Design, preparation and use of a trispecific antibody

Abstract

The present invention provides a design of a trispecific antibody, a preparation method and use thereof. The trispecific antibody of the present invention comprises (a) anti-Her2 monoclonal antibody Herceptin, (b) anti-CD16a (FcgRIIIa) single-chain antibody scFv, (c) SIRPa D1 protein, and (d) a flexible linker; the heavy chain C-terminus of the antibody Herceptin is connected to the anti-CD16a single-chain antibody through a linker, and the light chain C-terminus of the antibody Herceptin is connected to SIRPa D1 through a linker; the Fc segment of the antibody is modified to change its binding ability with the receptor and prolong the half-life of the protein; tumor cells expressing CD47 can be targeted by binding to SIRPa D1 and CD47 ligand; at the same time, a "don't eat me" signal can be blocked to activate macrophages to phagocytize tumor cells; anti-CD16a specifically binds to NK (Nature Killing) cells; therefore, macrophages and NK cells in the tumor microenvironment are recruited through SIRPa and anti-CD16a to kill tumor cells; the present invention ensures the specificity of anti-CD16a and does not cause side effects (neutropenia) caused by binding to CD16b on the surface of neutrophils.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese Patent Application No. 202210521832.2 filed on May 13, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. Technical Field

[0005] The present invention belongs to the field of biomedicine technology, and specifically relates to the design, preparation and use of a tri-specific antibody. Background Art

[0006] With the continuous development and deepening of oncology and immunology, tumor immunotherapy has become the most cutting-edge treatment in the field of anti-tumor. At present, the main research direction of tumor immunotherapy at home and abroad is immune checkpoint inhibitors. CD47 has always been considered by the industry as the most important target in the field of tumor immunity after PD-1 / PD-L1. At present, there are numerous candidate drugs targeting CD47 in the preclinical and clinical development stages around the world, but there is still no approved anti-CD47 therapy. [1] .

[0007] CD47 is widely expressed on the surface of many cancer cells, but it is also widely expressed on the surface of red blood cells to protect themselves from being phagocytosed. This means that while drugs targeting CD47 kill tumor cells, they will inevitably kill red blood cells by mistake, causing a decrease in the number of red blood cells and platelets, leading to severe anemia. [2] .

[0008] The present invention is a trispecific antibody targeting Her2, CD47 and CD16a. Herceptin has become the standard therapy for Her2-positive tumors such as breast cancer; the CD47-SIRPa "Don't eat me" pathway has been fully proven to be a target with significant clinical efficacy; CD16a is expressed on the surface of NK cells. The mechanism of action of the present invention includes but is not limited to the following three: (1) Tumors that are double positive for Her2 and CD47 can be pulled together with NK cells, and Perforin (perforin) released by NK cells damages the tumor cell membrane and forms a channel to allow a large amount of Granzyme B (granzyme B) from NK cells to enter the tumor cells and directly kill the tumor cells. [3](2) At the same time, because the CD47 of the tumor cells has been blocked by the SIRPa D1 binding of the tertiary antibody, the macrophages in the tumor microenvironment no longer receive the inhibitory signal of "Don't eat me", thereby activating the macrophages to engulf and kill tumor cells; (3) The anti-Her2 end can also directly block the formation of Her2 aggregates, thereby inhibiting the proliferation of Her2-positive tumor cells. [4] . Figure 2 The mechanism of action of the drug of the present invention is demonstrated. Bispecific antibodies targeting Her2 and CD47 have been reported, but trispecific antibodies targeting Her2-CD47-CD16a have not been reported and are First-In-Class.

[0009] Since human CD16 protein has two subtypes, CD16a and CD16b, CD16a is mainly expressed in NK cells, but also in monocytes and macrophages, and is an activating receptor; CD16b is expressed in neutrophils, and the two subtypes are highly homologous. Therefore, anti-CD16a antibodies can usually also bind to CD16b, often causing neutropenia. [5] The anti-CD16a in this invention specifically binds to the CD16a receptor but does not bind to CD16b, and therefore does not cause neutropenia. Summary of the invention

[0010] To solve the above problems, the present invention discloses the design, preparation and use of a trispecific antibody; the key technical problem that the present invention focuses on solving is to ensure the specificity of anti-CD16a, not to bind to CD16b on the surface of neutrophils, thereby not causing the side effect of neutropenia possessed by some anti-CD16a antibodies, while maintaining the high specificity and high affinity of Herceptin antibody and SIRPa D1 protein, and minimizing the side effects caused by nonspecific binding.

[0011] To achieve the above object, the technical solution of the present invention is as follows:

[0012] An object of the present invention is to provide a trispecific antibody or a trispecific antigen-binding fragment thereof having specificity for three antigens, wherein the antibody comprises (a) anti-Her2 monoclonal antibody Herceptin, (b) anti-CD16a single-chain antibody scFv, (c) SIRPa D1 protein, and (d) a flexible linker.

[0013] In one embodiment, Herceptin is used as the basic structure of a trispecific antibody, and an anti-CD16a single-chain antibody scFv or a SIRPa D1 protein is connected to the N-terminus or C-terminus of the Herceptin heavy chain through a flexible linker, and a SIRPa D1 protein or an anti-CD16a single-chain antibody scFv is connected to the N-terminus or C-terminus of the Herceptin light chain through a flexible linker.

[0014] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin heavy chain through a flexible linker.

[0015] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin heavy chain through a flexible linker.

[0016] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the N-terminus of the Herceptin heavy chain through a flexible linker.

[0017] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the N-terminus of the Herceptin heavy chain through a flexible linker.

[0018] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the N-terminus of the Herceptin light chain through a flexible linker.

[0019] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin light chain through a flexible linker.

[0020] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the N-terminus of the Herceptin light chain through a flexible linker.

[0021] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the N-terminus of the Herceptin heavy chain through a flexible linker.

[0022] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin light chain through a flexible linker.

[0023] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the N-terminus of the Herceptin light chain through a flexible linker.

[0024] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin heavy chain through a flexible linker.

[0025] In a specific embodiment, Herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin light chain through a flexible linker.

[0026] In one embodiment, the heavy chain variable region of the anti-Her2 monoclonal antibody Herceptin comprises: CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17 and CDR3 shown in SEQ ID NO:18; the light chain variable region comprises: CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20 and CDR3 shown in SEQ ID NO:21.

[0027] In a further embodiment, the heavy chain variable region of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:6.

[0028] In a further embodiment, the heavy chain constant region of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID NO:2 or 3, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:7.

[0029] In a further embodiment, the heavy chain of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID NO:4 or 5, and the light chain comprises the amino acid sequence shown in SEQ ID NO:8.

[0030] In one embodiment, the heavy chain variable region of the anti-CD16a single-chain antibody scFv comprises: CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:24; the light chain variable region comprises: CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:27.

[0031] In a further embodiment, the heavy chain variable region of the anti-CD16a single-chain antibody scFv comprises the amino acid sequence shown in SEQ ID NO: 28 or 29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30 or 31.

[0032] In a further embodiment, the anti-CD16a single-chain antibody scFv comprises the amino acid sequence shown in SEQ ID NO:11, 12 or 13.

[0033] In one embodiment, the SIRPa D1 protein comprises the amino acid sequence shown in SEQ ID NO:9 or 10.

