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

Abstract

The invention provides an anti-Trop2 antibody and anti-5T4 antibody-natural killer cell conjugate, a pharmaceutical composition containing the conjugate, a medical application of the conjugate and a preparation method of the conjugate.

Description

Technical Field

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

[0002] Since the early symptoms of tumors are not obvious, some tumors are discovered early but recur later, and the tumor itself is highly malignant, the number of patients with advanced tumors accounts for a relatively high proportion. Currently, the commonly used clinical treatments for solid tumors include surgery, radiotherapy, chemotherapy, conventional biological therapy, and traditional Chinese medicine treatment. These treatments often have certain toxic side effects. After surgical resection of the cancerous body, there may be complications of varying degrees, and there is a risk of recurrence after surgery. Radiotherapy and chemotherapy are the treatments with the greatest toxic side effects, which will cause a certain degree of damage to the patient's body. Although traditional Chinese medicine treatment has few toxic side effects, it often only plays an auxiliary role.

[0003] Conventional biological therapy also has many unavoidable toxic side effects. However, for patients with advanced solid tumors, most are no longer suitable for surgery, radiotherapy or concurrent chemoradiotherapy, and have failed systemic standard treatment.

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

[0006] 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.

[0007] 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.

[0008] 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.

[0009] In a fourth aspect, the present application provides use of the antibody-natural killer cell (NK cell) conjugate described in the first aspect or the cell population described in the second aspect in the preparation of a medicament for treating a tumor in an individual.

[0010] 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.

[0011] As non-limiting examples, the present application provides the following embodiments:

[0012] 1. 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 an antigen-binding fragment thereof and the anti-5T4 antibody or an antigen-binding fragment thereof are both coupled to the NK cell via a linker.

[0013] 2. The antibody-natural killer cell (NK cell) conjugate according to embodiment 1, wherein the anti-5T4 antibody or antigen-binding fragment thereof comprises:

[0014] a HCDR1 as set forth in SEQ ID NO: 15 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 15,

[0015] a HCDR2 as set forth in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 16,

[0016] a HCDR3 as set forth in SEQ ID NO: 17 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 17,

[0017] A LCDR1 as set forth in SEQ ID NO: 18 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 18,

[0018] A LCDR2 as set forth in SEQ ID NO: 19 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 19, and

[0019] 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;

[0020] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0021] 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.

[0022] 4. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-3, wherein the Trop2 antibody comprises:

[0023] A HCDR1 as shown in SEQ ID NO:5 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as shown in SEQ ID NO:5,

[0024] a HCDR2 as set forth in SEQ ID NO:6 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:6,

[0025] A HCDR3 as shown in SEQ ID NO:7 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence shown in SEQ ID NO:7,

[0026] A LCDR1 as set forth in SEQ ID NO:8 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:8,

[0027] A LCDR2 as set forth in SEQ ID NO:9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:9, and

[0028] 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;

[0029] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0030] 5. An antibody-natural killer cell (NK cell) conjugate as described in 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.

[0031] 6. An antibody-natural killer cell (NK cell) conjugate as described in any one of embodiments 1-5, wherein the anti-5T4 antibody and the Trop2 antibody are independently full-length antibodies, 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.

[0032] 7. An antibody-natural killer cell (NK cell) conjugate as described in 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.

[0033] 8. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-7, wherein:

[0034] The anti-5T4 antibody and the Trop2 antibody are independently full-length antibodies; and / or

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

[0036] The anti-5T4 antibody and the Trop2 antibody are independently monoclonal antibodies; and / or

[0037] The anti-5T4 antibody and the Trop2 antibody are independently of IgG1, IgG2 or IgG4 isotype; and / or

[0038] The anti-5T4 antibody and the Trop2 antibody independently comprise a light chain constant region of the κ subtype.

[0039] 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 + .

[0040] 10. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-9, wherein the CD16 + NKG2D + The proportion of NK cells is at least 90%, preferably, CD56 + The NK cell ratio is at least 95%.

[0041] 11. The antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-10, wherein:

[0042] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC); or

[0043] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood; or

[0044] The NK cells are obtained from in vitro induction, culture and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells (ESC).

[0045] 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 cell via a click chemistry reaction of a linker.

[0046] 13. An 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.

[0047] 14. An 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 by reacting an ester bond to an amide bond, for example, the active ester is a pentafluorophenyl ester, such as pentafluorophenyl pipecolic acid.

[0048] 15. An 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.

[0049] 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 as follows:

[0050]

[0051] Wherein n is an integer from 0 to 8.

[0052] 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:

[0053]

[0054] 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.

[0055] 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, and the structure is as follows:

[0056]

[0057] 20. The antibody-natural killer cell (NK cell) conjugate of embodiment 19, wherein the proportion of the α or β single configuration of the second linker is at least 90%.

[0058] 21. A cell population comprising the antibody-natural killer cell (NK cell) conjugate of any one of embodiments 1-20.

[0059] 22. The cell population as described in 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 number of cells accounts for at least 95%, preferably at least 98% of the total number of cells in the cell population.

[0060] 23. The cell population of embodiment 21, wherein:

[0061] CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population; and / or

[0062] CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population; and / or

[0063] CD3 + CD4 + and CD3 + CD8 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0064] 24. A cell population as described in any of embodiments 21-23, wherein the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total cell number in the cell population, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

[0065] 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.

[0066] 26. The pharmaceutical composition of embodiment 25, comprising sodium chloride and / or human serum albumin, optionally, the pharmaceutical composition is in the form of a sterile fresh preparation or a sterile frozen preparation.

[0067] 27. The pharmaceutical composition of embodiment 25 or 26, comprising trehalose, sucrose, dextran, DMSO or any combination thereof.

[0068] 28. A pharmaceutical composition as described in any one of embodiments 25-27, which is used to treat a tumor in an individual, optionally, the individual is a mammal, preferably a human.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 33. The use according to embodiment 32, 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.

[0074] 34. The use according to any one of embodiments 31 to 33, wherein the individual is a mammal, preferably a human.

[0075] 35. 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 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.

[0076] 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.

[0077] 37. The use according to embodiment 36, 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.

[0078] 38. The method of any one of embodiments 35-37, wherein the individual is a mammal, preferably a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 A schematic diagram of the structure of the antibody-coupled natural killer cells of the present application is shown.

[0081] Figure 2 A schematic diagram of the preparation process of the L2 linker of the present application is shown.

[0082] 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.

[0083] Figure 4 A schematic diagram of the preparation process of the N1 linker of the present application is shown.

[0084] 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.

[0085] Figure 6 A schematic diagram of the process flow for preparing the L2-Trop2 monoclonal antibody is shown.

[0086] Figure 7 A schematic diagram of the process flow for preparing the L2-5T4 monoclonal antibody is shown.

[0087] Figure 8 A schematic diagram of the coupling mechanism between N1 and NK cells is shown.

[0088] Fig. 9 A schematic diagram of the structure of UNK prepared in the present application is shown.

[0089] Fig.10The coupling mechanism and structural schematic diagram of IBR825 prepared in the present application are shown.

[0090] Fig.11 The killing rate of IBR825 prepared in the present application on BXPC3 cells is shown (n=3, ** represents P<0.01, **** represents P<0.0001).

[0091] Fig.12 The killing rate of IBR825 prepared in the present application on H292 cells is shown (n=3, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001).

[0092] Fig.13 The killing rate of IBR825 prepared in the present application on MDA-MB-468 cells is shown (n=3, **** represents P<0.0001).

