Targeting Her2 / CD3 / 4-1BB trispecific antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tri-specific antibody based on structure optimization protein activity and application of the antibody. Specifically, the invention relates to a trispecific antibody specifically combined with Her2, CD3 and 4-1BB, and the Her2 / CD3 / 4-1BB trispecific antibody comprises a Fab part of a monoclonal antibody specifically combined with CD3, an antibody fragment specifically combined with Her2 and an antibody fragment specifically recognized with 4-1BB. The invention also relates to an application of the antibody in preparing a medicine for treating cancers, and the antibody plays an important role in treating human tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a trispecific antibody based on structure-optimized protein activity and uses of the antibody. Background Art

[0002] In recent years, cancer immunotherapy has made great progress in mechanism research and practical application. Common immunotherapies include immune checkpoint inhibitors (PD-1 / L1), tumor vaccines (provenge, cimavax), cellular immune cell therapy (CAR-T) and nonspecific immunomodulators. Although immunotherapy has a good effect on the treatment of some malignant tumors, most malignant tumors still do not respond to immunotherapy. Therapies that utilize immune regulatory mechanisms such as cytokines, cancer vaccines and acquired immunotherapy can lead to tumor mutations in many patients (RosenbergSA. Interleukin-2 and the development of immunotherapy for the treatment of patients with cancer. Cancer J Sci Am. 2000 Feb; 6 Suppl 1: S2-7; Bassi P. BCG (Bacillus of Calmette Guerin) therapy of high-risk superficial bladder cancer. Surg Oncol. 2002 Jun; 11 (1-2): 77-83.). Due to these reactions, immunotherapy often cannot effectively eliminate mutated or infected cells. The reasons for the above failures can be divided into the following three main types: (1) impaired recognition of immune cells due to variable expression of antigens or reduced expression of class I major histocompatibility complex (MHCⅠ); (2) upregulation of inhibitory immune checkpoints and immunosuppressive microenvironment; (3) inability to effectively stimulate T cells due to lack of expression of co-stimulatory molecules on mutant or infected cells (Dong H, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10secretion. Nat Med. 1999 Dec; 5(12): 1365-9). A possible strategy to enhance the immune response is to provide co-stimulatory signals through auxiliary molecules to maintain the continuous killing and surveillance functions of antigen-specific T cells.

[0003] At present, great progress has been made in the research on co-stimulatory signals, mainly including B lymphocyte activation antigen (B7 / 4-1BB), intercellular adhesion molecules (IFA-1 / ICAM-1, ICAM-2), lymphocyte function-related antigen (CD2 / LFA-3), vascular endothelial adhesion molecule (VCAM-1), heat-stable antigen (HSA), etc. V, Trollmo C, Klareskog L. Modulating co-stimulation: a rational strategy in the treatment of rheumatoid arthritis [J]. Arthritis Res Ther. 2005; 7 (suppl 2): ​​S15-S20) As the development of PD-L1 / 4-1BB bispecific antibodies gradually matures and their applications expand, multifunctional antibodies with 4-1BB as co-stimulatory signals have also begun to receive attention (Alegre ML, Frauwirth KA, Thompson C BT-CELL REGULATION BY 4-1BB AND CTLA-4 [J]. Nature Reviews Immunology, 2001, 1 (3): 220-228.). 4-1BB, also known as CD137 or TNFRF9, is a member of the tumor necrosis factor (TNF) receptor family, along with OX40. 4-1BB is a type I transmembrane protein with a reading frame of 255 amino acids (NCBI: NP_001552), consisting of an N-terminal signal peptide of 17 amino acids, an extracellular region of 169 amino acids, a transmembrane region of 27 amino acids, and a C-terminal intracellular region of 42 amino acids. + and CD8 +Inducible co-stimulatory receptors expressed on T cells, NKT, NK cells, DC, macrophages, eosinophils, neutrophils and mast cells and Tregs. In the activation process of T cells, 4-1BB molecules can provide key co-stimulatory signals for them. After APC cells capture antigens, antigen peptides are presented on their surfaces, and B7-1, B7-2 and antigen peptides on APC become the initial activation signals of naive T cells. Under the stimulation of the initial activation signal, the 4-1BB molecule expression on the surface of T cells is upregulated (McCoy KD, Le Gros G. The role of CTLA-4 in the regulation of T cell immune responses [J]. Immunol Cell Biol. 1999; 77 (1): 1-10). 4-1BB recruits TNFR-related factors TRAF1 and TRAF2 to form heterotrimers, strengthens signal transduction through the c-Jun N-terminal kinase (JNK) pathway, the extracellular signal-regulated kinase (ERK) pathway, and through the β-catenin and AKT pathways, and regulates 4-1BB signals through the master transcription factor NF-κB, promoting the production and secretion of cytokines. Bcl-xL is a 30kDa anti-apoptotic protein belonging to the Bcl-2 family. Bcl-xL prevents apoptosis through two different mechanisms: inhibiting its apoptotic effect by heterodimerizing with apoptotic proteins, and helping to maintain normal membrane status under stress conditions by directly forming pores in the outer membrane of mitochondria. After activation, 4-1BB signaling can also promote the expression of BCL-xl, thereby inhibiting apoptosis of activated cells (Bart kowiak, Todd, and Michael A Curran. "4-1BB Agonists: Multi-Potent Potentiators of Tumor Immunity." Frontiers in Oncology vol. 5117. 8Jun. 2015).

[0004] At present, great progress has been made in the research on 4-1BB monoclonal antibodies. The anti-4-1BB agonist monoclonal antibody has been verified to have anti-tumor activity through animal models. It can selectively induce CD8 + It can promote the proliferation of T cells, upregulate the expression of pro-inflammatory cytokine IFN-γ, and enhance the killing effect of antigen-specific effector T cells, thereby promoting tumor clearance. Anti-4-1BB monoclonal antibody can also cause the proliferation of NK cells, thereby increasing CD8 +Cytotoxic activity of T cells. Anti-4-1BB agonistic antibodies can also cause the endothelial cells of the blood vessels of tumor cells to upregulate the expression of adhesion molecules, promoting activated T lymphocytes to infiltrate tumor tissues (Pan, Ping-Ying et al. "Regulation of dendritic cell function by NK cells: mechanisms underlying the synergismin the combination therapy of IL-12and 4-1BB activation." Journal of immunology vol.172,8 (2004): 4779-89). Both Pfizer and BMS have developed 4-1BB monoclonal antibodies, but the efficacy and safety data of monoclonal antibodies are not ideal, especially the presence of more severe hepatotoxicity. The reason may be that excessive activation of T cells can lead to potential systemic immune response risks. In order to avoid or slow down liver damage induced by excessive activation of T cells, current strategies focus on three aspects: 1. Limiting the exposure of CD137; 2. Targeting the tumor microenvironment; 3. Combining with immune checkpoint inhibitors such as PD-L1 (Kamata-Sakurai, Mika et al. Cancer discovery vol. 11, 1 (2021): 158-175). For example, for patients with PD-L1-positive melanoma, the combination of BMS's 4-1BB monoclonal antibody Urelumab (BMS-663513) achieved an ORR of 50%.

[0005] At present, 4-1BB type bispecific antibodies are mainly divided into two categories. One category is mainly used in combination with immune checkpoints such as PD-L1 and PD-1, which can relieve the inhibition of T cells by blocking immune checkpoints such as PD-L1 and promote T cell activation by activating 4-1BB to enhance the tumor suppressor function of T cells (Jeong S, et al. Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade. J Immunother. Cancer. 2021 Jul; 9 (7): e002428.). The other category is used in combination with different tumor targets, such as Her2, EGFR and B7-H3. For example, the human epidermal growth factor receptor (HER) tyrosine kinase family consists of HER1, Her2, HER3 and HER4, which play an important role in cell processes including growth, proliferation, differentiation and survival. Her2 is an atypical member of the ERBB family. Unlike other ERBB family members, Her2 has no ligand. Her2 usually forms heterodimers with HER3 or EGFR to initiate cell signal transduction. The overexpression of Her2 is not only related to the occurrence and development of tumors, but also an important clinical treatment monitoring and prognostic indicator, and an important target for the selection of tumor targeted therapy drugs. Her2 / 4-1BB dual antibody relies on the high expression of Her2 antigen on the tumor surface to promote the multimerization activation of 4-1BB, thereby promoting the activation and proliferation of T cells and inhibiting Her2 + The effect of this therapy on tumor cell growth (Hinner MJ, Clin Cancer Res. 2019 Oct 1; 25(19): 5878-5889.), but because it depends on the expression level of Her2 antigen, it often has no therapeutic effect on tumors with low antigen expression.

[0006] The development of trispecific T cell-engaging trispecific antibodies (TsAbs) has important application prospects for solving the limitations of the current clinical use of bispecific antibodies. In the tumor immune escape model, it was found that the inability and exhaustion caused by insufficient activation of T cells inhibited the therapeutic effect of antibodies, so introducing CD3 as the first activation signal is a good solution. However, for different antibodies involved in TsAbs, the location of the cell surface where the binding antigen epitope is located is different, which means that its structural design needs to be personalized. T cell-engaging trispecific antibodies represent a very effective way to redirect activated cytotoxic T cells to tumors (Runcie K, et al. Bi-specific and tri-specific antibodies-the next big thing in solid tumor therapeutics. Mol Med. 2018 Sep 24; 24 (1): 50.; Mullard A. Trispecific antibodies take to the clinic. Nat Rev Drug Discov. 2020 Oct; 19 (10): 657-658.). As part of the T cell receptor, CD3 is expressed in mature T cells and can transduce the activation signal generated by TCR recognizing antigens. TsAbs can simultaneously bind to surface tumor antigens and the CD3ε subunit of the T cell receptor, providing a physical connection between T cells and tumor cells to form an immune synapse, thereby effectively activating dormant T cells to kill tumor cells and achieve the effect of treating tumors. For the different antibodies involved in TsAbs, the locations of their binding antigen epitopes on the cell surface are different, which means that their structural design needs to be adjusted individually.Generally speaking, TsAbs-mediated binding of T cells and tumor cells involves the formation of immune synapses, which are highly complex and ordered supramolecular domains formed between T cells and tumor cells or antigen-presenting cells during T cell activation. They are a special structure where a central cluster TCR and antigen complex are bound by a circle of adhesion molecules of the integrin family (Wu L, et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nature Cancer 1, 86–98.; Yokosuka T, et al. The immunological synapse, TCR micro clusters, and T cell activation. Curr Top Microbiol Immunol. 2010; 340: 81-107.). During the formation of immune synapses, due to the structure of the TCR and antigen complex, the distance between T cells and target cells is about 15 nm. The spatial distance between TsAbs and different antigens may be equally important for the formation of the corresponding T cell immune synapse and T cell signal activation, but this spatial distance depends on completely different structural factors, including the location of the antigen epitope and the connection area between the antibody and the T cell membrane. The discovery and research progress of nanoantibodies help to optimize the steric hindrance in the interaction between target cells and effector T cells mediated by TsAbs (Hamers-Casterman C, et al. Naturally occurring antibodies devoid of lightchains. Nature. 1993 Jun 3; 363(6428): 446-8.). Unlike traditional antibodies, this type of antibody consists of only two heavy chains, only one heavy chain variable region (Variable Domain of Heavy Chain Antibody, VHH) and two conventional CH2 and CH3 regions. The trispecific antibody designed by combining 4-1BB nanoantibody with antibody fragments targeting tumor-specific antigens can avoid the risks of monoclonal antibodies and is safer and more effective than the combination of multiple specific immunomodulatory monoclonal antibodies. At the same time, it can provide co-stimulatory signals to fully and continuously activate T lymphocytes and recruit them around tumor cells. This trispecific antibody design can improve the safety, accuracy and efficacy of current immunotherapy and further expand the scope of application of immunotherapy.

