Anti-HER2 / CD16 / CCR4 trispecific antibody and preparation method and application thereof

Abstract

The invention relates to an anti-HER2 / CD16 / CCR4 trispecific antibody and a preparation method and application thereof.The trispecific antibody comprises a first antigen binding structural domain specifically bound with HER2, a second antigen binding structural domain specifically bound with CD16 and a third antigen binding structural domain specifically bound with CCR4, and the trispecific antibody can be bound with human HER2, CD16 and CCR4 at the same time, so that the trispecific antibody can be used for detecting human HER2, CD16 and CCR4. The anti-tumor activity is obviously enhanced. The trispecific antibody can more effectively activate NK cells, enhance ADCC effect, target HER2 positive tumor cells and remove immunosuppressive cells in a tumor microenvironment, and has the advantages of enhancing antitumor activity and overcoming immunosuppression.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a trispecific antibody against HER2 / CD16 / CCR4, its preparation method, and its application. Background Technology

[0002] Cancer immunotherapy, particularly antibody-based therapies, has made significant progress in recent years. Monoclonal antibodies (mAbs) have shown great potential in cancer treatment by specifically targeting tumor-associated antigens. However, single-target antibody therapies often face challenges such as limited efficacy and the development of drug resistance. Currently, several monospecific or bispecific antibodies targeting HER2, CD16, or CCR4 have entered clinical trials or been marketed. However, these antibodies still have some limitations: Monospecific antibodies can only target a single antigen, resulting in limited efficacy and a high risk of drug resistance; Bispecific antibodies, while targeting two antigens simultaneously, still cannot completely solve the problem of tumor immune escape. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a trispecific antibody against HER2 / CD16 / CCR4, its preparation method, and its applications.

[0004] The technical solution adopted in this invention is: a trispecific antibody against HER2 / CD16 / CCR4, comprising a first antigen-binding domain that specifically binds to HER2, a second antigen-binding domain that specifically binds to CD16, and a third antigen-binding domain that specifically binds to CCR4.

[0005] Preferably, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain each include two or more functional sequence fragments. Different antigen-binding domains, as well as different functional sequence fragments within the same antigen-binding domain, are connected by flexible linkers. The sequences of each flexible linker are the same, partially the same, or different.

[0006] Preferably, the flexible connector subsequence includes (GGGGS) n Or (GGGS) n n is an integer selected from 1 to 4.

[0007] Preferably, the first antigen-binding domain includes a light chain variable region as shown in SEQ ID No: 1 and a heavy chain variable region as shown in SEQ ID No: 2; the second antigen-binding domain includes a VH variable region and a CH constant region as shown in SEQ ID No: 3, and a VL variable region and a CL constant region as shown in SEQ ID No: 4; the third antigen-binding domain includes a light chain variable region as shown in SEQ ID No: 5 and a heavy chain variable region as shown in SEQ ID No: 6.

[0008] Preferably, the first antigen-binding domain includes Herceptin scFv, the second antigen-binding domain is Fab, and the third antigen-binding domain includes Mogamulizumab scFv.

[0009] A method for preparing trispecific antibodies against HER2 / CD16 / CCR4 was developed. The sequence structure of the trispecific antibody against HER2 / CD16 / CCR4 was designed, and an expression vector capable of expressing the trispecific antibody against HER2 / CD16 / CCR4 was constructed. After transfecting the expression vector into host cells, the cells were cultured, separated, and purified to obtain a protein solution containing the trispecific antibody against HER2 / CD16 / CCR4.

[0010] Preferably, the expression vector can be a pcDNA series vector or other protein expression vector; the host cell can be a mammalian cell, E. coli, or yeast.

[0011] Application of anti-HER2 / CD16 / CCR4 trispecific antibodies in the preparation of drugs for treating tumors.

[0012] Preferably, it is used for tumors that express HER2;

[0013] Preferably, the tumor is one or more of the following: breast cancer, gastric cancer, non-small cell lung cancer, colorectal cancer, biliary tract malignant tumor, esophageal adenocarcinoma, salivary gland cancer, ovarian cancer, endometrial cancer, and bladder cancer.

[0014] A plasmid that can express a trispecific antibody against HER2 / CD16 / CCR4;

[0015] Preferably, the nucleotide fragment with the sequence shown in SEQ ID No: 10 is constructed into the pcDNA 3.1 eukaryotic expression vector.