[0093] Fig.14 The killing rate of IBR825 prepared in the present application on Siha cells is shown (n=3, ** represents P<0.01, **** represents P<0.0001, ns represents no significant difference). DETAILED DESCRIPTION OF THE INVENTION

[0095] definition

[0096] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0097] Although the numerical ranges and parameter approximations shown in the broad scope of the present application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0098] 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, marmots, ground squirrels, etc.).

[0099] The term "antigen" as used herein is a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, polypeptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.

[0100] In a broad sense, "antibody" may refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses a complete antibody / full-length antibody, an antibody single chain, or any antigen-binding fragment (also referred to as an "antigen-binding portion") of an antibody. When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" may be understood as a complete body relative to the "antigen-binding fragment / antigen-binding portion", and the two together correspond to the broad concept of an antibody.

[0101] The term "agonist antibody" is an antibody that elicits a response, e.g., an antibody that mimics at least one functional activity of a target polypeptide. Agonist antibodies include antibodies that are ligand mimetics, e.g., where the ligand binds to a cell surface receptor, the binding induces cell signaling or activity through an intracellular cell signaling pathway, and where the antibody induces similar cell signaling or activation.

[0102] "Full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into multiple regions with high variability, called complementary determining regions (CDRs), interspersed with multiple more conservative regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or the subclasses described above), but the antibody does not need to belong to any particular class. According to the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be designated as different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional structure of immunoglobulins of different classes are well known. Chimeric or humanized antibodies are also encompassed in antibodies according to the present application. It is well known to those skilled in the art that the complementary determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are multiple common definitions of the CDR amino acid sequence of VH or VL, such as the Kabat definition, the IMGT definition, the Chothia definition, etc. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence in the VH and VL amino acid sequence can usually be determined according to different definitions. In an embodiment of the present application, the CDR amino acid sequence is defined using Kabat. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in a variety of ways.

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

[0104] As used herein, the terms "antigen binding fragment" or "antigen binding portion" or "antigen binding region" are used interchangeably and refer to a portion of an antibody that contains amino acid residues that interact with an antigen and confer specificity and affinity to the binding agent for the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2 or Fab', or any fragment that should be able to increase half-life by chemical modification, such as the addition of poly(alkylene) glycols such as polyethylene glycol ("PEGylation, PEGylation") (known as PEGylated fragments of 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 be composed of or contain a partial sequence of the heavy chain or light chain variable chain of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, and such a functional fragment will contain 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 in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody.

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

[0106] The term "Fc fragment", "Fc region", "Fc domain", "Fc part" or similar terms refers to a portion of the constant region of the heavy chain of an antibody, including the hinge region, the CH2 fragment and the CH3 fragment of the constant region. The Fc region of an antibody can be engineered or modified, including effector function-related modifications, such as to reduce or eliminate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions / mutations in the Fc region of the antibody.

[0107] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0108] Generally, in order to prepare monoclonal antibodies or functional fragments thereof, especially murine monoclonal antibodies or functional fragments thereof, reference can be made to the techniques described in the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the techniques for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0109] The term "conservative variant" or "conservative amino acid substitution" is a substitution that does not substantially affect or reduce the affinity of the protein. For example, an antibody may include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions and specifically bind to the target antigen. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parent amino acids, as long as the antibody specifically binds to the target antigen.

[0110] The term "isolated" biological components (e.g., nucleic acids, proteins (including antibodies), or organelles) have been substantially separated or purified from other biological components (i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., cells) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids.

[0111] The term "pharmaceutical composition" used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that is combined together to achieve a certain specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present application. For example, the ingredients contained in the pharmaceutical composition (for example, the antibody-cell conjugate according to the present application) can be applied to an individual as a whole, or separately to an individual. When the ingredients contained in the pharmaceutical composition are applied separately to an individual, the ingredients can be applied to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application can include conventional components of cell culture, especially NK cell culture, to maintain the activity of NK cells in the conjugate. Pharmaceutically acceptable carriers can also include water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any suitable route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0112] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the individual to whom it is administered. The actual amount administered, as well as the rate and time course of administration will depend on the individual condition and severity of the condition being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by general practitioners and other physicians, typically taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0113] 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). EC can be determined by ELISA and other methods. 50 value.

[0114] The term "identity / homology / consistency" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for alignment are well known in the art.

[0115] The inventors of the present application have obtained a class of non-genetically modified antibodies that can specifically target Trop2 and 5T4 antigens and have the ability to kill tumor cells by chemically coupling a specific full-length monoclonal antibody targeting human Trop2 and a specific full-length monoclonal antibody targeting human 5T4, as well as NK cells derived from the peripheral blood of allogeneic healthy donors through linkers L and N. Coupled natural killer cells (CAR-raNK) that are antibody-coupled. The CAR-raNK of the present application first specifically recognizes tumor cells that highly express Trop2 antigens and 5T4 antigens through anti-Trop2 antibodies and anti-5T4 antibodies, and then kills tumor cells through NK cells. Fig.10 Shown are schematic diagrams of the structures of exemplary anti-Trop2 antibodies and anti-5T4 antibody-natural killer cell conjugates of the present application.

[0116] Human Trop2 belongs to the TACSTD family. It is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). Trop2 is overexpressed in a variety of malignant tumors. It is an oncogene related to the occurrence, invasion and metastasis of malignant tumors and a signal transduction molecule that can regulate the growth of tumor cells. The Trop2 gene is located on the short arm of chromosome 1, specifically located at 1p32.1. The full length of the gene is 9072bp, with no introns and only one exon. The primary structure of the Trop2 protein is a 36kD polypeptide composed of 323 amino acids. It is a single-pass transmembrane surface glycoprotein. Trop2 may represent a new cell surface receptor that plays a role in regulating tumor cell growth. Trop2 plays a regulator role in cell self-renewal, proliferation and metastasis.

[0117] Human 5T4, also known as embryonic trophoblast glycoprotein (Trophoblast, 5T4), is an N-glycosylated transmembrane protein with a molecular weight of 72kDa. It is highly expressed in embryonic trophoblast cells, as well as on the surface of various solid tumors (non-small cell lung cancer, breast cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer) and tumor stem cells. It is low or not expressed in normal adult tissues. The extracellular region of the 5T4 protein contains multiple leucine repeat sequences, which are related to protein binding and can promote cell-stromal or cell-cell interactions.

[0118] NK cells recognize target antigens without major histocompatibility complex (MHC) restriction, and the risk of allogeneic NK cells causing graft-versus-host disease (GvHD) is extremely low. Therefore, allogeneic NK cell therapy can be performed clinically. In addition, the risk of NK cells causing cytokine release syndrome (CRS) is also extremely low. In 2020, Liu E et al. published a Phase I / II clinical trial of CAR-NK cells for the treatment of lymphoma in the New England Journal of Medicine. The results showed that after receiving CAR-NK cell infusion, 8 of the 11 subjects enrolled were relieved, 7 of whom achieved complete remission, and none of them had CRS, neurotoxicity or GvHD. It can be seen that the safety of CAR-NK cell therapy is relatively high. In addition, it is precisely based on the characteristics of NK cells recognizing target antigens without MHC restriction that NK cells can be prepared into universal products without being restricted by autologous cells, making the source of NK cells for this therapy very wide, such as allogeneic peripheral blood, umbilical cord blood, embryonic stem cells, human induced pluripotent stem cells and NK-92 cell lines.

