Anti-Trop2 antibody and anti-5T4 antibody-natural killer cell conjugates and uses thereof

Abstract

The present application provides anti-Trop2 antibody and anti-5T4 antibody-natural killer cell conjugates, pharmaceutical compositions comprising the conjugates, and pharmaceutical uses and preparation methods of the conjugates.

Description

Technical Field

[0001] The present application generally relates to the field of biopharmaceuticals. Specifically, the present application relates to anti-Trop2 antibody and anti-5T4 antibody-natural killer cell conjugates, pharmaceutical compositions containing the conjugates, and pharmaceutical uses and preparation methods of the conjugates. Background Art

[0002] According to national cancer statistics released by the National Cancer Center in January 2019, in 2015, there were approximately 3.929 million cases of malignant tumors and approximately 2.338 million deaths nationwide. Compared with historical data, the cancer burden continues to rise. Lung cancer, liver cancer, upper digestive system tumors, colorectal cancer, and female breast cancer remain the leading malignant tumors in my country. According to national cancer statistics released by the National Cancer Center in February 2022, in 2016, there were approximately 4.064 million cases of malignant tumors and approximately 2.4135 million deaths nationwide. Compared with 2015 data, the number of new cases and deaths from malignant tumors continues to rise, with lung cancer consistently being the most common cancer in my country.

[0003] The number of patients with advanced tumors is relatively high due to the lack of obvious early symptoms, the fact that some tumors are discovered early but recur later, and the high malignancy of the tumor itself. Currently, the commonly used clinical treatments for solid tumors include surgery, radiotherapy, chemotherapy, conventional biological therapy, and Traditional Chinese Medicine (TCM). These treatments often have certain toxic side effects. Surgical resection of the cancer may be accompanied by varying degrees of complications and the risk of recurrence after surgery. Radiotherapy and chemotherapy are the treatments with the most toxic side effects and will cause a certain degree of damage to the patient's body. Although Traditional Chinese Medicine (TCM) has fewer toxic side effects, it often only serves as an auxiliary aid. Conventional biological treatments also have many unavoidable toxic side effects. However, for patients with advanced solid tumors, the majority are no longer suitable for surgery, radiotherapy, or concurrent chemoradiotherapy, and have failed standard systemic 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 an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or its antigen-binding fragment and the anti-5T4 antibody or its antigen-binding fragment are both 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.

[0008] 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 tumors in an individual.

[0009] 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.

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

[0011] 1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibodies are anti-Trop2 antibodies or antigen-binding fragments thereof and anti-5T4 antibodies or antigen-binding fragments thereof, and the anti-Trop2 antibodies or antigen-binding fragments thereof and anti-5T4 antibodies or antigen-binding fragments thereof are coupled to the NK cells via a linker.

[0012] 2. The antibody-natural killer cell (NK cell) conjugate according to embodiment 1, wherein the anti-5T4 antibody or antigen-binding fragment thereof comprises:

[0013] a HCDR1 as set forth in SEQ ID NO: 15 or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 15,

[0014] a HCDR2 as set forth in SEQ ID NO: 16, or a HCDR2 having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 16,

[0015] a HCDR3 as set forth in SEQ ID NO: 17, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 17,

[0016] a LCDR1 as set forth in SEQ ID NO: 18, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 18,

[0017] a LCDR2 as set forth in SEQ ID NO: 19, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 19, and

[0018] a LCDR3 as set forth in SEQ ID NO: 20, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 20;

[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-5T4 antibody is as shown in SEQ ID NO: 11 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 13 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO: 13.

[0021] 4. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-3, wherein the Trop2 antibody comprises:

[0022] a HCDR1 as set forth in SEQ ID NO: 5 or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 5,

[0023] a HCDR2 as set forth in SEQ ID NO: 6, or a HCDR2 having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 6,

[0024] a HCDR3 as set forth in SEQ ID NO: 7, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 7,

[0025] a LCDR1 as set forth in SEQ ID NO: 8, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 8,

[0026] a LCDR2 as set forth in SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 9, and

[0027] a 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;

[0028] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0029] 5. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-4, wherein the amino acid sequence of the heavy chain variable region of the Trop2 antibody 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.

[0030] 6. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-5, wherein the anti-5T4 antibody and the Trop2 antibody are independently full-length antibodies, and optionally, the amino acid sequence of the heavy chain constant region of the anti-5T4 antibody is as shown in SEQ ID NO: 12 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO: 12, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 14 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO: 14.

[0031] 7. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-6, wherein the anti-5T4 antibody and the Trop2 antibody are independently full-length antibodies, optionally wherein the amino acid sequence of the heavy chain constant region of the anti-Trop2 antibody is as shown in SEQ ID NO:2 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO:2, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:4 or has at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in SEQ ID NO:4.

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

[0033] The anti-5T4 antibody and the Trop2 antibody are independently full-length antibodies; and / or

[0034] The anti-5T4 antibody and the Trop2 antibody are independently humanized antibodies or fully human antibodies; and / or

[0035] The anti-5T4 antibody and the Trop2 antibody are independently monoclonal antibodies; and / or

[0036] The anti-5T4 antibody and the Trop2 antibody are independently of IgG1, IgG2 or IgG4 isotype; and / or

[0037] The anti-5T4 antibody and the Trop2 antibody independently comprise a light chain constant region of the κ subtype.

[0038] 9. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-8, wherein the NK cell is CD16 + and / or NKG2D + , preferably CD16 + NKG2D + .

[0039] 10. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-9, wherein the CD16 + NKG2D + The NK cell ratio is at least 90%, preferably, CD56 + The NK cell ratio is at least 95%.

[0040] 11. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-10, wherein:

[0041] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC); or

[0042] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood; or

[0043] The NK cells are obtained by in vitro induction, culture and expansion of induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0044] 12. The antibody-natural killer cell (NK cell) conjugate according to any one of embodiments 1 to 11, wherein the anti-5T4 antibody and the Trop2 antibody are respectively coupled to the NK cells via a click chemistry reaction of a linker.

[0045] 13. The antibody-natural killer cell (NK cell) conjugate as described in embodiment 12, wherein the anti-5T4 antibody and the Trop2 antibody are coupled to the NK cell via a first linker and a second linker, a first copy of the first linker is coupled to the anti-5T4 antibody, a second copy of the first linker is coupled to the Trop2 antibody, 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.

[0046] 14. The antibody-natural killer cell (NK cell) conjugate as described in embodiment 13, wherein the first linker is an active ester, which can form a coupling with a lysine residue of the antibody through a reaction from an ester bond to an amide bond, for example, the active ester is a pentafluorophenyl ester, such as pentafluorophenyl pipecolic acid.

[0047] 15. The antibody-natural killer cell (NK cell) conjugate as described in embodiment 14, wherein the first linker further comprises a carbon-carbon triple bond structure capable of undergoing a cyclization reaction with an azide group to form a triazole five-membered ring, for example, the carbon-carbon triple bond structure is an octyne group.

[0048] 16. The antibody-natural killer cell (NK cell) conjugate according to embodiment 15, wherein the first linker is dibenzazepine octyne-glutaryl-amino ethylene glycol acetal-acetyl pipecolic acid pentafluorophenyl ester, the structure of which is shown below:

[0049] ,

[0050] Where n is an integer from 0 to 8.

[0051] 17. The antibody-natural killer cell (NK cell) conjugate according to embodiment 16, wherein the first linker is dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester, and the structure is as follows:

[0052] .

[0053] 18. The antibody-natural killer cell (NK cell) conjugate of embodiment 13, wherein the second linker is an azidoacetylated cyclohexosamine, for example, azidoacetylated cyclogalactosamine or azidoacetylated glucosamine.

[0054] 19. The antibody-natural killer cell (NK cell) conjugate according to embodiment 18, wherein the second linker is 1,3,4,6-oxy-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose, the structure of which is shown below:

[0055] .

[0056] 20. The antibody-natural killer cell (NK cell) conjugate of embodiment 19, wherein the proportion of the second linker in the α or β single configuration is at least 90%.

[0057] 21. A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-20.

[0058] 22. The cell population of embodiment 21, 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 cell number accounts for at least 95%, preferably at least 98% of the total cell number in the cell population.

[0059] 23. The cell population of embodiment 21, wherein:

[0060] CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population; and / or

[0061] CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population; and / or

[0062] CD3 + CD4 + and CD3 + CD8 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0063] 24. The cell population of any one of embodiments 21-23, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of the total cell number in the cell population, based on the cell number.

[0064] 25. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-20 or the cell population of any one of embodiments 21-24, and a pharmaceutically acceptable carrier.

