Bispecific antibodies and their applications

Abstract

The present invention provides a bispecific antibody and its application, in which IL15 and IL15Rα are introduced into the two chains of the first antibody of the bispecific antibody of the present invention, respectively (e.g., IL15 and IL15Rα replace CL1 and CH1, respectively), and the high affinity of IL15 and IL15Rα is utilized to form an IL15 / IL15Rα complex, thereby realizing correct pairing of the light chain / heavy chain of the first antibody and solving the problem of inaccurate matching of the light chain / heavy chain of the bispecific antibody. At the same time, mutations in the amino acid sequences of the variable domains VH1, VL1, IL15, and IL15Rα of the first antibody add one or more pairs of disulfide bonds between VH1 and VL1, and between IL15 and IL15Rα, further enhancing the binding activity between the light and heavy chains of the first antibody. This effectively overcomes issues during the bispecific antibody preparation process, such as incorrect light and heavy chain matching, large amounts of by-products, and poor stability, and ultimately allows for the preparation of a correctly paired bispecific multifunctional antibody targeting a cytokine. At the same time, this shortens the development cycle of bispecific antibodies and reduces production costs.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention belongs to the field of biomedicine and relates to bispecific antibodies, and the invention also relates to the applications of such bispecific antibodies.

[0002] [Background technology] Bispecific antibodies (BsAbs), also known as bifunctional antibodies, can simultaneously recognize and bind to two different antigens and epitopes, blocking two different signaling pathways to exert their effects. Compared with conventional antibodies, BsAbs have additional specific antigen-binding sites and offer the following therapeutic advantages:

[0003] Mediating immune cell killing of tumors: The key mechanism of action of bispecific antibodies is to mediate immune cell killing. Bispecific antibodies have two antigen-binding arms, one of which binds to the target antigen and the other to the target antigen on effector cells, which can activate the effector cells to target and kill tumor cells. Two bispecific antibody products currently approved for sale belong to this class: Trion Catumaxomab, developed by Pharma, can target the tumor surface antigen EpCAM and the T cell surface receptor CD3, while Blinatumomab, developed by Micromet and Amgen, can simultaneously bind to CD19 and CD3. Both achieve the goal of treating tumors by activating and recruiting killer T cells.

[0004] Dual-target signal blockade exerts unique or overlapping functions, effectively preventing drug resistance. Simultaneous binding of dual targets and blocking dual signal pathways is another important mechanism of action for bispecific antibodies. Receptor tyrosine kinases (RTKs) are the largest class of enzyme-linked receptors and play an important regulatory role in cell proliferation processes, including the HER family. RTKs are also important targets for tumor therapy because their abnormally high expression on the surface of tumor cells leads to their malignant proliferation. While single-target monoclonal antibodies against RTKs have been widely applied in tumor therapy, tumor cells can evade the immune system by switching signal pathways or activating intracellular signals through HER family members themselves or through homologous or heterodimers between different members. Therefore, using bispecific antibodies to simultaneously block two or more RTKs or their ligands can reduce tumor cell evasion and improve therapeutic efficacy.

[0005] Stronger specificity, targeting, and reduced off-target toxicity: By utilizing the property that the two antigen-binding arms of bispecific antibodies can bind to different antigens, the two antigen-binding arms can each bind to two antigens on the surface of cancer cells, effectively enhancing the binding specificity and targeting of the antibody to cancer cells and reducing side effects such as off-target toxicity.

[0006] Effectively reduce treatment costs: Take BiTE as an example, and compare it with conventional antibodies to see if it has a higher tissue penetration rate. Bispecific antibodies have strong competitive advantages in terms of tumor cell killing efficiency, off-target rate, and clinical indications, offering significant clinical advantages. In particular, their therapeutic efficacy can be 100-1000 times that of conventional antibodies, so the minimum dose can be reduced to 1 / 2000 of the original dose, significantly reducing the cost of drug treatment. Compared with combination therapy, the cost of bispecific antibodies is also much lower than the combination of two single drugs.

[0007] There are currently three bispecific antibody drugs approved for sale worldwide. In 2009, the European Union approved the first therapeutic bispecific antibody, Catumaxomab, developed by TrionPharma, targeting CD3 and EpCAM, for the treatment of malignant ascites. In 2014, the FDA granted accelerated approval to Blinatumomab, a bispecific antibody drug developed by Amgen based on BiTE® technology that targets CD3 and CD19, for the treatment of acute B-lymphoblastic leukemia. It is also the first approved drug targeting CD19. Amgen has more than a dozen BiTE® molecules in clinical development across a broad portfolio of hematological malignancies and solid tumors. AMG420, which targets BCMA / CD3, has received FDA-approved fast-track approval. Clinical trial results also demonstrate the superiority of the BiTE® molecule AMG212 (Pasotuxizumab), which targets prostate-specific membrane antigen (PSMA), in the treatment of solid tumors. In November 2017, the FDA granted accelerated approval to Roche's bispecific antibody emicizumab (targeting clotting factors X and IXa) for the treatment of hemophilia. It was approved for sale in China in December 2018, making it the first bispecific antibody approved in China.

[0008] Bispecific antibodies themselves have many advantages and can be applied in multiple therapeutic areas, such as cancer, chronic inflammatory diseases, autoimmune diseases, and infectious diseases. However, the main technical difficulty in preparing bispecific antibodies, especially asymmetric bispecific antibodies containing Fc regions (IgG-like), is obtaining correctly paired bispecific antibodies and simultaneously facing the problem of inaccurate HC / HC and LC / HC matching. Subsequently, KiH, ART-Ig, and BiMab technologies were developed to reduce the inaccurate HC / HC matching. CrossMAb technology (in which the variable domains VL and VH of a pair of heavy and light chains are replaced with each other, and the constant domains CL and CH1 are also replaced with each other), YBODY technology (one side uses a conventional Fab to create a monovalent unit Fab-Fc targeting antigen A, and the other side uses a single-chain antibody to create a single-chain unit ScFv-Fc targeting antigen B), WuXiBody (TCR constant regions replace the heavy chain constant region CH1 and the light chain constant region CL), and common light chain technology have been used to reduce inaccurate LC / HC matching. However, in the field of bispecific antibodies, there is still a need to further develop new technologies to solve the inaccurate LC / HC matching problem.

[0009] 1.Her2 Members of the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. The receptor family contains four distinct members, including epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4 or tyro2). Her2 is a transmembrane, surface-bound receptor tyrosine kinase that is normally involved in signaling pathways that lead to cell proliferation and differentiation.

[0010] Overexpression of Her2 can cause the impairment of normal cell function and is usually closely related to the occurrence and development of tumors. Homologous or heterologous polymerization of Her2 can cause phosphorylation of receptor tyrosine residues, triggering many signal transduction pathways and leading to cell proliferation and tumorigenesis. Her2 can be used as a prognostic and predictive biomarker. Gene amplification or overexpression occurs in approximately 15-30% of breast cancers and 10-30% of gastric and esophageal cancers. Her2 overexpression has also been observed in other tumors, such as ovarian, endometrial, bladder, lung, colon, and head and neck cancers.

[0011] Trastuzumab recognizes a membrane-proximal epitope in the extracellular domain IV of Her2. Specifically, the epitope consists of three rings (557-561, 570-573, and 593-603) at the C-terminus of the extracellular domain IV of Her2. Because the epitope is close to or may directly interact with the binding domain of its dimerization partner, trastuzumab binding to the epitope may induce steric hindrance that inhibits the dimerization process. Furthermore, trastuzumab binding may protect the extracellular domain of the Her2 receptor from protease attack and hydrolysis.

[0012] Currently, trastuzumab is used as a first-line treatment for breast cancer and effectively treats metastatic breast cancer with Her2 overexpression, with an objective response rate of 30-50%. However, it is less effective in treating metastatic breast cancer with low Her2 expression, and even when the antibody initially shows efficacy, many patients develop resistance within a year. Her2 can form ligand-dependent or ligand-independent heterodimers with other members of the Her2 family (Her1, Her3, and Her4), thereby activating downstream pathways and causing tumor cell proliferation. Trastuzumab's inability to inhibit heterodimer formation may be one of the reasons for the development of drug resistance.