[0119] The killing activity of NK cells is mainly through:

[0120] 1) Direct lysis of target cells: NK cells release cytotoxic granules such as perforin and granzyme through exocytosis, activating the caspase pathway to induce necrosis or apoptosis of target cells;

[0121] 2) Secretion of cytokines: Cytokine-mediated killing effect. NK cells can synthesize and secrete a variety of cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10 and G-CSF, to induce apoptosis of target cells;

[0122] 3) Induce apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, inducing apoptosis of CD95+ target cells and TRAIL receptor-positive target cells through a cascade reaction of endogenous enzymes;

[0123] 4) ADCC: antibody-dependent cell-mediated cytotoxicity;

[0124] 5) Immune checkpoint pathway: Use immune checkpoint blocking antibodies to activate NK cells.

[0125] The above multiple mechanisms of action, the potential for application as a universal product, and reliable safety make NK cell therapy an attractive immunotherapy.

[0126] Currently, the NK cells used for treatment include natural NK cells, CAR-NK cells, and ready-to-use allogeneic natural killer cells (ready-to-use allogenic NK, raNK, InBaiRui (Hangzhou) Biopharmaceutical Co., Ltd.). In the field of natural NK cell therapy, CYNK-001 developed by Cellularity has obtained the fast track qualification granted by the FDA for the treatment of acute myeloid leukemia. CAR-NK cells are similar to CAR-T cells, combining antigen targeting capabilities with the natural killing ability of NK cells, overcoming many limitations of other AML therapies, such as severe bone marrow suppression caused by chemotherapy, thereby constructing a new cell drug platform. In addition, the natural receptors of CAR-NK cells such as NKp46, NKp30, NKp44, NKG2D and CD226 are retained, and these receptors can independently recognize their ligands, retaining the basic killing function of NK cells.

[0127] 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:

[0128] (1) Linker L (e.g. L2) is an active ester (pentafluorophenyl ester) having an octyne group. The structure can be found in Figure 3 , the 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 ).

[0129] (2) Linker N (such as N1) is a derivative of N-acetylgalactosamine, which can be added to the NK cell suspension during cell culture, transferred into the cell through endocytosis, and finally modified on the NK cell surface glycoprotein in the form of N-acetylsialic acid derivatives through multiple steps of metabolism and synthesis, thereby forming universal NK (UNK) cells. The structure of N1, the connection mechanism between N1 and NK cells, and the schematic diagram of the structure of UNK are respectively shown in Figure 5 , Figure 8 and Fig. 9 .

[0130] (3) The azide group N3 of N1 on UNK forms a chemical covalent connection with the carbon-carbon triple bond (alkyne group) on L2 of the coupled antibody (click chemistry, references 1 to 10), thereby forming a complete antibody-coupled natural killer cell. The structure is shown in Fig.10 .

[0131] The linker L in the present 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, and the ester bond reacts to form an amide bond. In certain embodiments, the molecule contains a stable carbon-carbon triple bond structure that can undergo a cyclization reaction with an azide group to form a stable triazole five-membered ring.

[0132] The linker N in the present 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, which includes but is not limited to galactose and glucose. In certain embodiments, the molecule is transferred to a sialic acid-modified membrane protein through the cell's own metabolic pathway during the cell culture stage. In certain embodiments, the azidoacetyl group of the molecule can undergo a cyclization reaction with a carbon-carbon triple bond to form a stable triazole five-membered ring.

[0133] "Universal NK cells (UNK)" are natural killer cells from allogeneic donors, in which a linker N is added to the culture medium at the late stage of culture, and culture is continued for a period of time, so that the membrane proteins of these natural killer cells are modified by linker N. In certain embodiments, linker N is added on the 14th, 15th, and 16th days of culture. In certain embodiments, an excess of linker N is added to ensure a positive coupling rate.

[0134] "Conjugated-Antibody-Redirected raNK (CAR-raNK)" is formed by adding a conjugated antibody to UNK in the later stage of UNK culture. In certain embodiments, the conjugated antibody is added on the 15th, 16th, and 17th days of culture. In certain embodiments, the added conjugated antibody is a conjugate of a human IgG1 antibody against human Trop2 and a linker N. In certain embodiments, the conjugation positivity rate of antibody-conjugated natural killer cells exceeds 90%, and more preferably exceeds 95%.

[0135] PBMCs from allogeneic healthy donor peripheral blood are human peripheral blood mononuclear cells collected from the peripheral blood of allogeneic healthy donors and are the raw materials of allogeneic donor-derived natural killer cells. In certain embodiments, the HLA and KIR of PBMCs are screened. In certain embodiments, the variants of CD16a of PBMCs, i.e., 176V and 176F, are screened. In certain embodiments, most of the T cells are removed while separating PBMCs.

[0136] Preparation of Trop2-conjugated antibodies

[0137] In a specific embodiment, the sequence of the Trop2-conjugated antibody is derived from a phage library screening, and the target protein and Trop2 are used to identify the target protein. + After screening the binding activity of cells and confirming the cross-species study of humans, monkeys and mice, the best molecule was selected as the anti-Trop2 antibody. According to the DNA sequence of the anti-Trop2 antibody, a recombinant plasmid (IB12) with a GS gene that can express the anti-Trop2 monoclonal antibody was constructed.

[0138] The construction of monoclonal cell lines is based on protocols known to those skilled in the art. IB12 was electroporated into CHO cells, and after one round of screening of minipools and two rounds of screening after monoclonal plating, and confirmed by preliminary passage stability culture, a cell line that can stably express anti-Trop2 monoclonal antibodies was obtained as a primary cell bank (PCB).

[0139] Trop2 conjugated antibody, obtained by coupling the anti-Trop2 monoclonal antibody expressed from the original cell library with linker L:

[0140] Anti-Trop2 monoclonal antibodies were obtained based on a preparation scheme known to those skilled in the art. Cells from an original cell bank were taken, revived, amplified in a shake flask, and cultured on a 1L pilot scale, and then the untreated cell suspension (UPB) was collected and the cell culture supernatant was obtained by clarification and filtration. Then, anti-Trop2 monoclonal antibodies were obtained by three-step chromatography of Protein A affinity (PBS pH7.0 loading, 1M NaCl elution, 0.1M GlyHCl elution), anion exchange (PBS pH6.0 flow-through mode), and cation exchange (20mM acetic acid-sodium acetate pH5.0 loading, 0.5M NaCl linear gradient elution).

[0141] 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 through three steps. Figure 2 .

[0142] 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. Usually, one anti-Trop2 monoclonal antibody can be coupled to 1 to 4 L2 molecules, and preferably one anti-Trop2 monoclonal antibody can be coupled to 1 to 2 L2 molecules. The reaction can be terminated by adjusting the pH to about 5.0, and finally a Trop2-coupled antibody is obtained.

[0143] Preparation of 5T4-conjugated antibodies

[0144] In a specific embodiment, the sequence of the 5T4-conjugated antibody is obtained from phage library screening, and the optimal molecule is selected as the anti-5T4 antibody after screening for binding activity between the target protein and 5T4+ cells and confirmation of cross-species studies of humans, monkeys, and mice. According to the DNA sequence of the anti-5T4 antibody, a recombinant plasmid (IB12) with a GS gene that can express the anti-5T4 monoclonal antibody is constructed.

[0145] The construction of monoclonal cell lines is based on a scheme known to those skilled in the art. IB12 is electrotransfected into CHO cells, and after one round of screening of minipools and two rounds of screening after monoclonal plating, and confirmed by preliminary passage stability culture, a cell line that can stably express anti-5T4 monoclonal antibodies is obtained as a primary cell bank (PCB). Under GMP conditions, the primary cell bank is revived, amplified, packaged, and stored to establish a secondary bank, namely the master cell bank (MCB) and the working cell bank (WCB). The passage stability of the master cell bank has also been confirmed.