[0065] 26. The pharmaceutical composition according to embodiment 25, comprising sodium chloride and / or human serum albumin, optionally in the form of a sterile fresh preparation or a sterile frozen preparation.

[0066] 27. The pharmaceutical composition of embodiment 25 or 26, comprising trehalose, sucrose, dextran, DMSO or any combination thereof.

[0067] 28. The pharmaceutical composition of any one of embodiments 25-27, for use in treating a tumor in a subject, optionally a mammal, preferably a human.

[0068] 29. The pharmaceutical composition according to embodiment 28, wherein the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0069] 30. A pharmaceutical composition as described in embodiment 29, wherein the tumor is a malignant tumor, optionally, the malignant tumor is selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer such as small cell lung cancer and non-small cell lung cancer, and pancreatic cancer.

[0070] 31. Use of the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-20 or the cell population of any one of embodiments 21-24 in the preparation of a medicament for treating a tumor in an individual.

[0071] 32. The use according to embodiment 30, wherein the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0072] 33. The use according to embodiment 32, wherein the tumor is a malignant tumor, optionally selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer such as small cell lung cancer and non-small cell lung cancer, and pancreatic cancer.

[0073] 34. The use of any one of embodiments 31-33, wherein the individual is a mammal, preferably a human.

[0074] 35. A method of 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 of any one of embodiments 1-20, or the cell population of any one of embodiments 21-24, or the pharmaceutical composition of any one of embodiments 25-30.

[0075] 36. The method according to embodiment 35, wherein the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0076] 37. The use according to embodiment 36, wherein the tumor is a malignant tumor, optionally selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer such as small cell lung cancer and non-small cell lung cancer, and pancreatic cancer.

[0077] 38. The method of any one of embodiments 35-37, wherein the individual is a mammal, preferably a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 A schematic diagram of the structure of the antibody-coupled natural killer cells of the present application is shown.

[0079] Figure 2 The figure shows a schematic diagram of the process flow for preparing the L2 linker of the present application.

[0080] Figure 3 The schematic diagram of the structure of the L2 linker of the present application is shown. The Chinese name of the L2 linker is: (Z)-19-(1'-aza-2'-keto-dibenzo[b,f]cyclo-7'-octynyl)-3,6,9,12-tetraoxa-15-aza-16,20-diketo-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.

[0081] Figure 4 The figure shows a schematic diagram of the process flow for preparing the N1 linker of the present application.

[0082] Figure 5 The schematic diagram of the structure of the N1 linker of the present application is shown. The Chinese name of the N1 linker 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.

[0083] Figure 6A schematic diagram of the process flow for preparing the L2-Trop2 monoclonal antibody is shown.

[0084] Figure 7 A schematic diagram of the process flow for preparing the L2-5T4 monoclonal antibody is shown.

[0085] Figure 8 A schematic diagram of the coupling mechanism between N1 and NK cells is shown.

[0086] Figure 9 The schematic diagram of the structure of UNK prepared in this application is shown.

[0087] Figure 10 The coupling mechanism and structural diagram of IBR825 prepared in this application are shown.

[0088] Figure 11 The killing rate of IBR825 prepared in this application on BXPC3 cells (n=3, represents P < 0.01, represents P < 0.0001).

[0089] Figure 12 The killing rate of IBR825 prepared in this application on H292 cells (n=3, represents P < 0.01, represents P < 0.001, represents P < 0.0001).

[0090] Figure 13 The killing rate of IBR825 prepared in this application on MDA-MB-468 cells (n=3, represents P < 0.0001).

[0091] Figure 14 The killing rate of IBR825 prepared in this application on Siha cells (n=3, represents P < 0.01, represents P < 0.0001, ns represents no significant difference). Detailed Description of the Invention

[0092] definition

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0094] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum 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.

[0095] 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.).

[0096] As used herein, the term "antigen" 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.

[0097] In a broad sense, "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses intact antibodies / full-length antibodies, single antibody chains, or any antigen-binding fragment of an antibody (also referred to as an "antigen-binding portion"). When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" can be understood as the intact body relative to the "antigen-binding fragment / antigen-binding portion", and the two together correspond to the broad concept of antibody.

[0098] The term "agonist antibody" is an antibody that elicits a response, e.g., an antibody that mimics at least one functional activity of a polypeptide of interest. 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.

[0099] A "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 are further subdivided into highly variable regions known as complementarity determining regions (CDRs), interspersed with more conserved regions known as 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 the binding domain that interacts with the antigen. The constant region of an antibody mediates the binding of the immunoglobulin to tissues or factors of the host, 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 subclasses of the above), but antibodies do not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant domain of the heavy chain. In general, there are five major 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 called 、 、 、 ,as well as . The subunit structure and three-dimensional structure of different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also encompassed in the 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 many common ways to define 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 sequences can usually be determined according to different definitions. In the 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.

[0100] 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 be present in minor individuals.

[0101] As used herein, the terms "antigen-binding fragment" or "antigen-binding portion" or "antigen-binding region" are used interchangeably and refer to a portion of an antibody comprising 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 can increase half-life by chemical modification, such as the addition of poly(alkylene) glycols such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" is polyethylene glycol) or by incorporation into liposomes. Preferably, the functional fragment will consist of or comprise a portion of the heavy or light variable chain sequence of the antibody from which it is derived, sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. Such a functional fragment will comprise a minimum of 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) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab'); and (3) an Fv fragment having the VL and VH domains of a single arm of an antibody.

[0102] 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 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.

[0103] The terms "Fc fragment", "Fc region", "Fc domain", "Fc portion" or similar terms refer to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 segment and the CH3 segment of the constant region. The Fc region of an antibody can be engineered or modified, including modifications related to effector functions, such as to reduce or eliminate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody.

[0104] 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.

[0105] Generally, 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).

[0106] The term "conservative variant" or "conservative amino acid substitution" refers to those substitutions that do 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.

[0107] The term "isolated" refers to a biological component (e.g., a nucleic acid, protein (including antibodies), or organelle) that has 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., a cell) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified using standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0108] The term "pharmaceutical composition" as used herein refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are 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 this application. For example, the ingredients contained in the pharmaceutical composition (such as the antibody-cell conjugate according to the present application) can be applied to an individual as a whole, or separately. When the ingredients contained in the pharmaceutical composition are applied to an individual separately, the ingredients can be applied to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application can include conventional components for 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 appropriate 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.

[0109] 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, generally taking into account the condition being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0110] 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 50 It is also used to characterize the efficacy of a compound at the cellular level (including agonism and antagonism), and EC can be determined by methods such as ELISA. 50 value.

[0111] The term "identity / homology / identity" 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 and introduction of gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for alignment are well known in the art.

[0112] The inventors of this application have chemically coupled a full-length monoclonal antibody targeting human Trop2 and a full-length monoclonal antibody targeting human 5T4, along with allogeneic healthy donor peripheral blood-derived NK cells, via linkers L and N. This results in a non-genetically engineered antibody-coupled natural killer cell (CAR-raNK) that specifically targets Trop2 and 5T4 antigens and possesses the ability to kill tumor cells. The CAR-raNK of this application first uses anti-Trop2 and anti-5T4 antibodies to specifically recognize tumor cells that highly express Trop2 and 5T4 antigens, and then uses NK cells to kill tumor cells. Figure 10 Shown are schematic diagrams of the structures of exemplary anti-Trop2 antibody and anti-5T4 antibody-natural killer cell conjugates of the present application.

[0113] Human Trop2 belongs to the TACSTD family and is a cell surface glycoprotein encoded 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 various malignancies and is an oncogene associated with tumorigenesis, invasion, and metastasis. It is a signaling molecule that regulates tumor cell growth. The Trop2 gene is located on the short arm of chromosome 1, specifically at 1p32.1. The gene is 9072 bp long, with no introns and only one exon. The primary structure of the Trop2 protein is a 36 kD polypeptide consisting of 323 amino acids. It is a single-pass transmembrane surface glycoprotein. Trop2 may represent a new cell surface receptor that plays a role in regulating tumor cell growth. Trop2 plays a regulatory role in cell self-renewal, proliferation, and metastasis.

[0114] Human 5T4, also known as embryonic trophoblast glycoprotein (5T4), is an N-glycosylated transmembrane protein with a molecular weight of 72 kDa. It is highly expressed on the surface of embryonic trophoblast cells, as well as various solid tumors (non-small cell lung cancer, breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, and ovarian cancer) and cancer stem cells. Expression is low or absent in normal adult tissues. The extracellular region of the 5T4 protein contains multiple leucine repeat sequences that are involved in protein binding and promote cell-stromal and cell-cell interactions.