[0013] Pertuzumab is a humanized monoclonal antibody specifically designed to prevent HER2 receptor dimerization with other HER receptors (EGFR / HER1, HER3, and HER4) on the cell surface, a process thought to play a role in tumor growth and survival. Pertuzumab has demonstrated therapeutic efficacy against advanced prostate cancer, non-small cell lung cancer, ovarian cancer, and breast cancer, but its therapeutic efficacy depends on the level of Her2 expression. Pertuzumab identified a critical site in the Her2 extracellular domain II heterodimer. The identified epitope is located in the central segment 245-311 of subregion II, with key residues H245, V286, S288, L295, H296, and K311. Here, L295 and H296 are important sites mediating Her2 and Her3 heterodimerization, and the L295A / H296A double mutation can completely block Her2 / Her3 heterodimerization (Franklin, MC et al., Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex. Cancer Cell, 2004.5(4):pp.317-28). Therefore, pertuzumab can be used to effectively inhibit the formation of Her2 / Her3 heterodimers, but has not shown a significant inhibitory effect on the formation of EGFR / Her2 heterodimers.

[0014] 2.IL15 / IL15Ra IL-15 is a 14-15 kDa cytokine crucial for the function of NK cells, NKT cells, and memory CD8+ T cells. Although IL-15 is virtually absent in the body, it is transduced by binding to its receptor IL-15Rα, producing a highly potent complex, the IL-15 superagonist (IL-15SA), which is then transported to target cells. IL-15SA potently activates IL-15-responsive cells, particularly NK cells, thereby promoting antitumor and antiviral functions.

[0015] Researchers first identified IL-15 as a T lymphocyte growth factor in 1994 and IL-15 shares approximately 19% sequence identity with IL-2 and shares many highly similar biological properties. The three-dimensional structure of IL-15 resembles that of IL-2, consisting of a four-strand "up-down-down-down" helix bundle; other cytokines, such as IL-4, IL-7, and IL-9, also contain this conformation. IL-15 functions differently from other cytokines. IL-15 receptor α is expressed on IL-15-producing cells (e.g., macrophages and dendritic cells) and forms IL-15SA with IL-15, transmitting signals to NK, NKT, and memory CD8+ T cells that express IL-15Rβ (also known as IL-2Rβ) and a common γ chain (shared with IL-2, IL-4, IL-7, IL-9, and IL-21). It is likely this unique presentation that confers IL-15 the ability to mediate its unique functions. Murine IL-15 shares 70% amino acid sequence identity with human IL-15, and both human and mouse IL-15 share similar trans-expression patterns, signaling pathways, and biological activities. IL-15 is expressed in many cell types and tissues, including monocytes, macrophages, DCs, keratinocytes, fibroblasts, muscle cells, and neurons. IL-15, as a pleiotropic cytokine, plays an important role in both innate and adaptive immunity.

[0016] Trans-expressed IL-15 / IL-15Rα signals induce the recruitment and activation of JAK1 and JAK3 in response to the β and γ chains expressed on cells, and the activated JAK1 and JAK3 further phosphorylate STAT3 and STAT5. After phosphorylation, STAT3 and STAT5 form homologous dimers, translocate to the cell nucleus, and promote the transcription of target genes. IL-15 signaling stimulates a cascade of downstream reactions, inducing cell proliferation, reducing apoptosis, and enhancing immune cell activation and migration. In the absence of high-affinity IL-15Rα, IL-15 is expressed with intermediate affinity (Ka=1.10 9IL-15 binds solely to the β and γ receptor complex (IL-15Rβγ) of the IL-15 / M receptor, inducing the phosphorylation and activation of other tyrosine kinases (e.g., Lck, Fyn, Lyn, and Syk), and can also interact with the PI3K and MAPK pathways. Studies have shown that the metabolic checkpoint kinase mTOR can also be activated by high concentrations of IL-15, which is associated with enhanced NK cell proliferation and activation, and selective knockdown of mTOR blocks the maturation of bone marrow NK cells. IL-15's ability to promote NK cell proliferation is partly due to IL-15-mediated aerobic glycolysis. In the absence of IL-15, NK cell basal metabolism is very low, but increasing IL-15 concentrations can significantly enhance this physiological activity.

[0017] IL-15 shares immunological properties with IL-2, inducing T cell proliferation and survival, promoting NK cell proliferation and differentiation, and inducing the generation of cytotoxic T lymphocytes. However, unlike IL-2, IL-15 has no significant effect on Treg cells and does not induce capillary leak syndrome in mice or nonhuman primates (NHPs). Therefore, IL-15 is a preferred choice for tumor immunotherapy compared to IL-2. Rhesus IL-15 (rIL-15) was the first form of IL-15 used in in vivo experiments, and researchers believe that rIL-15 preferentially binds to cell-surface IL-15Rα. The heterodimeric IL-15 / IL-15Rα is a naturally occurring form of IL-15 released from cells and can respond independently of cell-stimulatory interactions. Novartis is currently conducting clinical trials of this form (NIZ985) in solid tumors. Developed by Cytune, RLI is a fusion protein consisting of IL-15 bound to the sushi domain of IL-15Rα, which functions as a soluble IL-15 agonist. IL-15 / IL-15Rα-Fc complexes are generated by mixing commercially available IL-15Rα-Fc chimeric fusion proteins with rIL-15, a compound widely used in preclinical studies.

[0018] However, for clinical use of cytokines, targeting is difficult when administered as a single agent, and high-concentration administration is required. However, they have the disadvantages of failing to achieve antitumor effects unless administered at high concentrations, resulting in immunosuppressive effects and high toxicity. Furthermore, immune system activation by non-targeted cytokines is gradual, resulting in widespread immune activation and potentially fatal side effects. Furthermore, cytokines are small proteins lacking the protective circulatory mechanisms of antibodies. Therefore, simple cytokines often have short half-lives, necessitating repeated administration of high doses over a short period of time. Currently, most clinically investigated drugs use PEGylation or Fc fusion to extend the half-life of cytokines. Although this has resulted in an extended half-life, the problem of insufficient cytokine targeting remains unresolved.

[0019] [Summary of the Invention] [Problem to be solved by the invention] [Means for solving the problem] A first aspect of the present invention provides a bispecific antibody comprising: a) a light chain and a heavy chain of a first antibody that specifically binds to a first antigen; and b) a light chain and a heavy chain of a second antibody that specifically binds to a second antigen, wherein the two chains in the first antibody are substituted with IL15 and IL15Rα, respectively (e.g., IL15 and IL15Rα replace CL1 and CH1, respectively), and the high affinity of IL15 and IL15Rα is utilized to form an IL15 / IL15Rα complex, thereby realizing correct pairing of the light chain / heavy chain of the first antibody and solving the problem of inaccurate matching of the light chain / heavy chain of the bispecific antibody. At the same time, mutations in the amino acid sequences of the variable domains VH1, VL1, IL15, and IL15Rα of the first antibody add one or more pairs of disulfide bonds between VH1 and VL1, and between IL15 and IL15Rα, further enhancing the binding activity between the light chain and heavy chain of the first antibody, thereby effectively overcoming the problem of inaccurate matching of the light chain and heavy chain of the bispecific antibody.

[0020] The bispecific antibody constructed in the present invention comprises a) a light chain and a heavy chain of a first antibody that specifically binds to a first antigen, and b) a light chain and a heavy chain of a second antibody that specifically binds to a second antigen, wherein the two chains in the first antibody comprise IL15 and IL15Rα, respectively, and are capable of forming an IL15 / IL15Rα complex.

[0021] In another preferred example, the IL15 comprises a mutant capable of binding to IL15Rα, and the IL15Rα comprises a mutant capable of binding to IL15. In another preferred example, the first antibody variable domains VH1 and VL1 are linked or polymerized, and the IL15 and IL15Rα are linked or polymerized.

[0022] In another preferred embodiment, said IL15 and IL15Rα replace the constant domains CH1 and CL of the first antibody. In another preferred example, the variable domains VL1 and VH1 of the first antibody are linked to the N-terminus of IL15 and IL15Rα.

[0023] In another preferred example, the variable domains VL1 and VH1 of the first antibody are linked to the C-terminus of IL15 and IL15Rα. In another preferred example, the variable domains VL1 and VH1 of said first antibody are swapped with each other.

[0024] In another preferred example, the first antibody light chain (from N-terminus to C-terminus) is polymerized in the same orientation as the first antibody heavy chain (from N-terminus to C-terminus). In another preferred example, the first antibody light chain (from C-terminus to N-terminus) is reverse polymerized with the first antibody heavy chain (from N-terminus to C-terminus).

[0025] In another preferred example, the variable domains VL1 and VH1 of the first antibody are linked to IL15 and IL15Rα via a low immunogenic amino acid linker sequence. In another preferred example, one or more pairs of disulfide bonds exist between the IL15 and IL15Rα.

