine release syndrome (CRS) is also extremely low. In 2020, Liu E et al. published the results of a Phase I / II clinical trial of CAR-NK cells for the treatment of lymphoma in the New England Journal of Medicine, showing that after receiving CAR-NK cell infusion, 8 of the 11 subjects enrolled in the group had their condition alleviated, 7 of whom achieved complete remission, and none of them developed CRS, neurotoxicity or GvHD. It can be seen that the application of CAR-NK cell therapy is relatively safe. In addition, it is precisely because of the characteristic of NK cells that they recognize target antigens without MHC restriction that NK cells can be prepared into universal products without being restricted by autologous cells, making the source of NK cells for this therapy very broad, such as allogeneic peripheral blood, umbilical cord blood, embryonic stem cells, human induced pluripotent stem cells and NK-92 cell lines.

[0124] The killing activity of NK cells is mainly through:

[0125] 1) Direct lysis of target cells: NK cells release cytotoxic granules such as perforin and granzyme through exocytosis, activating the caspase pathway to induce necrosis or apoptosis of target cells;

[0126] 2) Secretion of cytokines: Cytokine-mediated killing effect. NK cells can synthesize and secrete a variety of cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10 and G-CSF, to induce apoptosis of target cells;

[0127] 3) Induce apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, inducing apoptosis of CD95+ target cells and TRAIL receptor-positive target cells through a cascade reaction of endogenous enzymes;

[0128] 4) ADCC: antibody-dependent cell-mediated cytotoxicity;

[0129] 5) Immune checkpoint pathway: It expresses programmed death receptor 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4), etc., and exerts its effect by inhibiting immune checkpoints.

[0130] The above multiple mechanisms of action, the potential for application as a universal product, and reliable safety make NK cell therapy an attractive immunotherapy.

[0131] The antigen target selected in this application is Trop2, whose full name is human trophoblast cell surface antigen 2 (Trophoblast Cell-Surface Antigen 2). Trop2 belongs to the TACSTD family and is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. It is also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). Trop2 is overexpressed in a variety of malignant tumors. It is an oncogene related to the occurrence, invasion and metastasis of malignant tumors and a signal transduction molecule that can regulate the growth of tumor cells. The Trop2 gene is located on the short arm of chromosome 1, specifically located at 1p32.1. The full length of the gene is 9072bp, with no introns and only 1 exon. The primary structure of the Trop2 protein is a 36kD polypeptide composed of 323 amino acids. It is a single-pass transmembrane surface glycoprotein. Trop-2 may represent a novel cell surface receptor that plays a role in the regulation of tumor cell growth and plays a role as a regulator of cell self-renewal, proliferation, and metastasis.

[0132] In a first aspect, the present application provides an antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or the antigen-binding fragment thereof is coupled to the NK cell via a linker.

[0133] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof comprises:

[0134] A HCDR1 as shown in SEQ ID NO:5 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as shown in SEQ ID NO:5,

[0135] a HCDR2 as set forth in SEQ ID NO:6 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:6,

[0136] a HCDR3 as set forth in SEQ ID NO:7 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:7,

[0137] A LCDR1 as set forth in SEQ ID NO:8 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:8,

[0138] A LCDR2 as set forth in SEQ ID NO:9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:9, and

[0139] LCDR3 as set forth in SEQ ID NO: 10 or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 10;

[0140] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0141] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof is as 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 to the sequence shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as 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 to the sequence shown in SEQ ID NO:3.

[0142] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof 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 can 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, while still maintaining the function of the heavy chain variable region of the similar antibody. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 1 is added with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains the function of the heavy chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar antibody.

[0143] In some embodiments, the amino acid sequence of the light chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof 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 can also 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, while still maintaining the function of the light chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be 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 the function of the light chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 3, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar antibody.

[0144] In some embodiments, the anti-Trop2 antibody comprises a heavy chain variable region set forth in SEQ ID NO:1, a heavy chain constant region set forth in SEQ ID NO:2, a light chain variable region set forth in SEQ ID NO:3, and a light chain constant region set forth in SEQ ID NO:4.

[0145] In some embodiments, the anti-Trop2 antibody specifically binds to primate (eg, human or rhesus monkey, etc.) Trop2, for example, and does not bind to rodent (eg, rat or mouse) Trop2.

[0146] In some embodiments, the anti-Trop2 antibody is a whole antibody, a single chain antibody (scFv), or a bispecific antibody.

[0147] In some embodiments, the antigen-binding fragment of the anti-Trop2 antibody is Fab, Fab', Fv or F(ab') 2 .

[0148] In some embodiments, the anti-Trop2 antibody is a humanized antibody or a fully human antibody.

[0149] In some embodiments, the anti-Trop2 antibody is a monoclonal antibody.

[0150] In some embodiments, the anti-Trop2 antibody is of the IgG1, IgG2, or IgG4 isotype. In some embodiments, the anti-Trop2 antibody is of the IgG1 isotype.

[0151] In some embodiments, the anti-Trop2 antibody comprises a light chain constant region of the kappa subtype or the lambda subtype.

[0152] Given an antibody structure / sequence, the techniques for preparing the corresponding antibodies are within the skill of the art.