[0007] CAR-T (Chimeric Antigen Receptor-T Cell Immunotherapy) therapy is also called chimeric antigen receptor-T cell immunotherapy. This is a new type of precision targeted therapy for treating tumors. In recent years, it has achieved good results in clinical tumor treatment through optimization and improvement. It is a very promising new tumor immunotherapy method that can be accurate, fast, efficient, and may cure cancer. The second-generation CAR-T therapy uses gene modification technology to transfer genetic material with specific antigen recognition domains (such as anti-Her2-scFv, derived from trastuzumab, clone number 4D5) and T cell activation signals (4-1BB and CD3ζ intracellular activation domain) into T cells, so that T cells directly bind to specific antigens on the surface of tumor cells and are activated, directly killing tumor cells by releasing perforin, granzyme B, etc., and also recruiting endogenous immune cells in the human body to kill tumor cells by releasing cytokines, thereby achieving the purpose of treating tumors, and can also form immune memory T cells, thereby obtaining a specific anti-tumor long-term mechanism. At present, there are few ideal targets for solid tumors. As a member of the human epidermal growth factor receptor family, Her2 is lowly expressed or not expressed in normal tissues, but highly expressed in a variety of tumors (including breast cancer, lung cancer, prostate cancer, head and neck squamous cell carcinoma, gastrointestinal tumors and gynecological tumors), so it is a potential target for CAR-T therapy. As the first humanized monoclonal antibody targeting Her2, trastuzumab has influenced the diagnosis and treatment of breast cancer and is a major breakthrough in breast cancer drug treatment. In February 2013, a monoclonal antibody targeting Her2 and a microtubule inhibitor conjugate, Ado trastuzumab emtansine, was approved in the United States. In December 2018, the State Food and Drug Administration approved the marketing of Pertuzumab, a humanized monoclonal antibody targeting Her2, which blocks the cell cycle and induces apoptosis by inhibiting the ligand-dependent heterodimerization between Her2 and its family members. In addition, drugs such as lapatinib, pyrotinib, and neratinib that target Her2 or co-target receptors such as Her2 and EGFR have achieved good results in clinical applications. However, the above-approved drugs are currently only used for patients with tumors with high expression of Her2, while the prognosis is poor for patients with low expression of Her2 or antigen heterogeneity, as well as patients with Her2 antigen escape caused by targeted therapy. Although Her2-targeted CAR-T cell products are currently used to treat preclinical and clinical tumor patients, after receiving traditional CAR-T treatment, the Her2-positive cell antigen mutates, making it impossible for CAR-T cells to successfully identify tumor cells, resulting in immune escape. Similar problems of antigen modification also include the phenomenon of reduced expression levels of Her2 on the tumor surface.In addition, CAR-T therapy also faces some uncontrollable side effects (such as cytokine storm, risk of uncontrolled proliferation) as well as complex preparation process and high price. These unfavorable factors limit the further application of CAR-T therapy. Therefore, by designing 4-1BB trispecific antibodies to activate T cells in patients, and using tumor recognition functional domains to promote T cells to kill tumor cells, this treatment idea enables the initial T cells in patients to simulate the function of CAR-T cells, and the process is simple and the quality is controllable, which is a potential alternative to CAR-T therapy.

[0008] In summary, in order to achieve the best activation and killing effect of T cells mediated by TsAbs on antigen-expressing tumor cells, it is necessary to investigate the different connection positions of 4-1BB nanoantibodies, and establish a method to maintain T cell activation through CD3 and 4-1BB and mediate T cell targeting of Her2 through structural design optimization and screening. + Optimal TsAbs for tumor killing. Summary of the invention

[0009] Multi-target antibody drugs have become the key to solving similar problems mentioned above. Therefore, in order to find more effective treatment methods, the present invention provides a new type of tri-specific antibody, which will provide patients with more medication options.

[0010] The first aspect of the present invention provides an anti-Her2 / CD3 / 4-1BB trispecific antibody based on structural optimization design, wherein the Her2 / CD3 / 4-1BB trispecific antibody comprises a Fab portion of a monoclonal antibody that specifically binds to CD3, an antibody fragment that specifically binds to Her2, and an antibody fragment that specifically binds to 4-1BB.

[0011] The antibody fragment that specifically binds to Her2 is anti-Her2-VHH, the antibody fragment that specifically binds to 4-1BB is anti-4-1BB-VHH, and the Fab portion of the monoclonal antibody that specifically binds to CD3 is CD3 Fab; Her2-VHH and 4-1BB-VHH are respectively connected to the CD3 Fab fragment via a connecting peptide.

[0012] The connecting peptide is selected from one or more of a flexible connecting peptide, a rigid connecting peptide or a helical connecting peptide;

[0013] The flexible linker peptide is selected from one or more of (G4S)3Linker, 218Linker or 212Linker;

[0014] The rigid connecting peptide is selected from PD Linker;

[0015] The helical coiled linker peptide is selected from Coiled Coil Linker;

[0016] In some preferred embodiments, the CD3-HC amino acid sequence is shown as SEQ ID NO: 1; the CD3-LC amino acid sequence is shown as SEQ ID NO: 2; the Her2-VHH sequence is shown as SEQ ID NO: 3; and the 4-1BB-VHH sequence is shown as SEQ ID NO: 4.

[0017] In some preferred embodiments, the (G4S)3Linker amino acid sequence is shown in SEQ ID NO: 5; the 218Linker amino acid sequence is shown in SEQ ID NO: 6; the 212Linker amino acid sequence is shown in SEQ ID NO: 7; the PD Linker amino acid sequence is shown in SEQ ID NO: 8; the Coiled Coil Linker amino acid sequence is shown in SEQ ID NO: 9 and SEQ ID NO: 19;

[0018] Preferably, the Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NOs: 10-12, and a light chain amino acid sequence as shown in SEQ ID NOs: 2, 13-18.

[0019] More preferably, the Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 13; and / or

[0020] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 14; and / or

[0021] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 15; and / or

[0022] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 16; and / or

[0023] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 17; and / or

[0024] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 18; and / or

[0025] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 13; and / or

[0026] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 16; and / or

[0027] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 17; and / or

[0028] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 12, and a light chain amino acid sequence as shown in SEQ ID NO: 2; and / or

[0029] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 12, and a light chain amino acid sequence as shown in SEQ ID NO: 13.

[0030] Further preferably, the Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 16; and / or

[0031] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 17; and / or

[0032] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 18; and / or

[0033] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 13; and / or

[0034] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 16; and / or

[0035] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 17; and / or

[0036] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 12, and a light chain amino acid sequence as shown in SEQ ID NO: 2; and / or

[0037] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 12, and a light chain amino acid sequence as shown in SEQ ID NO: 13.

[0038] More preferably, the Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 18; and / or

[0039] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 16; and / or

[0040] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 11, and a light chain amino acid sequence as shown in SEQ ID NO: 17; and / or

[0041] The Her2 / CD3 / 4-1BB trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 12, and a light chain amino acid sequence as shown in SEQ ID NO: 13.

[0042] The second aspect of the present invention discloses a method for constructing a dimeric trispecific antibody molecule that targets immune effector T cell antigen CD3 and mediates T cell relocation to tumor cells. The construction method of the trispecific antibody is based on the Fab of the anti-CD3 monoclonal antibody (clone SP34), and the second domain and the third domain are nanoantibodies (clone 2Rs15d) targeting the Her2 positive target and nanoantibodies (anti-4-1BB-VHH, clone P1-1E) targeting the T cell surface co-stimulatory CD137 molecule, respectively, and the two are connected to the CD3 Fab fragment through a Linker. The construction method comprises the following steps:

[0043] (1) Two nanobodies, anti-Her2-VHH or Her2-VHH / 4-1BB-VHH, are simultaneously linked to the heavy chain domain of the CD3 Fab structure.

[0044] (2) The anti-4-1BB-VHH is linked to the light chain domain of the CD3 Fab structure or the light chain domain is not modified.

[0045] (3) The recombinant heavy and light chains obtained in step (1) and step (2) are linked via disulfide bonds in CH1 and CL in CD3-Fab to form a heterodimer, i.e., an anti-Her2 / CD3 / 4-1BB trispecific antibody.

[0046] The step (1) can be constructed or connected in the following manner:

[0047] (a) connecting the anti-Her2-VHH to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain via a connecting peptide; or

[0048] (b) connecting the anti-Her2-VHH to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain via a connecting peptide, and connecting the anti-4-1BB-VHH to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain via a connecting peptide; or

[0049] (c) Anti-Her2-VHH was connected to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a connecting peptide, and anti-4-1BB-VHH was connected to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a connecting peptide, and then anti-4-1BB-VHH was tandemly connected to the anti-4-1BB-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a connecting peptide.

[0050] The step (2) can be constructed or connected in the following manner:

[0051] (a) connecting the anti-4-1BB-VHH to the N-terminus of the CD3 Fab light chain variable region (VL) domain via a connecting peptide; or

[0052] (b) 4-1BB-VHH is chimeric between the CD3-LC constant region (CL) domains 171S-173D via a connecting peptide, and 172K is deleted; or

[0053] (c) connecting the anti-4-1BB-VHH to the C-terminus of the CD3 Fab light chain constant region (CL) domain via a linker peptide; or

[0054] (d) connecting the anti-4-1BB-VHH to the N-terminus of the CD3 Fab light chain variable region (VL) domain via a connecting peptide, and then tandemly connecting the anti-4-1BB-VHH to the anti-4-1BB-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain via a connecting peptide; or

[0055] (e) connecting the anti-4-1BB-VHH to the C-terminus of the CD3 Fab light chain constant region (CL) domain via a connecting peptide, and then tandemly connecting the anti-4-1BB-VHH to the anti-4-1BB-VHH region at the C-terminus of the CD3 Fab light chain constant region (CL) domain via a connecting peptide; or

[0056] (f) can be constructed or connected through the following three steps: (i) connecting anti-4-1BB-VHH to the C-terminus of the CD3Fab light chain constant region (CL) domain through a connecting peptide, (ii) concatenating anti-4-1BB-VHH to the C-terminus of the anti-4-1BB-VHH domain of the antibody obtained in (i) through a connecting peptide, and then concatenating anti-4-1BB-VHH to the C-terminus of the anti-4-1BB-VHH domain of the antibody obtained in (ii) through a connecting peptide. The recombinant light chain forms an antibody fragment with a structure of CD3 VL—CD3 Fab light chain constant region (CL) domain C-terminus—connecting peptide—4-1BB-VHH—connecting peptide—4-1BB-VHH—connecting peptide—4-1BB-VHH.