[0016] A eukaryotic 293F suspension host cell contains a plasmid capable of expressing a trispecific antibody against HER2 / CD16 / CCR4, and is capable of expressing the trispecific antibody against HER2 / CD16 / CCR4.

[0017] The beneficial effects of this invention are: the trispecific antibody against HER2 / CD16 / CCR4 can simultaneously target HER2, CD16 and CCR4, more effectively activate NK cells, enhance ADCC effect, target HER2 positive tumor cells, and clear immunosuppressive cells in the tumor microenvironment, thus having the advantages of enhancing anti-tumor activity and overcoming immunosuppression. Attached Figure Description

[0018] Figure 1 A schematic diagram of the structure of a specific antibody according to an embodiment three of the present invention;

[0019] Figure 2 Image of plasmid expressing trispecific antibodies;

[0020] Figure 3 Schematic diagram of SDS-PAGE electrophoresis of three specific antibodies;

[0021] Figure 4 The binding ability of the trispecific antibody of this invention to the HER2 protein was measured by ELISA;

[0022] Figure 5 The binding ability of the trispecific antibody of this invention to CCR4 protein was measured by ELISA;

[0023] Figure 6 SPR was used to measure the binding ability of the trispecific antibodies of this invention to the target proteins; A: binding ability to HER2 protein; B: binding ability to CCR4 protein; C: binding ability to CD16 protein;

[0024] Figure 7 FACS detection of the binding ability of trispecific antibodies to HER2-positive cell lines;

[0025] Figure 8 FACS was used to detect the binding ability of trispecific antibodies to Treg-positive cell lines.

[0026] Figure 9 Trispecific antibody-mediated detection of ADCC activity in HER2-positive tumor cell lines; A: Effect on NCI-N87; B: Effect on SK-BR-3;

[0027] Figure 10 Detection of the ability of three specific antibodies to block Treg cell migration;

[0028] Figure 11 Detection of the killing ability of three specific antibodies against Treg. Detailed Implementation

[0029] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0030] This invention relates to a trispecific antibody against HER2 / CD16 / CCR4, its preparation method, and its application. The trispecific antibody includes a first antigen-binding domain that specifically binds to HER2, a second antigen-binding domain that specifically binds to CD16, and a third antigen-binding domain that specifically binds to CCR4. Based on its structural characteristics, this trispecific antibody against HER2 / CD16 / CCR4 can simultaneously bind to HER2, CD16, and CCR4, significantly enhancing its antitumor activity.

[0031] HER2 (human epidermal growth factor receptor 2) is a tyrosine kinase receptor that is overexpressed in various cancers, including breast, gastric, and ovarian cancer. HER2 overexpression is closely associated with tumor growth, invasion, and metastasis. CD16 (FcγRIII) is a low-affinity IgG Fc receptor primarily expressed on immune effector cells such as natural killer (NK) cells and macrophages. Binding of CD16 to the antibody Fc fragment can trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP), thereby killing tumor cells. CCR4 (CC chemokine receptor type 4) is a seven-transmembrane G protein-coupled receptor that is highly expressed on regulatory T cells (Tregs) and Th2 cells. Binding of CCR4 to its ligands CCL17 and CCL22 can promote the recruitment of Treg cells to the tumor microenvironment and suppress anti-tumor immune responses.

[0032] The trispecific antibody is formed by linking Herceptin scFv, anti-CD16 Fab, and Mogamulizumab scFv using flexible linkers to form a single-chain antibody. Herceptin scFv and Mogamulizumab scFv are respectively linked to both ends of the anti-CD16 single-chain antibody using flexible linkers. The light chain variable region and heavy chain variable region of Herceptin scFv, and the light chain variable region and heavy chain variable region of Mogamulizumab scFv are linked using flexible linkers. The anti-CD16 single-chain antibody consists of a heavy chain variable region VH, a heavy chain constant region CH, a light chain variable region VL, and a light chain constant region CL, with CH and VL linked by flexible linkers. The multiple flexible linkers in the trispecific antibody may be identical, partially identical, or different. The amino acid sequence of the flexible linker is shown in any of SEQ ID No. 7-9; the corresponding nucleotide sequence is shown in SEQ ID No. 17-19.