[0034] In a specific embodiment, the amino acid sequence of one chain of the trispecific antibody is:

[0035] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAHIWWDDDKRYSPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARINPAYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK(SEQ ID NO:32)

[0036] Or:

[0037] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGSASAPTLKLEEGEFSEARVQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:33)

[0038] Or:

[0039] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT

[0040] RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL

[0041] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0042] VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0043] EAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0044] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0045] YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0046] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSEVQLV

[0047] QSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQ

[0048] KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0049] GGGGSGGGGSGGGGSGGGGSQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQR

[0050] PGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:34)

[0051] Or:

[0052] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT

[0053] RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL

[0054] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0055] VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0056] ELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0057] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0058] YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0059] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSEVQLV

[0060] QSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQ

[0061] KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0062] GGGGSGGGGSGGGGSGGGGSQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQR

[0063] PGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:35)

[0064] Or:

[0065] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT

[0066] RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL

[0067] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

[0068] VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0069] ELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0070] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0071] YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:36)

[0072] The amino acid sequence of the other chain is:

[0073] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS

[0074] RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSD

[0075] EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

[0076] LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEEELQVIQP

[0077] DKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK(SEQ ID

[0078] NO:37)

[0079] Or:

[0080] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS

[0081] RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSD

[0082] EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

[0083] LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEEELQVIQP

[0084] DKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK(SEQ ID

[0085] NO:38)

[0086] In one aspect, the present invention prepares a trispecific antibody that specifically binds to Her2, CD16a and CD47, and the resulting antibody is named TriAB01, TriAB02, TriAB03 or TriAB04. In another aspect, the present invention prepares a bispecific antibody that specifically binds to Her2 and CD47, and the resulting antibody is named TriAB-C. The amino acid sequence of one chain of the trispecific antibody TriAB01 is SEQ ID NO:32, and the amino acid sequence of the other chain is SEQ ID NO:37. The amino acid sequence of one chain of the trispecific antibody TriAB02 is SEQ ID NO:33, and the amino acid sequence of the other chain is SEQ ID NO:37. The amino acid sequence of one chain of the trispecific antibody TriAB03 is SEQ ID NO:34, and the amino acid sequence of the other chain is SEQID NO:37. The amino acid sequence of one chain of the trispecific antibody TriAB04 is SEQ ID NO:35, and the amino acid sequence of the other chain is SEQ ID NO:37. The amino acid sequence of one chain of the bispecific antibody TriAB-C is SEQ ID NO: 36, and the amino acid sequence of the other chain is SEQ ID NO: 37. In some embodiments, the trispecific antibody that specifically binds to Her2, CD16a, and CD47 is preferably TriAB04.

[0087] In some embodiments, the monoclonal antibody Herceptin introduces L234A or L235A mutations into the Fc segment, and simultaneously introduces M252Y, S254T or T256E mutations into the Fc segment.

[0088] In some embodiments, the Fc segment of the monoclonal antibody Herceptin is wild-type, and M252Y, S254T or T256E mutations are introduced into the Fc segment.

[0089] In some embodiments, the present invention provides an isolated nucleic acid molecule encoding the trispecific antibody or trispecific antigen-binding fragment thereof described herein.

[0090] In some embodiments, the present invention provides a vector comprising a nucleic acid sequence encoding the trispecific antibody or trispecific antigen-binding fragment thereof described herein.

[0091] Another object of the present invention is to provide a method for preparing the above-mentioned trispecific antibody, comprising the following steps:

[0092] (1) The structure of the present invention is as follows Figure 1As shown, it is a symmetrical structure. The anti-CD16a single-chain antibody scFv or SIRPa D1 protein is connected to the N-terminus or C-terminus of the heavy chain of the antibody Herceptin through a flexible linker. The scFv consists of a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL are connected together by a flexible peptide linker. In the scFv, the order of the domains can be VH-linker-VL or VL-linker-VH. SIRPa D1 or anti-CD16a single-chain antibody scFv is connected to the N-terminus or C-terminus of the light chain of the Herceptin antibody through a flexible linker. L234A / L235A mutations are introduced into the Fc segment of the antibody Herceptin to reduce the binding ability to the receptors FcgRIII and FcgRIIa, and M252Y / S254T / T256E mutations are introduced into the Fc segment to extend the half-life of the protein;

[0093] (2) cloning the DNA fragment obtained in step (1) into a pcDNA series vector or other vectors for mammalian cell expression systems by conventional molecular biological means, cloning the DNA fragment obtained in step (1) into another pcDNA series vector or other vectors for mammalian cell expression systems, including but not limited to, wherein the vector of the expression system includes a fusion DNA sequence connected to appropriate transcription and translation regulatory sequences;

[0094] (3) Transfecting the recombinant vector obtained in step (2) into mammalian cells to express and purify the fusion protein to obtain trispecific antibodies. The transfection method can be chemical reagent transfection or electroporation transfection, and the mammalian cells can be HEK293 cells or CHO (Chinese Hamster Ovary) cells or derivatives of the above cells or other expression systems (such as E. coli and yeast). The obtained antibodies are named TriAB01 / TriAB02 / TriAB03 / TriAB04 / TriAB-C.

[0095] In certain embodiments, the step (1) specifically includes the following steps:

[0096] (a) synthesizing the DNA sequences of the heavy chain and light chain of the Herceptin antibody and the anti-CD16a single-chain antibody scFv;

[0097] (b) designing primers to amplify the heavy chain of Herceptin antibody and anti-CD16a single-chain antibody scFv respectively using the plasmid in (a) as a template;

[0098] (c) using the DNA fragment in (b) as a template, performing Overlap PCR with an upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv DNA sequence to obtain a DNA sequence;

[0099] (d) designing mutation primers with the mutation point as the boundary, using the fragment in (c) as the template, using the upstream primer and the downstream mutation primer for amplifying the heavy chain DNA sequence of the Herceptin antibody to amplify the fragment before the mutation point, and using the upstream mutation primer and the downstream primer for amplifying the anti-CD16a single-chain antibody scFv to amplify the fragment after the mutation point;

[0100] (e) using the two fragments obtained in (d) as templates, using the upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and the downstream primer for amplifying the anti-CD16a single-chain antibody scFv to amplify the mutated fragments;

[0101] (f) obtaining another DNA fragment in the same manner;

[0102] (g) connecting the fragments prepared in (e) and (f) to pcDNA series vectors or other vectors used in, including but not limited to, mammalian cell expression systems, transforming into E. coli competent cells DH5a or other competent cells, and picking single clones to prepare plasmids;

[0103] (h) The plasmid prepared in (g) was transfected into HEK293 cells and cultured in a shaker at 37°C, 8% CO2, and 125 rpm. After 7 days of transient expression, the supernatant was purified by protein A affinity chromatography to obtain the recombinant antibody, and the antibody concentration was determined by UV280 binding theoretical extinction coefficient.

[0104] Another object of the present invention is to provide the use of the above-mentioned trispecific antibody in the preparation of anti-tumor drugs. The trispecific antibody can be used to treat any tumor that expresses Her2 and CD47 simultaneously, or expresses Her2 or CD47 alone, or low-expression Her2 or Her2-negative tumors, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, biliary cancer and ovarian cancer. The use also includes monotherapy for tumor treatment or combined with other tumor treatment methods.