[0057] The connecting peptide is selected from one or more of a flexible connecting peptide, a rigid connecting peptide or a helical connecting peptide;

[0058] The flexible linker peptide is selected from one or more of (G4S)3Linker, 218Linker or 212Linker;

[0059] The rigid connecting peptide is selected from PD Linker;

[0060] The helical coiled linker peptide is selected from Coiled Coil Linker;

[0061] Furthermore, the step (1) can be constructed or connected in the following manner:

[0062] (a) connecting anti-Her2-VHH to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain via a flexible linker peptide (G4S)3Linker. The recombinant heavy chain sequentially forms an antibody fragment with the structure of CD3 VH—CD3CH1—(G4S)3Linker—Her2-VHH from the N-terminus to the C-terminus; or

[0063] (b) connecting anti-Her2-VHH to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker, and connecting anti-4-1BB-VHH to the N-terminal 1E of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker. The recombinant heavy chain sequentially forms an antibody fragment with the structure of 4-1BB-VHH—PD Linker—CD3VH—CD3 CH1—(G4S) 3Linker—Her2-VHH from the N-terminus to the C-terminus; or

[0064] (c) Anti-Her2-VHH was connected to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker, and anti-4-1BB-VHH was connected to the N-terminal 1E of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker, and then anti-4-1BB-VHH was serially connected to the anti-4-1BB-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker. The recombinant heavy chain sequentially forms an antibody fragment with the structure of 4-1BB-VHH—218Linker—4-1BB-VHH—PD Linker—CD3 VH—CD3CH1—(G4S) 3Linker—Her2-VHH from the N-terminus to the C-terminus.

[0065] Among them, the anti-Her2-VHH is connected to the C-terminal 228C of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker, and the CD3 Fab light chain is not modified. The prepared bispecific antibody structure can be labeled as Her2 / CD3 as the control antibody of the present invention.

[0066] Furthermore, the step (2) can be constructed or connected in the following manner:

[0067] (a) connecting anti-4-1BB-VHH to the N-terminal 1Q of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker. The recombinant light chain forms an antibody fragment with the structure of 4-1BB-VHH—PD Linker—CD3VL—CD3CL from the N-terminus to the C-terminus; or

[0068] (b) 4-1BB-VHH is embedded in the CD3-LC constant region (CL) domains 171S-173D via a Coiled Coil Linker, and 172K is deleted; or

[0069] (c) connecting anti-4-1BB-VHH to the C-terminal 217C of the CD3 Fab light chain constant region (CL) domain via a flexible linker peptide (G4S)3Linker. The recombinant light chain sequentially forms an antibody fragment with the structure of CD3 VL-CD3 CL-(G4S)3Linker-4-1BB-VHH from the N-terminus to the C-terminus; or

[0070] (d) connecting the anti-4-1BB-VHH to the N-terminal 1Q of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, and then connecting the anti-4-1BB-VHH to the anti-4-1BB-VHH region at the N-terminal of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218 Linker. The recombinant light chain sequentially forms an antibody fragment with the structure of 4-1BB-VHH—218 Linker—4-1BB-VHH—PD Linker—CD3 VL—CD3 CL from the N-terminus to the C-terminus; or

[0071] (e) connecting the anti-4-1BB-VHH to the C-terminal 217C of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker, and then connecting the anti-4-1BB-VHH to the anti-4-1BB-VHH region at the C-terminal of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide 218Linker. The recombinant light chain sequentially forms an antibody fragment with the structure of CD3 VL-CD3CL-(G4S) 3Linker-4-1BB-VHH-218Linker-4-1BB-VHH from the N-terminus to the C-terminus; or

[0072] (f) can be constructed or connected by the following three steps: (i) connecting anti-4-1BB-VHH to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker, (ii) connecting anti-4-1BB-VHH to the C-terminus of the anti-4-1BB-VHH domain of the antibody obtained in (i) through a flexible connecting peptide 218Linker, (iii) connecting anti-4-1BB-VHH to the C-terminus of the anti-4-1BB-VHH domain of the antibody obtained in (ii) through a flexible connecting peptide 212Linker. The recombinant light chain sequentially forms an antibody fragment with the structure of CD3 VL-CD3CL-(G4S) 3Linker-4-1BB-VHH-218Linker-4-1BB-VHH-212Linker-4-1BB-VHH from the N-terminus to the C-terminus.

[0073] In a preferred embodiment, the Her2 / CD3 / 4-1BB trispecific antibody in step (3) is constructed based on the construction in step (1) and combined with the construction method shown in step (2) to obtain a 4-1BB monovalent Her2 / CD3 / 4-1BB trispecific antibody, as shown below:

[0074] (1) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker; and anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, labeled as Her2 / CD3 / 4-1BB-LN;

[0075] (2) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker; and anti-4-1BB-VHH was embedded between the CD3 Fab light chain constant region (CL) domain 171S-173D through a helical coiled linker peptide Coiled Coil Linker, and 172K was deleted, labeled as Her2 / CD3 / 4-1BB-LG;

[0076] (3) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; and anti-4-1BB-VHH was connected to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker, labeled as Her2 / CD3 / 4-1BB-LC;

[0077] In a preferred embodiment, the Her2 / CD3 / 4-1BB trispecific antibody in step (3) is constructed with an anti-CD3 Fab structure as a skeleton, based on the construction in step (1), in combination with the construction method shown in step (2), to obtain a 4-1BB bivalent Her2 / CD3 / 4-1BB trispecific antibody, as shown below:

[0078] (4) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker; and anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain and the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, labeled as Her2 / CD3 / 4-1BB-HLN;

[0079] (5) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker; and anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, and then anti-4-1BB-VHH was connected in series to the anti-4-1BB-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker, labeled as Her2 / CD3 / 4-1BB-LDN;

[0080] (6) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S)3Linker, and anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker, and then anti-4-1BB-VHH was connected in series to the anti-4-1BB-VHH region at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker; the CD3 Fab light chain was not modified and was labeled as Her2 / CD3 / 4-1BB-HDN;

[0081] (7) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; and anti-4-1BB-VHH was connected to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker, and then the anti-4-1BB-VHH was connected in series to the C-terminus of the anti-4-1BB-VHH domain of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide 218Linker, and labeled as Her2 / CD3 / 4-1BB-LDC;

[0082] In a preferred embodiment, the Her2 / CD3 / 4-1BB trispecific antibody in step (3) is constructed based on the construction in step (1) and combined with the construction method shown in step (2) to obtain a 4-1BB trivalent Her2 / CD3 / 4-1BB trispecific antibody, as shown below:

[0083] (8) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; and anti-4-1BB-VHH was connected to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker, and then the anti-4-1BB-VHH was sequentially connected to the C-terminus of the 4-1BB-VHH domain at the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide 218Linker, and then the anti-4-1BB-VHH was sequentially connected to the CD3 through a flexible connecting peptide 212Linker. The C-terminus of the 4-1BB-VHH domain of the Fab light chain constant region (CL) domain forms a recombinant light chain with a structure of light chain variable region—light chain constant region—(G4S)3Linker—4-1BB-VHH—218Linker—4-1BB-VHH—212Linker—4-1BB-VHH from N-terminus to C-terminus, labeled Her2 / CD3 / 4-1BB-LTC;

[0084] (9) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker, and then anti-4-1BB-VHH was connected to the N-terminal 1E region of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker; anti-4-1BB-VHH was connected to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible linker peptide (G4S) 3Linker, and anti-4-1BB-VHH was sequentially connected in series to the C-terminus of the anti-4-1BB-VHH domain at the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible linker peptide 218Linker, and labeled as Her2 / CD3 / 4-1BB-HLDC;

[0085] (10) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S) 3Linker, and anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker, and then anti-4-1BB-VHH was sequentially connected in series to the N-terminus of the anti-4-1BB-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker; finally, anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, marked as Her2 / CD3 / 4-1BB-HDLN;

[0086] (11) Anti-Her2-VHH was connected to the C-terminus of the CD3 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S)3Linker, and anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker; then anti-4-1BB-VHH was connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, and then anti-4-1BB-VHH was serially connected to the N-terminus of the anti-4-1BB-VHH domain at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker, marked as Her2 / CD3 / 4-1BB-HLDN.

[0087] The third aspect of the present invention also provides a method for preparing the trispecific antibody of the present invention. The method comprises:

[0088] (1) obtaining a fusion gene of a trispecific antibody and constructing an expression vector of the trispecific antibody;

[0089] (2) transfecting the above expression vector into host cells by genetic engineering methods;

[0090] (3) culturing the host cell under conditions that allow production of the trispecific antibody;

[0091] (4) Isolating and purifying the produced antibody protein.

[0092] The expression vector in step (1) can be a eukaryotic cell expression vector pFuse, pSeqtag, pCMV, pcDNA, pFastBac1, pPIC9K, pcAGGS, etc. and a prokaryotic expression vector pET, pGEX, pMAL, pQE, pTrc, pBV, pTXB, preferably a eukaryotic expression vector.

[0093] The host cell in step (2) can be Escherichia coli, Bacillus thuringiensis, Pichia pastoris, insect cells, 293 suspension cells and Chinese insect ovary cells, preferably 293 suspension cells.

[0094] In a fourth aspect of the present invention, there is provided the use of the antibody in the manufacture of a medicament for treating cancer. The cancer includes but is not limited to breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, brain tumor, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma or lymphoma.

[0095] In one embodiment, it can be used to treat Her2-positive tumors. The Her2 / CD3 / 4-1BB trispecific antibody molecule can effectively kill Her2+ tumor cells in vitro, and the tumor killing effect in vitro is significantly higher than that of the Her2 / CD3 bispecific antibody.

[0096] The fifth aspect of the present invention provides a method for treating a tumor, which comprises administering the trispecific antibody of the present application to a subject in need thereof in an amount effective for treating the tumor.

[0097] The technical solution of the present invention has achieved beneficial technical effects:

[0098] 1. The trispecific antibody drug of the present invention achieves target therapy for both T cells and tumor cells, while maintaining the continuous activation of T cells with co-stimulatory signals. The trispecific antibody based on the Fab and VHH structure has stronger penetration ability in the solid tumor microenvironment than antibodies with Fc regions, and has a shorter half-life in the body, which is conducive to controlling the number of administrations and dosages, and effectively reducing side effects such as off-target and cytokine storms.

[0099] 2. Connecting the VHH antibody molecule to different positions of Fab through different connecting peptide Linkers has different effects: When constructing the trispecific antibody described in the present invention, the connection and functional distinction between the domains need to be considered comprehensively. When adding a domain to the C-terminus of Fab, it is important to note that the added domain can be expressed normally while ensuring the independence of the domain. Therefore, the present invention uses a flexible connecting peptide Linker; the present invention also provides a method for embedding the domain into the light / heavy chain constant region through a helical coiled connecting peptide (Coiled Coil Linker), which can more effectively ensure the independence and targeting between the three domains. By introducing VHH at different positions of Fab, the distance between the targeted tumor cells and T cells can be effectively adjusted, thereby forming an effective immune synapse and avoiding the influence of immunosuppressive signals on the activation and killing effect of T cells. Based on previous studies, we found that the best site for introducing Her2 targeting antibodies in CD3 targeting Fab is the C-terminus of Fab, and the best site for introducing 4-1BB targeting antibodies is the C-terminus of the Fab light chain.