[0146] 5T4-conjugated antibody, obtained by coupling the anti-5T4 monoclonal antibody expressed in the WCB pilot batch to the linker L:

[0147] Anti-5T4 monoclonal antibodies are obtained based on a pilot production (including cell culture and protein purification) protocol known to those skilled in the art. Cells from a working cell bank are taken, revived, expanded for 3 to 5 rounds, and cultured on a pilot scale, and then the untreated cell suspension (UPB) is collected and the cell culture supernatant is obtained by clarification and filtration. Then, through a protein purification process, anti-5T4 monoclonal antibodies are obtained through a three-step chromatography method of Protein A affinity, anion exchange, and cation exchange.

[0148] 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. Usually, one anti-5T4 monoclonal antibody can be coupled to 1 to 4 L2 molecules, and preferably one anti-5T4 monoclonal antibody can be coupled to 1 to 2 L2 molecules. The reaction can be terminated by adjusting the pH to about 5.0, and finally a 5T4-coupled antibody is obtained.

[0149] Preparation of CAR-raNK cells

[0150] In a specific embodiment, the preparation process of CAR-raNK cells includes the steps of preparing PBMC, culturing NK cells, preparing IBR825 cells (stock solution), etc.:

[0151] PBMCs are collected from healthy allogeneic donors. After separation of T cells and red blood cells, the cells are transferred to primary cell cryopreservation medium and the total number of viable cells is ≥ 6.0 × 10 7 PBMCs are obtained by packaging in the specification of 1 cell / tube. PBMCs are stored in liquid nitrogen at ≤-175℃ for a long time.

[0152] The PBMCs frozen in liquid nitrogen are taken out, revived in an adapted culture medium, and expanded by increasing the culture scale, i.e., increasing the culture medium. Preferably, NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their division and proliferation ability and activity.

[0153] When the NK cells are cultured for 15 to 16 days, the linker N, especially N1, is added to the culture medium and incubated for 12 to 18 hours. After N1 modifies the NK cells, UNK ( Figure 8 , Fig. 9 Then UNK reacts with Trop2-conjugated antibody and 5T4-conjugated antibody in a certain ratio in the culture medium ( Fig.10 ), where the ratio of 5T4-conjugated antibody to Trop2-conjugated antibody ranges from 1:10 to 10:1, and finally IBR825 is obtained. In pilot-scale production, the IBR825 stock solution (DS) is obtained at this time, and then it is divided into infusion bags through cell filling equipment to obtain IBR825 finished product (DP). Each bag of finished product can be infused into the patient once.

[0154] Usually, one peripheral blood lymphocyte collected from a healthy donor can produce 100-150 doses of finished product according to the above process.

[0155] 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.

[0156] In some embodiments, the anti-5T4 antibody or antigen-binding fragment thereof comprises:

[0157] A HCDR1 as shown in SEQ ID NO: 15 or having a sequence at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 15,

[0158] a HCDR2 as set forth in SEQ ID NO: 16 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 16,

[0159] a HCDR3 as set forth in SEQ ID NO: 17 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 17,

[0160] A LCDR1 as set forth in SEQ ID NO: 18 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO: 18,

[0161] A LCDR2 as set forth in SEQ ID NO: 19 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO: 19, and

[0162] 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;

[0163] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0164] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-5T4 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:11, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:13.

[0165] 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 shown in 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 shown in 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 maintaining the function of the heavy chain variable region of the similar antibody. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 11 is added with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains the function of the heavy chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 11, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar antibody.

[0166] 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 shown in 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 shown in SEQ ID NO: 13 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function of the light chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 13, and the resulting amino acid sequence still maintains the function of the light chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 13, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar antibody.

[0167] In some embodiments, the anti-5T4 antibody is a full-length antibody.

[0168] In some embodiments, 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:14.

[0169] In some embodiments, the amino acid sequence of the heavy chain constant region of the anti-5T4 antibody differs from the amino acid sequence shown in 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 shown in 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 shown in SEQ ID NO: 12 is added with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains the function of the heavy chain 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.

[0170] In some embodiments, the amino acid sequence of the light chain constant region of the anti-5T4 antibody differs from the amino acid sequence shown in 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 shown in SEQ ID NO: 14 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function of the light chain 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 also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14, and the resulting amino acid sequence still maintains 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.

[0171] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof comprises:

[0172] A HCDR1 as shown in SEQ ID NO:5 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as shown in SEQ ID NO:5,

[0173] a HCDR2 as set forth in SEQ ID NO:6 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:6,

[0174] A HCDR3 as shown in SEQ ID NO:7 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence shown in SEQ ID NO:7,

[0175] A LCDR1 as set forth in SEQ ID NO:8 or having at least 80%, 85%, 90%, 95% or 99% identity to a sequence as set forth in SEQ ID NO:8,

[0176] A LCDR2 as set forth in SEQ ID NO:9 or having at least 80%, 85%, 90%, 95% or 99% identity to the sequence set forth in SEQ ID NO:9, and

[0177] 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;

[0178] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0179] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:1, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:3, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:3.

[0180] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof differs from the amino acid sequence shown in SEQ ID NO: 1 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 1 can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function of the heavy chain variable region of the similar antibody. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 1 is added with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains the function of the heavy chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar antibody.

[0181] In some embodiments, the amino acid sequence of the light chain variable region of the anti-Trop2 antibody or antigen-binding fragment thereof differs from the amino acid sequence shown in SEQ ID NO: 3 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 3 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function of the light chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 3, and the resulting amino acid sequence still maintains the function of the light chain variable region of the similar antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 3, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar antibody.

[0182] In some embodiments, the anti-Trop2 antibody is a full-length antibody.

[0183] In some embodiments, 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO:4.

[0184] In some embodiments, the amino acid sequence of the heavy chain constant region of the anti-Trop2 antibody differs from the amino acid sequence shown in 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 shown in 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 shown in SEQ ID NO: 2 is added with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains the function of the heavy chain 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.

[0185] In some embodiments, the amino acid sequence of the light chain constant region of the anti-Trop2 antibody differs from the amino acid sequence shown in 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 shown in SEQ ID NO: 4 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function of the light chain 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 also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 4, and the resulting amino acid sequence still maintains 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 basically maintains the function of the light chain constant region of the similar antibody.

[0186] 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.

[0187] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each full-length antibodies.

[0188] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each humanized antibodies or fully human antibodies.

[0189] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each monoclonal antibodies.

[0190] 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.

[0191] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody each comprise a light chain constant region of the kappa subtype.

[0192] In some embodiments, the NK cells are CD16 + and / or NKG2D + In some embodiments, the NK cells are CD16 + NKG2D + .

[0193] 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%).

[0194] 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.

[0195] In some embodiments, NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC), which is also an exemplary method in the embodiments of the present application. PBMC is one of the main sources of NK cells, with the advantages of relatively easy collection, easy in vitro expansion, and no toxic side effects. However, the proportion of NK cells in PBMC is only 10%-15%. The method of expanding NK cells derived from PBMC includes using a combination of cytokines, feeder cells or membrane particles to stimulate the in vitro expansion of NK cells. These different expansion systems show different levels of NK cell expansion efficiency. In some embodiments, HLA and KIR of PBMC are screened. In some embodiments, variants of CD16a of PBMC, i.e., 176V and 176F, are screened. In some embodiments, one or more cytokines are used to maintain or activate the activity of natural killer cells during culture. In some embodiments, one or more immunoglobulins or fusion proteins are used to inhibit the proliferation of B cells, macrophages and other immune cells.