[0115] NK cells recognize target antigens without major histocompatibility complex (MHC) restriction, making the risk of allogeneic NK cells causing graft-versus-host disease (GvHD) extremely low. Therefore, allogeneic NK cell therapy is feasible in clinical practice. Furthermore, 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 lymphoma treatment in the New England Journal of Medicine. Among the 11 enrolled subjects, eight achieved disease remission after receiving CAR-NK cell infusion, including complete remission in seven patients. None experienced CRS, neurotoxicity, or GvHD. This demonstrates the high safety profile of CAR-NK cell therapy. Furthermore, the lack of MHC restriction in NK cell recognition of target antigens allows NK cells to be prepared as a universal product without the restriction of autologous cells. This allows for the use of a wide range of NK cell sources for this therapy, including allogeneic peripheral blood, umbilical cord blood, embryonic stem cells, human induced pluripotent stem cells, and the NK-92 cell line.

[0116] The killing activity of NK cells is mainly through:

[0117] 1) Direct lysis of target cells: NK cells release cytotoxic granules such as perforin and granzymes through exocytosis, activating the caspase pathway to induce necrosis or apoptosis of target cells;

[0118] 2) Cytokine secretion: 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 target cell apoptosis;

[0119] 3) Induction of apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, inducing apoptosis in CD95+ target cells and TRAIL receptor-positive target cells through a cascade reaction of endogenous enzymes;

[0120] 4) ADCC: antibody-dependent cell-mediated cytotoxicity;

[0121] 5) Immune checkpoint pathway: Use immune checkpoint blocking antibodies to activate NK cells.

[0122] The above multiple mechanisms of action, the potential for application as a universal product, and reliable safety make NK cell therapy an attractive immunotherapy.

[0123] Currently, NK cells used in therapeutics include natural NK cells, CAR-NK cells, and off-the-shelf allogeneic natural killer cells (ready-to-use allogenic NK cells, raNK, available from InBaiRui (Hangzhou) Biopharmaceutical Co., Ltd.). In the field of natural NK cell therapy, CYNK-001, developed by Cellularity, has received Fast Track designation from the FDA for the treatment of acute myeloid leukemia. Similar to CAR-T cells, CAR-NK cells combine antigen-targeting capabilities with the natural killing capacity of NK cells, overcoming many limitations of other AML therapies, such as severe chemotherapy-induced myelosuppression, thereby establishing a new cellular drug platform. Furthermore, CAR-NK cells retain their natural receptors, such as NKp46, NKp30, NKp44, NKG2D, and CD226, allowing them to independently recognize their ligands, preserving the essential killing function of NK cells.

[0124] In a specific embodiment, the CAR-raNK of the present application is composed of NK cells that highly express CD16 and NKG2D, linker N, linker L, anti-Trop2 monoclonal antibody and anti-5T4 monoclonal antibody, wherein:

[0125] (1) Linker L (e.g. L2) is an active ester (pentafluorophenyl ester) with an octyne group. The structure can be found in Figure 3 , linker L can be connected to a specific lysine (Lys) residue on the light chain of a monoclonal antibody through a chemical reaction to form a conjugated antibody ( Figure 6 and Figure 7 ).

[0126] (2) Linker N (e.g., N1) is a derivative of N-acetylgalactosamine that can be added to NK cell suspensions during cell culture. It is transferred into the cells through endocytosis and, through multiple steps of metabolism and synthesis, is ultimately modified on the NK cell surface glycoprotein in the form of an N-acetylated sialic acid derivative, thereby forming universal NK (UNK) cells. For the structure of N1, the connection mechanism between N1 and NK cells, and the schematic diagram of the structure of UNK, see Figure 5 、 Figure 8 and Figure 9 .

[0127] (3) The azide group N3 of N1 on UNK forms a chemical covalent link with the carbon-carbon triple bond (alkyne group) on L2 of the conjugated antibody (click chemistry, references 1-10), thereby forming a complete antibody-conjugated natural killer cell. The structure is shown in Figure 10 .

[0128] The linker L in this application is a water-stable molecule that specifically covalently binds to a specific residue of a monoclonal antibody. In certain embodiments, the molecule is an active ester with a tetraethylene glycol chain structure that slowly hydrolyzes in water. In certain embodiments, the molecule reacts with the amino group on the hydrophilic interface of the protein, reacting the ester bond to an amide bond. In certain embodiments, the molecule contains a stable carbon-carbon triple bond structure that can undergo a cyclization reaction with the azide group to form a stable triazole five-membered ring.

[0129] The linker N in this application is a water-stable molecule that specifically covalently binds to a sialic acid-modified membrane protein. In certain embodiments, the molecule is an azidoacetylated cyclohexosamine, including but not limited to galactose and glucose. In certain embodiments, the molecule is transferred to the sialic acid-modified membrane protein during cell culture via the cell's own metabolic pathways. In certain embodiments, the azidoacetyl group of the molecule can undergo a cyclization reaction with a carbon-carbon triple bond to form a stable five-membered triazole ring.

[0130] Universal NK cells (UNK) are cells derived from allogeneic donors. Linker N is added to the culture medium during the later stages of culture, and culture is continued for a period of time to allow the membrane proteins of these NK cells to be modified with linker N. In certain embodiments, linker N is added on days 14, 15, or 16 of culture. In certain embodiments, an excess of linker N is added to ensure a positive coupling rate.

[0131] Conjugated-Antibody-Redirected raNK (CAR-raNK) cells are formed by adding a conjugated antibody to the UNK cells during the later stages of culture. In certain embodiments, the conjugated antibody is added on days 15, 16, or 17 of culture. In certain embodiments, the added conjugated antibody is a conjugate of a human IgG1 anti-human Trop2 antibody and a linker N. In certain embodiments, the positive rate of antibody-conjugated NK cell conjugation exceeds 90%, and more preferably exceeds 95%.

[0132] Allogeneic healthy donor peripheral blood mononuclear cells (PBMCs) are human peripheral blood mononuclear cells (PBMCs) collected from the peripheral blood of healthy allogeneic donors and serve as the starting material for allogeneic donor-derived natural killer (NK) cells. In certain embodiments, PBMCs are screened for HLA and KIR. In certain embodiments, PBMCs are screened for CD16a variants, namely 176V and 176F. In certain embodiments, PBMCs are isolated while depleting the majority of their T cells.

[0133] Preparation of Trop2-conjugated antibodies

[0134] In a specific embodiment, the sequence of the Trop2-conjugated antibody is obtained from a phage library screening, and the target protein and Trop2 are combined to form a single antibody. + After screening for binding activity in various cells and confirming cross-species studies in humans, monkeys, and mice, the optimal molecule was selected as the anti-Trop2 antibody. Based on the DNA sequence of the anti-Trop2 antibody, a recombinant plasmid (IB12) containing the GS gene capable of expressing the anti-Trop2 monoclonal antibody was constructed.

[0135] Monoclonal cell lines were constructed using protocols known to those skilled in the art. IB12 was electroporated into CHO cells. After one round of screening using minipools and two rounds of screening using monoclonal plating, and initial passage and culture stability confirmation, a cell line stably expressing the anti-Trop2 monoclonal antibody was obtained, serving as the primary cell bank (PCB).

[0136] Trop2 conjugated antibody, obtained by coupling the anti-Trop2 monoclonal antibody expressed from the original cell library to the linker L:

[0137] Anti-Trop2 monoclonal antibodies were obtained using a preparation protocol known to those skilled in the art. Cells from a primary cell bank were revived, expanded in shake flasks, and cultured in a 1L pilot scale. The untreated cell suspension (UPB) was collected and clarified by filtration to obtain the cell culture supernatant. The anti-Trop2 monoclonal antibody was then obtained through a three-step chromatography process consisting of Protein A affinity chromatography (PBS pH 7.0 loading, 1M NaCl wash, 0.1M GlyHCl elution), anion exchange chromatography (PBS pH 6.0 flow-through mode), and cation exchange chromatography (20mM acetic acid-sodium acetate pH 5.0 loading, 0.5M NaCl linear gradient elution).

[0138] The linker L, especially L2, is obtained by esterification of the stable chemical molecules dibenzazepine octyne glutaric acid (DBCO) and aminotetraethylene glycol acetyl pipecolic acid methyl ester with pentafluorophenyl ester in three steps. Figure 2 .