[0026] In another preferred embodiment, the IL15 comprises the mutations shown below, and the counting method is counted from the first amino acid of IL15 shown in SEQ ID No. 1.

[0027] [Table 1] TIFF0007818838000002.tif78169

[0028] In another preferred example, the IL15 and IL15Rα comprise the combination of mutations shown below, and the counting method is as follows: counting from the first amino acid of IL15 shown in SEQ ID No. 1 and counting from the first amino acid of IL15Rα shown in SEQ ID No. 3.

[0029] [Table 2]

[0030] In another preferred example, there are one or more pairs of disulfide bonds between the variable domains VH1 and VL1 of the first antibody. In another preferred embodiment, the VH1 and VL1 comprise the following combinations of mutations based on EU numbering:

[0031] [Table 3]

[0032] In another preferred example, the first and second antibody heavy chains each comprise an A chain and a B chain having different mutations in the Fc segment, and the A chain and the B chain have the following combinations of mutations based on EU numbering:

[0033] [Table 4] TIFF0007818838000006.tif148168

[0034] In another preferred embodiment, the Fc segment is human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc and mutants thereof.

[0035] In another preferred example, one chain of the A and B chains of the Fc segment is capable of binding to Protein A, and the other chain is a mutant that is unable to bind to Protein A, and the mutation includes H435R or H435R / Y436F based on EU numbering.

[0036] In another preferred embodiment, the first antigen is CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD217, CD239, CD274, CD276, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, c-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, or NKG2D. ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, IL-6R, IL-2R, CCR4, VEGFR-2, CD6, CTLA-4, integrin α4, DNA / histone complex, PDGFRα, NeuGcGM3, IL-4Rα, IL-6Rα wherein the second antigen is a different epitope of the first antigen or another antigen as described above.

[0037] In another preferred embodiment, the first / second antibody is a chimeric, humanized or fully human antibody. In another preferred embodiment, the bispecific antibody has the structure shown in Formula I:

[0038] [ka]

[0039] where: Chain 1: VL1 or VH1 is linked via L1 to the N-terminus or C-terminus of IL15 or IL15Rα; Chain 2: arranged from the N-terminus to C in the following order: VH1 or VL1, L2, IL15Rα or IL15, L3, Fc; Chain 3: The second antibody heavy chain, arranged from N-terminus to C-terminus in the following order: VH2-CH1-Fc; Chain 4: The second antibody light chain, arranged from N-terminus to C-terminus in the following order: VL2-CL; "-" represents a peptide bond; L1, L2, and L3 are each independently a bond or linker sequence; In another preferred embodiment, the bispecific antibody has the structure shown in formula II:

[0040] [ka]

[0041] where: Chain 1: IL15 or IL15Rα is linked via L1 to the N-terminus or C-terminus of VL1 or VH1; Chain 2: arranged from the N-terminus to the C-terminus in the following order: IL15Rα or IL15, L2, VH1 or VL1, L3, Fc; Chain 3: The second antibody heavy chain, arranged from N-terminus to C-terminus in the following order: VH2-CH1-Fc; Chain 4: The second antibody light chain, arranged from N-terminus to C-terminus in the following order: VL2-CL; "-" represents a peptide bond; L1, L2 and L3 are each independently a bond or linker sequence.

[0042] In another preferred example, the above chain 1 and chain 2 comprise the combinations shown in the table below.

[0043] [Table 5] TIFF0007818838000010.tif206169

[0044] In another preferred embodiment, the bispecific antibody has the structure shown in formula III:

[0045] [ka]

[0046] where: Chain 1: arranged from the N-terminus to the C-terminus, VL1-L1-IL15, where L1 is a low immunogenic amino acid linker sequence; Chain 2: VH1-L2-IL15Rα-L3-Fc, arranged from the N-terminus to the C-terminus, where L2 and L3 are amino acid linker sequences with low immunogenicity; Chain 3: arranged from N-terminus to C-terminus in the order VH2-CH1-Fc; Chain 4: arranged from N-terminus to C-terminus in the order VL2-CL; "-" represents a peptide bond.

[0047] In another preferred example, L1, L2 and L3 comprise glycine (G) and serine (S) residues. In another preferred example, said L1, L2 and L3 comprise one or more GGGGS repeats.

[0048] In another preferred embodiment, the IL15 sequence is SEQ ID No. 1 or SEQ The IL15Rα sequence is as set forth in SEQ ID No. 2, and the IL15Rα sequence is as set forth in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9.

[0049] In another preferred example, the Fc sequence is as set forth in SEQ ID No. 18 or SEQ ID No. 19. In another preferred example, the first antigen and the second antigen are antigens that bind to two different epitopes of Her2, respectively.

[0050] In another preferred embodiment, the bispecific antibody has SEQ ID No. 10, SEQ It is obtained by fusion of the sequences of SEQ ID No. 11, SEQ ID No. 13 and SEQ ID No. 12.

[0051] In another preferred example, the first antigen and the second antigen are a CS1 antigen and a CD38 antigen, respectively. In another preferred embodiment, the bispecific antibody has SEQ ID No. 14, SEQ It is obtained by fusion of the sequences of SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No. 16.

[0052] A second aspect of the present invention is (a) a bispecific antibody according to the first aspect of the invention, and (b) a pharmaceutically acceptable carrier. do.

[0053] In another preferred embodiment, the pharmaceutical composition further comprises another drug for treating cancer (or tumor), such as a chemotherapeutic agent. A third aspect of the present invention provides the use of a bispecific antibody according to the first aspect of the invention and a pharmaceutical composition according to the second aspect of the invention in the preparation of a medicament for the treatment of cancer (or tumour), an infectious disease or an immunomodulatory disease.

[0054] A fourth aspect of the invention provides the use of a bispecific antibody according to the first aspect of the invention in the preparation of a medicament for inhibiting tumour growth. In another preferred example, the cancer or tumor comprises a cancer or tumor from a site such as colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, etc.

[0055] In another preferred example, the cancer (or tumor) comprises myeloid carcinoma, lymphoma, leukemia, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0056] A fifth aspect of the present invention provides the use of a bispecific antibody that binds a Her2 double epitope according to the first aspect of the invention in the preparation of a reagent or kit for diagnosing HER2-positive tumours (e.g. breast cancer, gastric cancer).

[0057] [Effects of the invention] Through experiments, the inventors of the present invention surprisingly discovered that a bispecific antibody introduces IL15 and IL15Rα (e.g., IL15 and IL15Rα replace CL1 and CH1, respectively) into the two chains of a first antibody, and utilizes the high affinity of IL15 and IL15Rα to form an IL15 / IL15Rα complex, thereby achieving correct pairing of the light chain / heavy chain of the first antibody and solving the problem of inaccurate matching of the light chain / heavy chain of the bispecific antibody. At the same time, mutations in the amino acid sequences of the variable domains VH1, VL1, IL15, and IL15Rα of the first antibody add one or more pairs of disulfide bonds between VH1 and VL1, and between IL15 and IL15Rα, further enhancing the binding activity between the light and heavy chains of the first antibody. This effectively overcomes issues during the bispecific antibody preparation process, such as incorrect light and heavy chain matching, large amounts of by-products, and poor stability, and ultimately allows for the preparation of a correctly paired bispecific multifunctional antibody targeting a cytokine. At the same time, this shortens the development cycle of bispecific antibodies and reduces production costs.

[0058] In another aspect, the bispecific antibody constructed in the present invention overcomes the problem of inaccurate light chain / heavy chain matching, and at the same time has IL-15 / IL-15Rα activity, can target cytokines to tumor sites, and specifically expand and activate T cells and NK cells in PMBCs at tumor sites, increasing the number of immune cells and the release of killer cytokines, thereby more effectively killing tumor cells and reducing the dosage.

[0059] In a third aspect, the present invention successfully constructs a correctly paired trastuzumab / pertuzumab / IL15 bispecific antibody and utilizes Her2 bispecific antibody targeting to target IL15 to tumor tissues, stimulate immune responses, and kill Her2-positive tumors through multiple mechanisms.

[0060] In a fourth aspect, the present invention successfully constructed a correctly paired elotuzumab / daratumumab / IL15 bispecific antibody that binds to the CS1 / CD38 antigen, which has the same CS1 antigen binding affinity as the CS1 monoclonal antibody, and at the same time, has the same CD38 antigen binding affinity as the CD38 monoclonal antibody, and has great potential in the treatment of hematological malignancies.

[0061] In order to more readily understand the present invention, certain technical and scientific terms are specifically defined below. Unless expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0062] term The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Boil. Chem., 243, p. 3558 (1968).