[0153] As a non-limiting example, the sequence of the anti-Trop2 monoclonal antibody can be obtained from phage library screening, and the anti-Trop2 monoclonal antibody is selected after being screened for binding activity between Trop2 and cells expressing Trop2, species cross-species studies, and affinity studies, wherein 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 technology, in which the IB12 plasmid is electrotransfected into CHO cells, and after one round of screening of the minipool and two rounds of screening after monoclonal plating, and confirmed by preliminary subculture stability culture, a cell line that can stably express anti-Trop2 monoclonal antibodies is obtained. Anti-Trop2 monoclonal antibodies are based on production schemes known in the art, including cell culture and protein purification. Take the working cells, after recovery, 3 to 5 rounds of amplification, pilot scale culture, and then collect the untreated cell suspension (UPB), and obtain the cell culture supernatant by clarification and filtration. Then, through the protein purification process, the anti-Trop2 monoclonal antibody is obtained through a three-step chromatography method of Protein A affinity, anion exchange and cation exchange.

[0154] In some embodiments, the NK cells are CD16 + and / or NKG2D + In some embodiments, the NK cells are CD16 + NKG2D + .

[0155] In some embodiments, the CD16 + NKG2D + The proportion of NK cells is at least 90%, for example, at least 95%, at least 98% or at least 99%. In some embodiments, the CD16 + NKG2D + The proportion of NK cells is at least 90%, and CD56 + The proportion of NK cells is at least 95% (eg, at least 96%, at least 97%, at least 98% or at least 99%).

[0156] There are various techniques for culturing, expanding and obtaining NK cells in vitro, and the general principles and methodologies thereof are known to those skilled in the art.

[0157] In some embodiments, NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC), which is also an exemplary method in the embodiments of the present application. PBMC is one of the main sources of NK cells, with the advantages of relatively easy collection, easy in vitro expansion, and no toxic side effects. However, the proportion of NK cells in PBMC is only 10%-15%. The method of expanding NK cells derived from PBMC includes using a combination of cytokines, feeder cells or membrane particles to stimulate the in vitro expansion of NK cells. These different expansion systems show different levels of NK cell expansion efficiency. In some embodiments, HLA and KIR of PBMC are screened. In some embodiments, variants of CD16a of PBMC, i.e., 176V and 176F, are screened. In some embodiments, one or more cytokines are used to maintain or activate the activity of natural killer cells during culture. In some embodiments, one or more immunoglobulins or fusion proteins are used to inhibit the proliferation of B cells, macrophages and other immune cells.

[0158] PBMCs can be obtained from apheresis peripheral blood lymphocytes from allogeneic healthy donors. After separation of T cells and red blood cells, the cells are transferred to primary cell cryopreservation medium and the total number of viable cells is ≥ 6.0 × 10 7 The PBMCs are packaged in the specification of 1 cell / branch to obtain PBMCs. PBMCs are stored for a long time in liquid nitrogen at ≤-175°C. The PBMCs frozen in liquid nitrogen are taken out and revived in an adapted culture medium. By expanding the culture scale (i.e., increasing the culture medium expansion), NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their ability to divide and proliferate and their activity. An exemplary preparation method can be found in Example 1 of the present application.

[0159] In some embodiments, NK cells are obtained by in vitro culture and expansion of NK cells from umbilical cord blood. There are generally two different methods to obtain a large number of NK cells from umbilical cord blood. One method is to expand NK cells in umbilical cord blood, and the other method is to induce umbilical cord blood CD34 + Hematopoietic stem / progenitor cells differentiate into NK cells and then expand.

[0160] In some embodiments, the NK cells are obtained from in vitro culture and expansion of NK cell lines. As an example, NK-92 is the first NK cell-based immunotherapy approved by the FDA for clinical trials and is a homogenous immortalized NK lymphoma cell.

[0161] In some embodiments, NK cells are obtained from the in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells (MSC).

[0162] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof and the NK cells are coupled together via a click chemistry reaction of a linker.

[0163] Click chemistry, also known as "linking chemistry" and "speed-matching combinatorial chemistry", is a synthetic concept introduced by chemist Barry Sharpless in 2001. Its main purpose is to quickly and reliably complete the chemical synthesis of various molecules by splicing small units. There are four main types of click chemistry reactions: cycloaddition reactions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, and carbon-carbon multiple bond addition reactions.

[0164] In some embodiments, the anti-Trop2 antibody or its antigen-binding fragment and the NK cells are coupled together by a first linker and a second linker, wherein the first linker is coupled to the anti-Trop2 antibody or its antigen-binding fragment, and the second linker is coupled to the NK cells, and the first linker and the second linker are coupled to form the antibody-NK cell conjugate.

[0165] In some embodiments, the first linker is an active ester that can be coupled to a lysine residue of an antibody by reacting an ester bond to an amide bond. For example, the active ester is a pentafluorophenyl ester, such as pentafluorophenyl pipecolic acid. The active ester can be a water-stable molecule that can specifically covalently bind to a lysine residue of an antibody. In some embodiments, the active ester is a tetraethylene glycol chain structure that slowly hydrolyzes in water. In some embodiments, the active ester reacts with an amino group on the hydrophilic interface of a protein to react an ester bond to an amide bond, thereby establishing a coupling mechanism.

[0166] In some embodiments, the first linker further comprises a carbon-carbon triple bond structure capable of undergoing a cyclization reaction with an azide group to form a triazole five-membered ring, for example, the carbon-carbon triple bond structure is an octyne group.

[0167] In some embodiments, the first linker is dibenzazepine octyne-glutaryl-aminoethylene glycol acetal-acetyl pipecolic acid pentafluorophenyl ester, the structure of which is shown below:

[0168]

[0169] Wherein n is an integer from 0 to 8.