[0105] Another object of the present invention is to provide the use of the trispecific antibody prepared according to the above method in the preparation of bispecific antibodies, trispecific antibodies or multispecific antibody drugs. Bispecific antibodies, trispecific antibodies or multispecific antibodies can be used for the treatment of any tumor that simultaneously expresses Her2 and CD47 or single expression of Her2 or CD47, or low expression of Her2 or Her2 negative, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, biliary cancer and ovarian cancer. Uses also include monotherapy for tumor treatment or combined with other tumor treatment methods.

[0106] Features of the present invention:

[0107] 1. To solve the defect of low specificity in targeting CD16a, the anti-CD16a scFv in the present invention specifically binds to CD16a but not to CD16b, thereby avoiding neutropenia caused by the binding of neutrophils and antibodies.

[0108] 2. Recruiting NK cells through anti-CD16a can also kill tumor cells when Fc function is insufficient. When Fc function is sufficient, the function of killing tumor cells is more powerful.

[0109] 3. Through the binding of SIRPa D1 and CD47 ligand, it can connect to tumor cells expressing CD47; at the same time, it can block the "Don't eat me" signal and activate macrophages to engulf tumor cells;

[0110] 4. Reduce the binding ability of the antibody Fc segment to the receptor FcgRIIIa by mutation, thereby avoiding neutropenia caused by the binding of neutrophils and antibodies.

[0111] 5. At the same time, M252Y / S254T / T256E mutations were introduced into the Fc segment to extend the half-life of the protein.

[0112] 6. The antibody structure is similar to traditional IgG, with a symmetrical structure design and no knob-into-hole mutation, which avoids the production of isomers. It has significant advantages in purification process development and production costs.

[0113] 7. Herceptin itself has the function of directly inhibiting tumor growth [6] .

[0114] 8. Under normal circumstances, cardiomyocytes express Her2. The main toxic side effect of Herceptin is damage to cardiomyocytes. Long-term use can reduce left ventricular ejection fraction (4-18%) or cause severe heart failure (4%). [7]. CD47 is expressed at a low level in myocardial cells, so the possibility of trispecific antibodies binding to and damaging the myocardium is low. It is worth noting that CD47 increases during myocarditis and myocardial ischemia-reperfusion. Many literatures have shown that CD47 antagonism can reduce myocarditis, alleviate myocardial ischemia-reperfusion damage, and reduce atherosclerosis. [8] Therefore, the antagonism of CD47 contained in the three antibodies of the present invention may help to reduce the damage of Her2 targeted therapy and alleviate toxic side effects.

[0115] 9. In most cases, only tumor cells express both Her2 and CD47 (double positive); normal cells usually only express Her2 or CD47. At a certain concentration, trispecific antibodies only bind to tumor cells, but not to normal cells expressing Her2 or CD47, thereby reducing damage to normal cells.

[0116] 10. Since this trispecific antibody simultaneously mobilizes macrophages and NK cells as effector cells, it also has a killing effect on tumors with low or no Her2 expression, similar to the bystander effect of Enhertu [9] . Since it does not contain small molecule toxins, the toxic side effects of this trispecific antibody will be significantly lower than Enhertu, which contains toxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 is a schematic diagram of the structure of the trispecific antibody of the present invention;

[0118] Figure 2 Schematic diagram of the action mechanism of the trispecific antibody of the present invention;

[0119] Figure 3 This is the SDS-PAGE electrophoresis diagram of the trispecific antibody of the present invention;

[0120] Figure 4-5 This is a graph showing the binding ability of the trispecific antibody of the present invention to the Her2 protein;

[0121] Figure 6-7 This is a graph showing the binding ability of the trispecific antibody of the present invention to the CD47 protein;

[0122] Figure 8-9 This is a graph showing the binding ability of the trispecific antibody of the present invention to CD16a protein;

[0123] Figure 10-11 This is a graph showing the binding ability of the trispecific antibody of the present invention to the CD16b protein;

[0124] Fig.12 This is a graph showing the ability of the trispecific antibody of the present invention to bind to SK-BR-3 cells;

[0125] Fig.13 This is a graph showing the ability of the trispecific antibody of the present invention to bind to SK-OV-3 cells.

[0126] Fig.14 This is a graph showing the ability of the trispecific antibody of the present invention to recruit macrophages for phagocytosis;

[0127] Fig.15 This is a detection diagram of blood coagulation induced by the trispecific antibody of the present invention;

[0128] Fig.16 This is a detection diagram of the internalization of Her2 induced by the trispecific antibody of the present invention;

[0129] Fig.17 This is a graph showing the affinity detection between the trispecific antibody of the present invention and the FcRn protein;

[0130] Fig.18 This is an ELISA test chart of the binding ability of the trispecific antibody of the present invention to FcRn protein;

[0131] Fig.19 is the ADCC activity of the trispecific antibody of the present invention against NCI-N87 cells;

[0132] Fig. 20 is the ADCC activity of the trispecific antibody of the present invention against OE19 cells;

[0133] Fig.21 is the ADCC activity of the trispecific antibody of the present invention against MDA-MB-231 cells;

[0134] Fig. 22 The ADCC activity of the trispecific antibody of the present invention against SK-OV-3 cells;

[0135] Fig.23 is the ADCC activity of the trispecific antibody of the present invention against BT-474 cells;

[0136] Fig.24 is the anti-tumor activity of the trispecific antibody of the present invention in the NCI-N87 xenograft model;

[0137] Fig.25 This is an evaluation of the efficacy of the trispecific antibodies of the present invention in the ExVivo organoid system. DETAILED DESCRIPTION OF THE INVENTION

[0139] Unless otherwise defined herein, all scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclature and techniques associated with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0140] Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods and embodiments are illustrative rather than restrictive. Other features and advantages of the present disclosure will be apparent from the following detailed description of the invention and the claims.

[0141] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0142] As used herein, the term "antibody" is used in its broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0143] The trispecific antibodies of the present disclosure can be separated and / or purified. As used herein, the term "separated" means that it has been removed from its natural environment. As used herein, the term "purified" means that the purity is improved, wherein "purity" is a relative term and is not necessarily interpreted as absolute purity. In an exemplary embodiment, the purity of the trispecific antibody is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% or about 100%. The bispecific antibodies or trispecific antibodies of the present disclosure can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. In one embodiment, affinity chromatography is used to purify the bispecific antibodies or trispecific antibodies of the present disclosure.

[0144] The term "bispecific antibody" as used herein is an antibody with two different antigen binding specificities. For example, the bispecific antibodies of the present disclosure can bind to Her2 and CD47 with sufficient affinity.

[0145] As used herein, the terms "trispecific antibody", "trispecific antibody that specifically binds to Her2, CD47 and CD16a", and "trispecific antibody against Her2, CD47 and CD16a" are used interchangeably and refer to a trispecific antibody that can bind to Her2, CD47 and CD16a with sufficient affinity, so that the trispecific antibody can be used as a therapeutic agent / diagnostic agent targeting cells expressing Her2 and / or CD47 or CD16a. The trispecific antibody of the present disclosure does not bind to CD16b or binds with very low affinity, thereby reducing or lowering side effects caused by binding to CD16b on the surface of neutrophils, such as neutropenia. In addition, the trispecific antibody of the present disclosure maintains the high specificity and high affinity of the Herceptin antibody and the SIRPa D1 protein, minimizing the side effects caused by nonspecific binding.

[0146] In some embodiments, the trispecific antibody has high affinity for the Her2 antigen, EC 50 The value is 0.1-0.3 nM, as detected by ELISA or FACS. In some embodiments, the trispecific antibody has a high affinity for the CD47 antigen, EC 50 The value is 0.5-3.5 nM, as detected by ELISA or FACS. In some embodiments, the trispecific antibody has a high affinity to the CD16a antigen, EC 50 Values ​​are 5-30 nM as detected by ELISA or FACS.