[0063] The "antibody" according to the present invention includes not only complete antibodies, but also fragments, polypeptide sequences, and derivatives and analogs thereof that have antigen-binding activity. The antigen-binding fragment refers to one or more portions of a full-length antibody, which retain the ability to bind to an antigen (e.g., HER2) and compete with the full antibody for specific binding to the antigen. Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), and chimeric antibodies comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding portions of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibody 2E12) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding portions of the antibody are screened for specificity in the same manner as for intact antibodies. The term "Fd fragment" refers to an antibody fragment composed of the VH and CH1 domains; the term "Fv fragment" refers to an antibody fragment composed of the VL and VH domains of a single arm of an antibody; and the term "dAb fragment" refers to an antibody fragment composed of the VH domain (Ward et al., Nature 1999, 144:111-114). 341:544-546 (1989)), the term "Fab fragment" refers to an antibody fragment composed of the VL, VH, CL and CH1 domains, and the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0064] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain essentially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative, or analog of the invention is (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted (such substituted amino acid residues may or may not be encoded by the genetic code), or (ii) a polypeptide having a substitution at one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a protein sequence, or a fusion protein formed with a 6His tag). Following the teachings herein, these fragments, derivatives and analogs are well within the purview of those skilled in the art.

[0065] As used herein, an "epitope" or "antigenic epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant." An epitope or antigenic determinant is usually composed of chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and usually has specific three-dimensional structural and charge characteristics. For example, an epitope usually contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique alternating structure, which may be "linear" or "conformational." See, for example, "Epitope Mapping Protocols in Methods in Molecular Biology," Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues of the protein that are separated from one another.

[0066] The "IL-15" referred to in the present invention may be any IL-15, such as human IL-15 or IL-15 from a non-human mammal or non-mammal, or a mutant capable of binding to IL15Rα. Examples include IL-15 from non-human mammals such as pigs, rabbits, monkeys, orangutans, and mice, and non-mammals such as chickens, preferably human IL-15. The term "mutant capable of binding to IL15Rα" refers to a mutant molecule obtained by substitution, addition, or deletion of one or more amino acids, which improves or decreases the affinity between IL-15 and its receptor, or increases or decreases its activity in stimulating T cells or NK cells. The "IL-15" referred to in the present invention is preferably in its mutant form, and more preferably has the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2.

[0067] The "IL-15Rα" referred to in the present invention may be IL-15Rα of any species, such as human IL-15Rα, non-human mammalian IL-15Rα, or non-mammalian IL-15Rα, or a mutant capable of binding to IL15Rα. Examples include non-human mammals such as pig, rabbit, monkey, orangutan, and mouse, and non-mammalian species such as chicken. A preferred example is human IL-15Rα, more preferably the extracellular domain fragment IL-15Rα of human IL-15Rα called ECD (UniProtKB database, accession number Q13261, 31-205aa). The term "mutant capable of binding to IL15Rα" refers to a functional mutant having the ability to bind to its ligand molecule, such as IL-15, formed by the deletion, insertion, or substitution mutation of one or more amino acids on IL-15Rα, and is preferably a human IL-15Rα molecule, more preferably a truncated form of the human IL-15Rα extracellular domain fragment, i.e., a molecule having human IL-15 receptor α activity, such as IL-15Rα (SEQ ID NO: 3-9), obtained by the deletion of one or more amino acids starting from the C-terminus of the extracellular domain fragment, preferably carrying a deletion mutant form of 65 to 178 amino acids.

[0068] The term "Fc segment" as used herein refers to the C-terminal region of an immunoglobulin that does not have antigen-binding activity. This is the site where an antibody molecule interacts with effector molecules and cells, and is a dimeric molecule comprising two disulfide-linked antibody heavy chain Fc region polypeptides. The Fc region can be produced in a trypsin-treated complete (full-length) antibody by papain digestion or IdeS digestion, or can be produced recombinantly. The "Fc portion" preferably comprises at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of an IgG.

[0069] Fc heterodimer mutation refers to a change in Fc structure or function caused by the substitution, insertion, or deletion of one or more amino acids at appropriate sites in the Fc. Mutationally engineered Fc variants can form space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, and other interactions. The interactions between Fc mutants contribute to the formation of stable heterodimers. Preferred mutations are designed as "knob-in-hole" mutations. Furthermore, the Fc described in the present invention can also contain other mutations that result in functional changes, such as glycosylation modification mutations, FcγR-binding region mutations (to adjust ADCC activity), and amino acid mutations that improve antibody stability. The Fc of the present invention includes human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, and human IgG4 Fc and their mutants, in which one chain can bind to protein A and the other chain is a mutant that cannot bind to protein A, and includes the mutations H435R or H435R / Y436F based on the EU numbering system.

[0070] The "heterodimer" described in the present invention is preferably a gene coexpression product. It is coexpressed in prokaryotic cells such as Escherichia coli or eukaryotic cells such as 293 and CHO. The term "coexpression" refers to the simultaneous expression of multiple genes in a cell, resulting in the simultaneous appearance of their products. These genes exist simultaneously and can be inhibited individually or together. In the present invention, coexpression in a single eukaryotic cell is preferred. Gene expression products obtained by coexpression favor the efficient and simple formation of complexes, which favors the formation of heterodimers in the present invention.

[0071] Mutational design techniques for Fc variants have been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion protein forms. Representative examples include the "Knob-in-Hole" morphology proposed by Cater et al. (Protein Engineering, vol. 9, no. 7, pp. 617-621, 1996); the formation of Fc-containing heterodimeric forms using electrostatic steering by Amgen engineers (US2010286374A1); the heterodimeric forms (SEED bodies) formed by IgG / IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection, pp. 1-8, 2010); bispecific molecules formed by Genmab's DuoBody (Science, 2007.317(5844)) platform technology; the formation of heterodimeric protein forms integrating different mechanisms of action by Xencor engineers integrating structural calculations and Fc amino acid mutations (mAbs3:6, 546-557; November / December 2011); and the formation of heterodimeric protein forms integrating different mechanisms of action by Suzhou ALPHAMAB. Obtaining heterodimeric protein forms includes ONCOLOGY Company's charge network-based Fc modification method (CN201110459100.7); as well as other genetic engineering methods based on Fc amino acid changes or functional modification to form heterodimeric functional proteins. The knob-in-hole structure of the Fc variant fragment described in this invention refers to the fact that the two Fc fragments are mutated separately and can be linked in a "knob-in-hole" fashion after mutation. Site-directed mutation modification of the Fc region is preferably performed using the "knob-in-hole" model proposed by Cater et al., so that the resulting first and second Fc variants can be combined via a "knob-in-hole" mechanism to form heterodimers. The selection of specific immunoglobulin Fc regions from specific immunoglobulin classes and subclasses is within the knowledge of those skilled in the art. Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are preferred, with human antibodies IgG1 and IgG4 being more preferred.One of the first Fc variant or the second Fc variant is randomly selected for a knob mutation and the other for a hole mutation, hi one embodiment, the first Fc variant is a knob mutation and the second Fc variant is a hole mutation.

[0072] The term "linker sequence" refers to one or more amino acid residues inserted into an immunoglobulin domain to provide sufficient mobility for the light and heavy chain domains to fold into the exchanged dual variable region immunoglobulin. In the present invention, a linker sequence is used to link IL-15 or IL-15Rα to the corresponding light or heavy chain to ensure correct protein folding and peptide stability. The "linking peptide" of the present invention is preferably a low immunogenic amino acid sequence, preferably (GGGGS)n, where n is 0, 1, 2, 3, 4, 5, or more, preferably 1 to 5.

[0073] The antibodies of the present invention may be used alone or may be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase)-modifying moiety, or any combination of these mentioned above.

