Targeting Her2 / CD3 / CD28 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 CD28, and the Her2 / CD3 / CD28 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 CD28. 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, tumor immunotherapy has gradually achieved great results in the treatment of various cancers, such as immune checkpoint blockade and engineering T cells to enhance T cell activation and killing ability. The number of approved immunotherapy drugs continues to increase, and a variety of treatment methods have been developed clinically and preclinically. However, in tumor immunotherapy, most malignant tumors still do not respond to immunotherapy, and 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, while Her2 gene amplification and protein overexpression exist in many tumors (including breast cancer, lung cancer, prostate cancer, head and neck squamous cell carcinoma, and gynecological tumors), so it is a potential target for immunotherapy. Her2 promotes tumor cell proliferation by inhibiting apoptosis; increases tumor cell invasiveness; promotes tumor angiogenesis and lymphangiogenesis, and can also inhibit anti-tumor immune response (Marchiò C, Annaratone L, Marques A, Casorzo L, Berrino E, Sapino A. Evolving concepts in Her2 evaluation in breast cancer: Heterogeneity, Her2-low carcinomas and beyond. Semin Cancer Biol. 2021 Jul; 72: 123-135. doi: 10.1016 / j.semcancer.2020.02.016. Epub 2020 Feb 26. PMID: 32112814.). Trastuzumab is the first humanized monoclonal antibody targeting Her2, breaking the treatment bottleneck of chemotherapy alone for Her2-positive patients, establishing trastuzumab as the first-line standard treatment for such patients, and opening the door to precise targeted treatment of Her2-positive cancers. Compared with chemotherapy alone, trastuzumab combined with chemotherapy can significantly prolong the patient's overall survival [OS, 13.8 months (95% CI 12-16) vs. 11.1 months (95% CI 10-13), HR 0.74, 95% CI 0.60-0.91, p=0.0046], and the objective response rate (ORR) also increased from 34.5% to 47.3% (Keam SJ. Trastuzumab Deruxtecan: First Approval. Drugs. 2020Apr; 80(5): 501-508. doi: 10.1007 / s40265-020-01281-4. PMID: 32144719.). In December 2018, the State Food and Drug Administration approved the marketing of Pertuzumab, 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 Her2, EGFR, and other receptors have achieved good results in clinical applications. However, for patients with low Her2 expression or antigen heterogeneity, as well as Her2 antigen escape caused by targeted therapy, the prognosis is poor. Whether it is pertuzumab combined with trastuzumab or lapatinib combined with capecitabine and oxaliplatin, the OS of patients has not been further prolonged.

[0003] Bispecific antibodies (BsAbs) are the representative immunotherapy that can break the deadlock of monoclonal antibody therapy. Compared with traditional monoclonal antibodies, bispecific antibodies have the advantages of more precise targeting and stronger therapeutic effects, bringing new opportunities for tumor immunotherapy. Since Brennan et al. constructed the first bispecific antibody by chemically coupling two monoclonal antibodies in 1985 (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science. 1985 Jul 5; 229 (4708): 81-3.), bispecific antibodies with different targets and different forms have been widely studied. With the rapid development of genetic engineering technology, people can control the size, affinity, half-life, stability, solubility and other characteristics of BsAbs through recombinant DNA and other means, and can construct chimeric or humanized Abs to meet different needs (Du J, et al. Engineering Bifunctional Antibodies with Constant Region Fusion Architectures. J Am Chem Soc. 2017 Dec 27; 139(51): 18607-18615.; Hollander N. Bispecific antibodies for cancer therapy. Immunotherapy. 2009 Mar; 1(2): 211-22.). A novel asymmetrical anti-Her2 / CD3 BsAbs exhibits potent cytotoxicity for Her2-positive tumor cells (Yu S, Zhang J, Yan Y, Yao X, Fang L, Xiong H, Liu Y, Chu Q, Zhou P, Wu KA novel asymmetrical anti-Her2 / CD3 bispecific antibody exhibits potent cytotoxicity for Her2-positive tumor cells. J Exp Clin Cancer Res. 2019 Aug 14; 38(1): 355. doi: 10.1186 / s13046-019-1354-1. PMID: 31412896; PMCID: PMC6694677.).T-DM1 (Ado-trastuzumabemtansine), an ADC drug approved for the treatment of Her2-positive breast cancer, has dual anti-tumor effects of targeting and cytotoxic killing. It is a representative of the second-generation ADC drugs and the first ADC drug used for solid tumors. It can be used as a standard treatment option for patients with Her2-positive advanced breast cancer who have failed trastuzumab and lapatinib treatment. And Zanidatamab BsAbs has achieved good clinical therapeutic effects in the treatment of locally advanced or metastatic cancers expressing Her2 or Her2 amplification (Meric-Bernstam F, Beeram M, Hamilton E, Oh DY, Hanna DL, Kang YK, Elimova E, Chaves J, Goodwin R, Lee J, Nabell L, Rha SY, Mayordomo J, El-Khoueiry A, Pant S, Raghav K, Kim JW, Patnaik A, Gray T, Davies R, Ozog MA, Woolery J, Lee KW. Zanidatamab, a novel bispecific antibody, for the treatment of locally advanced or metastatic Her2-expressing or Her2-amplified cancers: a phase 1, dose-escalation and expansion study. Lancet Oncol. 2022 Dec; 23(12): 1558-1570. doi: 10.1016 / S1470-2045(22)00621-0. Epub 2022 Nov 16. PMID: 36400106.). However, problems such as heavy chain mismatch, antibody inactivation, insufficient T cell activation, and non-human related immunogenicity limit the wider clinical application of BsAbs (Thakur A, et al. Bispecific antibody based therapeutics: Strengths and challenges. Blood Rev. 2018 Jul; 32(4): 339-347.).BsAbs can reduce drug resistance and serious adverse reactions by directly targeting immune cells to tumors. However, compared with liquid tumors, BsAbs are still difficult to treat solid tumors. Liquid tumors are usually more dispersed and therefore more accessible to circulating immune cells. However, it is difficult to break through the tumor microenvironment barrier and immune escape caused by T cell exhaustion in the treatment of solid tumors (Staerz UD, et al. Hybrid antibodies can target sites for attack by T cells. Nature. 1985 Apr 18-24; 314(6012): 628-31. doi: 10.1038 / 314628a0. PMID: 2859527.; Thakur A, et al. Bispecific antibody-based therapeutics: Strengths and challenges. Blood Rev. 2018 Jul; 32(4): 339-347. doi: 10.1016 / j.blre.2018.02.004. Epub 2018 2018Feb 20.PMID:29482895.). The problems of T cell proliferation exhaustion and inertia require the introduction of co-stimulatory domains to maintain the tumor-killing ability and functional persistence of antigen-specific T cells.

[0004] In recent years, great progress has been made in the study of co-stimulatory signals, mainly CD28, CD2, CD5, CD30, OX40, inducible costimulator (ICOS), LFA-1 and 4-1BB. As the second co-stimulatory signal, the most important is the binding of CD28 from the surface of T cells with the B7 family molecule CD80 / CD86 on the surface of APC. CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes. In the human immune system, it is expressed on about 80% of CD4 + T cells, 50% CD8 +In T cells, it is also expressed in neutrophils and eosinophils. The complete activation of initial T cells usually requires three signals. The first signal is the binding of antigen and antigen receptor, the second signal is the co-stimulatory signal, and the third signal is the cytokine signal. CD28, as a co-stimulatory molecule, participates in the reception and transmission of the second signal. In the process of T cell immune response and recognition effect, the first signal mediated by TCR and pMHC-antigen peptide complex is not enough to fully activate the initial Th cells or Tc cells. The membrane protein molecules on the surface of T cells and their ligands mediated co-stimulatory signals CD28-CD80 / CD86 must be activated. CD28 binds to its ligand B7, phosphorylates downstream PI3K and other molecules through the MYPPPY motif, and then activates the T cell activation signal pathway. CD28 signal plays a key role in many T cell activation and function processes, including cytoskeleton remodeling, cytokine production, survival and differentiation (Esensten JH, Helou YA, Chopra G, Weiss A, Bluestone JA. CD28 Costimulation: From Mechanism to Therapy. Immunity. 2016 May 17; 44 (5): 973-88. doi: 10.1016 / j.immuni.2016.04.020. PMID: 27192564; PMCID: PMC4932896.). For Th cells, receiving the first signal leads to upregulation of the expression of the important co-stimulatory molecule CD28 on the surface of Th cells. The second signal of T cell activation, such as nuclear transmission, depends on the binding of CD28 to the B7 molecule on the surface of APC cells. Co-stimulatory signaling lowers the threshold of T cell activation, thereby further enhancing T cell proliferation, differentiation, and tumor killing ability (Bashour KT, Gondarenko A, Chen H, Shen K, Liu X, Huse M, Hone JC, Kam LC. CD28 and CD3 have complementary roles in T-cell traction forces. Proc Natl Acad Sci US A. 2014 Feb 11; 111(6): 2241-6. doi: 10.1073 / pnas.1315606111. Epub 2014 Jan 27. PMID: 24469820; PMCID: PMC3926067.).