[0147] The terms "flexible linker", "flexible peptide linker" or "linker" as used herein refer to a peptide linker, preferably a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5-100, more preferably 10-50, and most preferably 10-20 amino acids in length. In one embodiment, the peptide linker is (GxS)n or (GxS)nGm, wherein G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6 and m = 0, 1, 2 or 3) or (x = 4 and n = 2, 3, 4 or 5 and m = 0, 1, 2 or 3), preferably x = 4 and n = 2 or 3, more preferably x = 4 and n = 3. In a preferred embodiment, the amino acid sequence of the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 14). In a preferred embodiment, the amino acid sequence of the peptide linker is GSASAPTLKLEEGEFSEARV (SEQ ID NO: 15).

[0148] As used herein, the term "amino acid mutation" encompasses amino acid substitutions, deletions, insertions and modifications. The trispecific antibodies may be substituted, deleted, inserted and modified to obtain the desired function. Amino acid mutations may be generated using genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Various names may be used herein to indicate amino acid mutations. For example, a mutation from leucine to alanine at position 234 of the Fc segment may be indicated as L234A.

[0149] The multispecific antibodies of the present disclosure may comprise one or more amino acid substitutions, which may be non-conservative amino acid substitutions, i.e., substitutions of one amino acid with another amino acid having different structural and / or chemical properties. The substitutions may also be conservative amino acid substitutions, which do not adversely affect or alter the basic properties of the protein / polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as an amino acid residue replaced by a physically or functionally similar residue (e.g., having similar size, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions preferably involve the substitution of the corresponding amino acid residue by another amino acid residue from the same side chain family.

[0150] Amino acid substitutions can be introduced into the multispecific antibodies of the disclosure and screened for desired activity, retained / improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC), etc. Both conservative and non-conservative amino acid substitutions are contemplated for preparation of antibody variants.

[0151] In one aspect of the present invention, the Herceptin Fc segment of the antibody can be mutated to reduce the binding ability to the receptors FcgRIII and FcgRIIa. In one embodiment, the L234A / L235A mutation is introduced into the Herceptin Fc segment of the antibody to reduce the binding ability to the receptors FcgRIII and FcgRIIa. In another aspect of the present invention, the Herceptin Fc segment of the antibody can be modified to extend the half-life of the protein. In one embodiment, the M252Y / S254T / T256E mutation is introduced into the Herceptin Fc segment of the antibody to extend the half-life of the protein. In one embodiment, the L234A / L235A mutation is introduced into the Herceptin Fc segment of the antibody to reduce the binding ability to the receptors FcgRIII and FcgRIIa, and the M252Y / S254T / T256E mutation is introduced into the Fc segment of the Fc segment to extend the half-life of the protein.

[0152] As used herein, the term "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a light chain variable region and a heavy chain variable region, wherein the light chain and heavy chain variable regions are connected via a linker, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the anti-CD16a binding domain is a scFv comprising a light chain and a heavy chain variable region of an amino acid sequence provided herein. The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0153] The heavy chain variable region and the light chain variable region are responsible for binding to the target antigen, and most of the sequence variability occurs in 6 complementary determining regions (CDRs), and each variable heavy chain (VH) and variable light chain (VL) domain has 3 CDRs. The complementary determining regions combine to form an antigen binding site. There are multiple schemes for identifying complementary determining regions, for example, the Kabat numbering system defines complementary determining regions based on sequence variability at the antigen binding region of the VH and VL domains, the Chothia numbering system defines "hypervariable loops" based on the position of the structural loop regions in the VH and VL domains, and the IMGT numbering system is based on the amino acid sequence alignment of germline V genes. For a given variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues constitute a specific CDR. The CDR region can be numbered according to the kabat numbering system, the Chothia numbering system, or the IMGT numbering system, etc. Those of ordinary skill in the art can use this type of numbering system for any variable region sequence, independent of any experimental data outside the sequence itself. Unless otherwise indicated, the VH CDR1 , VH CDR2, VH CDR3 and VL CDR1 , VL CDR2 and VL CDR3 amino acid sequences described herein are numbered according to the IMGT numbering system.

[0154] The present invention further provides isolated nucleic acid molecules encoding trispecific antibodies or trispecific antigen-binding fragments thereof as described herein. "Isolated nucleic acid molecules encoding trispecific antibodies" or "isolated nucleic acid molecules encoding trispecific antibodies or trispecific antigen-binding fragments thereof" refer to one or more nucleic acid molecules encoding antibody heavy chains and / or light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell. Nucleic acid molecules encoding the trispecific antibodies of the present invention can be expressed as a single polynucleotide encoding a complete trispecific antigen-binding molecule or as a plurality of (e.g., two or more) co-expressed polynucleotides. The polypeptides encoded by the co-expressed polynucleotides can be combined, for example, via disulfide bonds or other means to form functional trispecific antibodies.

[0155] In some aspects, the present invention relates to an expression vector comprising an isolated nucleic acid molecule encoding a trispecific antibody of the present invention, and a prokaryotic or eukaryotic host cell comprising an isolated nucleic acid molecule or vector encoding a trispecific antibody of the present invention. In one embodiment, the host cell comprises a vector (e.g., transformed or transfected with the vector), the vector comprising a polynucleotide encoding a trispecific antibody of the present invention (or a trispecific antigen-binding fragment thereof). The term "host cell" as used herein refers to any cell system that can be engineered to express a trispecific antibody of the present invention or a trispecific antigen-binding fragment thereof. Host cells suitable for expressing the trispecific antibodies of the present invention are well known in the art.

[0156] The present invention also provides a method for generating a trispecific antibody, comprising the following steps:

[0157] (1) connecting the anti-CD16a single-chain antibody scFv to the C-terminus or N-terminus of the Herceptin antibody heavy chain through a flexible linker; connecting the SIRPa D1 protein to the C-terminus or N-terminus of the Herceptin antibody light chain through a flexible linker; introducing mutations in the Herceptin antibody Fc segment;

[0158] (2) cloning the first DNA fragment obtained in step (1) into a pcDNA series vector or other vectors used in, but not limited to, mammalian cell expression systems; cloning the second DNA fragment obtained in step (1) into another pcDNA series vector or other vectors used in, but not limited to, mammalian cell expression systems, to obtain a recombinant vector;

[0159] (3) Transfecting the recombinant vector obtained in step (2) into mammalian cells or other expression systems to express and purify the fusion protein to obtain the trispecific antibody.

[0160] In one embodiment, in step (1), L234A or L235A mutation is introduced into the Fc segment of the Herceptin antibody, and M252Y, S254T or T256E mutation is simultaneously introduced into the Fc segment.

[0161] In one embodiment, the Fc segment of the Herceptin antibody in step (1) is wild type, and M252Y, S254T or T256E mutations are introduced into the Fc segment.