[0170] In some embodiments, the first linker is dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester, the structure of which is shown below:

[0171]

[0172] Dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester is also referred to as L2 linker in this application. For the synthesis route of L2 linker, see Figure 4 , see the structural formula Figure 5 , including three main synthetic steps: amide condensation, hydrolysis reaction and synthesis of active ester.

[0173] Step 1 - Amide condensation

[0174] Dibenzazepine octyne glutaric acid (L2-1) and aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-2) are commercially available, and the intermediate dibenzazepine octyne glutaryl aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-3) is obtained by chemical condensation reaction. L2-3 is a stable compound.

[0175] Specifically, compound L2-1 and compound L2-2 (1:1.1, L2-2 in excess) were dissolved in a dichloromethane (DCM) solution, hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) were added respectively, and finally triethanolamine (TEA) was added. The mixture was stirred at room temperature for 4 to 12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified with a silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil compound L2-3.

[0176] Step 2-Hydrolysis

[0177] The intermediate L2-3 obtained in step 1 is subjected to hydrolysis reaction to obtain the intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetyl pipecolic acid (L2-4). The hydrolysis reaction is usually a quantitative reaction and is directly used in the next step of synthesis without further purification.

[0178] Specifically, compound L2-3 is dissolved in a mixed solution of methanol and water, and a 1 mol / L lithium hydroxide (LiOH) aqueous solution is added after cooling to 0°C. The mixed solution is stirred at 0°C to room temperature for 4-12 hours, acidified with 1 mol / L HCl to a pH between 2-3, extracted with ethyl acetate (EA) three times, and dried to obtain a yellow oil, namely the intermediate dibenzazepine octyne glutaramidoacetal tetraethylene glycol acetyl pipecolic acid (L2-4).

[0179] Step 3-Synthesis of active ester

[0180] The intermediate L2-4 obtained in step 2 (without purification) is condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2, ie, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester.

[0181] Specifically, compound L2-4 is dissolved in tetrahydrofuran (THF), cooled to 0°C, and then 2-pentafluorophenol, hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) are added. The mixture is stirred at 0°C to room temperature for 4 to 12 hours, extracted with ethyl acetate (EA) three times, dried, and purified by silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester (L2), the structural formula of which is shown in Figure 5 .

[0182] Subsequently, the L2 linker can be coupled to the anti-Trop2 monoclonal antibody in a PBS pH 7.2-7.4 system (preferably HEPES pH 7.2) for site-directed coupling reaction (see schematic diagram). Figure 8 ). Usually, one anti-Trop2 monoclonal antibody can be coupled to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to about 5.0 to obtain an L2-coupled antibody.

[0183] In some embodiments, the second linker is an azidoacetylated cyclohexosamine, for example, an azidoacetylated galactosamine or an azidoacetylated glucosamine. In some embodiments, the second linker is a water-stable molecule that can specifically covalently bind to a sialic acid-modified membrane protein. In some embodiments, the second linker is transferred to a sialic acid-modified membrane protein through 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.

[0184] In some embodiments, the second linker is 1,3,4,6-oxo-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose, the structure of which is shown below:

[0185]

[0186] In some embodiments, the proportion of the α or β single configuration of 1,3,4,6-oxo-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose is at least 90%, for example, at least 95%, at least 98% or at least 99%.

[0187] 1,3,4,6-O-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose is also referred to as N1 linker in this application. The synthetic route of N1 linker is shown in Figure 6 , see the structural formula Figure 7 , including two main synthetic steps: aminoazide acetylation and hydroxyl acetylation.

[0188] Step 1 - Acetylation of aminoazide

[0189] 1.2 times of a-azidoacetic acid (compound 1), 2 times of hydroxybenzotriazole (HOBt) and triethylamine (Et 3 N), added to the N,N-dimethylformamide (DMF) solution of D-galactosamine hydrochloride (compound 2), added a small amount of methanol (MeOH) to dissolve, reacted at room temperature for 12 hours, poured the reaction mixture into dichloromethane (DCM) / methanol (MeOH) and oscillated, added ether to precipitate the oily product (compound 3), poured out the ether layer, repeated twice, and vacuum dried. Without further purification, directly proceeded to the next step.

[0190] Step 2 - Hydroxyacetyl esterification

[0191] The compound 3 obtained above was added with anhydrous pyridine (Pyr.) and acetic anhydride (Ac 2 O), add 4-dimethylaminopyridine (DMAP) catalyst at room temperature, react for 12 hours, HPLC detection shows that the reaction is almost complete, concentrate, and obtain a racemic mixture of tetraacetyl-N-azidoacetyl-a, b-D-galactosamine. The solid is precipitated from ethyl acetate / petroleum ether, mainly b-configuration product, with a purity of >85%. Further purification by silica gel column (dichloromethane: methanol = 20:1) obtains N1 product with a single optical isomer purity greater than 90%. The a, b-isomers can isomerize to each other in the cell, and after multiple steps of metabolism and synthesis, they are converted to N-azidoacetyl sialic acid, and finally expressed on the surface glycoprotein of NK cells. For the structural formula of N1 linker, see Figure 7 .

[0192] When the NK cells are cultured for 15 to 16 days, N1 linkers are added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (referred to as "UNK" in the embodiments of the present application) (see the connection mechanism between N1 linkers and NK cells and the structural schematic diagram of UNK). Fig. 9 and 10 ).

[0193] Finally, to obtain antibody-NK cell conjugates, UNK and L2-conjugated antibodies can be conjugated in culture medium (reaction mechanism see Fig.11 ) to obtain antibody-NK cell conjugate.