[0100] 3. Through literature summary and preliminary experimental results, we successfully selected antibody clones targeting Her2, 4-1BB and CD3, and expressed them respectively in eukaryotic expression systems through molecular cloning technology. After in vitro verification, it was confirmed that the selected antibodies all have good binding specificity and affinity with the target antigens.

[0101] 4. Through structural optimization and activity comparison, it is confirmed that these trispecific antibodies have the effect of improving T cell functional activity and immune killing target cells. These beneficial effects rely on the activation effect of the CD3-targeted antibody skeleton on T cells, which shows that CD3-Fab has unique advantages in the design of trispecific antibodies. In addition, based on the CD3-targeted antibody as a skeleton, it has better operational simplicity and functional stability for the introduction of multi-valent 4-1BB targeted activation antibodies and the introduction of different tumor-associated antigen targeting antibodies. This advantage cannot be replaced by the optimization design of such trispecific antibodies using other target antibodies as a skeleton.

[0102] 5. Through structural optimization and activity verification, the optimal molecular structure of the Her2 / CD3 / 4-1BB trispecific antibody was determined. Compared with the currently effective bispecific antibodies, the Her2 / CD3 / 4-1BB trispecific antibody can not only effectively kill Her2-positive tumors, but also effectively promote T cell proliferation, anti-apoptotic activity, promote cytokine secretion and maintain mitochondrial homeostasis. It can effectively prevent tumor antigen immune escape and enhance the synergistic immunotherapy effect of regulating the microenvironment of solid tumors.

[0103] 6. By comparing the in vitro functions with those of CAR-T-Her2, the preferred Her2 / CD3 / 4-1BB-LTC trispecific antibody not only has a significant advantage in cytokine release, but also promotes the transformation of PBMC into central memory T cells in terms of phenotypic changes. This phenotype can effectively promote the proliferation and activation of T cells, and reduces the possibility of exhaustion and apoptosis compared to the effector cell phenotype of CAR-T-Her2, which has a significant advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 Schematic diagram of different trispecific antibodies designed based on structure-optimized activity;

[0105] Figure 2 ELISA binding data of monovalent Her2 / CD3 / 4-1BB trispecific antibody targeting Her2 antigen;

[0106] Figure 3 ELISA binding data of monovalent Her2 / CD3 / 4-1BB trispecific antibody targeting CD3 antigen;

[0107] Figure 4 ELISA binding data of monovalent Her2 / CD3 / 4-1BB trispecific antibody targeting 4-1BB antigen;

[0108] Figure 5 Comparison of activation abilities of monovalent Her2 / CD3 / 4-1BB trispecific antibodies;

[0109] Figure 6 Monovalent Her2 / CD3 / 4-1BB trispecific antibody cytokine release;

[0110] Figure 7 ELISA binding data of the bivalent Her2 / CD3 / 4-1BB trispecific antibody targeting CD3 antigen;

[0111] Figure 8 ELISA binding data of the bivalent Her2 / CD3 / 4-1BB trispecific antibody targeting 4-1BB antigen;

[0112] Fig. 9 Comparison of activation abilities of bivalent Her2 / CD3 / 4-1BB trispecific antibodies;

[0113] Fig.10 Bivalent Her2 / CD3 / 4-1BB trispecific antibody cytokine release;

[0114] Fig.11 Comparison of the anti-apoptotic ability of the bivalent Her2 / CD3 / 4-1BB trispecific antibody;

[0115] Fig.12 ELISA binding data of trivalent Her2 / CD3 / 4-1BB trispecific antibody targeting CD3 antigen;

[0116] Fig.13 ELISA binding data of the trivalent Her2 / CD3 / 4-1BB trispecific antibody targeting 4-1BB antigen;

[0117] Fig.14 Comparison of activation abilities of three-valent Her2 / CD3 / 4-1BB trispecific antibodies;

[0118] Fig.15 Trivalent Her2 / CD3 / 4-1BB trispecific antibody cytokine release;

[0119] Fig.16 Comparison of the anti-apoptotic ability of the three-valent Her2 / CD3 / 4-1BB trispecific antibodies;

[0120] Fig.17 Comparison of the proliferation-promoting abilities of Her2 / CD3 / 4-1BB trispecific antibodies with different titers;

[0121] Fig.18 Comparison of activation abilities of Her2 / CD3 / 4-1BB trispecific antibodies of different titers;

[0122] Fig.19 Comparison of the anti-apoptotic ability of Her2 / CD3 / 4-1BB trispecific antibodies with different titers;

[0123] Fig. 20 Comparison of the killing ability of Her2 / CD3 / 4-1BB trispecific antibodies with different titers on cells with high expression of Her2;

[0124] Fig.21 Comparison of the killing ability of Her2 / CD3 / 4-1BB trispecific antibodies with different titers on Her2 low-expressing cells.

[0125] Fig. 22 Comparison of cytokine release between Her2 / CD3 / 4-1BB-LTC antibody and CAR-T-Her2;

[0126] Fig.23 Comparison of the anti-apoptotic function of Her2 / CD3 / 4-1BB-LTC antibody and CAR-T-Her2;

[0127] Fig.24 Comparison of phenotypic changes between Her2 / CD3 / 4-1BB-LTC antibody and CAR-T-Her2; DETAILED DESCRIPTION

[0128] The present invention is further explained and illustrated in conjunction with the following examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. In the following examples, the materials used in the experiments can be purchased or prepared with reference to existing disclosed technologies; those without indicating the source and specifications are commercially available; various processes and methods not described in detail are conventional methods known in the art.

[0129] Example 1 Construction and eukaryotic expression of bi / trispecific antibodies

[0130] 1.1 Vector construction

[0131] Construction of CD3-HC-Her2-VHH and CD3-LC expression vectors:

[0132] CD3-HC comes from the Fab heavy chain of anti-CD3 monoclonal antibody (clone number: SP34), and CD3-LC comes from the Fab light chain of anti-CD3 monoclonal antibody (clone number: SP34). The heavy chain of the Her2 / CD3 bispecific antibody is connected by CD3-HC and Her2-VHH in the following manner: Her2-VHH is connected to the C-terminal 228C of the CD3-HC constant region (CH1) domain through a flexible linker peptide (G4S)3Linker; the light chain CD3-LC is not modified. The coding genes of the above CD3-HC-Her2-VHH and CD3-LC are synthesized separately by conventional molecular biological methods, and the obtained synthetic genes are inserted into the pcAGGS eukaryotic expression vector with Amp resistance by homologous recombination. According to the connection method, the bispecific antibody expressed later is labeled as Her2 / CD3. In addition to connecting the anti-Her2-VHH to CD3-HC through the (G4S)3 Linker selected in this experiment, a connecting peptide Linker known to those skilled in the art can also be used for replacement connection. The relevant sequences are shown in Table 1:

[0133] Table 1

[0134]

[0135]

[0136] Construction of 4-1BB monovalent Her2 / CD3 / 4-1BB trispecific antibody expression vector:

[0137] The heavy chain of the Her2 / CD3 / 4-1BB trispecific antibody is connected by CD3-HC and Her2-VHH in the following manner: Her2-VHH is connected to the C-terminus 228C of the CD3-HC constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; the light chain of the Her2 / CD3 / 4-1BB trispecific antibody is connected by CD3-LC and 4-1BB-VHH in the following manner: 4-1BB-VHH is connected to the N-terminus of the CD3-LC variable region (VL) domain through a rigid connecting peptide PD Linker; or 4-1BB-VHH is embedded between 171S-173D of the CD3-LC constant region (CL) domain through a helical coiled connecting peptide Coiled Coil Linker, and 172K is deleted; or 4-1BB-VHH is connected to the C-terminus of the CD3-LC constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker. The obtained gene is inserted into the pcAGGS eukaryotic expression vector with Amp resistance by homologous recombination. According to the connection mode of the light chain of the Her2 / CD3 / 4-1BB trispecific antibody, that is, the connection mode of CD3-LC and 4-1BB-VHH, the 4-1BB monovalent Her2 / CD3 / 4-1BB trispecific antibody expressed later is labeled as Her2 / CD3 / 4-1BB-LN, Her2 / CD3 / 4-1BB-LG, Her2 / CD3 / 4-1BB-LC; wherein Her2-VHH and 4-1BB-VHH can be connected to CD3-HC or CD3-LC by (G4S)3Linker, PD Linker and Coiled Coil Linker selected in this experiment, and can also be replaced by connecting peptide Linkers known to those skilled in the art.

[0138] The relevant sequences are shown in Table 2:

[0139] Table 2

[0140] name Amino acid sequence number (G4S)3 Linker SEQ ID NO: 5 PD Linker SEQ ID NO: 8 Coiled Coil Linker SEQ ID NO: 9, 19 CD3-HC-Her2 heavy chain SEQ ID NO: 10 Her2 / CD3 / 4-1BB-LN light chain SEQ ID NO: 13 Her2 / CD3 / 4-1BB-LG light chain SEQ ID NO: 14 Her2 / CD3 / 4-1BB-LC light chain SEQ ID NO: 15

[0141] Construction of 4-1BB bivalent Her2 / CD3 / 4-1BB trispecific antibody expression vector:

[0142] The heavy chain and light chain of the 4-1BB bivalent Her2 / CD3 / 4-1BB trispecific antibody are connected by CD3-HC, 4-1BB-VHH and Her2-VHH in the following manner: (1) The heavy chain is connected to the CD3-HC constant region (CH1) domain C-terminal 228C region by a flexible linker peptide (G4S) 3Linker. The light chain is connected to the CD3 Fab light chain variable region (VL) domain N-terminal by a rigid linker peptide PD Linker, and then the CD3 Fab light chain variable region (VL) domain N-terminal by a flexible linker peptide 218Linker. (2) The heavy chain is connected to the CD3 Fab light chain variable region (VL) domain N-terminal by a flexible linker peptide (G4S) 3Linker. The Fab heavy chain constant region (CH1) domain C-terminal 228C region, and the anti-4-1BB-VHH is connected to the CD3 Fab heavy chain variable region (VH) domain N-terminal through a rigid linker peptide PD Linker; the light chain is connected to the CD3-LC variable region (VL) domain N-terminal through a rigid linker peptide PD Linker 4-1BB-VHH; (3) the heavy chain is connected to the CD3 Fab heavy chain constant region (CH1) domain C-terminal 228C region through a flexible linker peptide (G4S) 3Linker anti-Her2-VHH is connected to the CD3 Fab heavy chain constant region (CH1) domain C-terminal 228C region, and the anti-4-1BB-VHH is connected to the CD3 Fab heavy chain variable region (VH) domain N-terminal through a rigid linker peptide PD Linker, and then the anti-4-1BB-VHH is connected in series to the CD3 through a flexible linker peptide 218Linker. The anti-4-1BB-VHH region at the N-terminus of the Fab heavy chain variable region (VH) domain; the light chain is not modified; (4) the heavy chain is Her2-VHH connected to the C-terminal 228C region of the CD3-HC constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; the light chain is anti-4-1BB-VHH connected to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S) 3Linker, and then the anti-4-1BB-VHH is sequentially connected in series to the C-terminus of the 4-1BB-VHH domain at the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide 218Linker.