[0162] In a specific embodiment, the step (1) specifically comprises the following steps:

[0163] (a) synthesizing the heavy chain of Herceptin antibody and anti-CD16a single-chain antibody scFv DNA sequence;

[0164] (b) designing primers to amplify the heavy chain of Herceptin antibody and anti-CD16a single-chain antibody scFv respectively using the plasmid in (a) as a template;

[0165] (c) using the DNA fragment in (b) as a template, performing Overlap PCR with an upstream primer for amplifying the heavy chain DNA sequence of the antibody Herceptin and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv DNA sequence to obtain a DNA sequence;

[0166] (d) using the mutation point M252Y, S254T or T256E as the boundary, designing mutation primers, using the fragment in (c) as a template, using the upstream primer and the downstream mutation primer for amplifying the heavy chain DNA sequence of the Herceptin antibody to amplify the fragment before the mutation point, and using the upstream mutation primer and the downstream primer for amplifying the anti-CD16a single-chain antibody scFv to amplify the fragment after the mutation point;

[0167] (e) using the two fragments obtained in (d) as templates, using the upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and the downstream primer for amplifying the anti-CD16a single-chain antibody scFv to amplify the mutated DNA fragment;

[0168] (f) obtaining a second DNA fragment in the same manner;

[0169] (g) connecting the fragments prepared in (e) and (f) to pcDNA series vectors or other vectors used in, including but not limited to, mammalian cell expression systems, transforming into E. coli competent cells DH5a or other competent cells, and picking single clones to prepare plasmids;

[0170] (h) The plasmid prepared in (g) was transfected into HEK293 cells and cultured in a shaker at 37°C, 8% CO2, and 125 rpm. After 7 days of transient expression, the supernatant was purified by protein A affinity chromatography to obtain the recombinant antibody, and the antibody concentration was determined by UV280 binding theoretical extinction coefficient.

[0171] The bispecific antibodies, trispecific antibodies or multispecific antibodies described in the present disclosure can be used, for example, to treat or diagnose cancer, such as treating any tumor that simultaneously expresses Her2 and CD47 or single expression of Her2 or CD47, or low expression of Her2 or Her2-negative tumors, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, bile duct cancer and ovarian cancer. Therefore, in some embodiments, the present disclosure relates to the use of trispecific antibodies in the preparation of anti-tumor drugs. In some embodiments, the present disclosure relates to the use of trispecific antibodies produced using the methods described above in the preparation of anti-tumor drugs. In certain embodiments, the present invention provides a method for treating an individual with cancer using a trispecific antibody, the method comprising administering a therapeutically effective amount of a trispecific antibody to the individual, the cancer including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, bile duct cancer and ovarian cancer. In a further embodiment, the method further comprises administering other tumor treatment methods to the individual. In the above embodiments, the individual is preferably a mammal, more preferably a human. The trispecific antibodies of the present invention can be administered in vivo to an individual in need thereof by various routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal.

[0172] The term "treatment" as used herein refers to therapeutic treatment, the purpose of which is to slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For the purposes described herein, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder or disease; stabilization (i.e., no worsening) of the condition, disorder or disease state; delayed onset or slowed progression of the condition, disorder or disease; improvement of the condition, disorder or disease state. Treatment also includes extending survival compared to the expected survival without treatment. In other embodiments, "treatment" refers to preventive measures, wherein the purpose is, for example, in a person susceptible to the disease (e.g., an individual carrying genetic markers for a disease such as breast cancer), delaying the onset of an undesirable physiological condition, disorder or disease or reducing its severity. Example

[0173] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0174] Example 1

[0175] Molecular construction and production of trispecific antibodies

[0176] The antibody light and heavy chain genes were amplified by PCR using conventional molecular biology techniques, and the light and heavy chain genes were connected to the pcDNA3.4 vector using homologous recombination technology. The plasmids of the positive clones after sequencing were extracted and transfected into Expi293F cells, and cultured for 7 days in a shaker at 37°C / 5% CO2 / 125rpm. After the culture was completed, the supernatant was collected and purified by protein A affinity chromatography to obtain the purified trispecific antibody, and the antibody concentration was determined by UV280 binding theoretical extinction coefficient. The results of SDS-PAGE electrophoresis are shown in Figure 3 The three specific antibodies were successfully expressed and the purity of SDS-PAGE reached more than 80% after one-step affinity chromatography.

[0177] Example 2

[0178] ELISA to detect the binding of trispecific antibodies to Her2 protein

[0179] Coated with 2ug / ml Her2 protein (10004-H08H1, Sino Biological), 100ul / well, overnight at 4℃; blocked with 3% skim milk powder at 37℃ for 1h; added 100ul of trispecific antibodies and other control samples of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h; then added goat anti-human IgG / HRP, incubated at 37℃ for 1h, and after color development for 10min, read OD450 on the microplate reader. Results are shown in Figure 4 .

[0180] Coated with 2ug / ml Her2 protein, 100ul / well at 4℃ overnight; blocked with 3% skim milk powder at 37℃ for 1h and washed 3 times with PBST; added 100ul of trispecific antibodies and other control samples of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h and washed 3 times with PBST; then added 100ul of goat anti-human IgG-HRP diluted 1:8000, incubated at 37℃ for 1h and washed 6 times, added 100ul of TMB to each well for 5min of color development and then added 50ul of stop solution, and read OD450 on the microplate reader. Results are shown in Figure 5 The trispecific antibody has high affinity to the Her2 antigen.

[0181] Example 3

[0182] ELISA to detect the binding of trispecific antibodies to CD47 protein

[0183] Coated with 2ug / ml CD47 protein (12283-H08H, Sino Biological), 100ul / well, overnight at 4℃; blocked with 3% skim milk powder at 37℃ for 1h; added 100ul of trispecific antibodies of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h; then added goat anti-human IgG / HRP, incubated at 37℃ for 1h, and after color development for 10min, read OD450 on the microplate reader. Results are shown in Figure 6 .

[0184] Coated with 2ug / ml CD47 protein, 100ul / well at 4℃ overnight; blocked with 3% skim milk powder at 37℃ for 1h and washed 3 times with PBST; added 100ul of trispecific antibodies and other control samples of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h and washed 3 times with PBST; then added 100ul of goat anti-human IgG-HRP diluted 1:8000, incubated at 37℃ for 1h and washed 6 times, added 100ul TMB to each well for 5min color development and then added 50ul stop solution, and read OD450 on the microplate reader. Results are shown in Figure 7 The trispecific antibody has high affinity to the CD47 antigen.

[0185] Example 4

[0186] ELISA to detect the binding of trispecific antibodies to CD16a protein

[0187] Coated with 2ug / ml CD16a protein (10389-H08C, Sino Biological), 100ul / well, overnight at 4℃; blocked with 3% skim milk powder at 37℃ for 1h; added 100ul of trispecific antibodies of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h; then added goat anti-human IgG / HRP, incubated at 37℃ for 1h, and after color development for 10min, read OD450 on the microplate reader. Results are shown in Figure 8 .

[0188] Coated with 2ug / ml CD16a protein, 100ul / well at 4℃ overnight; blocked with 3% skim milk powder at 37℃ for 1h; added 100ul of trispecific antibodies and other control samples of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h; washed 3 times with PBST after incubation at 37℃ for 1h; then added 100ul of goat anti-human IgG-HRP diluted 1:8000, incubated at 37℃ for 1h, washed 6 times, added 100ul of TMB color development to each well for 5min, and then added 50ul of stop solution, and read OD450 on the microplate reader. Results are shown in Fig. 9 The trispecific antibody can specifically bind to CD16a.