[0196] PBMCs can be obtained from apheresis peripheral blood lymphocytes from allogeneic healthy donors. After separation of T cells and red blood cells, the cells are transferred to primary cell cryopreservation medium and the total number of viable cells is ≥ 6.0 × 10 7 The PBMCs are packaged in the specification of 1 cell / branch to obtain PBMCs. PBMCs are stored for a long time in liquid nitrogen at ≤-175°C. The PBMCs frozen in liquid nitrogen are taken out and revived in an adapted culture medium. By expanding the culture scale (i.e., increasing the culture medium expansion), NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their ability to divide and proliferate and their activity. An exemplary preparation method can be found in Example 1 of the present application.

[0197] In some embodiments, NK cells are obtained by in vitro culture and expansion of NK cells from umbilical cord blood. There are generally two different methods to obtain a large number of NK cells from umbilical cord blood. One method is to expand NK cells in umbilical cord blood, and the other method is to induce umbilical cord blood CD34 + Hematopoietic stem / progenitor cells differentiate into NK cells and then expand.

[0198] In some embodiments, the NK cells are obtained from in vitro culture and expansion of NK cell lines. As an example, NK-92 is the first NK cell-based immunotherapy approved by the FDA for clinical trials and is a homogenous immortalized NK lymphoma cell.

[0199] In some embodiments, NK cells are obtained from the in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells (ESC).

[0200] In some embodiments, the anti-5T4 antibody and the anti-Trop2 antibody are each coupled to NK cells via a click chemistry reaction of a linker.

[0201] Click chemistry, also known as "linking chemistry" and "speed-matching combinatorial chemistry", is a synthetic concept introduced by chemist Barry Sharpless in 2001. Its main purpose is to quickly and reliably complete the chemical synthesis of various molecules by splicing small units. There are four main types of click chemistry reactions: cycloaddition reactions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, and carbon-carbon multiple bond addition reactions.

[0202] 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.

[0203] In some embodiments, the first linker is an active ester that can be coupled to a lysine residue of an antibody by reacting an ester bond to an amide bond. For example, the active ester is a pentafluorophenyl ester, such as pentafluorophenyl pipecolic acid. The active ester can be a water-stable molecule that can specifically covalently bind to a lysine residue of an antibody. In some embodiments, the active ester is a tetraethylene glycol chain structure that slowly hydrolyzes in water. In some embodiments, the active ester reacts with an amino group on the hydrophilic interface of a protein to react an ester bond to an amide bond, thereby establishing a coupling mechanism.

[0204] 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.

[0205] In some embodiments, the first linker is dibenzazepine octyne-glutaryl-aminoethylene glycol acetal-acetyl pipecolic acid pentafluorophenyl ester, the structure of which is shown below:

[0206]

[0207] Wherein n is an integer from 0 to 8.

[0208] In some embodiments, the first linker is dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester, the structure of which is shown below:

[0209]

[0210] 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 can be found in Figure 2 , see the structural formula Figure 3 , including three main synthetic steps: amide condensation, hydrolysis reaction and synthesis of active ester.

[0211] Step 1 - Amide condensation

[0212] Dibenzazepine octyne glutaric acid (L2-1) and aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-2) are commercially available, and the intermediate dibenzazepine octyne glutaryl aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-3) is obtained by chemical condensation reaction. L2-3 is a stable compound.

[0213] Specifically, compound L2-1 and compound L2-2 (1:1.1, L2-2 in excess) were dissolved in a dichloromethane (DCM) solution, hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) were added respectively, and finally triethanolamine (TEA) was added. The mixture was stirred at room temperature for 4 to 12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified with a silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil compound L2-3.

[0214] Step 2-Hydrolysis

[0215] The intermediate L2-3 obtained in step 1 is subjected to hydrolysis reaction to obtain the intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetyl pipecolic acid (L2-4). The hydrolysis reaction is usually a quantitative reaction and is directly used in the next step of synthesis without further purification.

[0216] Specifically, compound L2-3 is dissolved in a mixed solution of methanol and water, and a 1 mol / L lithium hydroxide (LiOH) aqueous solution is added after cooling to 0°C. The mixed solution is stirred at 0°C to room temperature for 4-12 hours, acidified with 1 mol / L HCl to a pH between 2-3, extracted with ethyl acetate (EA) three times, and dried to obtain a yellow oil, namely the intermediate dibenzazepine octyne glutaramidoacetal tetraethylene glycol acetyl pipecolic acid (L2-4).

[0217] Step 3-Synthesis of active ester

[0218] The intermediate L2-4 obtained in step 2 (without purification) is condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2, ie, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester.

[0219] Specifically, compound L2-4 is dissolved in tetrahydrofuran (THF), cooled to 0°C, and then 2-pentafluorophenol, hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) are added. The mixture is stirred at 0°C to room temperature for 4 to 12 hours, extracted with ethyl acetate (EA) three times, dried, and purified by silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester (L2), the structural formula of which is shown in Figure 2 .

[0220] Subsequently, the L2 linker can be subjected to site-directed coupling reaction with the anti-5T4 antibody and the anti-Trop2 antibody in a PBS pH 7.2-7.4 system (preferably HEPES pH 7.2) (see schematic diagram). Figure 6 and 7 ). Generally, one anti-5T4 antibody or one anti-Trop2 antibody can be coupled to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to about 5.0 to obtain an L2-coupled antibody.

[0221] In some embodiments, the second linker is an azidoacetylated cyclohexosamine, for example, an azidoacetylated cyclogalactosamine or an azidoacetylated glucosamine. In some embodiments, the second linker is a water-stable molecule that can specifically covalently bind to a sialic acid-modified membrane protein. In some embodiments, the second linker is transferred to a sialic acid-modified membrane protein through the cell's own metabolic pathway during the cell culture stage. In some embodiments, the azidoacetyl group of the second linker can undergo a cyclization reaction with the carbon-carbon triple bond of the first linker to form a stable triazole five-membered ring.

[0222] 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:

[0223]

[0224] In some embodiments, the proportion of the α or β single configuration of 1,3,4,6-oxo-tetraacetyl-2-azidoacetamide-2-deoxy-a,bD-galactose is at least 90%, for example, at least 95%, at least 98% or at least 99%.

[0225] 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 Fig.10 , see the structural formula Fig.11 , including two main synthetic steps: aminoazide acetylation and hydroxyl acetylation.

[0226] Step 1 - Acetylation of aminoazide

[0227] 1.2 times of a-azidoacetic acid (compound 1), 2 times of hydroxybenzotriazole (HOBt) and triethylamine (Et3N) were added to the N,N-dimethylformamide (DMF) solution of D-galactosamine hydrochloride (compound 2), and a small amount of methanol (MeOH) was added to dissolve it. The reaction mixture was reacted at room temperature for 12 hours, and the reaction mixture was poured into dichloromethane (DCM) / methanol (MeOH) and shaken to mix. Ether was added to precipitate an oily product (compound 3), and the ether layer was poured out. This was repeated twice and vacuum dried. The next step was directly carried out without further purification.

[0228] Step 2 - Hydroxyacetyl esterification

[0229] The compound 3 obtained above was added with 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. The reaction was almost complete when detected by HPLC. 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 b-configuration product with a purity of >85%. The product N1 with a single optical isomer purity of more than 90% was further purified by silica gel column (dichloromethane: methanol = 20:1). The two isomers a and b- can isomerize to each other in the cell, and after multiple steps of metabolism and synthesis, they are converted into N-azidoacetyl sialic acid, and finally expressed on the surface glycoprotein of NK cells. For the structural formula of the N1 linker, see Fig.11 .