[0143] The obtained gene is inserted into the pcAGGS eukaryotic expression vector with Amp resistance by homologous recombination. According to the combination of light and heavy chains of the Her2 / CD3 / 4-1BB trispecific antibody, i.e., CD3-LC and CD3-HC, and the connection mode of 4-1BB-VHH, the 4-1BB bivalent Her2 / CD3 / 4-1BB trispecific antibody expressed later is labeled as Her2 / CD3 / 4-1BB-LDN, Her2 / CD3 / 4-1BB-HLN, Her2 / CD3 / 4-1BB-HDN, Her2 / CD3 / 4-1BB-LDC; wherein Her2-VHH and 4-1BB-VHH can be connected to CD3-HC or CD3-LC by (G4S)3Linker, PD Linker and Coiled CoilLinker selected in this experiment, and can also be replaced by connecting peptide Linkers known to those skilled in the art.

[0144] The relevant sequences are shown in Table 3:

[0145] Table 3

[0146]

[0147]

[0148] Construction of 4-1BB trivalent Her2 / CD3 / 4-1BB trispecific antibody expression vector:

[0149] The heavy chain and light chain of the 4-1BB trivalent Her2 / CD3 / 4-1BB trispecific antibody are connected by CD3-HC, 4-1BB-VHH and Her2-VHH in the following manner: (1) The heavy chain is connected by Her2-VHH to the C-terminal 228C region of the CD3-HC constant region (CH1) domain through a flexible connecting peptide (G4S)3Linker; the light chain is connected by anti-4-1BB-VHH to the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide (G4S)3Linker, and then the anti-4-1BB-VHH is connected in series to the C-terminus of the 4-1BB-VHH domain at the C-terminus of the CD3 Fab light chain constant region (CL) domain through a flexible connecting peptide 218Linker, and then the anti-4-1BB-VHH is connected in series to the CD3 through a flexible connecting peptide 212Linker. The Fab light chain constant region (CL) domain C-terminus of the 4-1BB-VHH domain; (2) the heavy chain is connected to the CD3 Fab heavy chain constant region (CH1) domain C-terminus 228C region through a flexible linker peptide (G4S) 3Linker to connect the anti-Her2-VHH to the CD3 Fab heavy chain variable region (VH) domain N-terminus through a rigid linker peptide PD Linker; the light chain is connected to the CD3 Fab light chain constant region (CL) domain C-terminus through a flexible linker peptide (G4S) 3Linker to connect the anti-4-1BB-VHH to the CD3 Fab light chain constant region (CL) domain C-terminus, and then the anti-4-1BB-VHH is connected in series to the CD3 Fab light chain constant region (CL) domain C-terminus of the 4-1BB-VHH domain through a flexible linker peptide 218Linker; (3) the heavy chain is connected to the CD3 Fab light chain constant region (CL) domain C-terminus through a flexible linker peptide (G4S) 3Linker to connect the anti-Her2-VHH to the CD3 The Fab heavy chain constant region (CH1) domain C-terminal 228C region, and the anti-4-1BB-VHH is connected to the CD3 Fab heavy chain variable region (VH) domain N-terminal through a rigid linker peptide PD Linker, and then the anti-4-1BB-VHH is connected in series to the anti-4-1BB-VHH region at the CD3 Fab heavy chain variable region (VH) domain N-terminal through a flexible linker peptide 218Linker; the light chain is 4-1BB-VHH connected to the CD3-LC variable region (VL) domain N-terminal through a rigid linker peptide PD Linker; (4) the heavy chain is anti-Her2-VHH connected to the CD3 Fab heavy chain constant region (CH1) domain C-terminal 228C region through a flexible linker peptide (G4S) 3Linker, and the anti-4-1BB-VHH is connected to the CD3 Fab heavy chain variable region (VH) domain N-terminal through a rigid linker peptide PD Linker;The light chain is a rigid linker peptide PD Linker that connects the anti-4-1BB-VHH to the N-terminus of the CD3 Fab light chain variable region (VL) domain, and then a flexible linker peptide 218 Linker that connects the anti-4-1BB-VHH to the anti-4-1BB-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain. ;

[0150] The obtained gene was inserted into the pcAGGS eukaryotic expression vector with Amp resistance by homologous recombination. According to the combination of light and heavy chains of Her2 / CD3 / 4-1BB trispecific antibody, i.e. CD3-LC and CD3-HC, and the connection mode of 4-1BB-VHH, the 4-1BB trivalent Her2 / CD3 / 4-1BB trispecific antibody expressed later was labeled as Her2 / CD3 / 4-1BB-LTC, Her2 / CD3 / 4-1BB-HLDC Her2 / CD3 / 4-1BB-HDLN, Her2 / CD3 / 4-1BB-HLDN; wherein Her2-VHH and 4-1BB-VHH can be connected to CD3-HC or CD3-LC by (G4S)3Linker, PD Linker and Coiled CoilLinker selected in this experiment, and can also be replaced by connecting peptide Linkers known to those skilled in the art.

[0151] The relevant sequences are shown in Table 4:

[0152] Table 4

[0153] name Amino acid sequence number (G4S)3 Linker SEQ ID NO: 5 218 Linker SEQ ID NO: 6 212 Linker SEQ ID NO: 7 PD Linker SEQ ID NO: 8 Her2 / CD3 / 4-1BB-HDLN heavy chain SEQ ID NO: 12 Her2 / CD3 / 4-1BB-HLDN heavy chain SEQ ID NO: 11 Her2 / CD3 / 4-1BB-HLDC heavy chain SEQ ID NO: 11 Her2 / CD3 / 4-1BB-LTC heavy chain SEQ ID NO: 10 Her2 / CD3 / 4-1BB-HDLN light chain SEQ ID NO: 13 Her2 / CD3 / 4-1BB-HLDN light chain SEQ ID NO: 16 Her2 / CD3 / 4-1BB-HLDC light chain SEQ ID NO: 17 Her2 / CD3 / 4-1BB-LTC light chain SEQ ID NO: 18

[0154] The schematic diagram of the trispecific antibody designed based on structure optimization activity of the present invention is as follows Figure 1 ( Figure 1 A. Figure 1 B).

[0155] 1.2 Eukaryotic expression of proteins

[0156] Inoculate 1.5×10 6 / mL 293 suspension cells were placed in 200 mL of culture medium in a 500 mL shake flask and cultured in a shaker incubator at 37°C, 165 rpm, and 5% CO2 for 24 h. Cell counts were performed after 24 h and the cell density was adjusted to 3×10 6 / mL, mix the light chain and heavy chain plasmids constructed in step 1.1 above in 10mL Opti-MEM and let stand at room temperature for 5min, add 500μL (1:2.5) PEI 40000 in 10mL Opti-MEM and mix gently and let stand at room temperature for 5min, mix the plasmid and PEI mixture, mix gently and let stand at room temperature for 20min. Add the above mixture dropwise to 200mL 293 suspension cell culture medium, shake the culture bottle gently while adding, mix well and put it in a shaker, and transfect for 72h. After the transfection is completed, centrifuge at 100g for 5min, take the supernatant, add 100mL culture medium to the culture bottle, resuspend and culture for 48h, centrifuge at 3000rpm for 5min, take the supernatant, mix the 293 suspension cell supernatants collected twice, and freeze at -20℃.

[0157] Example 2 Purification of trispecific antibodies

[0158] 2.1 Protein G affinity purification

[0159] Take 400 mL of the cell supernatant of 1.2 in the above Example 1, centrifuge at 15000 rpm and 4°C for 30 min, collect the supernatant, filter with a 0.45 μm filter membrane, and place on ice for later use. Take 4 mL of Protein G (20% ethanol / Protein G 1:1) in the chromatography column, rinse with Binding buffer 3 times, and use a gasket to press the resin surface. Balance the Protein G column with 20 mL of Binding buffer. Load once every 10 mL, and allow the sample to pass through the Protein G column at a uniform speed (about 0.5 mL / min). Wash the Protein G column with 40 mL of Binding buffer at a uniform speed (about 1 mL / min). First, add 10% of the volume of the eluent to the elution tube collection tube. Add Elution buffer to the column and elute once with 5 mL until the protein concentration cannot be quantified. The collected protein samples were concentrated using an Amicon Ultra-15 centrifugal filter and centrifuged at 3000 rpm for 20 minutes at 4°C to quantify the protein concentration.

[0160] Example 34-1BB monovalent Her2 / CD3 / 4-1BB trispecific antibody biological function evaluation

[0161] 3.1 Antigen binding validation of monovalent Her2 / CD3 / 4-1BB trispecific antibodies

[0162] The Her2 ECD-Fc, CD3 ECD-Fc and 4-1BB ECD-Fc antigen proteins were diluted to 10 μg / mL with PBS and coated in 96-well ELISA plates, 100 μL / well, and coated overnight at 4°C. The liquid in the plate was discarded, and the plate was washed twice with PBST, and 200 μL / well of 5% skim milk in PBST solution was added, blocked at room temperature for 2 hours, and the blocking solution in the well was drained. The Her2 / CD3 / 4-1BB antibodies with different structures were diluted 10 times with blocking solution in 8 gradients, with the starting antibody concentration of 100 nM, 2 replicates for each concentration gradient, 100 μL / well was added to the plate, and incubated at room temperature for 2 hours. The plate was washed 3 times with PBST and drained, and the HRP-labeled anti-kappa chain antibody was diluted 1:5000 with 5% skim milk in PBST and added to the plate, 100 μL / well, and incubated at room temperature for 1 hour. Wash the plate three times with PBST and drain, add 100 μL of TMB colorimetric solution to each well, color develop for 10 min at room temperature in the dark, then add 50 μL of 2M H2SO4 to each well to terminate the color development reaction, and use a microplate reader to detect the absorbance value at OD450nm. Make a four-parameter nonlinear regression graph with the sample concentration value as the horizontal axis and the absorbance value as the vertical axis and calculate the EC50 value.