[0074] Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product. [Brief explanation of the drawings]

[0075] [Figure 1] Structural form 1 of a bispecific antibody. [Figure 2] This is structural form 2 of a bispecific antibody. [Figure 3] Structural form 3 of a bispecific antibody. [Figure 4] Structural form 4 of a bispecific antibody. [Figure 5]Structural form of bispecific antibodies5. [Figure 6] Structural form of a bispecific antibody. [Figure 7] Structural form of a bispecific antibody. [Figure 8] Structural form of bispecific antibodies8. [Figure 9] Reduced and non-reduced bands of QP34563457 protein analyzed by SDS-PAGE electrophoresis. [Figure 10] HPLC-SEC analysis of QP34563457 protein purity. [Figure 11a] Peptide map analysis of QP34563457 protein. Figure 11a shows 36% sequence coverage of QP34563457 protein to the trastuzumab VL-IL15 chain (SEQ ID NO: 10). [Figure 11b] Peptide map analysis of QP34563457 protein. Figure 11b shows 56% sequence coverage of QP34563457 protein for trastuzumab VH-IL15Ra-Fc chain (SEQ ID NO:11). [Figure 11c] Peptide map analysis of QP34563457 protein. Figure 11c shows 64% sequence coverage of QP34563457 protein for pertuzumab VL-CL chain (SEQ ID NO: 12). [Figure 11d] Peptide map analysis of QP34563457 protein. Figure 11d shows 56% sequence coverage of QP34563457 protein for pertuzumab VH-CH1-Fc chain (SEQ ID NO:13). [Figure 12] ELISA detection of molecules such as QP34563457 bound to Her2-Fc fusion proteins. [Figure 13] ELISA detection of molecules such as QP34563457 bound to Her2M2-Fc fusion protein. [Figure 14]ELISA detection of molecules such as QP34623463 bound to the CD38 protein. [Figure 15] ELISA detection of molecules such as QP34623463 bound to CS1 protein. [Figure 16] FACS detection of molecules such as QP34563457 bound to SK-BR-3 cells. [Figure 17] FACS detection of molecules such as QP34563457 bound to BT-474 cells. [Figure 18] FACS detection of molecules such as QP34563457 bound to SK-OV-3 cells. [Figure 19] 1 shows growth inhibition curves of molecules such as QP34563457 against human breast cancer cells SK-BR-3. [Figure 20] 1 shows the growth inhibition curves of molecules such as QP34563457 against human breast cancer cells BT-474. [Figure 21] 1 shows the growth inhibition curves of molecules such as QP34563457 against human ovarian cancer cells SK-OV-3. [Figure 22] QP34563457 et al. mediated PBMC killing of human breast cancer cells SK-BR-3 cells. [Figure 23] QP34563457 et al. mediated PBMC killing of human breast cancer cell BT-474 cells. [Figure 24] QP34563457 et al. mediated PBMC killing of human ovarian cancer cells SK-OV-3 cells. [Figure 25] This is an experiment on molecules such as QP34563457 that detect proliferation in Mo7e cells. [Figure 26] 1 shows the tumor growth curve of the SK-OV-3 in vivo drug effect model. DETAILED DESCRIPTION OF THE INVENTION

[0076] In the following embodiments, experimental methods for which no specific conditions are given are generally performed according to conventional conditions or conditions or methods suggested by raw material or product manufacturers, such as molecular cloning, laboratory manuals, Cold Spring Harbor Laboratory, modern molecular biology techniques, cell biology, etc. Reagents for which no specific source is given are commercially available conventional reagents.

[0077] Example 1: Cloning and expression of fusion proteins The protein sequence is as follows: Anti-Her2 (Her2 extracellular domain IV region) VL-IL15SEQ ID NO:10 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGGSGGGGSG GGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Anti-Her2 (Her2 extracellular domain IV region) VH-IL15Ra-Fc (Knob) SEQ ID NO: 11 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti Her2′(Her2 extracellular domain II region) VL-CL SEQ ID NO:12 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC anti Her2′(Her2 extracellular domain II region) VH-CH1-CH2-CH3(Hole) SEQ ID NO:13 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK anti CS1 VL-IL15SEQ ID NO:14 DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS anti CS1 VH-IL15Rα-Fc(Knob) SEQ ID NO:15 EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti CD38 VL-CL SEQ ID NO:16 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC anti CD38 VH-CH1-CH2-CH3(Hole)SEQ ID NO:17 EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK Cloning and construction methods: The clone designs are shown in Table 1. Carriers encoding anti-Her2 (Tmab) VL-IL15 (SEQ ID NO: 10), anti-Her2 (Tmab) VH-IL15Ra-Fc (Knob) (SEQ ID NO: 11), anti-CS1 VL-IL15 (SEQ ID NO: 14), and anti-CS1 VH-IL15Ra-Fc (Knob) (SEQ ID NO: 15) were constructed, respectively. The plasmid contains DHFR as a screening marker and can be used to screen for stable clones. It also contains a carrier light chain sequence (SEQ ID NO: 12) encoding an anti-tumor-specific antigen antibody and a carrier heavy chain sequence encoding an anti-tumor-specific antigen antibody (wherein the Fc contains hole mutations, i.e., (T366S, L368A, Y407V) anti Her2' VL(Pmab)-CL (SEQ ID NO: 12), anti Her2' (Pmab) VH-CH1-CH2-CH3(Hole) (SEQ ID NO: 13), anti CD38 VL-CL (SEQ ID NO: 16), and anti CD38 VH-CH1-CH2-CH3(Hole) (SEQ ID NO: 17)). The plasmid also contains GS as a screening marker and can be used to screen for stable clones. A schematic diagram of the protein molecule structure is shown in Figure 6. In other embodiments, the protein molecule may be in other structural forms, as shown in Figures 2 to 8, respectively.

[0078] [Table 6]

[0079] table 2 Control antibodies trastuzumab, pertuzumab, elotuzumab, and daratumumab are designed and constructed as shown in .

[0080] [Table 7] TIFF0007818838000014.tif251169TIFF0007818838000015.tif243169TIFF0007818838000016.tif255169TIFF000 7818838000017.tif255168TIFF0007818838000018.tif254168TIFF0007818838000019.tif254169TIFF0007818838 000020.tif254169TIFF0007818838000021.tif254169TIFF0007818838000022.tif251168TIFF0007818838000023. tif253168TIFF0007818838000024.tif253169TIFF0007818838000025.tif254169TIFF0007818838000026.tif58169

[0081] Transient expression of target molecules Expi CHO-S cells were inoculated into Forti CHO medium (Gibco, A1148301) supplemented with 8 mM GlutaMax and cultured at 37°C, 120 rpm, and 8% CO2. The day before transfection, the cell density was adjusted to 3 x 10E6 / mL and cultured in a shaker at 37°C, 120 rpm, and 8% CO2. On the day of transfection, a sample was taken and counted, and the cell density was diluted to 6 x 10E6 / mL. 40 mL per flask was placed in a 125 mL shaker flask. 20 μg of the corresponding plasmid was added, mixed with 4.8 mL of Opti MEM, and 120 μL of Polyplus-FectoPRO transfection reagent was added. The DNA and transfection reagent were mixed evenly and allowed to stand at room temperature for 10 minutes. The mixture was then slowly added to the cells, mixed evenly, and cultured in a shaker. Add 2 mL of Feed PFF05 (OPM, F81279-001) and 1 mL of 30% glucose solution to each flask on days 1, 4, 6, and 8. On the first day of transfection, reduce the temperature to 32°C and the CO2 concentration to 5%. Collect samples on day 13, centrifuge at 8000 rpm for 20 minutes, and collect the supernatant for purification.

[0082] Example 2: Purification of fusion proteins Purification by Protein A affinity chromatography Pass at least 3 CV of equilibration solution through the column (20 ml actual volume). Ensure that the pH and conductivity of the solution exiting the final device match those of the equilibration solution. At a flow rate of 1 ml / min. Pass the supernatant of the culture medium through the column after centrifugation. Load 40 ml of sample at a flow rate of 0.33 ml / min. Pass at least 3 CV of equilibration solution through the column (20 ml actual volume). Ensure that the pH and conductivity of the solution exiting the final device match those of the equilibration solution. At a flow rate of 0.33 ml / min. Pass the eluent through the column. Start collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU. Stop collection when UV280 falls to 15 mAU. At a flow rate of 1 ml / min. After sample collection is complete, adjust the PAC-EP to neutral with a pH adjusting solution.

[0083] CH1-XL affinity chromatography The sample treated with Protein A was centrifuged at 8000 rpm for 15 minutes, the supernatant was removed, and at least 3 CV of equilibration solution was passed through the column. The actual volume was 20 ml. The pH and conductivity of the solution leaving the final device were the same as those of the equilibration solution, and the flow rate was 1 ml / min. The supernatant after centrifugation was passed through the sample loop and passed through the column at a flow rate of 0.33 ml / min. At least 3 CV of equilibration solution was passed through the column. The actual volume was 20 ml. The pH and conductivity of the solution leaving the final device were the same as those of the equilibration solution, and the flow rate was 1 ml / min. The flow rate was 0.33 ml / min, and the eluent was passed through the column. When UV280 rose to 10 mAU, collection of the elution peak (PAC-EP) began. When UV280 fell to 10 mAU, collection stopped. The flow rate was 1 ml / min. After sample collection was complete, the PAC-EP was adjusted to neutral with a pH adjusting solution.