[0139] L2 and anti-Trop2 monoclonal antibody can be site-specifically coupled in a PBS pH 7.2~7.4 system ( Figure 6 ), preferably in HEPES pH 7.2. Typically, one anti-Trop2 monoclonal antibody can be conjugated to 1–4 L2 molecules, and more preferably, one anti-Trop2 monoclonal antibody can be conjugated to 1–2 L2 molecules. The reaction can be terminated by adjusting the pH to around 5.0, yielding the Trop2-conjugated antibody.

[0140] Preparation of 5T4-conjugated antibodies

[0141] In a specific embodiment, the sequence of the 5T4-conjugated antibody is obtained from phage library screening. The optimal molecule is selected as the anti-5T4 antibody after screening for binding activity between the target protein and 5T4+ cells and confirmation by cross-species studies in humans, monkeys, and mice. Based on the DNA sequence of the anti-5T4 antibody, a recombinant plasmid (IB12) containing the GS gene capable of expressing the anti-5T4 monoclonal antibody is constructed.

[0142] The construction of a monoclonal cell line was performed using protocols well known to those skilled in the art. IB12 was electroporated into CHO cells. After one round of screening using minipools and two rounds of screening using monoclonal plating, and initial stability verification through passage culture, a cell line stably expressing the anti-5T4 monoclonal antibody was obtained, serving as the primary cell bank (PCB). Under GMP conditions, the primary cell bank was then revived, expanded, packaged, and stored to establish secondary banks: the master cell bank (MCB) and the working cell bank (WCB). The stability of the master cell bank was also verified through passage culture.

[0143] 5T4-conjugated antibody, obtained by coupling the anti-5T4 monoclonal antibody expressed in the WCB pilot batch to the linker L:

[0144] The anti-5T4 monoclonal antibody was obtained using a pilot production protocol (including cell culture and protein purification) well known to those skilled in the art. Cells from a working cell bank were revived, expanded for 3-5 rounds, and cultured at a pilot scale. The untreated cell suspension (UPB) was then collected and clarified and filtered to obtain the cell culture supernatant. The anti-5T4 monoclonal antibody was then purified using a three-step protein A affinity, anion exchange, and cation exchange chromatography process.

[0145] L2 and anti-5T4 monoclonal antibody can be site-specifically coupled in a PBS pH 7.2~7.4 system ( Figure 7 ), preferably in HEPES pH 7.2. Typically, one anti-5T4 monoclonal antibody can be conjugated to 1–4 L2 molecules, and more preferably, one anti-5T4 monoclonal antibody can be conjugated to 1–2 L2 molecules. The reaction can be terminated by adjusting the pH to around 5.0, yielding the 5T4-conjugated antibody.

[0146] Preparation of CAR-raNK cells

[0147] In a specific embodiment, the preparation process of CAR-raNK cells includes the following steps: preparation of PBMCs, culture of NK cells, preparation of IBR825 cells (stock solution), etc.

[0148] PBMCs are collected from healthy allogeneic donors. After separation of T cells and red blood cells, the cells are transferred to primary cell freezing medium and the total number of viable cells is ≥6.0×10 7 PBMCs are obtained by aliquoting into tubes of cells / tube. PBMCs can be stored long-term in liquid nitrogen at ≤-175°C.

[0149] PBMCs frozen in liquid nitrogen are removed and revived in an adapted culture medium. The culture scale is expanded, i.e., the culture medium is increased. Preferably, NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their ability to divide, proliferate, and be active.

[0150] When NK cells are cultured for 15-16 days, linker N, especially N1, is added to the culture medium and incubated for 12-18 hours. Figure 8 、 Figure 9 Then UNK reacts with Trop2-conjugated antibody and 5T4-conjugated antibody in a certain ratio in the culture medium ( Figure 10 ), where the 5T4-conjugated antibody:Trop2-conjugated antibody ratio ranges from 1:10 to 10:1, ultimately yielding IBR825. In pilot-scale production, the resulting IBR825 stock solution (DS) is then dispensed into infusion bags using cell-based packaging equipment, yielding the finished IBR825 product (DP). Each bag is sufficient for a single infusion in a patient.

[0151] Typically, one peripheral blood lymphocyte apheresis sample from a healthy donor can produce 100-150 doses of finished product according to the above process.

[0152] 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 an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or its antigen-binding fragment and the anti-5T4 antibody or its antigen-binding fragment are both coupled to the NK cell via a linker.

[0153] In some embodiments, the anti-5T4 antibody or antigen-binding fragment thereof comprises:

[0154] a HCDR1 as set forth in SEQ ID NO: 15 or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 15,

[0155] a HCDR2 as set forth in SEQ ID NO: 16, or a HCDR2 having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 16,

[0156] a HCDR3 as set forth in SEQ ID NO: 17, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 17,

[0157] a LCDR1 as set forth in SEQ ID NO: 18, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 18,

[0158] a LCDR2 as set forth in SEQ ID NO: 19, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 19, and

[0159] a LCDR3 as set forth in SEQ ID NO: 20, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 20;

[0160] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0161] In some embodiments, the anti-5T4 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 11, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 11, and a light chain variable region amino acid sequence as set forth in SEQ ID NO: 13, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 13.

[0162] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-5T4 antibody or antigen-binding fragment thereof differs from the amino acid sequence of SEQ ID NO: 11 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 of SEQ ID NO: 11 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 retaining 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 of SEQ ID NO: 11 is added by 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 retains 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: 11, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar antibody.

[0163] In some embodiments, the amino acid sequence of the light chain variable region of the anti-5T4 antibody or antigen-binding fragment thereof differs from the amino acid sequence of SEQ ID NO: 13 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 of SEQ ID NO: 13 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 retaining 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 be added to the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 13 while still retaining 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: 13, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar antibody.

[0164] In some embodiments, the anti-5T4 antibody is a full-length antibody.

[0165] In some embodiments, the anti-5T4 antibody has a heavy chain constant region amino acid sequence as set forth in SEQ ID NO: 12, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 12, and a light chain constant region amino acid sequence as set forth in SEQ ID NO: 14, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 14.

[0166] In some embodiments, the amino acid sequence of the heavy chain constant region of the anti-5T4 antibody differs from the amino acid sequence of SEQ ID NO: 12 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 of SEQ ID NO: 12 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 constant region of the similar antibody. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 12 is supplemented by 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 constant 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: 12, as long as the altered amino acid sequence substantially maintains the function of the heavy chain constant region of the similar antibody.

[0167] In some embodiments, the amino acid sequence of the light chain constant region of the anti-5T4 antibody differs from the amino acid sequence of SEQ ID NO: 14 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 of SEQ ID NO: 14 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 light chain constant 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 to the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 14 while still maintaining the function of the light chain constant 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: 14, as long as the altered amino acid sequence substantially maintains the function of the light chain constant region of the similar antibody.

[0168] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof comprises:

[0169] a HCDR1 as set forth in SEQ ID NO: 5 or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 5,

[0170] a HCDR2 as set forth in SEQ ID NO: 6, or a HCDR2 having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 6,

[0171] a HCDR3 as set forth in SEQ ID NO: 7, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 7,

[0172] a LCDR1 as set forth in SEQ ID NO: 8, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 8,

[0173] a LCDR2 as set forth in SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 9, and

[0174] a 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;

[0175] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0176] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 1, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1, and a light chain variable region amino acid sequence as set forth in SEQ ID NO: 3, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3.

[0177] 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 of 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 of 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 of SEQ ID NO: 1 is added by 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.

[0178] 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 of 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 of SEQ ID NO: 3 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 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 be added to the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 3 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 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.

[0179] In some embodiments, the anti-Trop2 antibody is a full-length antibody.

[0180] In some embodiments, the amino acid sequence of the heavy chain constant region of the anti-Trop2 antibody is as set forth in SEQ ID NO:2, or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:2, and the amino acid sequence of the light chain constant region is as set forth in SEQ ID NO:4, or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:4.

[0181] In some embodiments, the amino acid sequence of the heavy chain constant region of the anti-Trop2 antibody differs from the amino acid sequence of SEQ ID NO: 2 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 of SEQ ID NO: 2 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 constant region of the similar antibody. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 2 is added by 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 constant 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: 2, as long as the altered amino acid sequence substantially maintains the function of the heavy chain constant region of the similar antibody.

[0182] In some embodiments, the amino acid sequence of the light chain constant region of the anti-Trop2 antibody differs from the amino acid sequence of SEQ ID NO: 4 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 of SEQ ID NO: 4 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 light chain constant 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 to the C-terminal or N-terminal region of the amino acid sequence of SEQ ID NO: 4 while still maintaining the function of the light chain constant 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: 4, as long as the altered amino acid sequence substantially maintains the function of the light chain constant region of the similar antibody.