[0163] The results of the comparison of binding ability with Her antigen are as follows Figure 2 As shown in Table 5, compared with the control group CD3-Fab, the trispecific antibodies with different structures can effectively bind to the Her2 ECD-Fc antigen protein; further analysis of the EC50 values ​​found that compared with the other two structural antibodies, the trispecific antibody of Her2 / CD3 / 4-1BB-LG has good antigen binding ability. The EC50 values ​​of Her antigen binding are shown in Table 5:

[0164] Table 5

[0165] Antibody Name EC50(nM) Her2 / CD3 / 4-1BB-LN 4.178 Her2 / CD3 / 4-1BB-LG 3.035 Her2 / CD3 / 4-1BB-LC 5.125 Her2 / CD3 5.439 CD3-Fab Not Detected

[0166] The results of the comparison of binding ability with CD3 antigen are as follows Figure 3 As shown in Table 6, trispecific antibodies with different structures can effectively bind to CD3 ECD-Fc antigen protein; further analysis of EC50 values ​​revealed that Her2 / CD3 / 4-1BB-LN and Her2 / CD3 / 4-1BB-LG have similar abilities to bind to CD3 antigen. Compared with the other two structural antibodies, the ability of Her2 / CD3 / 4-1BB-LC trispecific antibody to bind to CD3 antigen is reduced. EC50 values ​​for CD3 antigen binding are shown in Table 6:

[0167] Table 6

[0168] Antibody Name EC50(nM) Her2 / CD3 / 4-1BB-LN 0.0736 Her2 / CD3 / 4-1BB-LG 0.0870 Her2 / CD3 / 4-1BB-LC 0.2040 Her2 / CD3 0.0911

[0169] The results of the comparison of binding ability with 4-1BB antigen are as follows Figure 4 As shown in Table 7, trispecific antibodies with different structures can effectively bind to 4-1BB ECD-Fc antigen protein; further analysis of the EC50 values ​​revealed that Her2 / CD3 / 4-1BB-LC trispecific antibodies have the strongest binding ability. The EC50 values ​​for 4-1BB antigen binding are shown in Table 7:

[0170] Table 7

[0171] Antibody Name EC50(nM) Her2 / CD3 / 4-1BB-LN 0.3928 Her2 / CD3 / 4-1BB-LG 0.7567 Her2 / CD3 / 4-1BB-LC 0.1071 Her2 / CD3 Not Detected

[0172] 3.2 Comparison of the ability of monovalent Her2 / CD3 / 4-1BB trispecific antibodies to mediate PBMC activation.

[0173] The PBMC cells were taken out of liquid nitrogen and quickly thawed in a 37°C water bath. The thawed cells were diluted with RPMI 1640 medium, centrifuged at 1000rpm for 5min, the supernatant was removed, the PBMC cells were resuspended in the medium, and the above centrifugation was repeated once. Cell count, PBMC cell density was adjusted to 1 million / mL; 100μL / well; tumor cells (SKBR3) were digested, and the cell density was adjusted to 0.5 million / mL using RPMI 1640, and 100μL / well was used for sample addition. Tumor cell co-incubation group configuration: PBMC and SKBR3 were mixed in equal volumes (E:T=2:1); tumor cell-free group configuration: PBMC were mixed with an equal volume of culture medium (100μL culture medium). Drug addition: Use the same concentration (1nM final concentration) of antibodies, 20μL / well. After culturing at 37°C for 24h, the expression of activation signals (CD25) was detected. The results are as follows. Figure 5 ( Figure 5 A. Figure 5 As shown in Figure 2 (B), the Her2 / CD3 / 4-1BB-LC trispecific antibody of the preferred embodiment has a significant advantage in CD25 expression on CD4+ T cells compared with the Her2 / CD3 / 4-1BB-LN and Her2 / CD3 / 4-1BB-LG structures.

[0174] 3.3 Comparison of cytokine release by monovalent Her2 / CD3 / 4-1BB trispecific antibodies

[0175] The specific implementation steps are the same as 3.2. PBMCs were thawed from liquid nitrogen and the cell density was adjusted to (1 million / mL). HCC1954 tumor cells were digested and the density was adjusted to (0.5 million / mL). After equal volume mixing, the cells were re-plated in a 24-well plate at a volume of 1 mL / well. Her2 / CD3 / 4-1BB trispecific antibodies of different structures were added at a concentration of 1 nM. The supernatant was collected after 48 hours. The cytokine release was compared using Invitrogen human IL-2 (TNF-α, IFN-γ) uncoated ELISA Kit. The results are shown in Figure 6 ( Figure 6 A. Figure 6 B. Figure 6 As shown in Figure C), the Her2 / CD3 / 4-1BB-LC trispecific antibody has significant advantages over the Her2 / CD3 / 4-1BB-LN and Her2 / CD3 / 4-1BB-LG structures in the release of IL-2, IFN-γ and TNF-α. Her2 / CD3 / 4-1BB-LC can be used as a structure for further optimization.

[0176] Example 44-1BB Bivalent Her2 / CD3 / 4-1BB Trispecific Antibody Biological Function Evaluation

[0177] 4.1 Antigen binding validation of dual-valent Her2 / CD3 / 4-1BB trispecific antibodies

[0178] CD3 ECD-Fc and 4-1BB ECD-Fc were diluted to 10 μg / mL with PBS and coated in a 96-well ELISA plate at 100 μL / well at 4°C overnight. Discard the liquid in the plate, wash the plate twice with PBST, add 200 μL / well of 5% skim milk in PBST solution, block at room temperature for 2 hours, and drain the blocking solution in the well. Dilute Her2 / CD3 / 4-1BB with different structures 10 times with blocking solution in 8 gradients, with the starting antibody concentration of 100 nM, 3 replicates for each concentration gradient, 100 μL / well added to the plate, and incubate at room temperature for 2 hours. Wash the plate 3 times with PBST and drain, dilute the HRP-labeled anti-kappa chain antibody 1:5000 with 5% skim milk in PBST, and add it to the plate at 100 μL / well, and incubate at room temperature for 1 hour. Wash the plate three times with PBST and drain, add 100 μL of TMB colorimetric solution to each well, color develop for 10 min at room temperature in the dark, then add 50 μL of 2M H2SO4 to each well to terminate the color development reaction, and use a microplate reader to detect the absorbance value at OD450nm. Make a four-parameter nonlinear regression graph with the sample concentration value as the horizontal axis and the absorbance value as the vertical axis and calculate the EC50 value.

[0179] The results of the comparison of binding ability with CD3 antigen are as follows Figure 7As shown in Table 8, the bivalent Her2 / CD3 / 4-1BB can effectively bind to the CD3 ECD-Fc antigen protein; further analysis of the EC50 values ​​revealed that the Her2 / CD3 / 4-1BB LDC trispecific antibody has the weakest ability to bind to the CD3 antigen compared to the other three structures. The CD3 antigen binding EC50 values ​​are shown in Table 8:

[0180] Table 8

[0181] Antibody Name EC50(nM) Her2 / CD3 / 4-1BB-HLN 0.1390 Her2 / CD3 / 4-1BB-LDC 0.3844 Her2 / CD3 / 4-1BB-LDN 0.1233 Her2 / CD3 / 4-1BB-HDN 0.1427 Her2 / CD3 Not Detected

[0182] The results of the comparison of binding ability with 4-1BB antigen are as follows Figure 8 As shown in Table 9, trispecific antibodies with different structures can effectively bind to 4-1BB ECD-Fc antigen protein; further analysis of EC50 values ​​revealed that Her2 / CD3 / 4-1BB-HLN and Her2 / CD3 / 4-1BB-LDN have the strongest binding ability, and Her2 / CD3 / 4-1BB-LDC and Her2 / CD3 / 4-1BB-HDN have similar binding abilities. The EC50 values ​​of 4-1BB antigen binding are shown in Table 9:

[0183] Table 9

[0184] Antibody Name EC50(nM) Her2 / CD3 / 4-1BB-HLN 0.1179 Her2 / CD3 / 4-1BB-LDC 0.3583 Her2 / CD3 / 4-1BB-LDN 0.1300 Her2 / CD3 / 4-1BB-HDN 0.3738

[0185] 4.2 Comparison of the ability of bivalent Her2 / CD3 / 4-1BB trispecific antibodies to mediate PBMC activation.

[0186] The PBMC cells were taken out of liquid nitrogen and quickly thawed in a 37°C water bath. The thawed cells were diluted with RPMI1640 medium, centrifuged at 1000rpm for 5min, the supernatant was removed, the PBMC cells were resuspended in the medium, and the above centrifugation was repeated once. Cell count, PBMC cell density was adjusted to 1 million / mL; 100μL / well; tumor cells (SKBR3) were digested, and the cell density was adjusted to 0.5 million / mL using RPMI 1640, and 100μL / well was used for sample addition. Tumor cell co-incubation group configuration: PBMC and SKBR3 were mixed in equal volumes (E:T=2:1); tumor cell-free group configuration: PBMC were mixed with an equal volume of culture medium, (+100μL culture medium). Drug addition: Use the same concentration (1nM final concentration) of antibodies, 20μL / well. After culturing at 37°C for 24h, the expression of activation signals (CD25) was detected. The results are as follows Fig. 9 ( Fig. 9 A. Fig. 9As shown in Figure 2(B), the Her2 / CD3 / 4-1BB-LDC trispecific antibody has a significant advantage in CD25 expression on activated CD4+ T cells compared with Her2 / CD3 / 4-1BB-LDN and Her2 / CD3 / 4-1BB-HDN antibodies, but has no significant difference with Her2 / CD3 / 4-1BB-HLN.

[0187] 4.3 Comparison of cytokine release by dual-valent Her2 / CD3 / 4-1BB trispecific antibodies

[0188] Thaw PBMC from liquid nitrogen, adjust the cell density to (1 million / mL), digest HCC1954 tumor cells, adjust the density to (0.5 million / mL), mix equal volumes and spread 1 mL / well volume back into 24-well plates, add Her2 / CD3 / 4-1BB trispecific antibodies of different structures at a concentration of 1 nM, and collect the supernatant after 48 hours. Use Invitrogen human IL-2 (TNF-α, IFN-γ) uncoated ELISA Kit to compare cytokine release. Fig.10 ( Fig.10 A. Fig.10 B. Fig.10 As shown in C), the Her2 / CD3 / 4-1BB-LDC trispecific antibody has a significant advantage in IL-2 release over Her2 / CD3 / 4-1BB-LDN, Her2 / CD3 / 4-1BB-HDN and Her2 / CD3 / 4-1BB-HLN antibodies. Her2 / CD3 / 4-1BB-LDC can be used as a structure for further optimization.

[0189] 4.4 Comparison of the anti-apoptotic ability of the bivalent Her2 / CD3 / 4-1BB trispecific antibodies

[0190] PBMCs were thawed from liquid nitrogen and the cell density was adjusted to (1 million / mL). HCC1954 tumor cells were digested and the density was adjusted to (0.5 million / mL). After equal volumes were mixed, 1 mL / well was plated back into a 24-well plate. Her2 / CD3 / 4-1BB trispecific antibodies of different structures were added at a concentration of 1 nM. After incubation at 37°C for 48 hours, the cells were collected by centrifugation and stained with flow cytometry antibodies PerCPcy5.5-anti human CD4; Pacific blue anti-human CD8; FITC anti-human BCL-xL. BCL-xL was analyzed by flow cytometry. + The results were compared with the percentage of anti-apoptosis effect. Fig.11 ( Fig.11 A. Fig.11As shown in B), the Her2 / CD3 / 4-1BB-LDC trispecific antibody has a significant advantage in anti-apoptosis ability over the Her2 / CD3 / 4-1BB-LDN and Her2 / CD3 / 4-1BB-HDN structures. Her2 / CD3 / 4-1BB-LDC can be used as a further optimized structure.