[0033] Table 1

[0034]

[0035] In some embodiments of the present invention, the anti-HER2 monoclonal antibody includes Herceptin scFv, the anti-CD16 single-chain antibody includes Fab, and the anti-CCR4 monoclonal antibody includes Mogamulizumab scFv. The constructed trispecific antibodies can include the following connection methods: Using the anti-CD16 single-chain antibody Fab as the basic structure of the trispecific antibody, the anti-HER2 monoclonal antibody Herceptin scFv is linked to the N-terminus of the variable region of the heavy chain of the anti-CD16 single-chain antibody Fab via a flexible linker, and the anti-CCR4 monoclonal antibody Mogamulizumab scFv is linked to the C-terminus of the constant region of the light chain of the anti-CD16 single-chain antibody Fab via a flexible linker; or, using the anti-CD16 single-chain antibody Fab as the basic structure of the trispecific antibody, the anti-CCR4 monoclonal antibody Mogamulizumab scFv is linked to the N-terminus of the variable region of the heavy chain of the anti-CD16 single-chain antibody Fab via a flexible linker, and the anti-HER2 monoclonal antibody Herceptin scFv is linked to the C-terminus of the constant region of the light chain of the anti-CD16 single-chain antibody Fab via a flexible linker; or, using the anti-HER2 monoclonal antibody Herceptin... The basic structure of a trispecific antibody is as follows: The anti-CD16 single-chain antibody Fab is linked to the N-terminus of the heavy chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv via a flexible linker, and the anti-CCR4 monoclonal antibody Mogamulizumab scFv is linked to the C-terminus of the light chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv via a flexible linker; or, the anti-HER2 monoclonal antibody Herceptin scFv is used as the basic structure of a trispecific antibody, with the anti-CCR4 monoclonal antibody Mogamulizumab scFv linked to the N-terminus of the heavy chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv via a flexible linker, and the anti-CD16 single-chain antibody Fab linked to the C-terminus of the light chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv via a flexible linker; or, the anti-CCR4 monoclonal antibody Mogamulizumab scFv is used as the basic structure of a trispecific antibody, with the anti-HER2 monoclonal antibody Herceptin scFv linked to the N-terminus of the heavy chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv via a flexible linker; or, the anti-CCR4 monoclonal antibody Mogamulizumab scFv is used as the basic structure of a trispecific antibody, with the anti-HER2 monoclonal antibody Herceptin scFv linked to the N-terminus of the heavy chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv via a flexible linker. The scFv is linked to the N-terminus of the heavy chain variable region of the anti-CCR4 monoclonal antibody Mogamulizumab scFv via a flexible linker, and the anti-CD16 single-chain antibody Fab is linked to the C-terminus of the light chain variable region of the anti-CCR4 monoclonal antibody Mogamulizumab scFv via a flexible linker.Alternatively, using the anti-CCR4 monoclonal antibody Mogamulizumab scFv as the basic structure of a trispecific antibody, the anti-CD16 single-chain antibody Fab is linked to the N-terminus of the heavy chain variable region of the anti-CCR4 monoclonal antibody Mogamulizumab scFv via a flexible linker, and the anti-HER2 monoclonal antibody Herceptin scFv is linked to the C-terminus of the light chain variable region of the anti-CCR4 monoclonal antibody Mogamulizumab scFv via a flexible linker.

[0036] The method for preparing the above-mentioned trispecific antibody against HER2 / CD16 / CCR4 includes the following steps: First, design the sequence structure of the trispecific antibody against HER2 / CD16 / CCR4, construct an expression vector capable of expressing the trispecific antibody against HER2 / CD16 / CCR4, transfect the expression vector into host cells, culture them, and then separate and purify them to obtain a protein solution containing the trispecific antibody against HER2 / CD16 / CCR4.

[0037] The expression vector can be a pcDNA series vector or other protein expression vectors. The vector of the expression system includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences. To facilitate the isolation and purification of trispecific antibodies, signal peptides and / or tags can be added to both ends of the sequence when designing the sequence structure of anti-HER2 / CD16 / CCR4 trispecific antibodies, such as adding a Kozak signal peptide or adding a 6×His tag at the C-terminus. The host cell can be mammalian cells or other expression systems (such as E. coli and yeast). The mammalian cell can be HEK293 cells or CHO (Chinese Hamster Ovary) cells or derived cells of the above. Due to their rich post-translational modification functions, mammalian cell expression systems yield trispecific antibodies with better affinity. Transfection can be performed using chemical reagents or electroporation.