[0005] The lack of T cell costimulatory signals will lead to a decrease in T cell activation ability and the occurrence of tumor cell immune escape. Therefore, CD28 antibodies have gradually been widely used and have become targeted therapies for malignant tumors, transplant rejection, autoimmunity and infectious diseases, as well as a series of new indications. However, in 2010, Hansel et al. found that CD28 monoclonal antibody TGN1412 had a life-threatening cytokine release syndrome during the first human study, so it is necessary to further study CD28 antibodies in terms of safety, specificity, and inhibition of immune escape (Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJ. The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov. 2010 Apr; 9 (4): 325-38. doi: 10.1038 / nrd3003. Epub 2010 Mar 22. PMID: 20305665.). In order to solve the above problems, CD28 bispecific antibodies and trispecific antibodies were further developed. T cell-engaging trispecific antibodies (TsAbs) represent a therapeutic approach that specifically targets tumors to activate cytotoxic T cells (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 taken to the clinic. Nat Rev Drug Discov. 2020 Oct; 19(10): 657-658.). A trispecific antibody SAR441236 reported in 2017 can independently bind to three different epitopes of the HIV envelope and has been shown to have a certain antiviral ability in animal models (Mullard A. Trispecific antibodies taken to the clinic. Nat Rev Drug Discov. 2020 Oct; 19(10): 657-658.). TsAbs are engineered to have three specific binding sites, which can not only activate T cells but also target multiple tumor surface antigens to achieve specific killing of tumor cells.

[0006] At present, the development and application of CD28 bispecific antibodies and trispecific antibodies have received widespread attention. CD28 bispecific antibodies (TSA×CD28) designed with specific targets of CD28 and tumor-specific antigen (TSA) can enhance the anti-tumor activity of CD3 bispecific antibodies, and have low activity and no toxic side effects when used alone in humanized immunocompetent mouse models or primates (Skokos D, et al. A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies. Sci Transl Med. 2020 Jan 8; 12(525):eaaw7888.doi:10.1126 / scitranslmed.aaw7888.PMID:31915305.). CD28 antibodies can also enhance the inhibitory effect of immune checkpoints. The new CD28 co-stimulatory bispecific antibody combined with the anti-PD-1 therapy Cemiplimab can significantly enhance anti-tumor activity in multiple cancer models, produce long-term T cell anti-tumor memory, and is not related to systemic cytokine release syndrome (Waite JC, et al. Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1immunotherapy. Sci Transl Med. 2020Jun 24; 12(549): eaba2325. doi: 10.1126 / scitranslmed.aba2325. PMID: 32581132.). In 2022, Nature published a TsAbs targeting Her2, CD3 and CD28, which stimulated the regression of breast cancer in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumor cell cycle progression.(Seung E, Xing Z, Wu L, Rao E, Cortez-Retamozo V, Ospina B, Chen L, Beil C, Song Z, Zhang B, Levit M, Deng G, Hebert A, Kirby P, Li A, Poulton EJ, Vicente R, Garrigou A, Piepenhagen P, Ulinski G, Sanicola-Nadel M, Bangari DS, Qiu H, Pao L, Wiederschain D, Wei R, Yang ZY, Nabel GJ. A trispecific antibody targeting Her2 and T cells inhibits breast cancer growth via CD4 cells. Nature. 2022 Mar; 603(7900): 328-334.). However, existing CD28 TsAbs still have problems such as structural rigidity, toxicity mediated by the whole antibody Fc fragment, and single target recognition region potency.

[0007] The development of TsAbs can break through the limitations of BsAbs and have more prominent advantages in immunotherapy. It can be seen from the tumor immune escape model of BsAbs that the introduction of CD3, the first signal of T cell activation, can achieve basic activation of T cells and reduce the incapacity and exhaustion of T cells. Since TsAbs involve different targets and the locations of their binding antigen epitopes on the cell surface are different, it means that their structural design needs to be adjusted according to the interaction between the antibody and the target cell. 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 microclusters, and T cell activation. Curr Top Microbiol Immunol. 2010; 340: 81-107.). 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 immune synapses, thereby effectively activating quiescent T cells to kill tumor cells and achieve the effect of treating tumors. During the formation of immune synapse, the distance between T cells and target cells is about 15nm due to the structure of TCR and antigen complex. Phosphorylases CD45 and CD148 are excluded from the synapse due to their external functional domains much longer than 15nm, thus allowing the TCR-induced tyrosine phosphorylation process to proceed smoothly in the absence of negative regulatory factors.Studies have shown that mutant CD45 proteins with shorter external functional regions weaken T cell-related signals, which also verifies the importance of the above-mentioned molecular exclusion in T cell signaling (Basu R, et al. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. Cell. 2016 Mar 24; 165 (1): 100-110.; Basu R, et al. Mechanical Communication at the Immunological Synapse. Trends Cell Biol. 2017 Apr; 27 (4): 241-254.). The spatial distance between TsAbs and different antigens may also be important for the corresponding T cell immune synapse formation and T cell signaling activation, but this spatial distance depends on completely different structural factors, including the antigen epitope, the interaction region between the antibody and the T cell.

[0008] In summary, in order to enhance the T cell activation ability of TsAbs and mediate the killing effect of T cells on antigen-expressing tumor cells, it is necessary to combine anti-CD28 with anti-Her2 recognition fragments, and use anti-CD3 to provide the first signal of T cell activation. Through the optimization and screening of structural design, optimized TsAbs based on enhancing T cell activation ability and mediating T cell targeted tumor killing are established to achieve antibody structure optimization, reduce drug metabolism time and Fc-mediated toxicity, and multi-valent design of target recognition region, so as to establish a method of maintaining T cell activity and persistence through CD3 and CD28, mediating T cell targeting Her2 + 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 / CD28 trispecific antibody based on structural optimization design, wherein the Her2 / CD3 / CD28 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 recognizes CD28.

[0011] The Her2 / CD3 / CD28 trispecific antibody connects CD3-HC, CD3-LC, CD28-VHH and / or Her2-VHH via a connecting peptide.

[0012] 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 CD28-VHH sequence is shown as SEQ ID NO: 4.

[0013] In some preferred embodiments, the connecting peptide is selected from one or more of a flexible connecting peptide, a rigid connecting peptide or a helical coiled connecting peptide; the flexible connecting peptide is selected from one or more of (G4S)3Linker, 218Linker or 212Linker; the rigid connecting peptide is selected from PD Linker; the helical coiled connecting peptide is selected from Coiled Coil Linker.

[0014] In some embodiments, the amino acid sequence of the connecting peptide is as shown in Table 1:

[0015] Table 1

[0016]

[0017] Preferably, the Her2 / CD3 / CD28 trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NOs: 10-13, and a light chain amino acid sequence as shown in SEQ ID NOs: 2, 14-18.

[0018] More preferably, the Her2 / CD3 / CD28 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

[0019] The Her2 / CD3 / CD28 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

[0020] The Her2 / CD3 / CD28 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

[0021] The Her2 / CD3 / CD28 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

[0022] The Her2 / CD3 / CD28 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

[0023] The Her2 / CD3 / CD28 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: 14; and / or

[0024] The Her2 / CD3 / CD28 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

[0025] The Her2 / CD3 / CD28 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

[0026] The Her2 / CD3 / CD28 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: 14; and / or

[0027] The Her2 / CD3 / CD28 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: 17; and / or

[0028] The Her2 / CD3 / CD28 trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO:13, and a light chain amino acid sequence as shown in SEQ ID NO:2.