[0183] In some embodiments, the forms of the antigen-binding fragment of the anti-5T4 antibody and the antigen-binding fragment of the anti-Trop2 antibody are independently selected from single-chain antibody (scFv) or Fab fragment.

[0184] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each full-length antibodies.

[0185] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each humanized antibodies or fully human antibodies.

[0186] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each monoclonal antibodies.

[0187] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each of the IgG1, IgG2, or IgG4 isotype. In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each of the IgG1 isotype.

[0188] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody each comprise a light chain constant region of the kappa subtype.

[0189] In some embodiments, the NK cells are CD16 + and / or NKG2D + In some embodiments, the NK cells are CD16 + NKG2D + .

[0190] 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%).

[0191] 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.

[0192] In some embodiments, NK cells are obtained by in vitro culture and expansion of peripheral blood mononuclear cells (PBMCs), which is also an exemplary method in the Examples of this application. PBMCs are one of the main sources of NK cells, offering advantages such as relatively easy collection, ease of in vitro expansion, and no toxic side effects. However, the proportion of NK cells in PBMCs is only 10%-15%. Methods for expanding PBMC-derived NK cells include using a combination of cytokines, feeder cells, or membrane particles to stimulate in vitro expansion of NK cells. These different expansion systems exhibit varying levels of NK cell expansion efficiency. In some embodiments, PBMCs are screened for HLA and KIR. In some embodiments, PBMCs are screened for CD16a variants, i.e., 176V and 176F. In some embodiments, one or more cytokines are used to maintain or activate natural killer cell activity 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.

[0193] PBMCs can be obtained from apheresis of peripheral blood lymphocytes from allogeneic healthy donors. After separation of T cells and red blood cells, the cells are transferred to primary cell freezing medium and the total number of viable cells is ≥6.0×10 7 PBMCs are packaged in the size of 10 cells / tube to obtain PBMCs. PBMCs are stored long-term in liquid nitrogen at ≤ -175°C. PBMCs frozen in liquid nitrogen are removed and revived in adapted culture medium. Their ability to divide, proliferate, and activate is maintained by expanding the culture scale (i.e., increasing the culture medium for expansion) and adding NK cell-related cytokines, including but not limited to IL-2 and IL-15. An exemplary preparation method can be found in Example 1 of this application.

[0194] In some embodiments, NK cells are obtained by in vitro culture and expansion of NK cells derived from umbilical cord blood. There are generally two different methods 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 CD34 + Hematopoietic stem / progenitor cells differentiate into NK cells and then expand.

[0195] In some embodiments, the NK cells are obtained from in vitro culture and expansion of an NK cell line. 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.

[0196] In some embodiments, NK cells are obtained from in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0197] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each conjugated to NK cells via a click chemistry reaction of a linker.

[0198] Click chemistry, also known as "linking chemistry" or "speed-matching combinatorial chemistry," is a synthetic concept introduced by chemist Barry Sharpless in 2001. Its main purpose is to rapidly and reliably synthesize a wide variety of molecules by piecing together small building blocks. There are four main types of click chemistry reactions: cycloadditions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, and addition reactions across carbon-carbon multiple bonds.

[0199] In some embodiments, the anti-5T4 antibody and the Trop2 antibody are coupled to the NK cell via a first linker and a second linker, wherein a first copy of the first linker is coupled to the anti-5T4 antibody, a second copy of the first linker is coupled to the Trop2 antibody, 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.

[0200] In some embodiments, the first linker is an active ester capable of forming a coupling with a lysine residue of an antibody via a reaction from an ester bond to an amide bond. For example, the active ester is a pentafluorophenyl ester, such as pentafluorophenyl pipecolate. 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 has a tetraethylene glycol chain structure that undergoes slow hydrolysis in water. In some embodiments, the active ester reacts with amino groups on the hydrophilic interface of the protein, converting the ester bond to an amide bond, thereby establishing a coupling mechanism.

[0201] 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.

[0202] In some embodiments, the first linker is dibenzazepinoctyne-glutaryl-aminoethylene glycol acetal-acetylpiperidinic acid pentafluorophenyl ester, the structure of which is shown below:

[0203] ,

[0204] Where n is an integer from 0 to 8.

[0205] In some embodiments, the first linker is dibenzazepinoctyne-glutaryl-aminotetraethylene glycol-acetylpiperidin pentafluorophenyl ester, the structure of which is shown below:

[0206] .

[0207] Dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester is also referred to as L2 linker in this application. The synthetic route of L2 linker is shown in Figure 2 , see the structural formula Figure 3 , including three main synthetic steps: amide condensation, hydrolysis reaction and synthesis of active ester.

[0208] Step 1 - Amide condensation

[0209] Dibenzazepine octyne glutaric acid (L2-1) and aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-2) are commercially available. Through a chemical condensation reaction, the intermediate dibenzazepine octyne glutaramidotetraethylene glycol acetyl pipecolic acid methyl ester (L2-3) is obtained. L2-3 is a stable compound.

[0210] Specifically, compounds L2-1 and L2-2 (1:1.1, with L2-2 in excess) were dissolved in dichloromethane (DCM). Hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) were added, respectively, followed by triethanolamine (TEA). The mixture was stirred at room temperature for 4–12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified using a silica gel column (dichloromethane:methanol = 20:1) to obtain compound L2-3 as a yellow oil.

[0211] Step 2-Hydrolysis

[0212] Intermediate L2-3 obtained in step 1 is hydrolyzed to obtain intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetyl pipecolic acid (L2-4). The hydrolysis reaction is generally quantitative and is used directly in the next step without further purification.

[0213] Specifically, compound L2-3 is dissolved in a mixture of methanol and water, cooled to 0°C, and then a 1 mol / L aqueous lithium hydroxide (LiOH) solution is added. The mixture is stirred at 0°C to room temperature for 4-12 hours, acidified with 1 mol / L HCl to a pH between 2 and 3, extracted three times with ethyl acetate (EA), and dried to obtain a yellow oil, the intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetylpiperidinic acid (L2-4).

[0214] Step 3-Synthesis of active ester

[0215] The intermediate L2-4 obtained in step 2 (without purification) is condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2, i.e., dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetylpiperidinic acid pentafluorophenyl ester.

[0216] Specifically, compound L2-4 was dissolved in tetrahydrofuran (THF), cooled to 0°C, and then 2-pentafluorophenol, hydroxybenzotriazole (HOBt), and dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred at 0°C to room temperature for 4-12 hours, extracted three times with ethyl acetate (EA), dried, and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain a yellow oil, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetylpiperidinic acid pentafluorophenyl ester (L2). The structural formula is shown in Figure 2 .

[0217] Subsequently, the L2 linker can be subjected to site-directed coupling reaction with anti-5T4 antibody and anti-Trop2 antibody in PBS pH 7.2-7.4 system (preferably HEPES pH 7.2) (see schematic diagram). Figure 6 and 7 Typically, one anti-5T4 antibody or one anti-Trop2 antibody can be conjugated to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to around 5.0 to obtain the L2-conjugated antibody.

[0218] In some embodiments, the second linker is an azidoacetylated cyclohexosamine, for example, azidoacetylated cyclogalactosamine or 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 the sialic acid-modified membrane protein during the cell culture phase through the cell's own metabolic pathway. 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.

[0219] 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:

[0220] .

[0221] In some embodiments, 1,3,4,6-oxo-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose The proportion of a single configuration is at least 90%, for example, at least 95%, at least 98% or at least 99%.

[0222] 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 10 , see the structural formula Figure 11 , including two main synthetic steps: aminoazide acetylation and hydroxyl acetylation.

[0223] Step 1 - Acetylation of aminoazido

[0224] 1.2 times the amount of α-azidoacetic acid (Compound 1), 2 times the amount of hydroxybenzotriazole (HOBt), and triethylamine (Et3N) were added to a solution of D-galactosamine hydrochloride (Compound 2) in N,N-dimethylformamide (DMF). A small amount of methanol (MeOH) was added for solubilization. The reaction mixture was allowed to react at room temperature for 12 hours. The reaction mixture was poured into a mixture of dichloromethane (DCM) / methanol (MeOH) and shaken to mix. Ether was added to precipitate the oily product (Compound 3). The ether layer was decanted and the reaction was repeated twice before vacuum drying. The mixture was directly carried out to the next step without further purification.