[0038] In some embodiments of the present invention, the method for preparing trispecific antibodies specifically includes the following steps:

[0039] (a) DNA sequence synthesis of single-chain antibody fragments, including anti-HER2 monoclonal antibody Herceptin scFv, anti-CD16 monoclonal antibody heavy chain variable region, heavy chain constant region, anti-CD16 monoclonal antibody light chain variable region, light chain constant region, and anti-CCR4 monoclonal antibody Mogamulizumab scFv; anti-HER2 monoclonal antibody Herceptin scFv is linked to the N-terminus of the anti-CD16 monoclonal antibody Fab heavy chain variable region via a flexible linker, and anti-CCR4 monoclonal antibody Mogamulizumab scFv is linked to the C-terminus of the anti-CD16 monoclonal antibody Fab light chain constant region via a flexible linker; scFv consists of a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL are linked together by a flexible peptide linker. In scFv, the domain order can be VH-linker-VL or VL-linker-VH;

[0040] (b) Design homologous primers to amplify the target fragment using the plasmid in (a) as a template;

[0041] (c) Design primer pairs for reverse PCR amplification of pcDNA series vectors;

[0042] (d) Homologous recombination of the fragments prepared in (b) and (c) is ligated into pcDNA series vectors or other vectors used for mammalian cell expression systems, including but not limited to mammalian cell expression systems, and transformed into E. coli competent cells DH5a or other competent cells. Single clones are picked and plasmids are prepared.

[0043] (e) The plasmid prepared in (d) was transfected into HEK293 cells and cultured in a shaker at 37°C, 5% CO2, and 140 rpm. After transient expression for 5-7 days, the supernatant was purified by Ni Smart Beads affinity chromatography to obtain recombinant antibody, and the target protein was verified by SDS-PAGE electrophoresis and Western Blot.

[0044] The prepared anti-HER2 / CD16 / CCR4 trispecific antibody can be used in anti-tumor drugs, especially suitable for the treatment of tumors that simultaneously express HER2 and CCR4. These tumors can be one or more of the following: breast cancer, gastric cancer, non-small cell lung cancer, colorectal cancer, biliary tract malignancies, esophageal adenocarcinoma, salivary gland cancer, ovarian cancer, endometrial cancer, and bladder cancer. This anti-HER2 / CD16 / CCR4 trispecific antibody can be used as a monotherapy for tumors or in combination with other tumor treatments. The HER2 / CD16 / CCR4 trispecific antibody aims to overcome the limitations of existing technologies and provide a more effective and durable cancer immunotherapy strategy. By simultaneously targeting HER2, CD16, and CCR4, this trispecific antibody can more effectively activate the immune system, kill tumor cells, and reverse the immunosuppressive state of the tumor microenvironment, thus bringing new hope to cancer patients.

[0045] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0046] Example 1: Preparation of a trispecific antibody against HER2 / CD16 / CCR4

[0047] 1.1 Design of Trispecific Antigen-Binding Domains

[0048] Design a gene sequence capable of expressing a trispecific antibody that simultaneously binds to HER2, CD16, and CCR4. The scFv fragment of the anti-HER2 monoclonal antibody Herceptin was selected as the first antigen-binding domain, the scFv fragment of the anti-CD16 monoclonal antibody as the second antigen-binding domain, and the scFv fragment of the anti-CCR4 monoclonal antibody Mogamulizumab as the third antigen-binding domain. The amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody Herceptin scFv is shown in SEQ ID No. 2, the nucleotide sequence is shown in SEQ ID No. 12, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 11; the amino acid sequence of the heavy chain variable region and the heavy chain constant region of the anti-CD16 single-chain antibody Fab is shown in SEQ ID No. 3, the nucleotide sequence is shown in SEQ ID No. 13, the amino acid sequence of the light chain variable region and the light chain constant region is shown in SEQ ID No. 4, and the nucleotide sequence is shown in SEQ ID No. 14; the amino acid sequence of the heavy chain variable region of the anti-CCR4 monoclonal antibody Mogamulizumab scFv is shown in SEQ ID No. 6, the nucleotide sequence is shown in SEQ ID No. 16, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 15.