[0029] Further preferably, the Her2 / CD3 / CD28 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

[0030] The Her2 / CD3 / CD28 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

[0031] The Her2 / CD3 / CD28 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: 14; and / or

[0032] The Her2 / CD3 / CD28 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

[0033] The Her2 / CD3 / CD28 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

[0034] The Her2 / CD3 / CD28 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: 14; and / or

[0035] The Her2 / CD3 / CD28 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: 17; and / or

[0036] The Her2 / CD3 / CD28 trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO:13, and a light chain amino acid sequence as shown in SEQ ID NO:2.

[0037] More preferably, the Her2 / CD3 / CD28 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

[0038] The Her2 / CD3 / CD28 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

[0039] The Her2 / CD3 / CD28 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: 14; and / or

[0040] The Her2 / CD3 / CD28 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: 17; and / or

[0041] The Her2 / CD3 / CD28 trispecific antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO:13, and a light chain amino acid sequence as shown in SEQ ID NO:2.

[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 trispecific antibody is constructed using the Fab of the first domain-anti-CD3 monoclonal antibody (clone SP34) as the structural basis, and the second domain and the third domain are nanoantibodies (clone 2Rs15d) targeting Her2 positive targets and nanoantibodies (clone P2-6G) targeting T cell surface co-stimulatory CD28 molecules, respectively, and the two are connected to the CD3 Fab fragment through a linker.

[0043] The construction method includes the following steps:

[0044] (1) Two nanobodies, anti-Her2-VHH or Her2-VHH / CD28-VHH, are simultaneously linked to the heavy chain domain of the CD3 Fab structure.

[0045] (2) Anti-CD28-VHH is linked to the light chain domain of CD3 Fab structure or the light chain domain is not modified.

[0046] (3) In the present invention, the recombinant heavy chain and the recombinant light chain are combined through the disulfide bonds in CH1 and CL in CD3-Fab to form a heterodimer, namely, an anti-Her2 / CD3 / CD28 trispecific antibody.

[0047] The step (1) can be constructed or connected in the following manner:

[0048] (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

[0049] (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-CD28-VHH to the N-terminal 1E of the CD3 Fab heavy chain variable region (VH) domain via a connecting peptide; or

[0050] (c) Anti-Her2-VHH was connected to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain via a connecting peptide, and anti-CD28-VHH was connected to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain via a connecting peptide, and then anti-CD28-VHH was tandemly connected to the anti-CD28-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain via a connecting peptide.

[0051] (d) Anti-Her2-VHH is connected to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a connecting peptide, and anti-CD28-VHH is connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a connecting peptide, and then anti-CD28-VHH is sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a connecting peptide, and then anti-CD28-VHH is sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a connecting peptide.

[0052] The step (2) can be constructed or connected in the following manner:

[0053] (a) connecting anti-CD28-VHH to the N-terminal 1Q of the CD3 Fab light chain variable region (VL) domain via a connecting peptide; or

[0054] (b) anti-CD28-VHH is chimerized between the 171S-173D domains of the CD3 Fab light chain constant region (CL) via a connecting peptide, and 172K is deleted; or

[0055] (c) connecting the anti-CD28-VHH to the C-terminal 217C of the CD3 Fab light chain constant region (CL) domain via a linker peptide; or

[0056] (d) connecting the anti-CD28-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-CD28-VHH to the anti-CD28-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain via a connecting peptide; or

[0057] (e) Anti-CD28-VHH is connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a connecting peptide, and then the anti-CD28-VHH is sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a connecting peptide, and then the anti-CD28-VHH is sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a connecting peptide.

[0058] The connecting peptide is selected from one or more of a flexible connecting peptide, a rigid connecting peptide or a helical connecting peptide;

[0059] The flexible linker peptide is selected from one or more of (G4S)3Linker, 218Linker or 212Linker;

[0060] The rigid connecting peptide is selected from PD Linker;

[0061] The helical coiled linker peptide is selected from Coiled Coil Linker;

[0062] Furthermore, the step (1) can be constructed or connected in the following manner:

[0063] (a) connecting the 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; or

[0064] (b) 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, and connecting anti-CD28-VHH to the N-terminal 1E of the CD3 Fab heavy chain variable region (VH) domain via a rigid linker peptide PD Linker; or

[0065] (c) connecting anti-Her2-VHH to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S)3Linker, and connecting anti-CD28-VHH to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a rigid connecting peptide PD Linker, and then connecting anti-CD28-VHH in series to the anti-CD28-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a flexible connecting peptide 218Linker; or

[0066] (d) Anti-Her2-VHH was connected to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S)3Linker, and anti-CD28-VHH was connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a rigid connecting peptide PD Linker. Anti-CD28-VHH was then sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible connecting peptide 218Linker, and anti-CD28-VHH was again sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible connecting peptide 212Linker.

[0067] Furthermore, the step (2) can be constructed or connected in the following manner:

[0068] (a) connecting anti-CD28-VHH to the N-terminal 1Q of the CD3 Fab light chain variable region (VL) domain via a rigid linker peptide PD Linker; or

[0069] (b) Anti-CD28-VHH is chimeric between the CD3 Fab light chain constant region (CL) domains 171S-173D via a Coiled Coil Linker, and 172K is deleted; or

[0070] (c) connecting anti-CD28-VHH to the C-terminal 217C of the CD3 Fab light chain constant region (CL) domain via a flexible linker peptide (G4S)3Linker; or

[0071] (d) connecting the anti-CD28-VHH to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker, and then connecting the anti-CD28-VHH in series to the anti-CD28-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker; or

[0072] (e) Anti-CD28-VHH was connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain through a rigid connecting peptide PD Linker, and the anti-CD28-VHH was sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible connecting peptide 218Linker, and then anti-CD28-VHH was sequentially connected in series to the N-terminus of the anti-CD28-VHH domain connected to the N-terminus of the CD3 Fab light chain variable region (VL) domain 218Linker through a flexible connecting peptide 212linker.

[0073] In a preferred embodiment, the Her2 / CD3 / CD28 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 Her2 / CD3 / CD28 monovalent 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-CD28-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 / CD28 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-CD28-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 / CD28 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-CD28-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 / CD28 LC;

[0077] In a preferred embodiment, the Her2 / CD3 / CD28 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 Her2 / CD3 / CD28 bivalent 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-CD28-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 / CD28HLN;

[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-CD28-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-CD28-VHH was sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker, and labeled as Her2 / CD3 / CD28 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-CD28-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-CD28-VHH was sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker, marked as Her2 / CD3 / CD28 HDN;

[0081] In a preferred embodiment, the Her2 / CD3 / CD28 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 Her2 / CD3 / CD28 trivalent trispecific antibody, as shown below:

[0082] (7) 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-CD28-VHH was connected to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker; anti-CD28-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-CD28-VHH was sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker, and marked as Her2 / CD3 / CD28 HNLDN;

[0083] (8) 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, anti-CD28-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-CD28-VHH was sequentially connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker; anti-CD28-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 / CD28 HDNLN;

[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 anti-CD28-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 the anti-CD28-VHH was sequentially connected to the anti-CD28-VHH region at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker, and then the anti-CD28-VHH was sequentially connected to the N-terminus of the anti-CD28-VHH domain through a flexible linker peptide 212linker, marked as Her2 / CD3 / CD28 HTN;

[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-CD28-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 the anti-CD28-VHH was serially connected to the anti-CD28-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker, and then the anti-CD28-VHH was serially connected to the N-terminus of the anti-CD28-VHH domain through a flexible linker peptide 212linker, marked as Her2 / CD3 / CD28 LTN;

[0086] In a preferred embodiment, the Her2 / CD3 / CD28 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 Her2 / CD3 / CD28 tetravalent trispecific antibody, as shown below:

[0087] (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; anti-CD28-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 the anti-CD28-VHH was connected in series to the anti-CD28-VHH region at the N-terminus of the CD3 Fab light chain variable region (VL) domain through a flexible linker peptide 218Linker; and anti-CD28-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 the anti-CD28-VHH was connected in series to the N-terminus of the anti-CD28-VHH domain at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker, marked as Her2 / CD3 / CD28 HDNLDN.

[0088] The third aspect of the present invention also provides a method for preparing the trispecific antibody of the present invention. The method comprises:

[0089] (1) obtaining a fusion gene of a trispecific antibody and constructing an expression vector of the trispecific antibody;

[0090] (2) transfecting the above expression vector into host cells by genetic engineering methods;

[0091] (3) culturing the host cell under conditions that allow production of the trispecific antibody;

[0092] (4) Isolating and purifying the produced antibody protein.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] In one embodiment, the invention can be used to treat Her2-positive tumors. The Her2 / CD3 / CD28 trispecific antibody molecule can significantly kill Her2-positive tumor cells in vitro.