[0230] When the NK cells are cultured for 15 to 16 days, N1 linkers are added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (referred to as "UNK" in the embodiments of the present application) (see the connection mechanism between N1 linkers and NK cells and the structural schematic diagram of UNK). Figure 8 and 9 ).

[0231] Finally, to obtain antibody-NK cell conjugates, UNK and L2-conjugated antibodies can be conjugated in culture medium (reaction mechanism see Fig.10 ) to obtain antibody-NK cell conjugate.

[0232] Subsequent processes also include preparing antibody-NK cell conjugate preparations to effectively prolong the stability of the antibody-NK cell conjugates. The preparation formula may contain ingredients such as sodium chloride and human serum albumin to maintain isotonicity, and may also contain ingredients such as trehalose, sucrose, dextran, and DMSO to maintain the cell's low-temperature resistance and the activity of proteins and enzymes.

[0233] 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.

[0234] In some embodiments, CD3 - CD56 + CD16 + In some embodiments, the number of cells in the cell population is at least 95% of the total number of cells in the cell population. - CD56 + CD16 + In some embodiments, the number of cells in the cell population is at least 98% of the total number of cells in the cell population. - CD56 + NKG2D + In some embodiments, the number of cells in the cell population is at least 95% of the total number of cells in the cell population. - CD56 + NKG2D + The number of cells accounts for at least 98% of the total number of cells in the cell population.

[0235] In some embodiments, CD3 + CD56 + The number of cells does not exceed 5% of the total number of cells in the cell population.

[0236] In some embodiments, CD3 - CD19 + The number of cells does not exceed 2% of the total number of cells in the cell population.

[0237] 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.

[0238] In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 90% of the total number of cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 95% of the total number of cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 98% of the total number of cells in the cell population by cell number. In some embodiments, the antibody-natural killer cell (NK cell) conjugate accounts for at least 99% of the total number of cells in the cell population by cell number.

[0239] 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.

[0240] In some embodiments, the pharmaceutical composition is in the form of a sterile fresh preparation or a sterile frozen preparation.

[0241] In some embodiments, the pharmaceutical composition comprises sodium chloride and / or human serum albumin.

[0242] In some embodiments, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0243] In some embodiments, the pharmaceutical composition is used to treat a tumor in a subject.

[0244] In some embodiments, the individual is a mammal.

[0245] In a preferred embodiment, the mammal is a human.

[0246] In some specific embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0247] In some specific embodiments, the tumor is a malignant tumor.

[0248] 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.

[0249] In a fourth aspect, the present application provides use of the antibody-natural killer cell (NK cell) conjugate described in the first aspect or the cell population described in the second aspect in the preparation of a medicament for treating a tumor in an individual.

[0250] In some embodiments, the individual is a mammal.

[0251] In a preferred embodiment, the mammal is a human.

[0252] In some embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0253] In some specific embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 +) and / or high expression of 5T4 (5T4 + ) tumors.

[0254] In some specific embodiments, the tumor is a malignant tumor.

[0255] 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.

[0256] 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.

[0257] In some specific embodiments, the tumor is a tumor cell that highly expresses Trop2 (Trop2 + ) and / or high expression of 5T4 (5T4 + ) tumors.

[0258] In some specific embodiments, the tumor is a malignant tumor.

[0259] 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.

[0260] In some embodiments, the individual is a mammal.

[0261] In a preferred embodiment, the mammal is a human.

[0262] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments. Example

[0263] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0264] Example 1 - Preparation of antibody-NK cell conjugates

[0265] The preparation of the antibody-NK cell conjugate of this embodiment can be roughly divided into the following steps:

[0266] (1) Preparation of anti-Trop2 monoclonal antibodies and anti-5T4 monoclonal antibodies;

[0267] (2) Preparation of NK cells;

[0268] (3) Preparation of antibody linker (referred to as L2 linker in this example);

[0269] (4) Preparation of NK cell linkers (referred to as N1 linkers in this example);

[0270] (5) an antibody linker (L2 linker) is applied to the antibody;

[0271] (6) applying an NK cell linker (N1 linker) to NK cells; and

[0272] (7) Conjugating two antibodies with linkers to NK cells.

[0273] (1) Preparation of anti-Trop2 monoclonal antibodies and anti-5T4 monoclonal antibodies

[0274] In brief, the sequence of the anti-Trop2 monoclonal antibody is obtained from phage library screening, and is obtained after being screened for binding activity between Trop2 and cells expressing Trop2, cross-species studies of humans, monkeys, and mice, and confirmed by affinity studies, and the anti-Trop2 monoclonal antibody is selected, wherein the anti-Trop2 monoclonal antibody includes the heavy chain variable region shown in SEQ ID NO: 1 (HCDR1-3 are SEQ ID NOs: 11, 6, and 7, respectively), the heavy chain constant region shown in SEQ ID NO: 2, the light chain variable region shown in SEQ ID NO: 3 (LCDR1-3 are SEQ ID NOs: 8, 9, and 10, respectively), and the light chain constant region shown in SEQ ID NO: 4. The DNA sequence of the anti-Trop2 monoclonal antibody is determined, and a recombinant plasmid (IB12) expressing the anti-Trop2 monoclonal antibody is constructed.

[0275] The construction of the monoclonal cell line is based on known technology, in which the IB12 plasmid is transfected into CHO cells by electroporation, and after one round of screening of the minipool and two rounds of screening after monoclonal plating, and confirmed by preliminary passage stability culture, a cell line that can stably express the anti-Trop2 monoclonal antibody is obtained.

[0276] The anti-Trop2 monoclonal antibody is based on a production scheme known in the art, including cell culture and protein purification. Working cells are taken, resuscitated, amplified for 3 to 5 rounds, and cultured on a pilot scale, and then the untreated cell suspension (UPB) is collected and the cell culture supernatant is obtained by clarification and filtration. Then, through the protein purification process, the anti-Trop2 monoclonal antibody is obtained through a three-step chromatography method of Protein A affinity, anion exchange, and cation exchange.

[0277] The sequence of the anti-5T4 monoclonal antibody is obtained from phage library screening, and the anti-5T4 monoclonal antibody is obtained after binding activity screening between 5T4 and cells expressing 5T4 and confirmation by affinity study, wherein the anti-5T4 monoclonal antibody includes the heavy chain variable region shown in SEQ ID NO: 11 (HCDR1-3 are SEQ ID NOs: 15, 16, and 17, respectively), the heavy chain constant region shown in SEQ ID NO: 12, the light chain variable region shown in SEQ ID NO: 13 (LCDR1-3 are SEQ ID NOs: 18, 19, and 20, respectively), and the light chain constant region shown in SEQ ID NO: 14. The DNA sequence of the anti-5T4 monoclonal antibody is determined, and a recombinant plasmid (IB12) expressing the anti-5T4 monoclonal antibody is constructed.

[0278] The construction of the monoclonal cell line is based on known technology, in which the IB12 plasmid is transfected into CHO cells by electroporation, and after one round of screening of the minipool and two rounds of screening after monoclonal plating, and confirmed by preliminary passage stability culture, a cell line that can stably express the anti-5T4 monoclonal antibody is obtained.