[0194] Subsequent processes also include preparing antibody-NK cell conjugate preparations to effectively prolong the stability of the antibody-NK cell conjugates. The preparation formula may contain ingredients such as sodium chloride and human serum albumin to maintain isotonicity, and may also contain ingredients such as trehalose, sucrose, dextran, and DMSO to maintain the cell's low-temperature resistance and the activity of proteins and enzymes.

[0195] In a second aspect, the present application provides a cell population comprising the antibody-natural killer cell (NK cell) conjugate described in the first aspect.

[0196] In some embodiments, CD3 - CD56 + CD16 + In some embodiments, the number of cells in the cell population is at least 95% of the total number of cells in the cell population. - CD56 + CD16 + In some embodiments, the number of cells in the cell population is at least 98% of the total number of cells in the cell population. - CD56 + NKG2D + In some embodiments, the number of cells in the cell population is at least 95% of the total number of cells in the cell population. - CD56 + NKG2D + The number of cells accounts for at least 98% of the total number of cells in the cell population.

[0197] In some embodiments, CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population.

[0198] In some embodiments, CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0199] In some embodiments, CD3 + CD4 + and CD3 + CD8 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0200] In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total number of cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 95% of the total number of cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 98% of the total number of cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 99% of the total number of cells in the cell population by cell number.

[0201] In a third aspect, the present application provides a pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate described in the first aspect or the cell population described in the second aspect, and a pharmaceutically acceptable carrier.

[0202] Where conditions permit, the pharmaceutical composition can be provided directly to the patient in the form of a sterile fresh preparation. If long-term storage (e.g., for multiple administrations) or transportation is required, the pharmaceutical composition can be provided in the form of a sterile frozen preparation, which is thawed and revived when used.

[0203] In some embodiments, the pharmaceutical composition comprises sodium chloride and / or human serum albumin.

[0204] In some embodiments, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0205] In some embodiments, the pharmaceutical composition is used to treat a tumor in an individual.

[0206] In some embodiments, the tumor is a tumor that overexpresses Trop2 + In some embodiments, the tumors with high expression of Trop2 (Trop2 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express Trop2.

[0207] In some embodiments, the tumor is a solid tumor.

[0208] In some embodiments, the tumor is a hematological tumor.

[0209] In some embodiments, the tumor is a malignant tumor.

[0210] In some embodiments, the tumor is cancer.

[0211] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial 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, and head and neck cancer, and metastases of the above cancers.

[0212] In some embodiments, the cancer is selected from the group consisting of lung cancer (eg, 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 or uterine cancer.

[0213] In a fourth aspect, the present application provides use of the antibody-natural killer cell (NK cell) conjugate described in the first aspect or the cell population described in the second aspect in the preparation of a medicament for treating a tumor in an individual.

[0214] In some embodiments, the tumor is a tumor that overexpresses Trop2 + In some embodiments, the tumors with high expression of Trop2 (Trop2 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express Trop2.

[0215] In some embodiments, the tumor is a solid tumor.

[0216] In some embodiments, the tumor is a hematological tumor.

[0217] In some embodiments, the tumor is a malignant tumor.

[0218] In some embodiments, the tumor is cancer.

[0219] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial 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, and head and neck cancer, and metastases of the above cancers.

[0220] In some embodiments, the cancer is selected from the group consisting of lung cancer (eg, 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 or uterine cancer.

[0221] In a fifth aspect, the present application provides a method for treating a tumor in an individual, the method comprising administering to the individual an effective amount of the antibody-natural killer cell (NK cell) conjugate described in the first aspect, the cell population described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0222] In some embodiments, the tumor is a tumor that overexpresses Trop2 + In some embodiments, the tumors with high expression of Trop2 (Trop2 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 +) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor that highly expresses Trop2 (Trop2 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express Trop2.

[0223] In some embodiments, the tumor is a solid tumor.

[0224] In some embodiments, the tumor is a hematological tumor.

[0225] In some embodiments, the tumor is a malignant tumor.

[0226] In some embodiments, the tumor is cancer.

[0227] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial 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, and head and neck cancer, and metastases of the above cancers.

[0228] In some embodiments, the cancer is selected from the group consisting of lung cancer (eg, 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 or uterine cancer.

[0229] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments. Example

[0230] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0231] Example 1 - Preparation of anti-Trop2 antibody-NK cell conjugates

[0232] The preparation of the antibody-NK cell conjugate of this embodiment can be roughly divided into the following steps:

[0233] (1) Preparation of anti-Trop2 monoclonal antibodies;

[0234] (2) Preparation of NK cells;

[0235] (3) Preparation of antibody linker (referred to as L2 linker in this example);

[0236] (4) Preparation of NK cell linkers (referred to as N1 linkers in this example);

[0237] (5) an antibody linker (L2 linker) is applied to the antibody;

[0238] (6) applying an NK cell linker (N1 linker) to NK cells; and

[0239] (7) Conjugating antibodies and NK cells respectively carrying linkers.

[0240] (1) Preparation of anti-Trop2 monoclonal antibodies

[0241] In brief, the sequence of the anti-Trop2 monoclonal antibody is obtained from phage library screening, and is obtained after being screened for binding activity between Trop2 and cells expressing Trop2, cross-species studies of humans, monkeys, and mice, and confirmed by affinity studies, and the anti-Trop2 monoclonal antibody is selected, wherein 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.

[0242] The construction of the monoclonal cell line is based on known technology, in which the IB12 plasmid is transfected into CHO cells by electroporation, and after one round of screening of the minipool and two rounds of screening after monoclonal plating, and confirmed by preliminary passage stability culture, a cell line that can stably express the anti-Trop2 monoclonal antibody is obtained.