[0225] Step 2 - Hydroxyacetyl esterification

[0226] Compound 3 obtained above was added to anhydrous pyridine (Pyr.) and acetic anhydride (Ac2O), and 4-dimethylaminopyridine (DMAP) catalyst was added at room temperature. The reaction was allowed to react for 12 hours. HPLC analysis showed that the reaction was almost complete. The mixture was concentrated to obtain a racemic mixture of tetraacetyl-N-azidoacetyl-a,b-D-galactosamine. The solid was precipitated from ethyl acetate / petroleum ether, mainly the b-configuration product with a purity of >85%. Further purification was performed on a silica gel column (dichloromethane:methanol = 20:1) to obtain the N1 product with a single optical isomer purity of >90%. The a,b-isomers can isomerize to each other in the cell and, after multiple steps of metabolism and synthesis, are converted to N-azidoacetylsialic acid, which is ultimately expressed on the surface glycoprotein of NK cells. The structural formula of the N1 linker can be found in [ 1 ]. Figure 11 .

[0227] 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 this application) (see the schematic diagram of the connection mechanism between N1 linkers and NK cells and the structure of UNK for details). Figure 8 and 9 ).

[0228] Finally, to obtain antibody-NK cell conjugates, UNK can be conjugated with L2-conjugated antibodies in culture medium (for reaction mechanism, see Figure 10 ), and obtain antibody-NK cell conjugates.

[0229] Subsequent processes also include preparing antibody-NK cell conjugate preparations to effectively prolong the stability of the antibody-NK cell conjugate. The formulation may contain ingredients such as sodium chloride and human serum albumin to maintain isotonicity, as well as ingredients such as trehalose, sucrose, dextran, and DMSO to maintain the cells' cold tolerance and protein and enzyme activity.

[0230] 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.

[0231] 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 cell number accounts for at least 98% of the total cell number in the cell population.

[0232] In some embodiments, CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population.

[0233] In some embodiments, CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0234] 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.

[0235] In some embodiments, the antibody-natural killer cell (NK cell) conjugate comprises at least 90% of the total cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate comprises at least 95% of the total cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate comprises at least 98% of the total cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate comprises at least 99% of the total cells in the cell population by cell number.

[0236] 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.

[0237] In some embodiments, the pharmaceutical composition is in the form of a sterile fresh preparation or a sterile frozen preparation.

[0238] In some embodiments, the pharmaceutical composition comprises sodium chloride and / or human serum albumin.

[0239] In some embodiments, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0240] In some embodiments, the pharmaceutical composition is used to treat a tumor in a subject.

[0241] In some embodiments, the individual is a mammal.

[0242] In a preferred embodiment, the mammal is a human.

[0243] In some specific embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0244] In some specific embodiments, the tumor is a malignant tumor.

[0245] In some specific embodiments, the malignancy is selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer such as small cell lung cancer and non-small cell lung cancer, and pancreatic cancer.

[0246] 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 tumors in an individual.

[0247] In some embodiments, the individual is a mammal.

[0248] In a preferred embodiment, the mammal is a human.

[0249] In some embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0250] In some specific embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 +) and / or high expression of 5T4 (5T4 + ) tumors.

[0251] In some specific embodiments, the tumor is a malignant tumor.

[0252] In some specific embodiments, the malignancy is selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer such as small cell lung cancer and non-small cell lung cancer, and pancreatic cancer.

[0253] 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.

[0254] In some specific embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0255] In some specific embodiments, the tumor is a malignant tumor.

[0256] In some specific embodiments, the malignancy is selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer such as small cell lung cancer and non-small cell lung cancer, and pancreatic cancer.

[0257] In some embodiments, the individual is a mammal.

[0258] In a preferred embodiment, the mammal is a human.

[0259] 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 limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described.

[0260] Example

[0261] The present application will be further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application.

[0262] Example 1 - Preparation of Antibody-NK Cell Conjugates

[0263] The preparation of the antibody-NK cell conjugate of this example can be roughly divided into the following steps:

[0264] (1) Preparation of anti-Trop2 monoclonal antibodies and anti-5T4 monoclonal antibodies;

[0265] (2) Preparation of NK cells;

[0266] (3) Preparation of antibody linker (referred to as L2 linker in this example);

[0267] (4) Preparation of NK cell linkers (referred to as N1 linkers in this example);

[0268] (5) Antibody linker (L2 linker) is applied to the antibody;

[0269] (6) applying an NK cell linker (N1 linker) to NK cells; and

[0270] (7) Conjugating two antibodies with linkers to NK cells.

[0271] (1) Preparation of anti-Trop2 monoclonal antibodies and anti-5T4 monoclonal antibodies

[0272] Briefly, the sequence of the anti-Trop2 monoclonal antibody was obtained from phage library screening. The antibody was selected through binding activity screening between Trop2 and Trop2-expressing cells, cross-species studies with humans, monkeys, and mice, and affinity studies. The antibody comprises the heavy chain variable region set forth in SEQ ID NO:1 (HCDRs 1-3 are SEQ ID NOs: 11, 6, and 7, respectively), the heavy chain constant region set forth in SEQ ID NO:2, the light chain variable region set forth in SEQ ID NO:3 (LCDRs 1-3 are SEQ ID NOs: 8, 9, and 10, respectively), and the light chain constant region set forth in SEQ ID NO:4. The DNA sequence of the anti-Trop2 monoclonal antibody was determined, and a recombinant plasmid (IB12) expressing the anti-Trop2 monoclonal antibody was constructed.

[0273] The construction of a monoclonal cell line is based on known technology, in which the IB12 plasmid is transfected into CHO cells by electroporation. After one round of screening in the minipool and two rounds of screening after monoclonal plating, and confirmation of stability through preliminary subculture, a cell line that can stably express the anti-Trop2 monoclonal antibody is obtained.

[0274] The anti-Trop2 monoclonal antibody is produced using a protocol known in the art, including cell culture and protein purification. Working cells are recovered, expanded for 3-5 rounds, and cultured at a pilot scale. The untreated cell suspension (UPB) is then collected and clarified and filtered to obtain the cell culture supernatant. The anti-Trop2 monoclonal antibody is then purified through a three-step protein A affinity, anion exchange, and cation exchange chromatography process.

[0275] The sequence of the anti-5T4 monoclonal antibody was obtained from phage library screening. The antibody was screened for binding activity between 5T4 and cells expressing 5T4 and confirmed by affinity studies. The antibody comprises a heavy chain variable region set forth in SEQ ID NO:11 (HCDRs 1-3 are SEQ ID NOs: 15, 16, and 17, respectively), a heavy chain constant region set forth in SEQ ID NO:12, a light chain variable region set forth in SEQ ID NO:13 (LCDRs 1-3 are SEQ ID NOs: 18, 19, and 20, respectively), and a light chain constant region set forth in SEQ ID NO:14. The DNA sequence of the anti-5T4 monoclonal antibody was determined, and a recombinant plasmid (IB12) expressing the anti-5T4 monoclonal antibody was constructed.

[0276] The construction of a monoclonal cell line is based on known technology, in which the IB12 plasmid is transfected into CHO cells by electroporation. After one round of screening in the minipool and two rounds of screening after monoclonal plating, and confirmation of stability through preliminary subculture, a cell line that can stably express the anti-5T4 monoclonal antibody is obtained.

[0277] The anti-5T4 monoclonal antibody is produced using a protocol known in the art, including cell culture and protein purification. Working cells are recovered, expanded for 3-5 rounds, and cultured at a pilot scale. The untreated cell suspension (UPB) is then collected and clarified and filtered to obtain the cell culture supernatant. The anti-5T4 monoclonal antibody is then purified through a three-step protein A affinity, anion exchange, and cation exchange chromatography process to obtain the anti-5T4 monoclonal antibody.

[0278] (2) Preparation of NK cells

[0279] PBMCs are collected from healthy allogeneic donors. After separation of T cells and red blood cells, the cells are transferred to primary cell freezing medium and the total number of viable cells is ≥6.0×10 7 PBMCs are obtained by aliquoting into tubes of cells / tube. PBMCs can be stored long-term in liquid nitrogen at ≤-175°C.

[0280] PBMCs frozen in liquid nitrogen are removed and revived in adapted culture medium. Their proliferation and activity are maintained by expanding the culture scale (i.e., increasing the culture medium for expansion) and adding NK cell-related cytokines, including but not limited to IL-2 and IL-15. Exemplary NK cell culture stages, process parameters, and process control indicators are shown in Table 1 below.

[0281] Table 1

[0282]

[0283] 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):

[0284] Table 2.

[0285]

[0286] (3) Preparation of Antibody Linker (referred to as L2 Linker in this Example)

[0287] For the synthetic route of L2 linker, see Figure 2 , see the structural formula Figure 3 , including three main synthetic steps: amide condensation, hydrolysis reaction and synthesis of active ester.