[0049] In the anti-HER2 monoclonal antibody Herceptin scFv, VH and VL are linked via SEQ ID No. 7; in the anti-CCR4 monoclonal antibody Mogamulizumab scFv, VH and VL are linked via SEQ ID No. 7; in the anti-CD16 single-chain antibody Fab, CH and VL are linked via SEQ ID No. 9; in the anti-HER2 monoclonal antibody Herceptin scFv and the anti-CD16 single-chain antibody Fab, they are linked via SEQ ID No. 8; in the anti-CD16 single-chain antibody Fab and the anti-CCR4 monoclonal antibody Mogamulizumab scFv, they are linked via SEQ ID No. 9. The trispecific antibody sequence is shown in SEQ ID NO: 10.

[0050] To improve protein expression efficiency and secretion into the extracellular space, a Kozak sequence and a signal peptide were added to the N-terminus of the target protein, and a 6×His tag was added to the C-terminus for subsequent protein purification. The amino acid sequence of the 6×His Tag is shown in SEQ ID No. 20.

[0051] Table 2

[0052]

[0053] 1.2 Expression and purification of trispecific antibodies

[0054] The target gene encoding the trispecific antibody was synthesized by the company (Qingke Biotechnology). The target gene was then cloned into a pcDNA 3.1 series mammalian expression vector using homologous recombination. The plasmid structure is shown below. Figure 2 As shown.

[0055] The constructed expression vector plasmid was transfected into HEK293 cells to obtain engineered bacteria capable of expressing trispecific antibodies. The plasmid containing the target gene (purchased from Sinocare) was transfected into HEK293 cells at a concentration of 1 μg / mL. The ratio of plasmid to cationic liposome PEI (purchased from TransGen) was 1:4. First, the plasmid and PEI reagent were diluted using PEI Buffer. The diluted PEI was slowly added to the diluted plasmid, gently mixed, and incubated at room temperature for 15-20 min. The mixture was then added to suspension-cultured 293F cells. At the time of transfection, the cells should be in good condition, plump, and at a density of 1-2 million / mL. Cells were cultured at 37°C, 5% CO2, and 140 rpm in a shaker. After 24 hours, SMS 293-SUPI (purchased from Sinocare) was added at a dose of 35 mL / L. Thereafter, feed was added every other day, and cell density was observed daily. The supernatant was collected after 5-7 days.

[0056] Ni Smart Beads (purchased from Tiandi Renhe) are primarily used for the capture and purification of histidine-tagged proteins secreted into eukaryotic culture supernatants. They enable efficient purification of target proteins even in the presence of EDTA and DTT. This medium can also be used for the purification of intracellularly expressed proteins in various expression systems, offering higher selectivity and better purity of the purified protein. The Ni Smart Beads were used to purify the target protein in the supernatant.

[0057] The buffers used for purification included Lysis Buffer: 20mM NaH2PO4, 500mM NaCl, pH 8.0; Wash Buffer: 20mM NaH2PO4, 500mM NaCl, 5mM imidazole, pH 8.0; and Elution Buffer: 20mM NaH2PO4, 500mM NaCl, 250mM imidazole, pH 8.0. The purification steps included: (1) equilibrating the column with 5 column volumes of Lysis Buffer, repeating 2-3 times; (2) adding the sample to the equilibrated gravity column, ensuring full contact between the medium and the sample, and collecting the eluent; (3) washing with 15 column volumes of Wash Buffer to remove non-specifically bound proteins, and collecting the wash solution; (4) eluting with 10 column volumes of Elution Buffer, collecting in fractions, one tube per column volume, and detecting by SDS-PAGE. The SDS-PAGE electrophoresis results after purification by Ni Smart Beads are as follows: Figure 3 As shown, the eluent was collected based on the electrophoresis results for subsequent experiments. The prepared trispecific antibody against HER2 / CD16 / CCR4 was named TNK16.

[0058] Example 2: Functional Verification of Trispecific Antibodies

[0059] 2.1 ELISA detection of the binding of trispecific antibodies to HER2 protein

[0060] The target protein was coated with antigen coating buffer (0.05 mol / L, pH 9.6 carbonate buffer, Na2CO3 1.59 g, NaHCO3 2.93 g, deionized water to 900 mL, pH adjusted to 9.6, volume brought to 1000 mL) and incubated overnight at 4°C. The protein was then blocked with 5% skim milk powder (purchased from BIOFROXX) at 37°C for 2 hours. Different concentrations (100000 ng / mL, 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 1.28 ng / mL, 0.256 ng / mL, 0.0512 ng / mL, 0 ng / mL) of trispecific antibody were added to the wells and incubated at 37°C. 2h; then add goat anti-human IgG (Fab) / HRP, incubate at 37℃ for 1h, add TMB substrate for color development, stop the reaction with stop solution, and then detect at 450nm using an ELISA reader.