[0243] The anti-Trop2 monoclonal antibody is based on a production scheme known in the art, including cell culture and protein purification. Working cells are taken, resuscitated, amplified for 3 to 5 rounds, and cultured on a pilot scale, and then the untreated cell suspension (UPB) is collected and the cell culture supernatant is obtained by clarification and filtration. Then, through the protein purification process, the anti-Trop2 monoclonal antibody is obtained through a three-step chromatography method of Protein A affinity, anion exchange, and cation exchange.

[0244] (2) Preparation of NK cells

[0245] PBMCs are collected from healthy allogeneic donors. After separation of T cells and red blood cells, the cells are transferred to primary cell cryopreservation medium and the total number of viable cells is ≥ 6.0 × 10 7 PBMCs are obtained by packaging in the specification of 1 cell / tube. PBMCs are stored in liquid nitrogen at ≤-175℃ for a long time.

[0246] The PBMCs frozen in liquid nitrogen are taken out and revived in an adapted culture medium. By expanding the culture scale (i.e., increasing the culture medium expansion), NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their ability to divide and proliferate and their activity. Exemplary NK cell culture stages, process parameters, and process control indicators are shown in Table 1 below.

[0247] Table 1

[0248]

[0249]

[0250]

[0251] The final cell purity quality control of NK cells meets the requirements in Table 2 below (the remaining cell density, biosafety, etc. requirements comply with industry standards):

[0252] Table 2.

[0253]

[0254] Figure 2 The results of flow cytometry detection of the above immunological markers of the final antibody-NK cell conjugate preparations are shown.

[0255] (3) Preparation of Antibody Linker (Referred to as L2 Linker in This Example)

[0256] For a synthetic route to the L2 linker, see Figure 4 , see the structural formula Figure 5 , including three main synthetic steps: amide condensation, hydrolysis reaction and synthesis of active ester.

[0257] Step 1 - Amide condensation

[0258] Dibenzazepine octyne glutaric acid (L2-1) and aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-2) are commercially available, and the intermediate dibenzazepine octyne glutaryl aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-3) is obtained by chemical condensation reaction. L2-3 is a stable compound.

[0259] Specifically, compound L2-1 and compound L2-2 (1:1.1, L2-2 in excess) were dissolved in a dichloromethane (DCM) solution, hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) were added respectively, and finally triethanolamine (TEA) was added. The mixture was stirred at room temperature for 4 to 12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified with a silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil compound L2-3.

[0260] Step 2-Hydrolysis

[0261] The intermediate L2-3 obtained in step 1 is subjected to hydrolysis reaction to obtain the intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetyl pipecolic acid (L2-4). The hydrolysis reaction is usually a quantitative reaction and is directly used in the next step of synthesis without further purification.

[0262] Specifically, compound L2-3 is dissolved in a mixed solution of methanol and water, and a 1 mol / L lithium hydroxide (LiOH) aqueous solution is added after cooling to 0°C. The mixed solution is stirred at 0°C to room temperature for 4-12 hours, acidified with 1 mol / L HCl to a pH between 2-3, extracted with ethyl acetate (EA) three times, and dried to obtain a yellow oil, namely the intermediate dibenzazepine octyne glutaramidoacetal tetraethylene glycol acetyl pipecolic acid (L2-4).

[0263] Step 3-Synthesis of active ester

[0264] The intermediate L2-4 obtained in step 2 (without purification) is condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2, ie, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester.

[0265] Specifically, compound L2-4 is dissolved in tetrahydrofuran (THF), cooled to 0°C, and then 2-pentafluorophenol, hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) are added. The mixture is stirred at 0°C to room temperature for 4 to 12 hours, extracted with ethyl acetate (EA) three times, dried, and purified by silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester (L2), the structural formula of which is shown in Figure 5 .

[0266] (4) Preparation of NK cell linker (referred to as N1 linker in this example)

[0267] For the synthetic route of N1 linker, see Figure 6 , see the structural formula Figure 7 , including two main synthetic steps: aminoazide acetylation and hydroxyl acetylation.

[0268] Step 1 - Acetylation of aminoazide

[0269] 1.2 times of a-azidoacetic acid (compound 1), 2 times of hydroxybenzotriazole (HOBt) and triethylamine (Et 3 N), added to the N,N-dimethylformamide (DMF) solution of D-galactosamine hydrochloride (compound 2), added a small amount of methanol (MeOH) to dissolve, reacted at room temperature for 12 hours, poured the reaction mixture into dichloromethane (DCM) / methanol (MeOH) and oscillated, added ether to precipitate the oily product (compound 3), poured out the ether layer, repeated twice, and vacuum dried. Without further purification, directly proceeded to the next step.

[0270] Step 2 - Hydroxyacetyl esterification

[0271] The compound 3 obtained above was added with anhydrous pyridine (Pyr.) and acetic anhydride (Ac 2 O), add 4-dimethylaminopyridine (DMAP) catalyst at room temperature, react for 12 hours, HPLC detection shows that the reaction is almost complete, concentrate, and obtain a racemic mixture of tetraacetyl-N-azidoacetyl-a, b-D-galactosamine. The solid is precipitated from ethyl acetate / petroleum ether, mainly b-configuration product, with a purity of >85%. Further purification by silica gel column (dichloromethane: methanol = 20:1) obtains N1 product with a single optical isomer purity greater than 90%. The a, b-isomers can isomerize to each other in the cell, and after multiple steps of metabolism and synthesis, they are converted to N-azidoacetyl sialic acid, and finally expressed on the surface glycoprotein of NK cells. For the structural formula of N1 linker, see Figure 7 .