[0288] Step 1 - Amide condensation

[0289] Dibenzazepine octyne glutaric acid (L2-1) and aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-2) are commercially available. Through a chemical condensation reaction, the intermediate dibenzazepine octyne glutaramidotetraethylene glycol acetyl pipecolic acid methyl ester (L2-3) is obtained. L2-3 is a stable compound.

[0290] Specifically, compounds L2-1 and L2-2 (1:1.1, with L2-2 in excess) were dissolved in dichloromethane (DCM). Hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) were added, respectively, followed by triethanolamine (TEA). The mixture was stirred at room temperature for 4–12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified using a silica gel column (dichloromethane:methanol = 20:1) to obtain compound L2-3 as a yellow oil.

[0291] Step 2-Hydrolysis

[0292] Intermediate L2-3 obtained in step 1 is hydrolyzed to obtain intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetyl pipecolic acid (L2-4). The hydrolysis reaction is generally quantitative and is used directly in the next step without further purification.

[0293] Specifically, compound L2-3 is dissolved in a mixture of methanol and water, cooled to 0°C, and then a 1 mol / L aqueous lithium hydroxide (LiOH) solution is added. The mixture is stirred at 0°C to room temperature for 4-12 hours, acidified with 1 mol / L HCl to a pH between 2 and 3, extracted three times with ethyl acetate (EA), and dried to obtain a yellow oil, the intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetylpiperidinic acid (L2-4).

[0294] Step 3-Synthesis of active ester

[0295] The intermediate L2-4 obtained in step 2 (without purification) is condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2, i.e., dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetylpiperidinic acid pentafluorophenyl ester.

[0296] Specifically, compound L2-4 was dissolved in tetrahydrofuran (THF), cooled to 0°C, and then 2-pentafluorophenol, hydroxybenzotriazole (HOBt), and dicyclohexylcarbodiimide (DCC) were added. The mixture was stirred at 0°C to room temperature for 4-12 hours, extracted three times with ethyl acetate (EA), dried, and purified on a silica gel column (dichloromethane:methanol = 20:1) to obtain a yellow oil, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetylpiperidinic acid pentafluorophenyl ester (L2). The structural formula is shown in Figure 3 .

[0297] (4) Preparation of NK cell linkers (referred to as N1 linkers in this example)

[0298] For the synthetic route of N1 linker, see Figure 4 , see the structural formula Figure 5 , including two main synthetic steps: aminoazide acetylation and hydroxyl acetylation.

[0299] Step 1 - Acetylation of aminoazido

[0300] 1.2 times the amount of α-azidoacetic acid (Compound 1), 2 times the amount of hydroxybenzotriazole (HOBt), and triethylamine (Et3N) were added to a solution of D-galactosamine hydrochloride (Compound 2) in N,N-dimethylformamide (DMF). A small amount of methanol (MeOH) was added for solubilization. The reaction mixture was allowed to react at room temperature for 12 hours. The reaction mixture was poured into a mixture of dichloromethane (DCM) / methanol (MeOH) and shaken to mix. Ether was added to precipitate the oily product (Compound 3). The ether layer was decanted and the reaction was repeated twice before vacuum drying. The mixture was directly carried out to the next step without further purification.

[0301] Step 2 - Hydroxyacetyl esterification

[0302] Compound 3 obtained above was added to anhydrous pyridine (Pyr.) and acetic anhydride (Ac2O), and 4-dimethylaminopyridine (DMAP) catalyst was added at room temperature. The reaction was allowed to react for 12 hours. HPLC analysis showed that the reaction was almost complete. The mixture was concentrated to obtain a racemic mixture of tetraacetyl-N-azidoacetyl-a,b-D-galactosamine. The solid was precipitated from ethyl acetate / petroleum ether, mainly the b-configuration product with a purity of >85%. Further purification was performed on a silica gel column (dichloromethane:methanol = 20:1) to obtain the N1 product with a single optical isomer purity of >90%. The a,b-isomers can isomerize to each other in the cell and, after multiple steps of metabolism and synthesis, are converted to N-azidoacetylsialic acid, which is ultimately expressed on the surface glycoprotein of NK cells. The structural formula of the N1 linker can be found in [ 1 ]. Figure 5 .

[0303] (5) Applying the antibody linker (L2 linker) to the antibody

[0304] The L2 linker and anti-Trop2 monoclonal antibody can undergo site-specific coupling reaction in PBS pH 7.2-7.4 (preferably HEPES pH 7.2) (see the schematic diagram for details). Figure 6 Typically, one anti-Trop2 monoclonal antibody can be conjugated to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to around 5.0 to obtain an L2-conjugated Trop2 antibody, also known as a Trop2-conjugated antibody.

[0305] The L2 linker and anti-5T4 monoclonal antibody can undergo site-specific coupling reaction in PBS pH 7.2-7.4 (preferably HEPES pH 7.2) (see the schematic diagram for details). Figure 7 Typically, one anti-5T4 monoclonal antibody can be conjugated to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to around 5.0 to obtain an L2-conjugated 5T4 antibody, also known as a 5T4-conjugated antibody.

[0306] (6) Applying NK cell linkers (N1 linkers) to NK cells

[0307] 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 this application) (see the schematic diagram of the connection mechanism between N1 linkers and NK cells and the structure of UNK for details). Figure 8 and 9 ).

[0308] (7) Coupling antibodies and NK cells with linkers

[0309] Trop2-conjugated antibody and 5T4-conjugated antibody were mixed in a certain ratio and reacted with UNK in the culture medium (for reaction mechanism, see Figure 10 ), obtaining a stock solution (DS) of the antibody-NK cell conjugate. This anti-Trop2 monoclonal antibody and anti-5T4 monoclonal antibody-NK cell conjugate was named "IBR825" and will be used in subsequent examples to represent anti-Trop2 and anti-5T4 antibody-NK cell conjugates.

[0310] Subsequent processes also include preparing antibody-NK cell conjugate preparations to effectively prolong the stability of the antibody-NK cell conjugate. The formulation may contain ingredients such as sodium chloride and human serum albumin to maintain isotonicity, as well as ingredients such as trehalose, sucrose, dextran, and DMSO to maintain the cells' cold tolerance and protein and enzyme activity.

[0311] Example 2-Affinity and species cross-detection of Trop2-conjugated antibodies to Trop2

[0312] In this example, the affinity of the L2-conjugated antibody prepared in step (5) of Example 1 for Trop2 from different species was tested using the OctetR8 instrument (BLI method). Specifically, various Trop2 proteins were loaded onto the His-K probe and bound to the free L2-conjugated antibody in solution (diluted in PBS (pH 7.4)). Binding was determined based on the maximum response value. The dissociation constant KD value was calculated based on the association rate and dissociation rate. The results are as follows:

[0313] Table 3.

[0314]

[0315] As can be seen from the results in Table 3, 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.

[0316] Example 3 - Comparison of basic antibody properties between 5T4-conjugated antibodies and anti-5T4 monoclonal antibodies

[0317] In this example, multiple basic antibody properties of the 5T4-conjugated antibody prepared in step (7) of Example 1 were compared with those of the anti-5T4 monoclonal antibody prepared in step (1) of Example 1. The results are shown in Table 4 below, where the methodology of each test item follows conventional test methods in the art.

[0318] Table 4.

[0319]

[0320] As can be seen from the results in Table 4, the coupling of anti-5T4 monoclonal antibody to NK cells did not significantly affect the properties of the antibody itself, so that the desired properties of the antibody were retained.

[0321] Example 4- Evaluation of the in vitro cytotoxicity of the conjugate IBR825 against different cancer cells

[0322] In the evaluation of cell killing activity in this example, eight tumor cell lines were selected to detect the expression levels of 5T4 and Trop2. The cell line names, cancer types, and the measured expression levels of 5T4 and Trop2 (determined by flow cytometry) are shown in Table 5 below:

[0323] Table 5.

[0324]

[0325] Calcein acetoxymethy1 ester (Calcein-AM) is a cytoplasmic fluorescent marker that is inherently nonfluorescent. Upon entry into cells, it is hydrolyzed by endogenous esterases to produce calcein, a strongly negatively charged polar molecule that cannot penetrate the cell membrane. Calcein is retained within the cell and emits strong green fluorescence. Tumor cells labeled with Calcein-AM are co-incubated with IBR825 cells. Upon tumor cell lysis, Calcein is released into the culture medium. Cytotoxicity against IBR825 cells is determined by measuring changes in calcein fluorescence intensity in the culture supernatant using a microplate reader (compared to tumor cell control wells (representing 100% cell lysis)).