[0097] 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.

[0098] The technical solution of the present invention has achieved beneficial technical effects:

[0099] 1. The trispecific antibody drug of the present invention achieves target therapy for both T cells and tumor cells, and maintains the continuous activation of T cells with costimulatory signals. The trispecific antibody based on the Fab and VHH structure is simpler to express and purify than the antibody with Fc region, and has stronger penetration ability in the solid tumor microenvironment. The trispecific antibody based on the Fab and VHH structure has a longer half-life in the body than the ordinary single-chain antibody tandem structure, which is more conducive to reducing the number of administrations and dosages, and effectively reducing side effects such as off-target and cytokine storm.

[0100] 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 comprehensively considered. When adding a domain to the N-terminus of Fab, it is important to pay attention to the targeting of the Fab domain itself. In order to effectively isolate two different domains, the present invention uses a rigid connecting peptide Linker to ensure the targeting of the antibodies at both ends; 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 in 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 adjusting the formation of 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 into CD3-targeting Fab is the C-terminus of the Fab heavy chain, and the best site for introducing CD28-targeting antibodies is the N-terminus of the Fab light chain.

[0101] 3. We successfully selected antibody clones targeting Her2, CD28 and CD3, and expressed them in eukaryotic expression systems through molecular cloning technology. After in vitro verification, it was confirmed that the selected antibodies all had good binding specificity and affinity with the target antigens.

[0102] 4. Through structural optimization and activity verification, the optimized molecular structure of the Her2 / CD3 / CD28 trispecific antibody is clarified, and a structurally stable protein is further obtained through a eukaryotic expression system, which exhibits stable Her2, CD28 and CD3 target binding specificity and affinity in vitro, and can effectively mediate the activation and killing effect of T cells. Compared with the currently effective bispecific antibodies, the Her2 / CD3 / CD28 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 and health, effectively preventing tumor antigen immune escape, while enhancing the synergistic immunotherapy effect of regulating the microenvironment of solid tumors.

[0103] 5. A suitable expression system was selected and trispecific antibodies with different structures were successfully expressed. Through structural optimization and activity comparison, it was confirmed that these trispecific antibodies can show good binding stability and improve T cell functional activity and immune killing target cell effects. These beneficial effects rely on the activation effect of the CD3 targeting antibody skeleton on T cells, which shows that the design of trispecific antibodies based on CD3 Fab has unique advantages; in addition, based on the CD3 targeting antibody as a skeleton, it has good operational simplicity and functional stability for the introduction of multivalent CD28 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0105] Figure 2 Verification of the binding function of Her2 / CD3 / CD28 trispecific antibodies with different structures and their ability to bind to Her2 antigen;

[0106] Figure 3 Verification of the binding function of Her2 / CD3 / CD28 trispecific antibodies with different structures and monovalent antibodies to CD28 antigen;

[0107] Figure 4 Verification of the binding function of Her2 / CD3 / CD28 trispecific antibodies with different structures and monovalent antibodies to CD3 antigen;

[0108] Figure 5 Verification of the cytokine release ability of Her2 / CD3 / CD28 trispecific antibodies with different structures in PBMCs and Her2-positive HCC1954 tumor cells;

[0109] Figure 6Effect of different monovalent Her2 / CD3 / CD28 trispecific antibodies on CD8 in PBMCs incubated with Her2-positive HCC1954 tumor cells + T cells and CD4 + Comparison of T cell activation functions;

[0110] Figure 7 Verification of the binding function of Her2 / CD3 / CD28 trispecific antibodies with different structures and dual potency to Her2 antigen;

[0111] Figure 8 Verification of the binding function of Her2 / CD3 / CD28 trispecific antibodies with different structures and dual potency to CD28 antigen;

[0112] Fig. 9 Verification of the binding function of Her2 / CD3 / CD28 trispecific antibodies with different structures and dual potency to CD3 antigen;

[0113] Fig.10 Verification of the cytokine release ability of Her2 / CD3 / CD28 trispecific antibodies with different structures in PBMCs and Her2-positive HCC1954 tumor cells;

[0114] Fig.11 Comparison of the ability of Her2 / CD3 / CD28 trispecific antibodies with different structures to enhance T cell anti-apoptosis after incubation of PBMC with Her2-positive HCC1954 tumor cells;

[0115] Fig.12 Verification of the binding function of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies with different structures to Her2 antigen;

[0116] Fig.13 Verification of the binding function of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies with different structures to CD28 antigen;

[0117] Fig.14 Verification of the binding function of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies with different structures to CD3 antigen; Fig.15 Verification of the cytokine release ability of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies with different structures in PBMC and Her2-positive HCC1954 tumor cells;

[0118] Fig.16 Different structures of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies against CD8 + T cells and CD4 + Comparison of T cell activation functions;

[0119] Fig.17 Verification of the cytokine release ability of Her2 / CD3 / CD28 trispecific antibodies with different titers in PBMCs incubated with Her2-positive HCC1954 tumor cells;

[0120] Fig.18 Comparison of the ability of Her2 / CD3 / CD28 trispecific antibodies with different titers to enhance T cell anti-apoptosis after incubation of PBMC with Her2-positive HCC1954 tumor cells;

[0121] Fig.19 Effects of different titers of Her2 / CD3 / CD28 trispecific antibodies on CD8 + T cells and CD4 + Comparison of T cell activation functions;

[0122] Fig. 20 Comparison of tumor cell killing ability of Her2 / CD3 / CD28 trispecific antibodies with different titers;

[0123] Fig.21 Comparison of the ability of Her2 / CD3 / CD28 trispecific antibodies with different titers to promote PBMC proliferation;

[0124] Fig. 22 Comparison of the cytokine release capacity of Her2 / CD3 / CD28 LTN and SAR443216 in PBMCs incubated with Her2-positive HCC1954 tumor cells;

[0125] Fig.23 Effect of Her2 / CD3 / CD28 LTN and SAR443216 on CD8 in PBMCs incubated with Her2-positive HCC1954 tumor cells + T cells and CD4 + Comparison of T cell activation functions;

[0126] Fig.24 Comparison of the ability of Her2 / CD3 / CD28 LTN and SAR443216 to enhance T cell anti-apoptosis after incubation of PBMC with Her2-positive HCC1954 tumor cells;

[0127] Fig.25 Comparison of the tumor cell killing abilities of Her2 / CD3 / CD28 LTN and SAR443216. DETAILED DESCRIPTION

[0128] The present invention is further explained and illustrated below in conjunction with the embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0129] In the following examples, the materials used in the experiments can be purchased or prepared according to the 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.

[0130] Example 1 Construction and eukaryotic expression of bi / trispecific antibodies

[0131] 1.1 Vector construction

[0132] Construction of CD3-HC-Her2-VHH and CD3-LC expression vectors:

[0133] The heavy chain of the Her2 / CD3 bispecific antibody is connected by CD3-HC and Her2-VHH (2Rs15d) in the following manner: Her2-VHH is connected to the C-terminus of the CD3-HC constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; the light chain CD3-LC is not modified. The coding genes of the above-mentioned 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. Among them, in addition to being connected to CD3-HC through the (G4S) 3Linker selected in this experiment, the anti-Her2-VHH can also be replaced by a connecting peptide Linker known to those skilled in the art.

[0134] The relevant sequences are shown in Table 2:

[0135] Table 2

[0136] name Amino acid sequence number CD3-HC SEQ ID NO: 1 CD3-LC SEQ ID NO: 2 Her2-VHH SEQ ID NO: 3 (G4S)3Linker SEQ ID NO: 5 Her2 / CD3 SEQ ID NO: 10

[0137] Construction of CD28 monovalent Her2 / CD3 / CD28 trispecific antibody expression vector:

[0138] The heavy chain of the Her2 / CD3 / CD28 trispecific antibody is connected by CD3-HC and Her2-VHH (2Rs15d) in the following manner: Her2-VHH is connected to the C-terminus of the CD3-HC constant region (CH1) domain through a flexible connecting peptide (G4S) 3Linker; the light chain of the Her2 / CD3 / CD28 trispecific antibody is connected by CD3-LC and CD28-VHH (P2-6G) in the following manner: CD28-VHH is connected to the N-terminus of the CD3-LC variable region (VL) domain through a rigid connecting peptide PD Linker; or CD28-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 CD28-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 was 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 / CD28 trispecific antibody, that is, the connection mode of CD3-LC and CD28-VHH, the expressed CD28 monovalent Her2 / CD3 / CD28 trispecific antibody was labeled as Her2 / CD3 / CD28 LN, Her2 / CD3 / CD28 LG, and Her2 / CD3 / CD28 LC, respectively. Among them, Her2-VHH and CD28-VHH can be connected to CD3-HC or CD3-LC by the PDLinker, (G4S)3Linker and Coiled Coil Linker selected in this experiment, and can also be replaced by a connecting peptide Linker known to those skilled in the art.