[0272] (5) Applying the antibody linker (L2 linker) to the antibody

[0273] The L2 linker and anti-Trop2 monoclonal antibody can undergo site-specific coupling reaction in a PBS pH 7.2-7.4 system (preferably HEPES pH 7.2) (see schematic diagram for details). Figure 8 ). Usually, one anti-Trop2 monoclonal antibody can be coupled to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to about 5.0 to obtain an L2-coupled antibody.

[0274] (6) Applying NK cell linker (N1 linker) to NK cells

[0275] When the NK cells are cultured for 15 to 16 days, N1 linkers are added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (referred to as "UNK" in the embodiments of the present application) (see the connection mechanism between N1 linkers and NK cells and the structural schematic diagram of UNK). Fig. 9 and 10 ).

[0276] (7) Coupling antibodies and NK cells with linkers

[0277] UNK and L2-conjugated antibody are allowed to undergo coupling reaction in the culture medium (see reaction mechanism for details). Fig.11 ) to obtain a stock solution (DS) of the antibody-NK cell conjugate. The anti-Trop2 monoclonal antibody-NK cell conjugate was named "IBR822", and this name will be used in subsequent examples to represent the anti-Trop2 antibody-NK cell conjugate.

[0278] Subsequent processes also include preparing antibody-NK cell conjugate preparations to effectively prolong the stability of the antibody-NK cell conjugates. The preparation formula may contain ingredients such as sodium chloride and human serum albumin to maintain isotonicity, and may also contain ingredients such as trehalose, sucrose, dextran, and DMSO to maintain the cell's low-temperature resistance and the activity of proteins and enzymes.

[0279] The purity of NK cells in the antibody-NK cell conjugate preparation was tested by flow cytometry, and the result was over 98%, exceeding the standard requirement of 95% ( Figure 2 In addition, flow cytometry was used to detect the coupling positive rate of the antibody-NK cell conjugate stock solution and the preparation, and UNK was used as a control. The results showed that the coupling positive rate of the preparation exceeded 98% ( Figure 3 ), usually the standard requirement is 90%.

[0280] Example 2-Affinity and species cross-detection of L2-conjugated antibodies for Trop2

[0281] In this example, the OctetR8 instrument was used to detect the affinity of the L2-coupled antibody prepared in step (5) of Example 1 for Trop2 of different species (BLI method). Specifically, various Trop2 proteins were loaded on the His-K probe and bound to the free L2-coupled antibody in the solution (diluted in PBS (pH 7.4)). The maximum response value was used to determine whether the binding occurred. The dissociation constant KD value was calculated based on the association rate and dissociation rate. The results are as follows:

[0282] Table 3.

[0283] antigen Dissociation constant (mol) <![CDATA[Binding rate constant (mol -1 ·s -1 )]]> <![CDATA[Dissociation rate constant (s -1 )]]> Human Trop2 <![CDATA[2.26×10 -8 ]]> <![CDATA[5.64×10 5 ]]> <![CDATA[1.27×10 -2 ]]> Rhesus monkey Trop2 <![CDATA[<1×10 -12 ]]> <![CDATA[5.69×10 5 ]]> <![CDATA[<1×10 -7 ]]> Rat Trop2 The maximum response value is less than 0.01 N / A N / A Mouse Trop2 The maximum response value is less than 0.01 N / A N / A

[0284] It can be seen from the results in Table 3 that the antibodies obtained in the present application are specific to primate Trop2 such as humans or rhesus monkeys, and do not bind to rodent Trop2 such as rats or mice.

[0285] Example 3- Evaluation of the in vitro killing activity of the conjugate IBR822 against different cancer cells

[0286] In the evaluation of cell killing activity in this example, 11 tumor cell lines expressing Trop2 were selected. The cell line names, cancer types, and pre-determined Trop2 expression levels (determined by flow cytometry) are shown in Table 4 below:

[0287] Table 4.

[0288] Cell lines Trop2 expression rate Cancer Type NCI-N87 95.3％ Gastric cancer HeLa 93.8％ Human cervical cancer cells SiH 99.4％ Human uterine squamous cell carcinoma MDA-MB-468 99.9％ Human breast cancer cells NCI-H292 99.6％ Human mucoepidermoid carcinoma lung strain HCC827 86.8％ Non-small cell lung cancer NCI-H1975 98.5％ Human non-small cell lung cancer adenocarcinoma cells BxPc 99.8％ Pancreatic cancer Colo205 87.0％ Colon cancer AGS 99.6％ Gastric cancer Capan-2 88.8％ Human pancreatic cancer

[0289] First, the real-time label-free cell analysis technology was used to detect the cell killing activity of the conjugate IBR822 and the mixture of NK cells (obtained in step (2) of Example 1) and UNK cells (obtained in step (6) of Example 1) + L2 conjugated antibody (obtained in step (5) of Example 1) as a reference against four tumor cell lines (BxPC3, NCI-H1975, MDA-MB-468, AGS). A tumor cell control group was set up in the experiment. 8000 tumor cells were inoculated in each well of each group, and then NK cells, UNK cells and conjugate IBR822 were added (in terms of NK cells, 16000 cells were inoculated in each well, and L2 conjugated antibody was also added to the UNK cell group, and the amount of L2 conjugated antibody added was the same as the amount of antibody carried by the conjugate IBR822). The RTCA instrument was set to monitor and record the cell index (CI) value every 15 minutes, and the cell killing rate was calculated based on the change in the cell index.