[0326] The cytotoxicity of NK cells and three IBR825 cell lines against the four highly expressing tumor cell lines mentioned above—non-small cell lung cancer (NCI-H292), breast cancer (MDA-MB-468), pancreatic cancer (BXPC3), and cervical cancer (Siha)—was assessed using Calcein AM fluorescence labeling. The three IBR825 cell lines were treated with the same total amount of conjugated antibody at a 5T4:Trop2 ratio of 2:1, 1:1, and 1:2, respectively.

[0327] Tumor cells were stained with Calcein AM for 30 min and then washed three times. The concentration of tumor cells was adjusted to 1.5×10 5100 μL / well was seeded into a 96-well cell culture plate. NK cells and three types of IBR825 cells were adjusted to 1.5×106 cells / mL, 5×105 cells / mL, and 1.7×105 cells / mL using cell diluent. Each gradient was replicated in triplicate, and 100 μL / well was added to the 96-well plate, resulting in a final effector-target ratio of approximately 10:1, 3:1, and 1:1. Corresponding control groups were also prepared and incubated at 37°C, 5% CO2 for 4 hours.

[0328] On-plate detection: 120 μL / well of the supernatant was transferred to a black 96-well ELISA plate in parallel, and the fluorescence intensity was measured using 490 nm as the excitation wavelength and 535 nm as the emission wavelength.

[0329] Table 6 below shows the cytotoxicity of NK cells and three IBR825 cells against tumor cells BXPC3, NCI-H292, MDA-MB-468, and Siha at a 3:1 effector-target ratio. All three IBR825s had high cytotoxicity against all four tumor cell types, and were higher than those of NK cells. Furthermore, there was a dose-response relationship between the cytotoxicity and IBR825. See Table 6 for the results. Figure 11-14 .

[0330] Table 6.

[0331]

[0332] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the present application and does not limit the scope of the present application unless otherwise required. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present application.

[0333] All publications and patent applications cited in this specification are incorporated herein by reference, just as each individual publication or patent application is 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 to be illustrative rather than restrictive.

[0334] Informal Sequence Listing

[0335] Amino acid sequence of anti-Trop2 antibody

[0336]

[0337]

[0338] Amino acid sequence of anti-5T4 antibody

[0339]

[0340]

[0341] References

[0342] [1] H. Kayser, R. Zeitler, C. Kannischt, D. Crunnow, R. Nuck and W. Reutter. J. Biol. Chem, 1992, 267:16934.

[0343] [2] O.T. Keppler, R. Horstcorte, M. Pawlita, C. Schmidt and W. Reutter. Glycobiology, 2001, 11, 11R.

[0344] [3] C. Oetke, R. Brossmer, L.R. Mantey, S. Hinderlich, R. Isecke, W. Reutter, O.T. Keppler and M. Pawlita. J. Biol. Chem, 2002, 277:6688.

[0345] [4] C. Oetke, S. Hinderlich, R. Brossmer, W. Reutter, M. Pawlita and O.T. Keppler. Eur. J. Biochem, 2001, 268:4553.

[0346] [5] Elinan Saxon and Carolyn R. Bertozzi. Cell Surface Engineering by a Modified Staudinger Reaction[J]. Science, 2000, 278(5460):2007 - 2010.

[0347] [6]David J. Vocadlo, Howard C. Hang, Eun-Ju kim et al. A ChemicalApproach for Identifying O-GlcNAc-Modified Protiens in Cells[J]. PNAS, 2003,100(16):9116-9121.

[0348] [7]Jian Du, M Adam Meledeo, Zhiyun Wang, et al. MetabolicGlycoengineering: Sialic Acid and Beyond[J]. Glycobiology, 2009,19(12):1382-1401.

[0349] [8]Sarah J. Luchansky, Scarlett Goon and Carolyn R. Bertozzi.Expanding the Diversity of Unnatural Cell-Surfance Sialic Acids[J].ChemBioChem, 2004,5(3):371-374.

[0350] [9]Michelle R. Bond, Haochi Zhang, Peter D. Vu, et al.Photocrosslinking of Glycoconjugates Using Metabolically IncorporatedDiazirine-Containing Sugars [J]. NATURE PROTOCOLS,2009,4(7):1044-1063.

[0351]

[10] Sarah J. Luchansky, Howerd C. Hang, Eliana Saxon, et al.Constructing Azide-Labeled Cell Surfaces Using Polysaccharide BiosyntheticPathways[J]. Methods Enzymol,2003, 362:249-272.

Claims

1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibodies are anti-Trop2 antibody or antigen-binding fragment thereof and anti-5T4 antibody or antigen-binding fragment thereof, and the anti-Trop2 antibody or antigen-binding fragment thereof and the anti-5T4 antibody or antigen-binding fragment thereof are coupled to the NK cell via a linker, wherein the anti-5T4 antibody or antigen-binding fragment thereof comprises: HCDR1 as shown in SEQ ID NO: 15, HCDR2 as shown in SEQ ID NO: 16, HCDR3 as shown in SEQ ID NO: 17, LCDR1 as shown in SEQ ID NO: 18, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO:20; And wherein the Trop2 antibody 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 defined according to Kabat. The NK cells are CD16 + NKG2D + , wherein the anti-5T4 antibody and the Trop2 antibody are coupled to the NK cell via a first linker and a second linker, wherein a first copy of the first linker is coupled to the anti-5T4 antibody, a second copy of the first linker is coupled to the Trop2 antibody, 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-amino ethylene 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-5T4 antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

13.

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

3.

4. The antibody-natural killer cell (NK cell) conjugate of claim 3, wherein the anti-5T4 antibody is a full-length antibody, and the amino acid sequence of the heavy chain constant region of the anti-5T4 antibody is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

14.

5. The antibody-natural killer cell (NK cell) conjugate of claim 4, wherein the Trop2 antibody is independently a full-length antibody, and wherein the amino acid sequence of the heavy chain constant region of the anti-Trop2 antibody is as shown in SEQ ID NO: 2, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:

4.

6. The antibody-natural killer cell (NK cell) conjugate according to claim 1, wherein: The anti-5T4 antibody and the Trop2 antibody are independently humanized antibodies or fully human antibodies; and / or The anti-5T4 antibody and the Trop2 antibody are independently monoclonal antibodies; and / or The anti-5T4 antibody and the Trop2 antibody are independently of IgG1, IgG2 or IgG4 isotype; and / or The anti-5T4 antibody and the Trop2 antibody independently comprise a light chain constant region of the κ subtype.

7. 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 by in vitro induction, culture and expansion of induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

8. 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 shown below: 。 9. 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, the structure of which is shown below: 。 10 . The antibody-natural killer cell (NK cell) conjugate of claim 9 , wherein the proportion of the second linker in the α or β single configuration is at least 90%. A cell population comprising the antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 10.

12. The cell population of claim 11, wherein CD3 - CD56 + CD16 + The cell number accounts for at least 95% of the total cell number in the cell population, and / or CD3 - CD56 + NKG2D + The number of cells is at least 95% of the total number of cells in the cell population.

13. The cell population of claim 11, wherein CD3 - CD56 + CD16 + The cell number accounts for at least 98% of the total cell number in the cell population, and / or CD3 - CD56 + NKG2D + The cell number accounts for at least 98% of the total cell number in the cell population.

14. The cell population according to claim 11 or 12, 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.

15. The cell population of claim 11 or 12, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total cell number in the cell population based on cell number.

16. The cell population of claim 11 or 12, 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.

17. The cell population of claim 11 or 12, 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.

18. The cell population of claim 11 or 12, 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.

19. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 10 or the cell population according to any one of claims 11 to 18, and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, comprising sodium chloride and / or human serum albumin.

21. The pharmaceutical composition of claim 19, which is in the form of a sterile fresh preparation or a sterile frozen preparation.

22. The pharmaceutical composition of claim 19, comprising trehalose, sucrose, dextran, DMSO or any combination thereof.

23. Use of the antibody-natural killer cell (NK cell) conjugate according to any one of claims 1 to 10 or the cell population according to any one of claims 11 to 18 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 + ) and / or high expression of 5T4 (5T4 + ), wherein the tumor is a malignant tumor, wherein the malignant tumor is selected from gastric cancer, cervical cancer, uterine squamous cell carcinoma, breast cancer, mucoepidermoid carcinoma, lung cancer and pancreatic cancer. The use according to claim 23 , wherein the lung cancer is small cell lung cancer or non-small cell lung cancer.

25. The use of any one of claims 23-24, wherein the subject is a mammal.

26. The use of any one of claims 23-24, wherein the subject is a human.

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