[0189] Example 5

[0190] ELISA to detect the binding of trispecific antibodies to CD16b protein

[0191] Coated with 2ug / ml CD16b protein (11046-H08H, Sino Biological), 100ul / well, overnight at 4℃; blocked with 3% skim milk powder at 37℃ for 1h; added 100ul of each of the three specific antibodies of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h; then added goat anti-human IgG / HRP, incubated at 37℃ for 1h, and after color development for 10min, read OD450 on the microplate reader. Results are shown in Fig.10 .

[0192] Coated with 2ug / ml CD16b protein, 100ul / well at 4℃ overnight; blocked with 3% skim milk powder at 37℃ for 1h; added 100ul of trispecific antibodies and other control samples of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubated at 37℃ for 1h; washed 3 times with PBST after incubation at 37℃ for 1h; then added 100ul of goat anti-human IgG-HRP diluted 1:8000, incubated at 37℃ for 1h, washed 6 times, added 100ul of TMB color development to each well for 5min, and then added 50ul of stop solution, and read OD450 on the microplate reader. Results are shown in Fig.11 The trispecific antibody does not bind to CD16b, thus avoiding the toxic side effects caused by it.

[0193] Example 6

[0194] FACS detection of the binding ability of trispecific antibodies to cell SK-BR-3

[0195] 50ul SK-BR-3 cells were added to a 96-well round-bottom plate, with a cell number of 50,000 / well. Antibodies were diluted in gradients with FACS buffer (sterile PBS, 0.2% BSA), and added to the 96-well round-bottom plate at 50ul / well. Incubated at 4°C for 1h. Centrifuged at 2000rpm for 3min, supernatant was discarded, washed twice with FACS buffer, 100ul / well fluorescent secondary antibody (Southern Biotech, 2040-09) was added, with a final concentration of 1ug / ml, incubated at 4°C for 1h, centrifuged at 2000rpm for 3min, supernatant was discarded, washed twice with FACS buffer, and resuspended with 100ul / well FACS buffer, and detected by flow cytometry. Results are shown in Fig.12 .

[0196] Example 7

[0197] FACS detection of the binding ability of trispecific antibodies to cell SK-OV-3

[0198] 50ul SK-OV-3 cells were added to a 96-well round-bottom plate, with a cell number of 50,000 / well. Antibodies were diluted in gradients with FACS buffer (sterile PBS, 0.2% BSA), and added to the 96-well round-bottom plate at 50ul / well. Incubated at 4°C for 1h. Centrifuged at 2000rpm for 3min, supernatant was discarded, washed twice with FACS buffer, 100ul / well fluorescent secondary antibody (Southern Biotech, 2040-09) was added, with a final concentration of 1ug / ml, incubated at 4°C for 1h, centrifuged at 2000rpm for 3min, supernatant was discarded, washed twice with FACS buffer, and resuspended with 100ul / well FACS buffer, and detected by flow cytometry. Results are shown in Fig.13 .

[0199] Example 8

[0200] FACS detection of macrophage-induced phagocytosis experiment

[0201] Mouse macrophages RAW264.7 and OE-19 were cultured and expanded to the required cell amount, target cells OE-19 were labeled with CFSE, washed 3 times with PBS, and incubated with different concentrations of TriAB04 (2.5nM, 5nM, 10nM, 25nM, 50nM, 100nM) and TTI-622. After incubation at 37°C for 2 hours, the cells were washed 3 times with PBS, and the target cell solution containing the protein to be tested was transferred to a plate containing RAW264.7 cells with an effector-target ratio of 1:3. RAW264.7 cells were simultaneously stimulated with 1ug / mL LPS. After incubation for 2 hours, the cells were collected for flow cytometry analysis to detect the CFSE density in RAW264.7 cells.

[10] The results are shown in Fig.14 The trispecific antibody activated a large number of tumor cells for phagocytosis.

[0202] Example 9

[0203] Hemagglutination assay to detect trispecific antibody activity

[0204] The human RBC was centrifuged at 1500rpm for 5min, washed three times with DPBS, and re-added with DPBS to prepare a 10% RBC suspension. The antibody to be tested was prepared into samples of different concentrations (3uM, 1uM, 0.33uM, 0.11uM, 0.037uM, 0.012uM, 0.004uM, 0) using PBS buffer, and mixed with 50ul+50ul volume of human RBC and placed in a 96-well cell culture plate. After standing at 37℃ for 2h, the aggregation was observed.

[11] The results are shown in Fig.15 The trispecific antibody did not induce coagulation at any concentration.

[0205] Example 10

[0206] Internalization efficiency of trispecific antibodies in Her2-positive cells

[0207] Human ovarian cancer cells SK-OV-3 were plated at 5*10 5 The target cells were plated in a 24-well plate and incubated with 400 nM TriAB04 and positive control Herceptin for 1, 24, 48 and 72 hours, respectively. After the incubation, the cells were trypsinized and washed once with 0.5% BSA buffer. TriAB04 and Herceptin were incubated on ice for 45 minutes at 0, and PE anti-human IgG Fc Antibody secondary antibody was incubated on ice for 45 minutes. The cells were centrifuged and washed once with 0.5% BSA buffer. The cells were collected for flow cytometry analysis to detect the FITC fluorescence intensity (MFI) in the cells.

[12] .

[0208] Internalization rate = (1-MFI 测量值 / MFI t＝1 )*100%

[0209] Results Fig.16 The trispecific antibody was able to induce HER2 internalization at a level comparable to that of Herceptin.

[0210] Embodiment 11

[0211] Detection of the binding ability of trispecific antibodies to FcRn protein

[0212] 11.1 Fortebio Affinity Test

[0213] The antibody to be tested was loaded with ProA probe (concentration of 4ug / ml), and then bound and dissociated with different concentrations of FcRn (4ug / ml, 1ug / ml, 0.25ug / ml and 0), and the affinity between the molecule to be tested and FcRn was calculated. Fig.17 Compared with the wild type, the affinity of the mutated Fc to FcRn increased 10-fold.

[0214] 11.2 ELISA affinity test

[0215] Coat 2ug / ml FcRn protein 100ul / well at 4℃ overnight; block with 3% skim milk powder at 37℃ for 1h; add 100ul of trispecific antibodies and other control samples of different concentrations (10000ng / ml, 2500ng / ml, 625ng / ml, 156.25ng / ml, 39.06ng / ml, 9.77ng / ml, 2.44ng / ml, 0) to each well, incubate at 37℃ for 1h; wash 3 times with PBST after incubation at 37℃ for 1h; then add 100ul of goat anti-human IgG-HRP diluted 1:8000, incubate at 37℃ for 1h, wash 6 times, add 100ul TMB to each well for color development for 5min, then add 50ul stop solution, and read OD450 on the microplate reader. Results are shown in Fig.18 Compared with the wild type, the affinity of the mutated Fc to FcRn was significantly improved.

[0216] Example 12

[0217] ADCC activity of trispecific antibodies

[0218] 12.1 ADCC activity against human gastric cancer cell line NCI-N87

[0219] Culture and expand cells to the required amount, plate cells into 96-well plates at 4000 / well, adhere to the wall overnight, resuspend fresh human PBMC in 1640 culture medium, add to 96-well plates according to the effector-target ratio of 10:1, and administer the drug at a concentration gradient of 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively. After 5 days of continuous culture, wash the plate 3 times with PBS, use CCK-8 reagent to detect the number of viable cells, and calculate IC according to the drug efficacy measurement. 50 The results are shown in Fig.19 The anti-tumor activity of the trispecific antibody was superior to that of Herceptin.