[0279] The anti-5T4 monoclonal antibody is based on a production scheme known in the art, including cell culture and protein purification. Working cells are taken, resuscitated, expanded for 3 to 5 rounds, and cultured on a pilot scale, and then the untreated cell suspension (UPB) is collected and the cell culture supernatant is obtained by clarification and filtration. Then, through the protein purification process, the anti-5T4 monoclonal antibody is obtained through a three-step chromatography method of Protein A affinity, anion exchange, and cation exchange.

[0280] (2) Preparation of NK cells

[0281] PBMCs are collected from healthy allogeneic donors. After separation of T cells and red blood cells, the cells are transferred to primary cell cryopreservation medium and the total number of viable cells is ≥ 6.0 × 10 7 PBMCs are obtained by packaging in the specification of 1 cell / tube. PBMCs are stored in liquid nitrogen at ≤-175℃ for a long time.

[0282] The PBMCs frozen in liquid nitrogen are taken out and revived in an adapted culture medium. By expanding the culture scale (i.e., increasing the culture medium expansion), NK cell-related cytokines, including but not limited to IL2 and IL15, are added to maintain their ability to divide and proliferate and their activity. Exemplary NK cell culture stages, process parameters, and process control indicators are shown in Table 1 below.

[0283] Table 1

[0284]

[0285]

[0286] 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):

[0287] Table 2.

[0288]

[0289] (3) Preparation of Antibody Linker (referred to as L2 Linker in this Example)

[0290] For a synthetic route to the 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.

[0291] Step 1 - Amide condensation

[0292] Dibenzazepine octyne glutaric acid (L2-1) and aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-2) are commercially available, and the intermediate dibenzazepine octyne glutaryl aminotetraethylene glycol acetyl pipecolic acid methyl ester (L2-3) is obtained by chemical condensation reaction. L2-3 is a stable compound.

[0293] Specifically, compound L2-1 and compound L2-2 (1:1.1, L2-2 in excess) were dissolved in a dichloromethane (DCM) solution, hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) were added respectively, and finally triethanolamine (TEA) was added. The mixture was stirred at room temperature for 4 to 12 hours, quenched with water, extracted twice with dichloromethane (DCM), and purified with a silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil compound L2-3.

[0294] Step 2-Hydrolysis

[0295] The intermediate L2-3 obtained in step 1 is subjected to hydrolysis reaction to obtain the intermediate dibenzazepine octyne glutaramido tetraethylene glycol acetyl pipecolic acid (L2-4). The hydrolysis reaction is usually a quantitative reaction and is directly used in the next step of synthesis without further purification.

[0296] Specifically, compound L2-3 is dissolved in a mixed solution of methanol and water, and a 1 mol / L lithium hydroxide (LiOH) aqueous solution is added after cooling to 0°C. The mixed solution is stirred at 0°C to room temperature for 4-12 hours, acidified with 1 mol / L HCl to a pH between 2-3, extracted with ethyl acetate (EA) three times, and dried to obtain a yellow oil, namely the intermediate dibenzazepine octyne glutaramidoacetal tetraethylene glycol acetyl pipecolic acid (L2-4).

[0297] Step 3-Synthesis of active ester

[0298] The intermediate L2-4 obtained in step 2 (without purification) is condensed with pentafluorophenol and dicyclohexylcarbodiimide (DCC) to obtain L2, ie, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester.

[0299] Specifically, compound L2-4 is dissolved in tetrahydrofuran (THF), cooled to 0°C, and then 2-pentafluorophenol, hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) are added. The mixture is stirred at 0°C to room temperature for 4 to 12 hours, extracted with ethyl acetate (EA) three times, dried, and purified by silica gel column (dichloromethane: methanol = 20:1) to obtain a yellow oil, dibenzazepine octyne-glutaryl-aminotetraethylene glycol-acetyl pipecolic acid pentafluorophenyl ester (L2), the structural formula of which is shown in Figure 3 .

[0300] (4) Preparation of NK cell linker (referred to as N1 linker in this example)

[0301] 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.

[0302] Step 1 - Acetylation of aminoazide

[0303] 1.2 times of a-azidoacetic acid (compound 1), 2 times of hydroxybenzotriazole (HOBt) and triethylamine (Et3N) were added to the N,N-dimethylformamide (DMF) solution of D-galactosamine hydrochloride (compound 2), and a small amount of methanol (MeOH) was added to dissolve it. The reaction mixture was reacted at room temperature for 12 hours, and the reaction mixture was poured into dichloromethane (DCM) / methanol (MeOH) and shaken to mix. Ether was added to precipitate an oily product (compound 3), and the ether layer was poured out. This was repeated twice and vacuum dried. The next step was directly carried out without further purification.

[0304] Step 2 - Hydroxyacetyl esterification

[0305] The compound 3 obtained above was added with 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. The reaction was almost complete when detected by HPLC. 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 b-configuration product with a purity of >85%. The product N1 with a single optical isomer purity of more than 90% was further purified by silica gel column (dichloromethane: methanol = 20:1). The two isomers a and b- can isomerize to each other in the cell, and after multiple steps of metabolism and synthesis, they are converted into N-azidoacetyl sialic acid, and finally expressed on the surface glycoprotein of NK cells. For the structural formula of the N1 linker, see Figure 5 .

[0306] (5) Applying the antibody linker (L2 linker) to the antibody

[0307] The L2 linker and anti-Trop2 monoclonal antibody can undergo site-specific coupling reaction in a PBS pH 7.2-7.4 system (preferably HEPES pH 7.2) (see schematic diagram for details). Figure 6 ). Usually, one anti-Trop2 monoclonal antibody can be coupled to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to about 5.0 to obtain an L2-coupled Trop2 antibody, also known as a Trop2-coupled antibody.

[0308] The L2 linker and anti-5T4 monoclonal antibody can undergo site-specific coupling reaction in a PBS pH 7.2-7.4 system (preferably HEPES pH 7.2) (see schematic diagram for details). Figure 7 ). Usually, one anti-5T4 monoclonal antibody can be coupled to 1-4 (preferably 1-2) L2 linkers. The reaction can be terminated by adjusting the pH to about 5.0 to obtain an L2-coupled 5T4 antibody, also known as a 5T4-coupled antibody.

[0309] (6) Applying NK cell linker (N1 linker) to NK cells

[0310] When the NK cells are cultured for 15 to 16 days, N1 linkers are added to the culture medium and incubated for 12 to 18 hours to obtain N1 linker-modified NK cells (referred to as "UNK" in the embodiments of the present application) (see the connection mechanism between N1 linkers and NK cells and the structural schematic diagram of UNK). Figure 8 and 9 ).

[0311] (7) Coupling antibodies and NK cells with linkers

[0312] Trop2-conjugated antibody and 5T4-conjugated antibody were mixed in a certain ratio and reacted with UNK in the culture medium (see reaction mechanism for details). Fig.10 ), and a stock solution (DS) of antibody-NK cell conjugate was obtained. The anti-Trop2 monoclonal antibody and anti-5T4 monoclonal antibody-NK cell conjugate was named "IBR825", and this name was used in subsequent examples to represent anti-Trop2 antibody and anti-5T4 antibody-NK cell conjugate.

[0313] Subsequent processes also include preparing antibody-NK cell conjugate preparations to effectively prolong the stability of the antibody-NK cell conjugates. The preparation formula may contain ingredients such as sodium chloride and human serum albumin to maintain isotonicity, and may also contain ingredients such as trehalose, sucrose, dextran, and DMSO to maintain the cell's low-temperature resistance and the activity of proteins and enzymes.