[0139] The relevant sequences are shown in Table 3:

[0140] Table 3

[0141] name Amino acid sequence number CD3-HC SEQ ID NO: 1 CD3-LC SEQ ID NO: 2 Her2-VHH SEQ ID NO: 3 CD28-VHH SEQ ID NO: 4 Her2 / CD3 SEQ ID NO: 10 PD Linker SEQ ID NO: 8 Coiled Coil Linker SEQ ID NO: 9 (G4S)3Linker SEQ ID NO: 5 Her2 / CD3 / CD28LN light chain SEQ ID NO: 14 Her2 / CD3 / CD28LG light chain SEQ ID NO: 15 Her2 / CD3 / CD28LC light chain SEQ ID NO: 16

[0142] Construction of CD28 bivalent Her2 / CD3 / CD28 trispecific antibody expression vector:

[0143] The heavy chain and light chain of the bivalent Her2 / CD3 / CD28 trispecific antibody are connected by CD3-HC, CD28-VHH and Her2-VHH in the following manner: (1) The heavy chain is connected to the C-terminal 228C region of the CD3-HC constant region (CH1) domain by a flexible linker peptide (G4S)3Linker; the light chain is connected to the anti-CD28-VHH by a rigid linker peptide PD Linker to the N-terminal of the CD3 Fab light chain variable region (VL) domain, and then the anti-CD28-VHH is connected in series to the anti-CD28-VHH region at the N-terminal of the CD3 Fab light chain variable region (VL) domain by a flexible linker peptide 218Linker; (2) The heavy chain is connected to the anti-Her2-VHH by a flexible linker peptide (G4S)3Linker to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain, and then connected to the anti-CD28-VHH region at the N-terminal of the CD3 Fab light chain variable region (VL) domain by a rigid linker peptide PDLinker. Linker connects anti-CD28-VHH to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain; the light chain connects CD28-VHH to the N-terminus of the CD3-LC variable region (VL) domain through a rigid linker peptide PD Linker; (3) the heavy chain connects 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 connects anti-CD28-VHH to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker, and then connects anti-CD28-VHH in series to the anti-CD28-VHH region at the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker; the light chain CD3-LC is not modified in any way.

[0144] 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 the Her2 / CD3 / CD28 trispecific antibody, i.e., CD3-LC and CD3-HC, and the connection mode of CD28-VHH, the trispecific antibody expressed later was labeled as bivalent Her2 / CD3 / CD28, namely Her2 / CD3 / CD28 LDN, Her2 / CD3 / CD28 HLN, and Her2 / CD3 / CD28 HDN; wherein Her2-VHH and CD28-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.

[0145] The relevant sequences are shown in Table 4:

[0146] Table 4

[0147]

[0148]

[0149] Construction of CD28 trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibody expression vectors:

[0150] The heavy and light chains of the trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies were linked by CD3-HC, CD28-VHH and Her2-VHH in the following manner: (1) The heavy chain was linked to the CD3-HC constant region (CH1) domain C-terminal 228C region by a flexible linker peptide (G4S)3Linker; the light chain was linked to the CD3 Fab light chain variable region (VL) domain N-terminal by a rigid linker peptide PD Linker, and the CD3 Fab light chain variable region (VL) domain N-terminal by a flexible linker peptide 218Linker. The N-terminus of the CD28-VHH domain at the N-terminus of the Fab light chain variable region (VL) domain is connected to the N-terminus of the anti-CD28-VHH domain, and then the anti-CD28-VHH is connected in series to the N-terminus of the anti-CD28-VHH domain through the flexible connecting peptide 212Linker, and from the N-terminus to the C-terminus, the recombinant light chain domain of anti-CD28-VHH—212Linker—anti-CD28-VHH—218Linker—anti-CD28-VHH—PD Linker—CD3 Fab light chain variable region—CD3 Fab light chain constant region is formed in sequence; (2) the heavy chain is to connect the anti-Her2-VHH to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through the flexible connecting peptide (G4S)3Linker, and to connect the anti-CD28-VHH to the N-terminus of the CD3 Fab heavy chain variable region (VH) domain through the rigid connecting peptide PDLinker; the light chain is to connect the anti-CD28-VHH to the CD3 through the rigid connecting peptide PDLinker The anti-CD28-VHH is connected to the N-terminal of the Fab light chain variable region (VL) domain, and then the anti-CD28-VHH is connected in series to the CD28-VHH region at the N-terminal of the CD3 Fab light chain variable region (VL) domain through a flexible connecting peptide 218Linker; (3) the heavy chain is connected to the anti-Her2-VHH at the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S)3Linker, and the anti-CD28-VHH is connected to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a rigid connecting peptide PD Linker, and then the anti-CD28-VHH is connected in series to the anti-CD28-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a flexible connecting peptide 218Linker; the light chain is connected to the CD3-LC variable region (VL) domain N-terminal through a rigid connecting peptide PD Linker;(4) The heavy chain is composed of anti-Her2-VHH 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-CD28-VHH connected to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker. The anti-CD28-VHH is connected in series to the anti-CD28-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a flexible linker peptide 218Linker, and then the anti-CD28-VHH is connected in series to the N-terminal of the anti-CD28-VHH domain through a flexible linker peptide 212Linker, forming anti-CD28-VHH—212Linker—anti-CD28-VHH—218Linker—anti-CD28-VHH—PD Linker—CD3 Fab heavy chain variable region—CD3 Fab heavy chain constant region (CH1)—(G4S)3Linker—recombinant heavy chain domain of anti-Her2-VHH; light chain CD3-LC is not modified. (5) The heavy chain is composed of anti-Her2-VHH connected to the C-terminal 228C region of the CD3 Fab heavy chain constant region (CH1) domain through a flexible connecting peptide (G4S) 3 Linker, and anti-CD28-VHH connected to the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a rigid connecting peptide PD Linker, and then the anti-CD28-VHH is connected in series to the anti-CD28-VHH region at the N-terminal of the CD3 Fab heavy chain variable region (VH) domain through a flexible connecting peptide 218 Linker; the light chain is composed of anti-CD28-VHH connected to the N-terminal of the CD3 Fab light chain variable region (VL) domain through a rigid connecting peptide PD Linker, and then the anti-CD28-VHH is connected in series to the CD28-VHH region at the N-terminal of the CD3 Fab light chain variable region (VL) domain through a flexible connecting peptide 218 Linker;

[0151] 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 the Her2 / CD3 / CD28 trispecific antibody, i.e., CD3-LC and CD3-HC, and the connection mode of CD28-VHH, the expressed trispecific antibody was labeled as trivalent Her2 / CD3 / CD28, namely, Her2 / CD3 / CD28LTN, Her2 / CD3 / CD28 HNLDN, Her2 / CD3 / CD28 HDNLN, Her2 / CD3 / CD28HTN, and tetravalent Her2 / CD3 / CD28, namely, Her2 / CD3 / CD28 HDNLDN; wherein Her2-VHH and CD28-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.

[0152] The relevant sequences are shown in Table 5:

[0153] Table 5

[0154]

[0155]

[0156] The trispecific antibody structure designed based on structure optimization activity of the present invention is as follows Figure 1 shown.

[0157] 1.2 Eukaryotic expression of proteins

[0158] 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℃.