[0084] PNGase F digestion A 39.5 μl sample was taken, 1.98 μl of 10% SDS and 1.58 μl of 1 M DTT were added, and after mixing evenly, the sample was boiled at 100°C for 10 minutes. After cooling, 4.8 μl of 10% NP-40 and 1.0 μl of PNGase F were added, and after mixing evenly, the sample was soaked at 4°C overnight. After overnight, the sample was heat inactivated at 75°C for 10 minutes, and then the sample was subjected to SDS-PAGE electrophoresis and SEC to detect the purity of the protein.

[0085] SDS-PAGE electrophoresis analysis and HPLC-SEC analysis Five micrograms of each sample was taken before and after digestion and diluted to 48 μl with 1x PBS. 12 μl of 5x Protein Loading Dye was added and heated at 95°C for 10 minutes. A pH 8.3 Tris-HCl buffer solution (containing 0.1% SDS) was poured into the electrophoresis chamber, and the samples were added to the sample chambers using a pipette gun, 60 μl of each sample chamber. The voltage was set to 140 V and the time to 100 minutes to initiate electrophoresis. After electrophoresis, the gel was removed, immersed in staining solution for 30 minutes, and destained in a destaining station until the protein bands became clearly visible. Figure 9 shows the reduced and non-reduced bands of QP34563457 protein analyzed by SDS-PAGE electrophoresis. Figure 10 shows an HPLC-SEC analysis of QP34563457 protein purity.

[0086] Example 3: Peptide map analysis of protein QP34563457 In this example, LC-MS is used to identify the amino acid sequence of the QP34563457 protein. After denaturation and reduction of the QP34563457 protein, trypsin was added for enzymatic digestion and then analyzed by LC-MS / MS (LC instrument: Agilent 1290 Infinity II, column: Agilent Peptide Plus, MS instrument: Agilent 6545 Q-TOF). The obtained data were searched using Peaks, and the results of the Peaks search were filtered by strict card value to obtain reliable peptide segments. The sequence coverage of the QP34563457 protein for the trastuzumab VL-IL15 chain was 36% (Figure 11a, gray indicates the identified amino acid sequence), the trastuzumab VH-IL15Ra-Fc chain was 56% (Figure 11b, gray indicates the identified amino acid sequence), the pertuzumab VL-CL chain was 64% (Figure 11c, gray indicates the identified amino acid sequence), and the pertuzumab VL-CL chain was 64% (Figure 11c, gray indicates the identified amino acid sequence). Achieving 56% sequence coverage for the VH-CH1-Fc chain (Figure 11d, gray indicates identified amino acid sequences). LC-MS peptide mapping analysis revealed each fragment within the QP34563457 molecule, including trastuzumab VL, IL15, trastuzumab VH, IL15Ra, Fc, pertuzumab VL, CL, pertuzumab VH, CH1, Fc, etc.

[0087] Example 4: ELISA detection of QP34563457 binding to Her2 and its mutant activity Design of a Her2 mutant protein that binds only to Pertuzumab. Matthew C. Franklin published the complex structure of Pertuzumab Fab and Her2 extracellular structure in Cancer Cell. The team used alanine scanning to investigate which key amino acids in Her2 affect binding to Pertuzumab Fab. The Her2 mutant protein that binds only to Pertuzumab but not to trastuzumab was named Her2M. 2 (protein number QP3732). The amino acid sequences of Her2 wt (protein number QP3731) and Her2M2 (protein number QP3732) are as follows:

[0088] QP3731:(Her2 ECD-Fc) (SEQ ID NO:32) MEFGLSWLFLVAILKGVQCTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* QP3732(Her2M2-Fc): (SEQ ID NO:33) MEFGLSWLFLVAILKGVQCTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPAFcVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* Materials: Milk (BD, 232100), PBS (SANGON BIOTECH, B548117-0500); HRP-anti-human IgG (H+L) (Jackson, 109-035-088); TMB (Luoyang BAIAOTONG Experimental Materials Center, C060201); Elisa plate (Costa, 9018). 1x PBS buffer: Weigh and dissolve 8.00 g NaCl, 0.20 g KCl, 2.9 g NaHPO·12H2O, and 0.2 g KH2PO4 in 800 mL of ddH2O. After dissolving thoroughly, weigh the solution to 1 L, adjust the pH to 7.4, sterilize at high temperature, and store. Alternatively, purchase commercially available 10x or 20x PBS solutions and dilute them into 1x PBS buffer. Blocking solution: Weigh 5g of milk into PBS to prepare the blocking solution and use immediately. Stop solution (1mol / L H2SO4): Take 109mL of 98% concentrated H2SO4 and slowly add dropwise to 2000mL of ddH2O. Allow TMB to develop for 10 minutes at 37°C, place on a shaker (120rpm), 100µl / well.

[0089] Experimental procedure: Her2 and its mutant proteins QP3731 and QP3732 were coated at 1µg / ml overnight at 4°C, then washed three times with PBS. 200µL / well of 5% milk blocking solution was added and incubated at 37°C for 1 hour. After blocking, the plates were washed three times with PBS. The samples were then incubated and diluted 5-fold at 20µg / ml for a total of seven dilutions, with the final well being diluted 100-fold. 100µl was added per well, mixed thoroughly, and incubated for 1 hour. Three washes were performed with PBST. Enzyme-labeled antibody was added: HRP-anti-human Fab antibody was added at a dilution of 1:5000, 100µl per well, mixed thoroughly, incubated for 1 hour, and washed six times with PBST. Substrate development solution was added: 100µL / well of TMB substrate development solution was added, and the plates were placed on a shaker at 200 rpm for 10 minutes in the dark at 35°C.

[0090] Termination: After the color development is completed, stop solution is added quickly at a volume of 100 μL / well to terminate the reaction. Detection: Measure the OD value at A450 nm using a microplate reader, and analyze the results using graphpad prism software.

[0091] Experimental results: Trastuzumab can bind very well to Her2 wt but does not bind to Her2M2 at all, while QP34563457 and Pertuzumab can all bind very well to Her2 wt and Her2M2 (results are shown in Figures 12 and 13).

[0092] Example 5: ELISA detection of binding of QP34623463 to CD38 and CS1 activity Materials: Milk (BD, 232100), PBS (SANGON BIOTECH, B548117-0500); HRP-anti-human IgG (H+L) (Jackson, 109-035-088); TMB (Luoyang BAIAOTONG Experimental Materials Center, C060201); Elisa plate (Costa, 9018). 1x PBS buffer: Weigh out 8.00 g NaCl, 0.20 g KCl, 2.9 g NaHPO·12H2O, and 0.2 g KHPO and dissolve in 800 mL of ddH2O. After dissolving thoroughly, weigh out the solution to 1 L, adjust the pH to 7.4, sterilize at high temperature, and store. Alternatively, purchase commercially available 10x or 20x PBS solutions and dilute them into 1x PBS buffer. Blocking solution: Weigh out 5 g of milk and add it to PBS to prepare the blocking solution for immediate use. Stop solution (1 mol / L H2SO4): Take 109 mL of 98% concentrated H2SO4 and slowly add dropwise to 2000 mL of ddH2O. Allow TMB to develop for 10 minutes at 37°C, 100 μl / well, and place on a shaker (120 rpm).

[0093] Experimental procedure: CD38-Fc and CS1-Fc fusion proteins were coated at 1µg / ml each overnight at 4°C, then washed three times with PBS. 200µL / well of 5% milk blocking solution was added and incubated at 37°C for 1 hour. After blocking, the plates were washed three times with PBS. The samples were then incubated and diluted 5x at 20µg / ml for a total of seven dilutions, with the final well being diluted 100x. 100µl was added per well, mixed thoroughly, and incubated for 1 hour. Three washes were performed with PBST. Addition of enzyme-labeled antibody: HRP-anti-human Fab antibody was added at a dilution of 1:5000, 100µl per well, mixed thoroughly, incubated for 1 hour, and washed six times with PBST. Addition of substrate developer: 100µL / well of TMB substrate developer solution was added, and the plates were placed on a shaker at 200 rpm for 10 minutes in the dark at 35°C. Termination: After the color development is completed, stop solution is added quickly at a volume of 100 μL / well to terminate the reaction. Detection: Measure the OD value at A450 nm using a microplate reader, and analyze the results using graphpad prism software.