[0191] Example 54-1BB Trivalent Her2 / CD3 / 4-1BB Trispecific Antibody Biological Function Evaluation

[0192] 5.1 Antigen binding validation of trivalent Her2 / CD3 / 4-1BB trispecific antibodies

[0193] CD3 ECD-Fc and 4-1BB ECD-Fc antigen proteins were diluted to 10 μg / mL with PBS and coated in 96-well ELISA plates, 100 μL / well, and coated overnight at 4°C. Discard the liquid in the plate, wash the plate twice with PBST, add 200 μL / well of 5% skim milk in PBST solution, block at room temperature for 2 hours, and drain the blocking solution in the well. Dilute Her2 / CD3 / 4-1BB with different structures 10 times with blocking solution in 8 gradients, with the starting antibody concentration of 100 nM, 3 replicates for each concentration gradient, 100 μL / well added to the plate, and incubate at room temperature for 2 hours. Wash the plate 3 times with PBST and drain, dilute the HRP-labeled anti-kappa chain antibody 1:5000 with 5% skim milk in PBST, and add it to the plate, 100 μL / well, and incubate at room temperature for 1 hour. Wash the plate three times with PBST and drain, add 100 μL of TMB colorimetric solution to each well, color develop for 10 min at room temperature in the dark, then add 50 μL of 2M H2SO4 to each well to terminate the color development reaction, and use a microplate reader to detect the absorbance value at OD450nm. Make a four-parameter nonlinear regression graph with the sample concentration value as the horizontal axis and the absorbance value as the vertical axis and calculate the EC50 value.

[0194] The results of the comparison of binding ability with CD3 antigen are as follows Fig.12 As shown in Table 10, the trivalent Her2 / CD3 / 4-1BB can effectively bind to the CD3 ECD-Fc antigen protein; further analysis of the EC50 values ​​revealed that Her2 / CD3 / 4-1BB-HLDN has the strongest binding ability; the other three structural antibodies have similar binding abilities, and in comparison, the Her2 / CD3 / 4-1BB-LTC trispecific antibody has a medium affinity for binding to the CD3 antigen. The EC50 values ​​for CD3 antigen binding are shown in Table 10:

[0195] Table 10

[0196]

[0197]

[0198] The results of the comparison of binding ability with 4-1BB antigen are as follows Fig.13 As shown in Table 11, the trispecific antibodies with different structures can effectively bind to the 4-1BB ECD-Fc antigen protein; further analysis of the EC50 values ​​revealed that Her2 / CD3 / 4-1BB-HLDN had the strongest binding ability; the other three structural antibodies had similar binding abilities, and in comparison, the trispecific antibody of Her2 / CD3 / 4-1BB LTC had a medium affinity for binding to the CD3 antigen. The EC50 values ​​for 4-1BB antigen binding are shown in Table 11:

[0199] Table 11

[0200] Antibody Name EC50(nM) Her2 / CD3 / 4-1BB-HDLN 0.2260 Her2 / CD3 / 4-1BB-HLDN 0.0761 Her2 / CD3 / 4-1BB-HLDC 0.1628 Her2 / CD3 / 4-1BB-LTC 0.1467

[0201] 5.2 Comparison of the ability of trivalent Her2 / CD3 / 4-1BB trispecific antibodies to mediate PBMC activation.

[0202] The PBMC cells were taken out of liquid nitrogen and quickly thawed in a 37°C water bath. The thawed cells were diluted with RPMI 1640 medium, centrifuged at 1000rpm for 5min, the supernatant was removed, the PBMC cells were resuspended in the medium, and the above centrifugation was repeated once. Cell count, PBMC cell density was adjusted to 1 million / mL; 100μL / well; tumor cells (SKBR3) were digested, and the cell density was adjusted to 0.5 million / mL using RPMI 1640, 100μL / well when adding samples. Tumor cell co-incubation group configuration: PBMC and SKBR3 were mixed in equal volumes (E:T=2:1); tumor cell-free group configuration: PBMC were mixed with an equal volume of culture medium, (+100μL culture medium). Drug addition: Use the same concentration (1nM final concentration) of antibodies, 20μL / well. After culturing at 37°C for 24h, the expression of activation signals (CD25) was detected. The results are as follows Fig.14 ( Fig.14 A. Fig.14 As shown in B), among the trispecific antibodies, Her2 / CD3 / 4-1BB-LTC antibody has stronger activation ability than Her2 / CD3 / 4-1BB-HLDN, ​​Her2 / CD3 / 4-1BB-HDLN and Her2 / CD3 / 4-1BB-HLDC.

[0203] 5.3 Comparison of cytokine release by trivalent Her2 / CD3 / 4-1BB trispecific antibodies

[0204] PBMCs were thawed from liquid nitrogen and the cell density was adjusted to (1 million / mL). HCC1954 tumor cells were digested and the density was adjusted to (0.5 million / mL). After equal volume mixing, 1 mL / well was plated back into a 24-well plate. Her2 / CD3 / 4-1BB trispecific antibodies of different structures were added at a concentration of 1 nM. The supernatant was collected after 48 hours. The cytokine release was compared using the Invitrogen human IL-2 (TNF-α, IFN-γ) uncoated ELISA Kit. The results are shown in Fig.15 ( Fig.15 A. Fig.15 B. Fig.15 As shown in C), the Her2 / CD3 / 4-1BB-LTC trispecific antibody has significant advantages over other structures in the release of IL-2, TNF-α, and IFN-γ.

[0205] 5.4 Comparison of the anti-apoptotic ability of the trivalent Her2 / CD3 / 4-1BB trispecific antibodies

[0206] The specific implementation steps were the same as those in 5.3. PBMCs were thawed from liquid nitrogen and the cell density was adjusted to (1 million / mL). HCC1954 tumor cells were digested and the density was adjusted to (0.5 million / mL). After equal volumes were mixed, 1 mL / well was plated back into a 24-well plate. Her2 / CD3 / 4-1BB trispecific antibodies of different structures were added at a concentration of 1 nM. After culturing at 37°C for 48 hours, the cells were collected by centrifugation and stained with flow cytometry antibodies PerCP cy5.5-anti human CD4; Pacific blue anti-human CD8; FITC anti-human BCL-xL. BCL-xL was analyzed by flow cytometry. + The results were compared with the percentage of anti-apoptosis effect. Fig.16 ( Fig.16 A. Fig.16 As shown in B), compared with different trispecific antibodies, the trivalent Her2 / CD3 / 4-1BB-LTC has a higher expression level of the anti-apoptotic molecule BCL-XL on CD4+ T cells, which has a significant advantage over other structures.

[0207] Example 6 Comparison of the proliferation-promoting abilities of Her2 / CD3 / 4-1BB trispecific antibodies of different titers

[0208] Use (Ca 2+ Mg 2+) PBS was used to dilute Her2 / CD3, Her2 / CD3 / 4-1BB-LC, Her2 / CD3 / 4-1BB-LDC and Her2 / CD3 / 4-1BB-LTC to a concentration of 100 nM, and the antibodies were coated onto the culture plate at 4°C overnight. The next day, the plates were rinsed three times with DPBS, and the PBMC density was adjusted with RPMI 1640 medium, 1 million PBMC / well was added, and the plates were cultured in a 37°C cell culture incubator for 4 days. After 4 days, the medium was changed and the cell number was detected by flow cytometry. The results are shown in Fig.17 As shown, compared with the Her2 / CD3 bispecific antibody, the proliferation-promoting ability of the trispecific antibody was significantly improved; among the trispecific antibodies, the trivalent Her2 / CD3 / 4-1BB-LTC trispecific antibody had a stronger proliferation-promoting ability than the monovalent and bivalent trispecific antibodies.

[0209] Example 7 Comparison of activation abilities of Her2 / CD3 / 4-1BB trispecific antibodies of different titers

[0210] The PBMC cells were taken out of liquid nitrogen and quickly thawed in a 37°C water bath. The thawed cells were diluted with RPMI 1640 medium, centrifuged at 1000rpm for 5min, the supernatant was removed, the PBMC cells were resuspended in the medium, and the above centrifugation was repeated once. Cell count, PBMC cell density was adjusted to 1 million / mL; 100μL / well; tumor cells (HCC1954) were digested, and the cell density was adjusted to 0.5 million / mL with RPMI 1640, and 100μL / well was used for sample addition. Tumor cell co-incubation group configuration: PBMC and HCC1954 were mixed in equal volumes (E:T=2:1); tumor cell-free group configuration: PBMC were mixed with an equal volume of culture medium, (+100μL culture medium). Drug addition: Use antibodies of the same concentration (100pM final concentration). After culturing at 37°C for 24h, the expression of activation signals (CD25) was detected. The results are as follows. Fig.18 ( Fig.18 A. Fig.18 As shown in Figure 2B), compared with the Her2 / CD3 bispecific antibody, the activation ability of the trispecific antibody was significantly improved; among the trispecific antibodies, the trivalent Her2 / CD3 / 4-1BB-LTC trispecific antibody had a stronger activation-promoting ability compared with the monovalent and bivalent trispecific antibodies.

[0211] Example 8 Comparison of anti-apoptotic ability of Her2 / CD3 / 4-1BB trispecific antibodies with different titers

[0212] The specific implementation steps are the same as those in Example 7. PBMCs were thawed from liquid nitrogen and the cell density was adjusted to (1 million / mL). HCC1954 tumor cells were digested and the density was adjusted to (0.5 million / mL). After equal volumes were mixed, 1 mL / well was plated back into a 24-well plate. Her2 / CD3 / 4-1BB trispecific antibodies of different structures were added at a concentration of 1 nM. After culturing at 37°C for 48 hours, the cells were collected by centrifugation and stained with flow cytometry antibodies PerCP cy5.5-anti human CD4; Pacific blue anti-human CD8; FITC anti-human BCL-xL. BCL-xL was analyzed by flow cytometry. + The results were compared with the percentage of anti-apoptosis effect. Fig.19 ( Fig.19 A. Fig.19 As shown in Figure 2B), compared with the Her2 / CD3 bispecific antibody, the anti-apoptosis ability of the trispecific antibody was significantly improved; among the trispecific antibodies, the trivalent Her2 / CD3 / 4-1BB-LTC trispecific antibody had a stronger anti-apoptosis ability than the monovalent and bivalent trispecific antibodies.

[0213] Example 9 Comparison of in vitro killing activity of Her2 / CD3 / 4-1BB trispecific antibodies with different titers.

[0214] PBMC cells were taken out of liquid nitrogen and thawed quickly in a 37°C water bath. Thawed cells were diluted with RPMI 1640 medium (medium: cells>10:1), centrifuged at 1000rpm for 5min, supernatant was removed, PBMC cells were resuspended with medium, centrifuged once, cells were counted, and PBMC cell density was adjusted; tumor cells HCC1954 (Her2 high expression) and MDA MB 231 (Her2 low expression) were digested, stained with CFSE dye for 15min, washed three times with medium and density was adjusted to make the PBMC to tumor cell effector target ratio 2:1, and tumor cells and equal volume of PBMC were mixed as negative control groups. 200μL 1nM antibody was prepared in a U-shaped plate, 10-fold dilution of 6 concentration gradients, 10μL / well was transferred to a 96-well plate, and PBMC with adjusted density and tumor cells were added in a total of 100μL / well, and tumor cells were mixed with equal volume of PBMC as negative control group and incubated in a total of 100μL / well. After incubation in a 37°C incubator for 24 h, the tumor cells were digested and the number of CFSE+ tumor cells was detected by flow cytometry.