[0061] The results are as follows Figure 4As shown, data analysis was performed using GraphPad Prism, and EC50 was plotted and calculated. The EC50 value of the trispecific antibody and HER2 antigen was 21.87 pM, and the EC50 value of Trastuzumab and HER2 antigen was 4.84 nM. There was no significant difference in affinity between the two.

[0062] 2.2 ELISA detection of the binding of trispecific antibodies to CCR4 protein

[0063] The target protein was coated with antigen coating solution and incubated overnight at 4°C; then blocked with 5% skim milk powder at 37°C for 2 hours; different concentrations (100000 ng / mL, 20000 ng / mL, 4000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 1.28 ng / mL, 0.256 ng / mL, 0.0512 ng / mL, 0 ng / mL) of trispecific antibodies were added to the wells and incubated at 37°C for 2 hours; then goat anti-human IgG (Fab) / HRP was added and incubated at 37°C for 1 hour; TMB substrate was added for color development; and the incubation was terminated with stop solution. Detection was then performed at 450 nm using a microplate reader.

[0064] The results are as follows Figure 5 As shown, data analysis was performed using GraphPad Prism, and EC50 was plotted and calculated. The EC50 value of the trispecific antibody with CCR4 antigen was 26.26 pM, and the EC50 value of Mogamulizumab with CCR4 antigen was 12.09 nM. The difference in affinity between the two was three orders of magnitude. The reduced affinity for the CCR4 end ensured that the trispecific antibody would bind more to Tregs in the tumor microenvironment, reducing the depletion of Tregs in peripheral blood.

[0065] 2.3 SPR detection of the binding of trispecific antibodies to HER2, CCR4, and CD16 proteins

[0066] The binding ability of the trispecific antibodies prepared in Example 1 to HER2, CCR4, and CD16 proteins was detected using an SPR instrument.

[0067] After baseline calibration of the SPR instrument (BIACORE T200), the antigen was immobilized on a CM5 chip (purchased from Cytiva). Buffer (1×PBS) was filtered through a 0.22 μm filter and adjusted to the appropriate pH. The trispecific antibody to be tested was prepared and diluted to an appropriate concentration. The analyte solution was injected into the flow cell, allowing it to flow over the chip surface. The binding and dissociation processes of the analyte and ligand were observed, and the changes in resonance signals were recorded. The binding curve was fitted using software (such as Biacore Evaluation Software) to obtain kinetic parameters (such as binding rate constant ka, dissociation rate constant kd, and affinity constant KD). The strength and kinetic characteristics of molecular interactions were analyzed based on the fitting results. Results are as follows: Figure 6 As shown, the affinity constants of the trispecific antibody for HER2, CCR4, and CD16 are 2.44 × 10⁻⁶, respectively. -8 6.29×10 -8 3.15×10 -8 The results showed that the trispecific antibody had good affinity for all three antigens.

[0068] Example 3: Analysis of the binding ability of trispecific antibodies to target cells

[0069] 3.1 FACS detection of the binding of trispecific antibodies to HER2-positive cell lines

[0070] The HER2-overexpressing cell line NCI-N87 was cultured at 37°C and 5% CO2. When the cell density reached 70%, the culture medium was removed, the cells were washed with PBS, digested with trypsin, centrifuged by pipetting, resuspended, and added to 1.5 mL centrifuge tubes, with a cell count of 2 × 10⁶ cells / mL. 5 Dilute the antibody serially with FACS buffer (sterile PBS, 1% FBS) at 50 μL / well in centrifuge tubes and incubate at 4°C for 1 h. After centrifugation at 400 x g for 5 min, discard the supernatant, wash 3 times with FACS buffer, add 100 μL / well of fluorescent secondary antibody, incubate at 4°C for 0.5 h, centrifuge at 400 x g for 5 min, discard the supernatant, wash 3 times with FACS buffer, resuspend in 100 μL / well of FACS buffer, and analyze by flow cytometry.