[0094] Experimental results: QP34623463 can bind not only to CD38 antigen but also to CS1 antigen (as shown in Figures 14 and 15). Example 6: FACS detection of binding of QP34563457 to Her2-high expressing cells Experimental materials: BT474 cells (human breast cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. SK-BR-3 cells (human breast cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. SK-OV-3 cells (human ovarian cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0095] Experimental Method: Experimental process: BT474 cells were cultured in RPMI1640 (Gibco) medium containing 10% FBS, 0.11 g / L sodium pyruvate, and 2.5 g / L glucose; SK-BR-3 cells were cultured in DMEM (Gibco) medium containing 10% FBS; and SK-OV-3 cells were cultured in McCoy's 5a medium (Gibco) containing 10% FBS. The cells were cultured at 37°C in a 5% CO2 incubator, harvested by trypsin digestion, and seeded into a 96-well plate at 100,000 cells per well. They were blocked with 2% FBS / PBS on ice for 1 hour, incubated with different concentrations of protein QP34563457 and a control protein, placed on ice for 1 hour, washed three times with PBS, incubated with a 1:200 dilution of PE-anti-human Fc, washed three times with PBS, resuspended in 200 μL of PBS, and the mean fluorescence value was measured by FACS. The results were analyzed using GraphPad Prism software.

[0096] Experimental results: QP34563457 was able to bind very well to the Her2-high expressing cells BT474, SK-BR-3, and SK-OV-3, with binding capacities close to those of the positive control monoclonal antibodies trastuzumab and pertuzumab, whereas the negative control antibodies were unable to bind to the Her2-high expressing cells (as shown in Figures 16-18).

[0097] Example 7: Growth inhibition mediated by Her2 bispecific antibodies Experimental objective: To confirm the inhibitory effect of Her2 bispecific antibody on tumor cell proliferation Experimental materials: BT474 cells (human breast cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. SK-BR-3 cells (human breast cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. SK-OV-3 cells (human ovarian cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The Cell Proliferation and Toxicity Detection Kit (CCK-8) was purchased from MEILUNBio (catalog number MA0218), human IgG was purchased from Sigma (catalog number I4506), and other antibodies were prepared in-house.

[0098] Experimental process: BT474 cells were cultured in RPMI1640 (Gibco) medium containing 10% FBS, 0.11 g / L sodium pyruvate, and 2.5 g / L glucose; SK-BR-3 cells were cultured in DMEM (Gibco) medium containing 10% FBS; and SK-OV-3 cells were cultured in McCoy's 5a medium (Gibco) containing 10% FBS. After incubation in a 37°C, 5% CO2 incubator, cells were harvested by trypsin digestion, centrifuged, and resuspended in medium containing 1% FBS. 10,000 cells were seeded into a 96-well plate in 50 μl per well and cultured for 3 hours at 37°C. Each antibody was diluted 3-fold with medium, 50 μl per well was mixed evenly with the cell suspension, and cultured in a 37°C, 5% CO2 incubator for 3 days. 10 μl of CCK-8 reagent was added to each well of the 96-well plate and incubated in a 37°C, 5% CO2 incubator for 2 hours. The 96-well plate was then removed and the absorbance at 450 nm was measured using a microplate reader. Cell viability values ​​were calculated and plotted against the logarithm of the sample concentration. Results were analyzed using GraphPad Prism software. A four-parameter cell growth inhibition curve was fitted.

[0099] Experimental results: QP34563457 exhibited significant inhibitory effects on the proliferation of HER2-positive tumor cells BT474, SK-BR-3, and SK-OV-3, which were superior (synergistic) to the monoclonal antibodies trastuzumab or pertuzumab and their combination (as shown in Figures 19-21).

[0100] Example 8: ADCC effect mediated by HER2 bispecific antibodies Experimental objective: To confirm the ADCC effect of HER2 bispecific antibodies. Materials: PBMCs were purchased from Shanghai SAILYBio Co., Ltd.; BT474 cells (human breast cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; SK-BR-3 cells (human breast cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; SK-OV-3 cells (human ovarian cancer cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; and the Cytotox96 non-radioactive cytotoxicity assay detection kit was purchased from Promega (G1780).

[0101] Experimental Process: Resuscitate PBMCs, collect the cells, and stockpile them for use the next day. Target Cell Preparation: Digest SK-BR-3, BT-474, and SK-OV-3 cells with trypsin and process at 1000 rpm for 5 minutes. After washing twice with PBS, plate 20,000 cells per well in 50 μl of solution per well into a 96-well plate and incubate at 37°C for 2 hours in 5% CO2. Antibody Preparation: Serially dilute 10 concentrations of antibodies (80 μg / ml to 0.000512 μg / ml, 0 μg / ml) 1:4 in medium (RPMI 1640 containing 10% low-IgG FBS). Add 50 μl of each diluted antibody concentration to each well and incubate at 37°C for 15 minutes. PBMC Preparation: Centrifuge the resuscitated PBMCs on the first day, resuspend them in medium (RPMI 1640 containing 10% low-IgG FBS), and count them. Add 50 μl per well to the above well plate at a PBMC:target cell ratio of 30:1 and incubate at 37°C for 4 hours. LDH detection: For the maximum release and volume correction groups, add 10 μl of eluent 45 minutes before incubation and continue culturing in the incubator. After 4 hours of incubation, transfer 50 μl of supernatant to a microtiter plate and add 50 μl of reagent according to the Cytotox96 non-radioactive cytotoxicity assay detection kit (Promega, G1780) instructions. After 30 minutes of incubation at room temperature in the dark, add 50 μl of stop solution. Read the absorbance at 490 nm (reading should be completed within 1 hour after adding the stop solution). Calculation: Killing rate = (sample release - spontaneous release of target cells - spontaneous release of effector cells) / (maximum release of target cells - spontaneous release of target cells) x 100. Spontaneous release (relative to target cells incubated with effector cells in the absence of antibody) The maximum release (target cells eluted with 1% Triton X-100) is defined as 0% cytotoxicity, and the maximum release (target cells eluted with 1% Triton X-100) is defined as 100% cytotoxicity.

[0102] Experimental Results: QP34563457 maintained comparable ADCC activity against BT474, SK-BR-3, and SK-OV-3 tumor cells compared to the two HER2 monoclonal antibodies (as shown in Figures 22-24).

[0103] Example 9: Mo7e cell proliferation experiment Experimental materials: Mo7e cells (human giant cell leukemia cell line) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; Cell Proliferation and Toxicity Detection Kit (CCK-8) was purchased from MEILUNBio, catalog number MA0218; recombinant human GM-CSF was purchased from perprotech, catalog number 300-03; human IgG was purchased from Sigma, catalog number I4506; and other antibodies were prepared by ourselves.

[0104] Experimental process: Mo7e cells were cultured in a 5% CO2 incubator at 37°C using RPMI1640 medium containing 10% FBS, 2 mM L-glutamine, and 8 ng / ml GM-CSF. Mo7e cells were collected and centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, the cells were washed twice with RPMI1640 medium without GM-CSF, the cells were resuspended in RPMI1640 medium without GM-CSF, and counted. The cells were then plated at 2 × 10 in a 96-well plate. 4 Each well was inoculated with 80 μl of the antibody solution and incubated at 37°C in a 5% CO2 incubator for 1 hour. Each antibody to be tested was diluted 4-fold with culture medium and then uniformly mixed with the cell suspension at 20 μl per well. The mixture was incubated at 37°C in a 5% CO2 incubator for 3 days. 10 μl of CCK-8 reagent was added to each well of the 96-well plate to be tested and incubated at 37°C in a 5% CO2 incubator for 4 hours. The 96-well plate was then removed and the absorbance at 450 nm was measured using a microplate reader.

[0105] Experimental Results: Mo7e cell proliferation experiments confirmed that Mo7e cells had adequate IL15 activity (as shown in Figure 25). Example 10: Animal drug effect experiments The inhibitory effect of QP34563457 on tumor growth will be evaluated in the HER2-high-expressing human ovarian cancer cell SK-OV-3 model.

[0106] Experimental process: SK-OV-3 cells were cultured in McCoy's 5A + 10% FBS medium containing 10% fetal bovine serum. SK-OV-3 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for inoculation. 1 x 10 cells were inoculated into the right dorsal region of each BALB / C nude mouse. 6 SK-OV-3 cells were subcutaneously inoculated, and tumor growth was monitored periodically. 3 When the tumors reached a volume of 10 μg, the mice were randomly divided into groups according to tumor size and body weight and administered (the administration volume was 10 μg / g body weight), and the first administration day was defined as D0. The administration schedule was as follows:

[0107] [Table 8]

[0108] The body weight and tumor volume of the mice were observed and recorded at each administration, and the long and short diameters of the tumor were measured with a caliper to calculate the tumor volume (mm 3 )=0.5×(a×b 2 The tumor growth status is recorded and a tumor growth curve is plotted. Six days after the final administration, observations are made and experimental data are recorded.