[0215] The results are as follows Fig. 20 and Fig.21 As shown, Fig. 20The medium (HCC1954 high Her2 expression level) trivalent Her2 / CD3 / 4-1BB-LTC structure showed a stronger tumor inhibitory effect compared with the monovalent Her2 / CD3 / 4-1BB-LC and bivalent Her2 / CD3 / 4-1BB-LDC. The results are shown in Table 12.

[0216] Table 12

[0217] Antibody Name IC50(nM) Her2 / CD3 0.02121 Her2 / CD3 / 4-1BB-LC 0.02282 Her2 / CD3 / 4-1BB-LDC 0.03410 Her2 / CD3 / 4-1BB-LTC 0.01843

[0218] Fig.21 The medium (MDA MB 231 low Her2 expression) trivalent Her2 / CD3 / 4-1BB-LTC structure further widened the gap with other structures, and its IC50 reached 0.06033nM, which was lower than Her / CD3 and Her2 / CD3 / 4-1BB-LDC, with significant difference. The results are shown in Table 13.

[0219] Table 13

[0220]

[0221]

[0222] Example 10 Comparison of in vitro antitumor activity of Her2 / CD3 / 4-1BB-LTC trispecific antibody and CAR-T-Her2

[0223] 10.1 Comparison of cytokine release ability between Her2 / CD3 / 4-1BB-LTC trispecific antibody and CAR-T-Her2

[0224] The CAR-T-Her2 cells were removed from the culture dish, the cells were counted, and the PBMC cell density was adjusted to 0.4 million / mL. The PBMC cells were removed from the liquid nitrogen and quickly thawed in a 37°C water bath. The thawed cells were diluted with RPMI 1640 medium (medium: cells>10:1), centrifuged at 1000rpm for 5min, the supernatant was removed, the PBMC cells were resuspended with the medium, the above centrifugation was repeated once, the cells were counted, and the PBMC cell density was adjusted to 0.4 million / mL; the tumor cells HCC1954 (Her2 high expression) were digested and the tumor cell density was adjusted to 0.2 million / mL. Equal volumes of PBMC and CAR-T-Her2 cells were mixed with tumor cells respectively, so that the effector-target ratio of effector cells to tumor cells was 2:1. The preferred Her2 / CD3 / 4-1BB-LTC trispecific antibody was added to the antibody group at a concentration of 1nM, and an equal volume of PBS was added to the CAR-T group, and the supernatant was collected after 48 hours. The cytokine release was compared using the Invitrogen human IL-2 (TNF-α, IFN-γ) uncoated ELISA Kit. Fig. 22 ( Fig. 22 A. Fig. 22 B. Fig. 22 As shown in C), the Her2 / CD3 / 4-1BB-LTC trispecific antibody has a significant advantage over CAR-T-Her2 in TNF-α release.

[0225] 10.2 Comparison of anti-apoptosis ability between Her2 / CD3 / 4-1BB-LTC trispecific antibody and CAR-T-Her2

[0226] Remove the CAR-T-Her2 cells from the culture dish, count the cells, and adjust the PBMC cell density to 0.4 million / mL. Remove the PBMC cells from liquid nitrogen and thaw quickly in a 37°C water bath. Use RPMI 1640 medium to dilute the thawed cells (medium: cells>10:1), centrifuge at 1000rpm for 5min, remove the supernatant, resuspend the PBMC cells in the medium, repeat the centrifugation once, count the cells, and adjust the PBMC cell density to 0.4 million / mL; digest the tumor cells HCC1954 (Her2 high expression), and adjust the tumor cell density to 0.2 million / mL. Mix equal volumes of PBMC and CAR-T-Her2 cells with tumor cells, so that the effector-target ratio of effector cells to tumor cells is 2:1. The antibody group was added with the preferred Her2 / CD3 / 4-1BB-LTC trispecific antibody and Her2 / CD3 bispecific antibody at a concentration of 1 nM, and the CAR-T group was added with an equal volume of PBS. After 48 hours, the cells were collected and stained with flow cytometry antibodies PerCP cy5.5-anti human CD4; Pacific blue anti-humanCD8; FITC anti-human BCL-xL, and BCL-xL was analyzed by flow cytometry. + The results were compared with the percentage of anti-apoptosis effect. Fig.23 ( Fig.23 A. Fig.23 As shown in B), the trivalent Her2 / CD3 / 4-1BB-LTC trispecific antibody improves the anti-apoptosis ability of T cells and has significant advantages over CAR-T-Her2.

[0227] 10.3 Comparison of phenotypic changes between Her2 / CD3 / 4-1BB-LTC trispecific antibody and CAR-T-Her2

[0228] Remove the CAR-T-Her2 cells from the culture dish, count the cells, and adjust the PBMC cell density to 0.4 million / mL. Remove the PBMC cells from liquid nitrogen and thaw quickly in a 37°C water bath. Use RPMI 1640 medium to dilute the thawed cells (medium: cells>10:1), centrifuge at 1000rpm for 5min, remove the supernatant, resuspend the PBMC cells in the medium, repeat the centrifugation once, count the cells, and adjust the PBMC cell density to 0.4 million / mL; digest the tumor cells HCC1954 (Her2 high expression), and adjust the tumor cell density to 0.2 million / mL. Mix equal volumes of PBMC and CAR-T-Her2 cells with tumor cells, so that the effector-target ratio of effector cells to tumor cells is 2:1. The antibody group was added with the preferred Her2 / CD3 / 4-1BB-LTC trispecific antibody and Her2 / CD3 bispecific antibody at a concentration of 1 nM, and the CAR-T group was added with an equal volume of PBS. After 72 hours, the cells were collected and stained with flow cytometry antibodies PerCP cy5.5-anti human CD4; Pacific blue anti-humanCD8; FITC-anti-human CD45RO; APC anti-human CCR7. Analysis by flow cytometry The percentages of naive T cells (CCR7+; CD45RO-), TCM or central memory T cells (CCR7+; CD45RO+), TEM or effector memory T cells (CCR7-; CD45RO+), and TE or effector T cells (CCR7-; CD45RO-) were compared to compare the phenotypic change trends. Fig.24 ( Fig.24 A. Fig.24 B) shows that in CD8+T cells and CD4+T cells, the trispecific antibody Her2 / CD3 / 4-1BB-LTC has significant differences in the phenotype of central memory T cells compared with CAR-T-Her2. This difference means that Her2 / CD3 / 4-1BB-LTC makes T cells more viable. CAR-T-Her2 is widely distributed in the TE phenotype, which causes T cells to gradually lose lymph node homing receptors, proliferation ability, IL-2 production, self-renewal and survival ability. Therefore, compared with CAR-T-Her2, T cells under the action of Her2 / CD3 / 4-1BB-LTC have better survival ability and anti-tumor function.

[0229] In summary, we obtained the preferred Her2 / CD3 / 4-1BB-LC structure by examining the biological activity of different positions in the 4-1BB trispecific antibody; obtained the preferred Her2 / CD3 / 4-1BB-LDC structure by examining the relationship between the position and valence of the bivalent; finally, we examined the possible structural design of the trivalent and further optimized the design of the trivalent 4-1BB trispecific antibody. We compared the cytokine release, activation and anti-apoptosis ability, and found that the trivalent Her2 / CD3 / 4-1BB-LTC structure has significant advantages over other trivalent designs, indicating that the valence and structure of 4-1BB jointly affect the biological function of 4-1BB. In terms of in vitro killing ability, the Her2 / CD3 / 4-1BB-LTC structure showed significant differences between the monovalent optimal structure Her2 / CD3 / 4-1BB-LC and the bivalent optimal structure Her2 / CD3 / 4-1BB-LDC, indicating that Her2 / CD3 / 4-1BB-LTC is the preferred optimal 4-1BB trispecific antibody structure.

Claims

1. A Her2 / CD3 / 4-1BB trispecific antibody, characterized in that: The Her2 / CD3 / 4-1BB trispecific antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 10-12.

2. A Her2 / CD3 / 4-1BB trispecific antibody, characterized in that: The Her2 / CD3 / 4-1BB trispecific antibody has a light chain amino acid sequence as shown in SEQ ID NOs: 2, 13-18.

3. A Her2 / CD3 / 4-1BB trispecific antibody, characterized in that: The Her2 / CD3 / 4-1BB trispecific antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 13; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 10, and the light chain amino acid sequence shown in SEQ ID NO: 14; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 10, and the light chain amino acid sequence as shown in SEQ ID NO: 15; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 10, and the light chain amino acid sequence shown in SEQ ID NO: 16; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 10, and the light chain amino acid sequence shown in SEQ ID NO: 17; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 10, and the light chain amino acid sequence as shown in SEQ ID NO: 18; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 11, and the light chain amino acid sequence as shown in SEQ ID NO: 13; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 11, and the light chain amino acid sequence as shown in SEQ ID NO: 16; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 11, and the light chain amino acid sequence shown in SEQ ID NO: 17; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 12, and the light chain amino acid sequence as shown in SEQ ID NO: 2; and / or The heavy chain amino acid sequence is shown as SEQ ID NO:12, and the light chain amino acid sequence is shown as SEQ ID NO:

13.

4. A Her2 / CD3 / 4-1BB trispecific antibody, characterized in that: The Her2 / CD3 / 4-1BB trispecific antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 16; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 10, and the light chain amino acid sequence shown in SEQ ID NO: 17; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 10, and the light chain amino acid sequence as shown in SEQ ID NO: 18; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 11, and the light chain amino acid sequence as shown in SEQ ID NO: 13; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 11, and the light chain amino acid sequence as shown in SEQ ID NO: 16; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 11, and the light chain amino acid sequence shown in SEQ ID NO: 17; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 12, and the light chain amino acid sequence as shown in SEQ ID NO: 2; and / or The heavy chain amino acid sequence is shown as SEQ ID NO:12, and the light chain amino acid sequence is shown as SEQ ID NO:

13.

5. A Her2 / CD3 / 4-1BB trispecific antibody, characterized in that: The Her2 / CD3 / 4-1BB trispecific antibody has a heavy chain amino acid sequence as shown in SEQ ID NO: 10, and a light chain amino acid sequence as shown in SEQ ID NO: 18; and / or The heavy chain amino acid sequence as shown in SEQ ID NO: 11, and the light chain amino acid sequence as shown in SEQ ID NO: 16; and / or The heavy chain amino acid sequence shown in SEQ ID NO: 11, and the light chain amino acid sequence shown in SEQ ID NO: 17; and / or The heavy chain amino acid sequence is shown as SEQ ID NO:12, and the light chain amino acid sequence is shown as SEQ ID NO:

13.

6. Use of the antibody according to any one of claims 1 to 5 for treating Her2 / 4-1BB single-positive or double-positive tumors.

7. Use of the antibody according to any one of claims 1 to 5 for preparing a drug for treating Her2 / 4-1BB single-positive or double-positive tumors.

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