[0290] Table 5 below shows the killing effects of NK cells, UNK cells + L2 conjugated antibody mixture and conjugate IBR822 on tumor cells BxPC3, NCI-H1975, MDA-MB-468, and AGS, among which the killing rate of conjugate IBR822 on all four tumor cells reached more than 80%, which was higher than that of NK cells and UNK cells + L2 conjugated antibody mixture.

[0291] Table 5.

[0292]

[0293] Next, the Calcein AM release method was used to detect the dose-effect relationship of the conjugate IBR822 in killing tumor cells, using NCI-H292 cells as a representative. IBR822 was set at 3 effect-target ratio concentrations, with 6 replicates for each concentration. The fluorescence intensity change was detected by an ELISA instrument and the cell killing rate was calculated. The results are shown in Table 6 below.

[0294] Table 6.

[0295]

[0296] The results showed that the killing effect of IBR822 on tumor cells had a dose-effect relationship.

[0297] Example 4- Evaluation of the factor release function of conjugate IBR822

[0298] This example conducts functional studies on the cytokine secretion caused by the conjugate IBR822, and the cytokines studied include: IL-2 (interleukin-2), IL-6 (interleukin-6), IL-8 (interleukin-8), TNFα (tumor necrosis factor α), IFNγ (interferon gamma), CCL2 (chemokine 2), CCL3 (chemokine 3), CCL5 (chemokine 5). For the measurement of cytokines, commercial ELISA kits were used to measure various cell culture supernatants according to the manufacturer's operating instructions.

[0299] NCI-H292 was selected as a representative tumor cell line.

[0300] After the conjugate IBR822 was co-incubated with NCI-H292 cells (8000 cells / well) at different effector-target ratios (1:1, 3:1, 10:1, the corresponding control group did not contain tumor cells), the secretion levels of CCL2, CCL3, CCL5, IL2, IL6, IL8, IFNγ, and TNFα in the cell culture supernatant were determined by ELISA. The results are shown in Table 7.

[0301] Table 7.

[0302]

[0303] The results showed that the secretion levels of each factor were lower than the average level of healthy people.

[0304] Example 5 - Efficacy study of conjugate IBR822 in MDA-MB-468 tumor-bearing mouse model

[0305] NCG mice were inoculated with 4×10 6 After 100 MDA-MB-468 cells, wait until the tumor volume reaches 30-60 mm 3At about 1:10 pm, the animals were randomly divided into 5 groups according to the tumor volume, with 10 animals in each group:

[0306] Group 1 - Vehicle Control (Formulation Buffer),

[0307] Group 2 - IL-15,

[0308] Group 3-IL-15+IBR822 low dose (5.0×10 8 cells / kg),

[0309] Group 4: IL-15+IBR822 medium dose (1.0×10 9 cells / kg),

[0310] Group 5-IL-15+IBR822 high dose (1.5×10 9 cells / kg) group.

[0311] In the in vivo efficacy test, exogenous addition of IL-15 increased the duration of IBR822 in the animal body. IL-15 (0.18 mg / kg) was administered by intraperitoneal injection once a week for a total of 3 times. The other groups were administered by tail vein injection twice a week for a total of 6 times. The body weight and tumor volume were measured twice a week, and the NCG mice were killed on the 20th day to obtain the tumor mass for weighing.

[0312] The design of the efficacy experiment of the human breast cancer cell MDA-MB-468 transplanted tumor mouse model is shown in Table 8.

[0313] Table 8.

[0314]

[0315] -: Not applicable; the day of group administration is day 0;

[0316] BIW: twice weekly; W: week; iv: intravenous injection; ip: intraperitoneal injection.

[0317] The results show that:

[0318] 1) Body weight and clinical observation: In this example, all mice did not show obvious weight loss and abnormal behavior, indicating that the mice had good tolerance to IBR822 injection under this experimental condition;

[0319] 2) Tumor volume: On the 20th day after administration, the average tumor volume of Group 1 (vehicle control group) was 161.44±6.46mm 3Compared with the vehicle control group, the average tumor volumes of 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) were 160.18 ± 11.36 mm 3 、118.03±3.49mm 3 、107.83±5.29mm 3 and 105.43±4.27mm 3 , tumor growth inhibition rates (TGI) were 0.74%, 51.64%, 63.63% and 66.95%, respectively;

[0320] 3) Tumor weight: The average tumor weight of the vehicle 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 vehicle control group, the tumor weight reduction rates were -6.16%, 19.18%, 25.34% and 30.14%, respectively.

[0321] In general, compared with the vehicle control group, the tumor volume and weight of the IL-15+IBR822 low-dose group, IL-15+IBR822 medium-dose group, and IL-15+IBR822 high-dose group were significantly reduced (p<0.01), showing a significant tumor inhibition effect, and dose dependence was observed. For specific results, see Fig.12 , Fig.13 and Table 9.

[0322] Table 9.

[0323]

[0324] Compared with the vehicle control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0325] Tumor volume V = 0.5 × a × b 2 , a, b represent the long diameter and wide diameter of the tumor, respectively;

[0326] Tumor growth inhibition rate TGI (%) = (1-T / C) × 100%. T / C% is the relative tumor growth rate, T / C% = (T i -T 0 ) / (V i -V 0)×100%, that is, at a certain time point, Ti is the average tumor volume of the drug-treated group after the start of drug administration, T 0 is the average tumor volume of the drug-treated group at the first administration, V 0 is the average tumor volume of the vehicle control group at the first administration, V i is the average tumor volume of the vehicle control group after the start of drug administration; T / C = T RTV / C RTV × 100%, where T RTV is the RTV of the drug-treated group, C RTV is the RTV of the vehicle control group.