[0159] Example 2 Purification of trispecific antibodies

[0160] 2.1 Protein G affinity purification

[0161] Take 400mL of the cell supernatant of 1.2 in the above Example 1, centrifuge at 15000rpm, 4℃ for 30min, collect the supernatant, filter with a 0.45μm filter membrane, and place on ice for later use. Take 4mL of Protein G (20% ethanol / Protein G 1:1) in the chromatography column, rinse with Binding buffer 3 times, and press the resin surface with a gasket. Balance the Protein G column with 20mL of Binding buffer. Load once every 10mL, and let the sample pass through the Protein G column at a uniform speed (about 0.5mL / min). Wash the Protein G column with 40mL of Binding buffer at a uniform speed (about 1mL / min). First, add 10% of the volume of the eluent neutralization buffer to the elution tube collection tube, add Elution buffer to the column, elute once with 5mL until the protein concentration cannot be quantified, and quantify all protein concentrations. Use an Amicon Ultra-15 centrifugal filter to concentrate the collected protein samples, centrifuge at 3000rpm at 4℃ for 20 minutes, and quantify the protein concentration.

[0162] Example 3 Evaluation of biological functions of Her2 / CD3 / CD28 trispecific antibodies

[0163] 3.1 Validation of monovalent Her2 / CD3 / CD28 trispecific antibody antigen binding

[0164] Dilute Her2 ECD-Fc, CD28 ECD-Fc, and CD3 ECD-Fc antigens to 10 μg / mL with PBS and coat them 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 the antibody to be tested 10 times with blocking solution in 6 gradients, with the starting antibody concentration of 200 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.

[0165] The results are shown in Table 6 and Figure 2 As shown, by comparing EC50, it was found that Her2 / CD3 / CD28 antibodies with different structures retained their binding affinity and specificity to Her2 antigen.

[0166] As shown in Table 7 and Figure 3 As shown, by comparing EC50, it was found that Her2 / CD3 / CD28 trispecific antibodies with different structures have high affinity and specificity for CD28.

[0167] As shown in Table 8 and Figure 4 As shown, by comparing the EC50, it was found that the Her2 / CD3 / CD28 trispecific antibody had improved binding ability to the CD3 antigen compared with the Her2 / CD3 bispecific antibody, indicating that Her2 / CD3 / CD28 retained its binding affinity and specificity to the CD3 antigen.

[0168] Table 6 Binding ability of monovalent Her2 / CD3 / CD28 trispecific antibodies to Her2 antigen

[0169] Constructs EC50(nM) Her2 / CD3 / CD28 LN 0.727 Her2 / CD3 / CD28LG 0.796 Her2 / CD3 / CD28LC 1.644 Her2 / CD3 0.722 CD3 Fab Not determined

[0170] Table 7 Binding ability of monovalent Her2 / CD3 / CD28 trispecific antibodies to CD28 antigen

[0171] Constructs EC50(nM) Her2 / CD3 / CD28 LN 0.804 Her2 / CD3 / CD28LG 3.435 Her2 / CD3 / CD28LC 5.975 Her2 / CD3 Not determined CD3 Fab Not determined

[0172] Table 8 Binding ability of monovalent Her2 / CD3 / CD28 trispecific antibodies to CD3 antigen

[0173]

[0174]

[0175] 3.2 Verification of the cytokine release ability of monovalent Her2 / CD3 / CD28 trispecific antibody in PBMC and Her2-positive tumor cells incubated

[0176] 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 (0.2 million / mL). After equal volumes were mixed, 1 mL / well was plated back into a 24-well plate. Her2 / CD3 / CD28 trispecific antibodies of different structures were added at a concentration of 1 nM, and the supernatant was collected after 24 hours.

[0177] IL-2, IFN-γ and TNF-α ELISA detection kits were used for the detection of cytokines, as follows: Capture Antibody was coated on the ELISA plate and placed at room temperature for 4 hours or at 4°C overnight; after washing with Wash Buffer 3 times, the ELISA plate was blocked with ELISA / ELISAPOT Diluent (1×) at room temperature for 2 hours or at 4°C overnight. IL-2, IFN-γ and TNF-α standards were prepared and added, and the cell supernatant was diluted and added to the ELISA plate. After reacting at 37°C for 2 hours, the plate was washed 5 times with Wash Buffer. Detection Antibody solution was diluted and added, and incubated at room temperature for 1-2 hours; Wash Buffer was used to wash the plate 5 times, and Streptavidin-HRP solution was added. After 30 minutes to 1 hour at room temperature, the plate was washed 5 times. 1×TMD solution was added for color development, and Stop Solution was used to terminate the reaction. The absorbance at a wavelength of 450nm was detected using an ELISA reader.

[0178] The results are as follows Figure 5 ( Figure 5 A. Figure 5 B. Figure 5 As shown in C), compared with the Her2 / CD3 bispecific antibody, the trispecific antibody Her2 / CD3 / CD28 has a higher cytokine release level on T cells co-incubated with Her2-positive cells HCC1954 (Her2 3+). Among them, the monovalent Her2 / CD3 / CD28 LN structure has a higher cytokine release level, further proving that Her2 / CD3 / CD28 LN has a stronger T cell activation ability.

[0179] 3.3 Monovalent Her2 / CD3 / CD28 trispecific antibody against CD8 + T cells and CD4 + Comparison of T cell activation ability

[0180] The specific implementation steps are the same as 3.2. After 24 hours, the cells were collected by centrifugation and stained with flow cytometry antibodies PerCP cy5.5-anti humanCD4; Pacific blue anti-human CD8; APC anti-human CD69; FITC anti-human CD25. CD8 was analyzed by flow cytometry. + T cells and CD4 + T cell CD69 + CD25 + Percentage, compare the activation effect.

[0181] The activation results are as follows Figure 6 ( Figure 6 A. Figure 6 As shown in B), compared with the Her2 / CD3 bispecific antibody, the Her2 / CD3 / CD28 trispecific antibody with different structures has obvious + T cells and CD4 + The activation effect of T cells after co-culture with target cells is enhanced, among which Her2 / CD3 / CD28 LN is more effective for CD8 + T cells and CD4 + T cells have the highest degree of activation.

[0182] 3.4 Antigen binding validation of bivalent Her2 / CD3 / CD28 trispecific antibodies

[0183] The steps are the same as 3.1, and the results are shown in Table 9 and Figure 7 As shown, by comparing EC50, it was found that there was no significant difference in the binding ability of Her2 / CD3 / CD28 with different structures to Her2 antigen, and the Her2 / CD3 / CD28 antibody retained its binding affinity and specificity to the Her2 antigen.

[0184] As shown in Table 10 and Figure 8 As shown, by comparing EC50, it was found that Her2 / CD3 / CD28 trispecific antibodies with different structures have high affinity and specificity for CD28.

[0185] As shown in Table 11 and Fig. 9 As shown, by comparing EC50, it was found that the Her2 / CD3 / CD28 trispecific antibody had a higher binding ability to the CD3 antigen, and Her2 / CD3 / CD28 retained its binding affinity and specificity to the CD3 antigen.

[0186] Table 9 Binding ability of bivalent Her2 / CD3 / CD28 trispecific antibodies to Her2 antigen

[0187] Constructs EC50(nM) Her2 / CD3 / CD28 HLN 1.511 Her2 / CD3 / CD28LDN 2.27 Her2 / CD3 / CD28HDN 2.554

[0188] Table 10 Binding ability of dual-valent Her2 / CD3 / CD28 trispecific antibodies to CD28 antigen

[0189] Constructs EC50(nM) Her2 / CD3 / CD28 HLN 1.024 Her2 / CD3 / CD28 LDN 2.809 Her2 / CD3 / CD28 HDN 2.853

[0190] Table 11 Binding ability of dual-valent Her2 / CD3 / CD28 trispecific antibodies to CD3 antigen

[0191] Constructs EC50(nM) Her2 / CD3 / CD28 HLN 1.507 Her2 / CD3 / CD28 LDN 1.351 Her2 / CD3 / CD28 HDN 1.050

[0192] 3.5 Verification of the cytokine release ability of the bivalent Her2 / CD3 / CD28 trispecific antibody in PBMC and Her2-positive tumor cells

[0193] The specific implementation steps are the same as 3.2, the sample concentration is 100pM, and the results are as follows Fig.10 ( Fig.10 A. Fig.10 As shown in B), compared with the Her2 / CD3 bispecific antibody, the trispecific antibody Her2 / CD3 / CD28 has a higher cytokine release ability on T cells co-incubated with Her2-positive cells HCC1954 (Her2 3+). Among them, the bivalent Her2 / CD3 / CD28 LDN structure has a higher cytokine release level, further proving that Her2 / CD3 / CD28 LDN has a stronger T cell activation ability.