[0220] 12.2 ADCC activity against human esophageal cancer cell OE19

[0221] Culture and expand cells to the required amount, plate cells into 96-well plates at 4000 / well, adhere to the wall overnight, resuspend fresh human PBMC in 1640 culture medium, add to 96-well plates according to the effector-target ratio of 10:1, and administer the drug at a concentration gradient of 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively. After 5 days of continuous culture, wash the plate 3 times with PBS, use CCK-8 reagent to detect the number of viable cells, and calculate IC according to the drug efficacy measurement. 50 The results are shown in Fig. 20 The anti-tumor activity of the trispecific antibody was superior to that of Herceptin.

[0222] 12.3 ADCC activity against human breast cancer cells MDA-MB-231

[0223] Culture and expand cells to the required amount, plate cells into 96-well plates at 1500 / well, adhere to the wall overnight, resuspend fresh human PBMC in 1640 culture medium, add to 96-well plates according to the effector-target ratio of 10:1, and administer concentration gradients of 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively. After 5 days of continuous culture, wash the plates 3 times with PBS, use CCK-8 reagent to detect the number of viable cells, and calculate IC according to the drug efficacy measurement. 50 The results are shown in Fig.21 The anti-tumor activity of the trispecific antibody was superior to that of the control drug Herceptin and the combination of Herceptin and TTI-622.

[0224] 12.4 ADCC activity against human esophageal cancer cell SK-OV-3

[0225] Culture and expand cells to the required amount, plate cells into 96-well plates at 4000 / well, adhere to the wall overnight, resuspend fresh human PBMC in 1640 culture medium, add to 96-well plates according to the effector-target ratio of 10:1, and administer the drug at a concentration gradient of 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively. After 5 days of continuous culture, wash the plate 3 times with PBS, use CCK-8 reagent to detect the number of viable cells, and calculate IC according to the drug efficacy measurement. 50 The results are shown in Fig. 22 The anti-tumor activity of the trispecific antibody was superior to that of Herceptin.

[0226] 12.5 ADCC activity against human breast cancer cells BT-474

[0227] Culture and expand cells to the required amount, plate cells into 96-well plates at 4000 / well, adhere to the wall overnight, resuspend fresh human PBMC in 1640 culture medium, add to 96-well plates according to the effector-target ratio of 10:1, and administer the drug at a concentration gradient of 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively. After 5 days of continuous culture, wash the plate 3 times with PBS, use CCK-8 reagent to detect the number of viable cells, and calculate IC according to the drug efficacy measurement. 50 The results are shown in Fig.23 The trispecific antibody has similar anti-tumor activity to Herceptin.

[0228] Embodiment 13

[0229] Antitumor activity of trispecific antibodies in the NCI-N87 xenograft model

[0230] Human gastric cancer cells NCI-N87 were cultured in 1640 medium containing 10% FBS at 37°C and 5% CO2. The cells were cultured continuously and inoculated into 28 SCID mice (24 for grouping and 4 for backup) before the tenth generation. Each mouse was inoculated with approximately 8×10 6 NCI-N87 cells, inoculated in a volume of 100uL, inoculated on the right side of the mouse back near the armpit. The mice were anesthetized with 3-4% isoflurane before inoculation.

[0231] When the tumor grows to an average size of about 100-150 mm 3 Around 24 tumor-bearing mice will be randomly divided into 4 groups according to tumor volume and body weight, with 6 mice in each group. The day of group administration is defined as day 0. The grouping and administration schedule are shown in the following table:

[0232]

[0233]

[0234] Tumor volume: Tumor volume was measured twice a week for 4 consecutive weeks after grouping. The calculation method of tumor volume (V) is as follows: V = (length × width2) / 2. The calculation method of relative tumor volume (RTV) of each mouse is: RTV = Vt / V0, where Vt is the measured volume per day and V0 is the volume at the beginning of treatment.

[0235] Results Fig.24 In this xenograft model, the anti-tumor activity of the trispecific antibody was significantly superior to that of Herceptin.

[0236] Embodiment 14

[0237] Efficacy study of trispecific antibodies in the ExVivo organoid system

[0238] Pleural effusion samples from Her2 2+ breast cancer patients were collected and cells in the samples were separated, including tumor cells and immune cells. Candidate drugs and reference drugs were co-incubated with tumor cell immune cell complexes in vitro. After 4-5 days of treatment, the culture medium and cells were collected for analysis, including tumor cells and Granzyme B. The results are shown in Fig.25 In the ExVivo evaluation system of this sample, the anti-tumor activity of the trispecific antibody was significantly better than the combination of Herceptin and TTI-622.

[0239] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

[0240] References:

[0241] [1]Andre Veillete,Jun Chen.SIRPa-CD47 Immune Checkpoint Blockade in Anticancer Therapy.

[0242] [2]A.Neil Barclay.Signal regulatory protein alpha(SIRPa) / CD47interaction and function.

[0243] [3]Song-Yang Wu,Tong Fu,Yi-Zhou Jiang,Zhi-Ming Shao.Natural killercells in cancer biology and therapy.

[0244] [4]Marc Turini,Patrick Chames,Pierre Bruhns,Daniel Baty,BrigitteKerfelec.A FcgRIII-engaging bispecific antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy.

[0245] [5]Yawu Jing,Zhenya Ni,Jianming Wu,LeeAnn Higgins etal.Identification of an ADAM17cleavage region in human CD16(FcgRIII)and theengineering of a non-cleavable version of the receptors in NK cells.

[0246] [6]Xuesai Zhang,Jianhe Chen,Zhibing Weng,Qingrou Li et al.A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab andpertuzumab with a distinct mechanism of action.

[0247] [7]Robert S.Copeland-Halperin,Jenifer E.Liu,Anthony F.Yu.Cardiotoxicityof HER2-targeted therapies.

[0248] [8]Quanli Cheng,Junlian Gu,Binay Kumar Adhikari et al.Is CD47 apotentially promising therapeutic target in cardiovascular diseases?-Role ofCD47 in cardiovascular diseases.

[0249] [9]Yusuke Ogitani,Katsunobu Hagihara,Masataka Oitate,Hiroyuki Naitoet al.Bystander killing effect of DS-8201a,a novel anti-human epidermal growth factor receptor 2antibody-drug conjugate,in tumors with humanepidermal growth factor receptor 2heterogeneity.

[0250]

[10] Treatment of CD47+disease cells with SIRP alpha-Fc fusions.

[0251]

[11] CN110724672A.Current Processing System 105D11 Organic Chemistry.

[0252]

[12] US20210380716A1.Recombinant bifunctional protein targeting CD47and HER2.

Claims

1. A trispecific antibody having specificity for three antigens, characterized in that: The antibody comprises (a) anti-Her2 monoclonal antibody Herceptin, (b) anti-CD16a single chain antibody scFv, (c) SIRPa D1 protein, and (d) flexible linker, Herceptin is used as the basic structure of the trispecific antibody, and the anti-CD16a single-chain antibody scFv or SIRPa D1 protein is connected to the N-terminus or C-terminus of the Herceptin heavy chain through a flexible linker, and the SIRPa D1 protein or anti-CD16a single-chain antibody scFv is connected to the N-terminus or C-terminus of the Herceptin light chain through a flexible linker. The amino acid sequence of the heavy chain of the anti-Her2 monoclonal antibody Herceptin is shown in SEQ ID NO: 4 or 5, and the amino acid sequence of the light chain is shown in SEQ ID NO:

8. The amino acid sequence of the SIRPa D1 protein is shown in SEQ ID NO: 9 or 10, The amino acid sequence of the heavy chain variable region of the anti-CD16a single-chain antibody scFv is shown in SEQ ID NO: 28 or 29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30 or 31.