[0327] Therefore, under the experimental conditions of this example, 5.0×10 8 ~1.5×10 9 Animals treated with IB822 in the cell / kg range tolerated it well. IL-15+IBR822 administered by tail vein (twice a week, 6 times in total) significantly inhibited the growth of tumors in mice transplanted with human breast cancer cells MDA-MB-468 in a dose-dependent manner.

[0328] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present application and does not limit the scope of the present application unless otherwise required. Language in the specification should not be construed as indicating that any non-claimed element is essential to the practice of the present application.

[0329] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance the understanding of the application, and is not intended to limit the application to such theory, mechanism, proof or discovery in any way. Although the application has been shown and described in detail in the accompanying drawings and the preceding description, the application should be considered illustrative rather than restrictive.

Claims

1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or the antigen-binding fragment thereof is coupled to the NK cell via a linker, wherein the anti-Trop2 antibody or the antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO:7, LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:10; The amino acid sequences of HCDR and LCDR are based on the Kabat definition. The NK cells are CD16 + NKG2D + , wherein the anti-Trop2 antibody or antigen-binding fragment thereof and the NK cell are coupled together by a first linker and a second linker, wherein the first linker is coupled to the anti-Trop2 antibody or antigen-binding fragment thereof, and the second linker is coupled to the NK cell, and the first linker and the second linker are coupled to form the antibody-NK cell conjugate, The first linker is dibenzazepine octyne-glutaryl-aminoethylene glycol acetal-acetyl pipecolic acid pentafluorophenyl ester, and the structure is shown below: Where n is an integer from 0 to 8, and The second linker is azidoacetylated galactosamine or azidoacetylated glucosamine.

2. The antibody-natural killer cell (NK cell) conjugate of claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or its antigen-binding fragment is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

3.

3. The antibody-natural killer cell (NK cell) conjugate of claim 2, 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.

4. The antibody-natural killer cell (NK cell) conjugate according to claim 1, wherein: The anti-Trop2 antibody is a monoclonal antibody or a single-chain antibody (scFv); or The antigen-binding fragment of the anti-Trop2 antibody is Fab, Fab', Fv or F(ab')2; or The anti-Trop2 antibody is a humanized antibody or a fully human antibody; or The anti-Trop2 antibody is of IgG1, IgG2 or IgG4 isotype; or The anti-Trop2 antibody comprises a light chain constant region of a κ subtype or a λ subtype; or The anti-Trop2 antibody specifically binds to primate Trop2 but does not bind to rodent Trop2.

5. The antibody-natural killer cell (NK cell) conjugate according to claim 1, wherein The NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC); or The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood; or The NK cells are obtained from the in vitro culture and expansion of NK cell lines; or The NK cells are obtained from in vitro induction, culture and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells (MSC).

6. The antibody-natural killer cell (NK cell) conjugate according to claim 1, wherein the first linker is dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester, and the structure is as follows:

7. The antibody-natural killer cell (NK cell) conjugate according to claim 1, wherein the second linker is 1,3,4,6-oxy-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose, and the structure is as follows:

8. The antibody-natural killer cell (NK cell) conjugate of claim 7, wherein the proportion of the α or β single configuration of the second linker is at least 90%.

9. A cell population comprising the antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 8.

10. The cell population of claim 9, wherein CD3 - CD56 + CD16 + The number of cells accounts for at least 95% of the total number of cells in the cell population, and / or CD3 - CD56 + NKG2D + The number of cells accounts for at least 95% of the total number of cells in the cell population.

11. The cell population of claim 9, wherein CD3 - CD56 + CD16 + The number of cells accounts for 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 98% of the total number of cells in the cell population.

12. The cell population according to claim 9 or 10, wherein: CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population; and / or CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population; and / or CD3 + CD4 + and CD3 + CD8 + The number of cells does not exceed 2% of the total number of cells in the cell population.

13. The cell population of claim 9 or 10, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total cell number in the cell population in terms of cell number.

14. The cell population of claim 9 or 10, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 95% of the total cell number in the cell population based on cell number.

15. The cell population of claim 9 or 10, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 98% of the total cell number in the cell population based on cell number.

16. The cell population of claim 9 or 10, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 99% of the total cell number in the cell population based on cell number.

17. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 8 or the cell population according to any one of claims 9 to 16, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, comprising sodium chloride and / or human serum albumin.

19. The pharmaceutical composition according to claim 17 or 18, comprising trehalose, sucrose, dextran, DMSO or any combination thereof.

20. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is provided in the form of a sterile fresh preparation or a sterile frozen preparation.

21. Use of the antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 8 or the cell population according to any one of claims 9 to 16 in the preparation of a medicament for treating a tumor in an individual, wherein the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) tumors.

22. The use according to claim 21, wherein the tumor is a solid tumor.

23. The use according to claim 21, wherein the tumor is a malignant tumor.

24. The use according to claim 21, wherein the tumor is cancer.

25. The method of claim 23, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial 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, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, and head and neck cancer, and metastases thereof.

26. The use of claim 24, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, cervical cancer, and endometrial or uterine cancer.

27. The use according to claim 25 or 26, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma.

28. The use of claim 25, wherein the squamous cell carcinoma is squamous epithelial cell carcinoma.

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