[0071] The results are as follows Figure 7 As shown, the trispecific antibody showed a slight decrease in binding to HER2-overexpressing cell lines compared to the parental antibody, but still maintained a strong binding ability.

[0072] 3.2 FACS detection of the binding of trispecific antibodies to Treg-positive cell lines

[0073] 50 μL of cells were added to a 96-well circular-bottom plate, with a cell count of 2 × 10⁻⁶.5 / well, antibody was serially diluted with FACS buffer (sterile PBS, 1% FBS), and 50 μL / well was added to each well of a 96-well round-bottom plate. Incubation was performed at 4°C for 1 h. After centrifugation at 400 x g for 5 min, the supernatant was discarded, and the plate was washed 3 times with FACS buffer. 100 μL / well of fluorescent secondary antibody was added, and the plate was incubated at 4°C for 0.5 h. After centrifugation at 400 x g for 5 min, the supernatant was discarded, and the plate was washed 3 times with FACS buffer. The plate was then resuspended in 100 μL / well of FACS buffer and analyzed by flow cytometry. Results are as follows: Figure 8 As shown, both the trispecific antibody and the parental antibody can effectively bind to human Treg cells.

[0074] Example 4: Antibody-mediated detection of ADCC activity in HER2-positive tumor cell lines

[0075] The killing ability of trispecific antibodies was detected using HER2-positive cell lines SK-BR-3 and NCI-N87 as target cells.

[0076] PBMCs (purchased from Miaoshun Biotechnology) and target cells were mixed in a fixed ratio, and antibodies were added. After incubation for 48 hours, the supernatant was removed, and complete culture medium containing 10% CCK-8 solution was added to each well. Wells with appropriate amounts of cell culture medium and CCK-8 but without cells served as blank controls. After further incubation in a cell culture incubator for 1-2 hours, the absorbance at 450 nm was measured using a microplate reader. The cell killing rate was calculated using the formula: [OD(0 with drug) - OD(with drug)] / [OD(0 with drug) - OD(blank)] × 100%.

[0077] The results are as follows Figure 9 As shown, for HER2-positive cell lines SK-BR-3 and NCI-N87, the trispecific antibody and the parental antibody Trastuzumab exhibited similar killing levels. At the highest dose, the trispecific antibody killed 63.67% of SK-BR-3 cells, while the parental antibody Trastuzumab killed 52.64%. In the NCI-N87 cell line, the maximum dose of the trispecific antibody killed 60.28% of the cells, while the parental antibody killed only 39.04%.

[0078] Example 5: Antibody-mediated Treg cell migration inhibition assay

[0079] The Transwell assay was used to detect the ability of trispecific antibodies to block Treg cell migration.

[0080] Add 600 μL of SKBR3 tumor cell culture supernatant and 100 ng / mL CCL22 to a 24-well plate; pipette 30,000 Tregs into 200 μL of Treg-specific culture medium, mix well, and then evenly drop the mixture into the chambers; place the chambers in the well plate and check for air bubbles between the culture medium and the chambers; let stand for 15 min to avoid cell aggregation during sedimentation; set up different groups and add IgG, Mogamulizumab monoclonal antibody, and TNK16 respectively, with a final concentration of 10 μg / mL; then incubate in an incubator for 4 h; count the number of cells in the lower chamber using a hemocytometer.

[0081] The results are as follows Figure 10 As shown, compared with CCL22 alone, the combination of CLL22 and trispecific antibody significantly inhibited the chemotaxis of Treg cells, with an inhibitory effect similar to that of CLL22+Mogamulizumab. This indicates that the trispecific antibody can inhibit the chemokine-mediated chemotaxis of Treg cells to the tumor microenvironment.

[0082] Example 6: Antibody-mediated CCR4 + Treg cell killing experiment

[0083] Treg cells and PBMCs were added to 96-well plates at a ratio of 1:20. The plates were divided into IgG group, Mogamulizumab group, and trispecific antibody group. After incubation for 24 hours, the killing effect of the antibodies on Treg cells was detected by LDH assay.