2. The trispecific antibody according to claim 1, characterized in that The anti-CD16a single-chain antibody scFv comprises the amino acid sequence shown in SEQ ID NO:11, 12 or 13.

3. The trispecific antibody according to claim 1 or 2, characterized in that Using Herceptin as the basic structure of a trispecific antibody, SIRPa D1 protein and anti-CD16a scFv were linked in the following manner: The SIRPa D1 protein was connected to the C-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv was connected to the C-terminus of the Herceptin heavy chain via a flexible linker; The SIRPa D1 protein was connected to the N-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv was connected to the C-terminus of the Herceptin heavy chain via a flexible linker; The SIRPa D1 protein was connected to the C-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv was connected to the N-terminus of the Herceptin heavy chain via a flexible linker; The SIRPa D1 protein was linked to the N-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv was linked to the N-terminus of the Herceptin heavy chain via a flexible linker; The SIRPa D1 protein was linked to the N-terminus of the Herceptin heavy chain via a flexible linker, and the anti-CD16a scFv was linked to the N-terminus of the Herceptin light chain via a flexible linker; The SIRPa D1 protein was linked to the C-terminus of the Herceptin heavy chain via a flexible linker, and the anti-CD16a scFv was linked to the C-terminus of the Herceptin light chain via a flexible linker; The SIRPa D1 protein was connected to the C-terminus of the Herceptin heavy chain via a flexible linker, and the anti-CD16a scFv was connected to the N-terminus of the Herceptin light chain via a flexible linker; The SIRPa D1 protein was linked to the C-terminus of the Herceptin heavy chain via a flexible linker, and the anti-CD16a scFv was linked to the N-terminus of the Herceptin heavy chain via a flexible linker; The SIRPa D1 protein was connected to the N-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv was connected to the C-terminus of the Herceptin light chain via a flexible linker; The SIRPa D1 protein was connected to the C-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv was connected to the N-terminus of the Herceptin light chain via a flexible linker; The SIRPa D1 protein is connected to the N-terminus of the Herceptin heavy chain via a flexible linker, and the anti-CD16a scFv is connected to the C-terminus of the Herceptin heavy chain via a flexible linker; or The SIRPa D1 protein was connected to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv was connected to the C-terminus of the Herceptin light chain through a flexible linker.

4. The trispecific antibody according to claim 1 or 2, characterized in that The Herceptin antibody has L234A or L235A mutations introduced into the Fc segment, and M252Y, S254T or T256E mutations are also introduced into the Fc segment.

5. The trispecific antibody according to claim 1 or 2, characterized in that The Fc segment of the Herceptin antibody is wild type, and M252Y, S254T or T256E mutations are introduced into the Fc segment.

6. A trispecific antibody specific for three antigens, characterized in that: The antibody comprises (a) anti-Her2 monoclonal antibody Herceptin, (b) anti-CD16a single chain antibody scFv, (c) SIRPa D1 protein, and (d) flexible linker, Herceptin is used as the basic structure of the trispecific antibody, and the anti-CD16a single-chain antibody scFv or SIRPa D1 protein is connected to the N-terminus or C-terminus of the Herceptin heavy chain through a flexible linker, and the SIRPa D1 protein or anti-CD16a single-chain antibody scFv is connected to the N-terminus or C-terminus of the Herceptin light chain through a flexible linker, and The amino acid sequence of one chain is SEQ ID NO:32 or SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35 or SEQ ID NO:36, and the amino acid sequence of the other chain is SEQ ID NO:37 or SEQ ID NO:

38.

7. An isolated nucleic acid molecule encoding the trispecific antibody of any one of claims 1 to 6. A vector comprising the nucleic acid sequence according to claim 7.

9. A cell comprising the nucleic acid sequence of claim 7 or the vector of claim 8.

10. A method for preparing a trispecific antibody according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) connecting the anti-CD16a single-chain antibody scFv to the C-terminus or N-terminus of the Herceptin antibody heavy chain through a flexible linker; connecting the SIRPa D1 protein to the C-terminus or N-terminus of the Herceptin antibody light chain through a flexible linker; introducing mutations in the Herceptin antibody Fc segment; (2) cloning the first DNA fragment obtained in step (1) into a pcDNA series vector or other vectors used in, but not limited to, mammalian cell expression systems; cloning the second DNA fragment obtained in step (1) into another pcDNA series vector or other vectors used in, but not limited to, mammalian cell expression systems, to obtain a recombinant vector; (3) Transfecting the recombinant vector obtained in step (2) into mammalian cells or other expression systems to express and purify the fusion protein to obtain the trispecific antibody.

11. The method for preparing a trispecific antibody according to claim 10, characterized in that: The mammalian cells in step (3) include HEK293 cells, CHO cells, and cells derived from the above cells.

12. The method for preparing a trispecific antibody according to claim 10, characterized in that: In step (1), L234A or L235A mutations are introduced into the Fc segment of the Herceptin antibody, and M252Y, S254T or T256E mutations are simultaneously introduced into the Fc segment.

13. The method for preparing a trispecific antibody according to claim 10, characterized in that: In step (1), the Fc segment of the Herceptin antibody is wild type, and M252Y, S254T or T256E mutations are introduced into the Fc segment.

14. The method for preparing a trispecific antibody according to claim 10, characterized in that: The step (1) specifically includes the following steps: (a) synthesizing the heavy chain of Herceptin antibody and anti-CD16a single-chain antibody scFv DNA sequence; (b) designing primers to amplify the heavy chain of Herceptin antibody and anti-CD16a single-chain antibody scFv respectively using the plasmid in (a) as a template; (c) using the DNA fragment in (b) as a template, performing Overlap PCR with an upstream primer for amplifying the heavy chain DNA sequence of the antibody Herceptin and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv DNA sequence to obtain a DNA sequence; (d) using the mutation point M252Y, S254T or T256E as the boundary, designing mutation primers, using the fragment in (c) as a template, using the upstream primer and the downstream mutation primer for amplifying the heavy chain DNA sequence of the Herceptin antibody to amplify the fragment before the mutation point, and using the upstream mutation primer and the downstream primer for amplifying the anti-CD16a single-chain antibody scFv to amplify the fragment after the mutation point; (e) using the two fragments obtained in (d) as templates, using the upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and the downstream primer for amplifying the anti-CD16a single-chain antibody scFv to amplify the mutated DNA fragment; (f) obtaining a second DNA fragment in the same manner; (g) connecting the fragments prepared in (e) and (f) to pcDNA series vectors or other vectors used in, including but not limited to, mammalian cell expression systems, transforming into E. coli competent cells DH5a or other competent cells, and picking single clones to prepare plasmids; (h) The plasmid prepared in (g) was transfected into HEK293 cells and cultured in a shaker at 37°C, 8% CO2, and 125 rpm. After 7 days of transient expression, the supernatant was purified by protein A affinity chromatography to obtain the recombinant antibody, and the antibody concentration was determined by UV280 binding theoretical extinction coefficient.

15. Use of the trispecific antibody according to any one of claims 1 to 6 or the trispecific antibody prepared according to the method according to any one of claims 10 to 14 in the preparation of an anti-tumor drug.

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