[0194] 3.6 Comparison of the ability of dual-valent Her2 / CD3 / CD28 trispecific antibodies to enhance T cell anti-apoptosis

[0195] The specific implementation steps are the same as 3.2, the sample concentration is 100pM, and the cells are collected by centrifugation after 24 hours. The flow cytometry antibodies PerCPcy5.5-anti human CD4 and Pacific blue anti-human CD8 are stained on ice for 1 hour, the fixative is at room temperature for 30 minutes, and the permeabilization solution is prepared with 3μg / mL FITC anti-human BCL-xL. The cells are resuspended at room temperature for 30 minutes, washed once by FACS, and BCL-xL is analyzed by flow cytometry. + Percentages were used to compare the anti-apoptotic effects.

[0196] The results are as follows Fig.11 ( Fig.11 A. Fig.11As shown in Figure 2, compared with the Her2 / CD3 bispecific antibody, the Her2 / CD3 / CD28 trispecific antibody with different structures has a significant effect on enhancing the anti-apoptotic ability of T cells, among which the Her2 / CD3 / CD28 LDN in the bivalent mode has a better effect on enhancing the CD4 + T cells and CD8 + The anti-apoptosis ability of T cells helps to maintain mitochondrial homeostasis and has more obvious advantages compared to other structures.

[0197] 3.7 Validation of antigen binding of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies

[0198] The steps are the same as 3.1, and the results are shown in Table 12 and Fig.12 As shown, by comparing EC50, it was found that there was no significant difference in the binding ability of Her2 / CD3 / CD28 with different structures to Her2 antigen, and the Her2 / CD3 / CD28 antibody retained its binding affinity and specificity to the Her2 antigen.

[0199] As shown in Table 13 and Fig.13 As shown, by comparing EC50, it was found that Her2 / CD3 / CD28 trispecific antibodies with different structures have high affinity and specificity for CD28.

[0200] As shown in Table 14 and Fig.14 As shown, by comparing the EC50, it was found that Her2 / CD3 / CD28 retained its binding affinity and specificity to the CD3 antigen.

[0201] Table 12 Binding ability of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies to Her2 antigen

[0202] Constructs EC50(nM) Her2 / CD3 / CD28 LTN 3.577 Her2 / CD3 / CD28 HTN 2.350 Her2 / CD3 / CD28 HNLDN 2.713 Her2 / CD3 / CD28 HDNLN 5.017 Her2 / CD3 / CD28 HDNLDN 2.399

[0203] Table 13 Binding ability of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies to CD28 antigen

[0204] Constructs EC50(nM) Her2 / CD3 / CD28 LTN 1.494 Her2 / CD3 / CD28 HTN 1.290 Her2 / CD3 / CD28 HNLDN 0.871 Her2 / CD3 / CD28 HDNLN 1.194 Her2 / CD3 / CD28 HDNLDN 1.045

[0205] Table 14 Binding ability of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies to CD3 antigen

[0206]

[0207]

[0208] 3.8 Verification of the cytokine release ability of trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies in PBMC and Her2-positive tumor cells

[0209] The specific implementation steps are the same as 3.2, the sample concentration is 10pM, and the results are as follows Fig.15 ( Fig.15 A. Fig.15 B. Fig.15 As shown in C), the trispecific antibodies Her2 / CD3 / CD28 with different structures have a higher cytokine release ability on T cells co-incubated with Her2-positive cells HCC1954 (Her2 3+). Among them, the trivalent Her2 / CD3 / CD28 LTN structure has a higher cytokine release level, further proving that Her2 / CD3 / CD28 LTN has a stronger T cell activation ability.

[0210] 3.9 Trivalent and tetravalent Her2 / CD3 / CD28 trispecific antibodies against CD8 + T cells and CD4 + Comparison of T cell activation ability

[0211] The specific implementation steps are the same as 3.2, the sample concentration is 10pM, and the results are as follows Fig.16 ( Fig.16 A. Fig.16 As shown in B), the Her2 / CD3 / CD28 trispecific antibodies with different structures have obvious + T cells and CD4 + The activation effect of T cells after co-culture with target cells is enhanced, among which Her2 / CD3 / CD28 LTN is more important for CD8 + T cells and CD4 + T cell activation ability is the highest and has significant advantages over other structures.

[0212] 3.10 Verification of the cytokine release ability of Her2 / CD3 / CD28 trispecific antibodies with different titers in PBMC and Her2-positive tumor cells

[0213] The specific implementation steps are the same as 3.2, the sample concentration is 10pM, and the results are as follows Fig.17 ( Fig.17 A. Fig.17 B. Fig.17As shown in Figure C), compared with the Her2 / CD3 bispecific antibody, the trispecific antibody Her2 / CD3 / CD28 has a higher cytokine release ability for T cells co-incubated with Her2-positive cells HCC1954 (Her23+). Among them, the trivalent Her2 / CD3 / CD28LTN structure has the highest release level and significant advantage, further proving that Her2 / CD3 / CD28 LTN has a stronger T cell activation ability.

[0214] 3.11 Comparison of the ability of Her2 / CD3 / CD28 trispecific antibodies with different titers to enhance T cell anti-apoptosis

[0215] The specific implementation steps are the same as 3.2, the sample concentration is 10pM, after 24 hours, the cells are collected by centrifugation, and the flow cytometry antibodies PerCPcy5.5-anti human CD4 and Pacific blue anti-human CD8 are stained on ice for 1 hour, the fixative is at room temperature for 30 minutes, the permeabilization solution is prepared with 3μg / mL FITC anti-human BCL-xL, the cells are resuspended at room temperature for 30 minutes, washed once by FACS, and BCL-xL is analyzed by flow cytometry + Percentages were used to compare the anti-apoptotic effects.

[0216] The results are as follows Fig.18 ( Fig.18 A. Fig.18 As shown in Figure 2, compared with the Her2 / CD3 bispecific antibody, the Her2 / CD3 / CD28 trispecific antibodies with different valences have a significant effect on enhancing the anti-apoptotic ability of T cells. Among them, the Her2 / CD3 / CD28 LTN has a better effect on enhancing the anti-apoptotic ability of CD4 T cells. + T cells and CD8 + The anti-apoptosis ability of T cells helps to maintain mitochondrial homeostasis and has more obvious advantages compared to other structures.

[0217] 3.12 Effects of Her2 / CD3 / CD28 trispecific antibodies of different titers on CD8 + T cells and CD4 + Comparison of T cell activation ability

[0218] The specific implementation steps are the same as 3.2, the sample concentration is 10pM, and the results are as follows Fig.19 ( Fig.19 A. Fig.19 As shown in B), compared with the Her2 / CD3 bispecific antibody, the Her2 / CD3 / CD28 trispecific antibody with different structures has obvious + T cells and CD4 +The activation effect of T cells after co-culture with target cells is enhanced, among which Her2 / CD3 / CD28 LTN is more important for CD8 + T cells and CD4 + T cells have the highest degree of activation and have significant advantages over other structures.

[0219] 3.13 In vitro tumor killing activity of Her2 / CD3 / CD28 trispecific antibodies with different titers

[0220] 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, and PBMC cells were resuspended in medium; the above centrifugation was repeated once; cell counts were made to adjust the PBMC cell density; tumor cells were digested to adjust the density, K562-Her2 was labeled with CFSE, the PBMC and K562-Her2 effector-target ratio was adjusted to 5:1 and 2:1, and K562-Her2 was mixed with an equal volume of medium as a positive control group. 200μL of 100nM antibody was prepared in a U-shaped plate, 10-fold dilutions were made into 6 concentration gradients, 10μL / well was transferred to a 96-well plate, and PBMC with adjusted density and tumor cells were added at 100μL / well, and K562-Her2 was mixed with an equal volume of medium as a positive control group and incubated at 100μL / well for 24h to detect the number of live cells. The percentage of tumor cell killing was calculated by comparing the number of CFSE-positive cells in 7AAD-negative cells with the negative control group.

[0221] The results are as follows Fig. 20 As shown in the figure, by comparing the killing percentages, it was found that compared with the Her2 / CD3 bispecific antibody, the trispecific antibody Her2 / CD3 / CD28 had better cytotoxicity to the Her2-positive cells K562-Her2, especially the trivalent Her2 / CD3 / CD28 LTN had a higher ability to kill Her2-positive cells and showed a stronger tumor inhibitory effect; therefore, the superior structures with higher tumor cell toxicity between different valencies can be compared.