[0084] 20 μL of Assay Buffer was added to three wells containing cells for spontaneous release, and 20 μL of Assay Buffer was added to three cell-free wells containing only 1% low-serum medium as a background control. The plates were incubated in a CO2 incubator at 37°C for the required time. One hour before the scheduled assay, 20 μL of 10% Triton X-100 solution was added to three wells containing cells for maximum release, and 20 μL of LDH Positive Control was added to three cell-free wells containing only 1% low-serum medium as a positive control. The plates were mixed by pipetting several times and incubated in a CO2 incubator. The 96-well tissue culture plates were centrifuged at 400 g for 5 min. 100 μL of cell supernatant was transferred to a new 96-well assay plate, and 100 μL of LDH Reaction Solution was added to each well. The plates were incubated at 37°C for a maximum of 30 min, and the absorbance was read at 565 nm using a microplate reader. Cytotoxicity percentage (%) = (A sample - A spontaneous) / (A maximum release - A spontaneous) × 100.

[0085] The results are as follows Figure 11 As shown, at low concentrations, Mogamulizumab showed slightly better cytotoxicity than the trispecific antibody. At the highest dose of 400 nM, the percentage of cytotoxicity mediated by the trispecific antibody was 70.37%, while that of Mogamulizumab was only 53.85%. This indicates that the trispecific antibody has a better ability to kill CCR4+Treg cells under high-dose conditions.

[0086] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. An anti-HER2 / CD16 / CCR4 trispecific antibody, characterized in that: The first antigen binding domain specifically binds HER2, the second antigen binding domain specifically binds CD16, and the third antigen binding domain specifically binds CCR4.

2. The anti-HER2 / CD16 / CCR4 trispecific antibody of claim 1, wherein: The first, second, and third antigen binding domains each comprise two or more functional sequence fragments, which are connected by flexible linkers between different antigen binding domains and between different functional sequence fragments in the same antigen binding domain, and each flexible linker sequence is the same, partially the same, or different. Preferably, the flexible linker sequence comprises (GGGGS) n or (GGGS) n , n is an integer selected from 1-4.

3. The anti-HER2 / CD16 / CCR4 trispecific antibody according to claim 1 or 2, characterized in that: The first antigen binding domain comprises a light chain variable region with an amino acid sequence as shown in SEQ ID No: 1 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID No: 2; the second antigen binding domain comprises a VH variable region with an amino acid sequence as shown in SEQ ID No: 3 and a CH constant region, and a VL variable region with an amino acid sequence as shown in SEQ ID No: 4 and a CL constant region; and the third antigen binding domain comprises a light chain variable region with an amino acid sequence as shown in SEQ ID No: 5 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID No:

6.

4. The trispecific antibody against HER2 / CD16 / CCR4 according to claim 1 or 2, characterized in that: The first antigen binding domain comprises Herceptin scFv, the second antigen binding domain comprises Fab, and the third antigen binding domain comprises Mogamulizumab scFv.

5. A method of producing the trispecific antibody against HER2 / CD16 / CCR4 according to any one of claims 1 to 4, characterized in that: The sequence structure of the anti-HER2 / CD16 / CCR4 trispecific antibody is designed, an expression vector capable of expressing the anti-HER2 / CD16 / CCR4 trispecific antibody is constructed, and after the expression vector is transfected into host cells, the host cells are cultured, and then separated and purified to obtain a protein solution containing the anti-HER2 / CD16 / CCR4 trispecific antibody.

6. The method of claim 5, wherein: The expression vector can be a pcDNA series vector, and the host cells can be mammalian cells, E. coli, or yeast.

7. Use of the anti-HER2 / CD16 / CCR4 trispecific antibody of any one of claims 1-4 in the preparation of a medicament for treating a tumor.

8. Use according to claim 7, characterized in that: The tumor is capable of expressing HER2. Preferably, the tumor is one or more of breast cancer, gastric cancer, non-small cell lung cancer, colorectal cancer, biliary tract malignant tumor, esophageal adenocarcinoma, salivary gland cancer, ovarian cancer, endometrial cancer, and bladder cancer.

9. A plasmid, characterized by: The anti-HER2 / CD16 / CCR4 trispecific antibody of any one of claims 1-4 can be expressed. Preferably, a nucleotide fragment with the sequence as shown in SEQ ID No: 10 is constructed into a pcDNA 3.1 eukaryotic expression vector.

10. A eukaryotic 293F suspension host cell, characterized by: The plasmid of claim 9, and the anti-HER2 / CD16 / CCR4 trispecific antibody can be expressed.

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