[0314] Example 2-Affinity of Trop2-conjugated antibodies to Trop2 and cross-species detection

[0315] In this example, the OctetR8 instrument was used to detect the affinity of the L2-coupled antibody prepared in step (5) of Example 1 for Trop2 of different species (BLI method). Specifically, various Trop2 proteins were loaded on the His-K probe and bound to the free L2-coupled antibody in the solution (diluted in PBS (pH 7.4)). The maximum response value was used to determine whether the binding occurred. The dissociation constant KD value was calculated based on the association rate and dissociation rate. The results are as follows:

[0316] Table 3.

[0317]

[0318] It can be seen from the results in Table 3 that the antibodies obtained in the present application are specific to primate Trop2 such as humans or rhesus monkeys, and do not bind to rodent Trop2 such as rats or mice.

[0319] Example 3 - Comparison of basic antibody properties between 5T4 conjugated antibody and anti-5T4 monoclonal antibody

[0320] In this example, the 5T4 conjugated antibody prepared in step (7) of Example 1 was compared with the anti-5T4 monoclonal antibody prepared in step (1) of Example 1 for multiple basic antibody properties. The results are shown in Table 4 below, wherein the methodology of each test item follows the conventional test methods in the art.

[0321] Table 4.

[0322]

[0323]

[0324] It can be seen from the results in Table 4 that the coupling of anti-5T4 monoclonal antibody to NK cells does not significantly affect the properties of the antibody itself, so that the desired properties of the antibody are retained.

[0325] Example 4- Evaluation of the in vitro killing activity of the conjugate IBR825 against different cancer cells

[0326] In the evaluation of cell killing activity in this example, 8 tumor cell lines were selected to detect the expression levels of 5T4 and Trop2, respectively. The cell line names, cancer types, and the measured expression levels of 5T4 and Trop2 (measured by flow cytometry) are shown in Table 5 below:

[0327] Table 5.

[0328]

[0329] Calcein acetoxymethy1 ester (Calcein-AM) is a cytoplasmic fluorescent marker. It has no fluorescence itself. After entering the cell, it is hydrolyzed by endogenous esterase in the cell to generate a polar molecule calcein (Calcein) with a strong negative charge that cannot penetrate the cell membrane, and is thus retained in the cell. Calcein can emit strong green fluorescence. Tumor cells are labeled with it and co-incubated with IBR825 cells. When the tumor cells are lysed, Calcein is released into the culture medium. The cytotoxic activity of IBR825 cells is determined by detecting the change in Calcein fluorescence intensity in the culture supernatant with an ELISA instrument (compared with the tumor cell control well (representing 100% cell lysis)).

[0330] The above four highly expressed tumor cells, non-small cell lung cancer cells (NCI-H292), breast cancer cells (MDA-MB-468), prostate cancer cells (BXPC3), and cervical cancer cells (Siha), were selected, and the Calcein AM fluorescence labeling method was used to detect the killing activity of NK cells and three types of IBR825 cells against the above tumor cells. The three types of IBR825 cells were 5T4-conjugated antibody: Trop2-conjugated antibody at a ratio of 2:1, 1:1, and 1:2, respectively, when the total amount of conjugated antibody added remained unchanged.

[0331] The tumor cells were stained with Calcein AM for 30 min and then washed three times. The tumor cell concentration was adjusted to 1.5×10 5 / mL, inoculated in 96-well cell culture plates at 100μL / well. NK cells and three types of IBR825 cells were adjusted to 1.5×106 / mL, 5×105 / mL, and 1.7×105 / mL with cell diluent. Three replicates were made for each gradient, and 100μL / well was added to the 96-well plate, so that the final effect-target ratio was approximately: 10:1, 3:1, and 1:1. Corresponding control groups were also made and cultured at 37°C and 5% CO2 for 4h.

[0332] On-machine detection: transfer 120 μL / well of the supernatant to a black 96-well ELISA plate in parallel, and use 490 nm as the excitation wavelength and 535 nm as the emission wavelength to measure the fluorescence intensity.

[0333] Table 6 below shows the killing effects of NK cells and three IBR825 cells on tumor cells BXPC3, NCI-H292, MDA-MB-468 and Siha at a 3:1 effector-target ratio. The three IBR825s had a high killing rate on all four tumor cells, which was higher than that of NK cells. And there was a dose-effect relationship between the killing activity and IBR825. The results are shown in Figure 11-14 .

[0334] Table 6.

[0335]

[0336] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present application and does not limit the scope of the present application unless otherwise required. Language in the specification should not be construed as indicating that any non-claimed element is essential to the practice of the present application.

[0337] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance the understanding of the application, and is not intended to limit the application to such theory, mechanism, proof or discovery in any way. Although the application has been shown and described in detail in the accompanying drawings and the preceding description, the application should be considered illustrative rather than restrictive.

[0338] Informal Sequence Listing

[0339] Amino acid sequence of anti-Trop2 antibody

[0340]

[0341]

[0342]

[0343] Amino acid sequence of the anti-5T4 antibody

[0344]

[0345]

[0346] References

[0347] [1] H.Kayser, R.Zeitler, C.Kannischt, D.Crunnow, R.Nuck and W.Reutter. J.Biol.Chem, 1992, 267:16934.

[0348] [2] O.T.Keppler, R.Horstcorte,, M.Pawlita, C.Schmidt and W.Reutter. Glycobiology, 2001, 11, 11R.

[0349] [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.

[0350] [4] C.Oetke, S.Hinderlich, R.Brossmer, W.Reutter, M.Pawlita and O.T.Keppler. Eur.J.Biochem, 2001, 268:4553.

[0351] [5] Elinan Saxon and Carolyn R.Bertozzi. Cell Surface Engineering by a Modified Staudinger Reaction[J]. Science, 2000, 278(5460):2007 - 2010.

[0352] [6] David J.Vocadlo, Howard C.Hang, Eun-Ju kim et al. A Chemical Approach for Identifying O-GlcNAc-Modified Protiens in Cells[J]. PNAS, 2003, 100(16):9116 - 9121.

[0353] [7]Jian Du,M Adam Meledeo,Zhiyun Wang,et al.MetabolicGlycoengineering:Sialic Acid and Beyond[J].Glycobiology,2009,19(12):1382-1401.

[0354] [8]Sarah J.Luchansky,Scarlett Goon and Carolyn R.Bertozzi.Expandingthe Diversity of Unnatural Cell-Surfance Sialic Acids[J].ChemBioChem,2004,5(3):371-374.

[0355] [9]Michelle R.Bond,Haochi Zhang,Peter D.Vu,et al.Photocrosslinking ofGlycoconjugates Using Metabolically Incorporated Diazirine-Containing Sugars[J].NATURE PROTOCOLS,2009,4(7):1044-1063.

[0356]

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

Claims

1. An antibody-natural killer cell (NK cell) conjugate, wherein the antibody is an anti-Trop2 antibody or an antigen-binding fragment thereof and an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-Trop2 antibody or an antigen-binding fragment thereof and the anti-5T4 antibody or an antigen-binding fragment thereof are both coupled to the NK cell via a linker.

2. A cell population comprising the antibody-natural killer cell (NK cell) conjugate according to claim 1.

3. A pharmaceutical composition comprising the antibody-natural killer cell (NK cell) conjugate according to claim 1 or the cell population according to claim 2, and a pharmaceutically acceptable carrier.

4. Use of the antibody-natural killer cell (NK cell) conjugate according to claim 1 or the cell population according to claim 2 in the preparation of a medicament for treating tumors.

5. 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 of claim 1 or the cell population of claim 2 or the pharmaceutical composition of claim 3.

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