[0222] 3.14 Comparison of the ability of Her2 / CD3 / CD28 trispecific antibodies with different titers to promote PBMC proliferation

[0223] Use (Ca 2+ Mg 2+) Dilute Her2 / CD3, Her2 / CD3 / CD28 LN, Her2 / CD3 / CD28 LDN and Her2 / CD3 / CD28 LTN to 100 nM with PBS and cover the plate overnight at 4°C. The next day, rinse three times with DPBS, adjust the PBMC density with RPMI1640 medium, add 1 million PBMC / well, and culture in a cell culture incubator at 37°C for 4 days. After 4 days, change the medium and detect the cell number by flow cytometry. Count the cells every four days. The results are shown in Fig.21 As shown in the figure, compared with Her2 / CD3 bispecific antibodies, the ability of trispecific antibodies to promote proliferation is significantly improved; among trispecific antibodies, the trivalent Her2 / CD3 / CD28 LTN trispecific antibody has a stronger ability to promote PBMC proliferation compared with monovalent and bivalent trispecific antibodies. Therefore, Her2 / CD3 / CD28 LTN is the best optimized structure.

[0224] Example 4 Comparison of the ability of Her2 / CD3 / CD28 LTN and SAR443216

[0225] SAR443216 is a HER2 / CD3 / CD28 tri-specific T cell engager under development by Sanofi. Phase 1 clinical trials are currently underway overseas. It is expected to provide a new treatment for patients with HER2-expressing solid tumors (including HER2-low-expressing solid tumors).

[0226] 4.1 Comparison of cytokine release ability of Her2 / CD3 / CD28 LTN and SAR443216 in PBMC and Her2-positive tumor cells incubated

[0227] The specific implementation steps are the same as 3.2, the sample concentration is 100pM, the PBMC and HCC1954 effector-target ratios are 5:1 and 3:1, and the results are as follows Fig. 22 ( Fig. 22 A. Fig. 22 B. Fig. 22 As shown in C), compared with SAR443216, Her2 / CD3 / CD28 LTN has a higher ability to promote cytokine release in T cells co-incubated with Her2-positive cells HCC1954 (Her2 3+). In addition, trivalent Her2 / CD3 / CD28 LTN has the highest release level and significant advantage, further proving that Her2 / CD3 / CD28 LTN has a stronger T cell activation ability compared with the existing technology.

[0228] 4.2 Her2 / CD3 / CD28 LTN and SAR443216 on CD8 + T cells and CD4 + Comparison of T cell activation ability

[0229] The specific implementation steps were the same as 3.2, the sample concentration was 100 pM, the effector-target ratio of PBMC to HCC1954 was 5:1, 3:1 and 1:1, and the cells were collected by centrifugation after 24 hours, and stained with flow cytometry antibodies PerCP cy5.5-anti human CD4; Pacific blue anti-human CD8; APC anti-human CD69; FITC anti-human CD25, and CD8 was analyzed by flow cytometry. + T cells and CD4 + T cell CD69 + CD25 + Percentage, compare the activation effect.

[0230] The activation results are as follows Fig.23 ( Fig.23 A. Fig.23 As shown in B), compared with SAR443216, the Her2 / CD3 / CD28 LTN trispecific antibody has obvious + T cells and CD4 + The activation effect of T cells after co-culture with target cells is enhanced. It is also proved that Her2 / CD3 / CD28 LTN has a significant advantage in T cell activation ability compared with the existing technology.

[0231] 4.3 Comparison of Her2 / CD3 / CD28 LTN and SAR443216 in enhancing the anti-apoptosis ability of T cells

[0232] The specific implementation steps are the same as 3.2, the sample concentration is 100pM, the PBMC to HCC1954 effector-target ratio is 5:1, 3:1 and 1:1, after 24 hours, the cells are collected by centrifugation, and the flow cytometry antibodies PerCP cy5.5-anti human CD4 and Pacific blue anti-human CD8 are stained on ice for 1 hour, the fixative is at room temperature for 30 minutes, the permeabilization solution is prepared with 3μg / mL FITC anti-human BCL-xL, the cells are resuspended at room temperature for 30 minutes, washed once by FACS, and BCL-xL is analyzed by flow cytometry + Percentages were used to compare the anti-apoptotic effects.

[0233] The results are as follows Fig.24 ( Fig.24 A. Fig.24 As shown in B), compared with SAR443216, the Her2 / CD3 / CD28 LTN trispecific antibody has a significant CD8 + T cells and CD4 +The results showed that compared with the existing technology, Her2 / CD3 / CD28 LTN has a significant advantage in promoting the anti-apoptosis ability of T cells and is more helpful in maintaining mitochondrial homeostasis.

[0234] 4.4 Comparison of in vitro tumor killing activity between Her2 / CD3 / CD28 LTN and SAR443216

[0235] The specific implementation steps are the same as 3.13, and the results are as follows Fig.25 As shown, by comparing the killing percentages, it was found that compared with SAR443216, the Her2 / CD3 / CD28 LTN trispecific antibody had better cytotoxicity and higher Her2-positive cell killing ability against Her2-positive cells K562-Her2, showing a stronger tumor inhibitory effect; and it was proved that compared with the existing technology, Her2 / CD3 / CD28 LTN has a significant advantage in tumor cell killing ability.

[0236] In summary, by examining the effects of different positions of CD28 in trispecific antibodies on the biological activity of antibodies, the optimal Her2 / CD3 / CD28 LN structure was obtained. On this basis, the bivalent CD28 tri-antibody was optimized and designed, and the relationship between the position and valence of the bivalent was examined to obtain the optimal Her2 / CD3 / CD28 LDN structure. The trivalent and tetravalent related structures were further designed. By comparing the cytokine release ability, the activation ability of PBMC and the anti-apoptosis ability, the trivalent Her2 / CD3 / CD28 LTN structure had significant advantages over other structures, indicating that the valence and structure of CD28 antibodies jointly affect the biological function of CD28 antibodies. In the comparison of in vitro killing ability and the ability to enhance PBMC proliferation, Her2 / CD3 / CD28LTN showed significant advantages over the single-valent optimal structure Her2 / CD3 / CD28 LN and the bivalent optimal structure Her2 / CD3 / CD28LDN. Furthermore, we compared the optimal structures Her2 / CD3 / CD28 LTN and SAR443216 trispecific antibodies in terms of their ability to promote cytokine release, promote T cell activation, enhance T cell anti-apoptosis, and kill tumor cells. Her2 / CD3 / CD28 LTN has significant advantages over SAR443216 in all the above aspects, which fully proves that Her2 / CD3 / CD28 LTN is the preferred optimal CD28 trispecific antibody structure.

Claims

1. A Her2 / CD3 / CD28 trispecific antibody, characterized in that: The Her2 / CD3 / CD28 trispecific antibody has a heavy chain amino acid sequence as shown in SEQ ID NOs: 10-13.

2. A Her2 / CD3 / CD28 trispecific antibody, characterized in that: The Her2 / CD3 / CD28 trispecific antibody has a light chain amino acid sequence as shown in SEQ ID NOs: 2, 14-18.

3. A Her2 / CD3 / CD28 trispecific antibody, characterized in that: The Her2 / CD3 / CD28 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: 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: 14; 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 as shown in SEQ ID NO: 12, and the light chain amino acid sequence as shown in SEQ ID NO: 14; 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: 17; and / or The heavy chain amino acid sequence is shown as SEQ ID NO:13, and the light chain amino acid sequence is shown as SEQ ID NO:

2.

4. A Her2 / CD3 / CD28 trispecific antibody, characterized in that: The Her2 / CD3 / CD28 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: 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: 14; 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 as shown in SEQ ID NO: 12, and the light chain amino acid sequence as shown in SEQ ID NO: 14; 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: 17; and / or The heavy chain amino acid sequence is shown as SEQ ID NO:13, and the light chain amino acid sequence is shown as SEQ ID NO:

2.

5. A Her2 / CD3 / CD28 trispecific antibody, characterized in that: The Her2 / CD3 / CD28 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 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: 14; 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: 17; and / or The heavy chain amino acid sequence is shown as SEQ ID NO:13, and the light chain amino acid sequence is shown as SEQ ID NO:

2.

6. Use of the antibody according to any one of claims 1 to 5 for treating Her2 / CD28 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 / CD28 single-positive or double-positive tumors.

Images