[0109] Experimental Results: The results showed that tastuzumab, pertuzumab, trastuzumab + pertuzumab, and QP34563457 were all able to significantly inhibit the growth of SK-OV-3 tumors, and tumors showed regression after six doses (TGItv>100%), demonstrating that QP34563457 still has significant tumor growth inhibitory activity in the huPBMC-graft NCG mouse model at a lower dose (10% of the monoclonal antibody dose) (shown in Figure 26 and Table 4).

[0110] [Table 9]

[0111] Although specific embodiments of the present invention have been described in detail, it should be understood that those skilled in the art, based on all the teachings disclosed, can make various modifications and substitutions to those details, and all of these modifications are within the scope of the present invention, the full scope of which is given by the appended claims and their equivalents.

Claims

1. A bispecific antibody, a) a first antibody that specifically binds to a first antigen; b) a second antibody that specifically binds to a second antigen; the first antibody comprises two polypeptides, chain 1 and chain 2, the second antibody comprises two polypeptides, chain 3 and chain 4, chain 3 is a heavy chain of the second antibody, chain 4 is a light chain of the second antibody, and the first antibody and the second antibody polymerize via chain 2 and chain 3; wherein the first antibody comprises IL15 and IL15Rα, the IL15 and IL15Rα being located in two polypeptides of the first antibody, respectively, and capable of forming an IL15 / IL15Rα complex; The IL15 is a wild-type IL15 or a mutant capable of binding to IL15Rα, and the IL15Rα is a wild-type IL15Rα or a mutant capable of binding to IL15, There are one or more pairs of disulfide bonds between the IL15 and the IL15Rα, The IL15 comprises the following mutations, counting from the first amino acid of IL15 shown in SEQ ID No. 1: N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q / Q108E, or N4D / D61N / E64Q / Q108E, Alternatively, the IL15 and the IL15Rα comprise the following combination of mutations, and the counting method is counting from the first amino acid of IL15 shown in SEQ ID No. 1 to the first amino acid of IL15Rα shown in SEQ ID No. 3; Combination 1: IL-15 has a V49C mutation and IL15Rα has a S40C mutation; Combination 2: IL-15 has the L52C mutation and IL15Rα has the S40C mutation; Combination 3: IL-15 has the E89C mutation and IL15Rα has the K34C mutation; Combination 4: IL-15 has a Q48C mutation and IL15Rα has a G38C mutation; Combination 5: IL-15 has the E53C mutation and IL15Rα has the L42C mutation; Combination 6: IL-15 has a C42S mutation and IL15Rα has an A37C mutation; Combination 7: IL-15 has an L45C mutation and IL15Rα has a G38C mutation; or Combination 8: IL-15 has an L45C mutation and IL15Rα has an A37C mutation; The bispecific antibody,

2. The chain 2 of the first antibody and the chain 3 of the second antibody each comprise an Fc segment, and the Fc segment is a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc. The bispecific antibody of claim 1.

3. The Fc segments of the first and second antibodies comprise A and B chains with different mutations, which promote interaction of the A and B chains to form a heterodimer, and the A and B chains are characterized in that they have the following combinations of mutations or sequences based on EU numbering: Combination 1: Chain A has a T366Y mutation and Chain B has a Y407T mutation; Combination 2: Chain A has the T366W mutation and Chain B has the T366S / L368A / Y407V mutation; Combination 3: Chain A has S354C / T366W mutations and Chain B has Y349C / T366S / L368A / Y407V mutations; Combination 4: Chain A has S364H / F405A mutations and Chain B has Y349T / T394F mutations; Combination 5: Chain A has T350V / L351Y / F405A / Y407V mutations and Chain B has T350V / T366L / K392L / T394W mutations; Combination 6: Chain A has K392D / K409D mutations and Chain B has E356K / D399K mutations; Combination 7: Chain A has D221E / P228E / L368E mutations and Chain B has D221R / P228R / K409R mutations; Combination 8: Chain A has K360E / K409W mutations and Chain B has Q347R / D399V / F405T mutations; Combination 9: Chain A has K360E / K409W / Y349C mutations and Chain B has Q347R / D399V / F405T / S354C mutations; Combination 10: Chain A has K370E / K409W mutations and Chain B has E357N / D399V / F405T mutations; Combination 11: Chain A has the F405L mutation and Chain B has the K409R mutation; Combination 12: Chain A has K360D / D399M / Y407A mutations and Chain B has E345R / Q347R / T366V / K409V mutations; Combination 13: Y349S / K370Y / T366M / K409V mutations in chain A and E356G / E357D / S364Q / Y407A mutations in chain B; Combination 14: Chain A has the L351D / L368E mutations and Chain B has the L351K / T366K mutations; Combination 15: the amino acid sequence of chain A is shown in SEQ ID No. 20 and the amino acid sequence of chain B is shown in SEQ ID No. 21; Combination 16: Chain A has the L368D / K370S mutation and Chain B has the E357Q / S364K mutation; Combination 17: chain A has S354C / T366W / K409A mutations and chain B has Y349C / T366S / L368A / Y407V / F405K mutations; Combination 18: chain A has S354C / T366W / F405K / K360E / Q347E mutations and chain B has Y349C / T366S / L368A / Y407V / Q347R / T394W mutations; Combination 19: Chain A has T366W / K409A mutations and Chain B has T366S / L368G / Y407A / F405K mutations; Combination 20: Chain A has T366W / F405K mutations and Chain B has T366S / L368G / Y407A / K409A mutations; Combination 21: Chain A has Q347A / S364K / T366V / K370T / K392Y / F405S / Y407V / K409W / T411N mutations and Chain B has Q347E / Y349A / L351F / S364T / T366V / K370T / T394D / V397L / D399E / D401Q / F405A / Y407S / K409R / T411R mutations; or Combination 22: K274Q / N276K / Y300F / A339T / Q347A / S364K / T366V / K370T / N384S / K392Y / V397M / F405S / Y407V / K409W / T411N / V422I / H435R / Y436F mutations in chain A and Q347E / Y349A / L351F / S364T / T366V / K370T / T394D / V397L / D399E / D401Q / F405A / Y407S / K409R / T411R mutations in chain B; The bispecific antibody of claim 2.

4. In the A and B chains of the Fc segment, one chain is capable of binding to Protein A, and the other chain is a mutant that cannot bind to Protein A, and the mutation includes H435R or H435R / Y436F based on EU numbering. The bispecific antibody of claim 3.

5. having the structure shown in Formula I or Formula II: 【Chemistry 1】 where: Chain 1: VL1 or VH1 is linked via L1 to the N-terminus or C-terminus of IL15 or IL15Rα; Chain 2: arranged from N-terminus to C in the following order: VH1 or VL1, L2, IL15Rα or IL15, L3, Fc; Chain 3: The second antibody heavy chain, arranged from N-terminus to C-terminus in the following order: VH2-CH1-Fc; Chain 4: The second antibody light chain, arranged from N-terminus to C-terminus in the order VL2-CL; VH1, VL1 represent the variable domains of the first antibody; VH2, VL2 represent the variable domains of the second antibody; CH1, CL represent the constant domains of the second antibody; "-" represents a peptide bond; L1, L2, and L3 are each independently a bond or a linker sequence; the Fc in chain 2 and chain 3 is an A chain or a B chain with different mutations, which promotes interaction of the A chain and the B chain to form a heterodimer; 【Chemistry 2】 where: Chain 1: IL15 or IL15Rα is linked via L1 to the N-terminus or C-terminus of VL1 or VH1; Chain 2: arranged from N-terminus to C-terminus in the following order: IL15Rα or IL15, L2, VH1 or VL1, L3, Fc; Chain 3: The second antibody heavy chain, arranged from N-terminus to C-terminus in the following order: VH2-CH1-Fc; Chain 4: The second antibody light chain, arranged from N-terminus to C-terminus in the order VL2-CL; VH1, VL1 represent the variable domains of the first antibody; VH2, VL2 represent the variable domains of the second antibody; CH1, CL represent the constant domains of the second antibody; "-" represents a peptide bond; L1, L2, and L3 are each independently a bond or a linker sequence; The Fc in chain 2 and chain 3 is an A chain or a B chain with different mutations, which promotes the interaction of the A chain and the B chain to form a heterodimer. The bispecific antibody according to any one of claims 1 to 4.

6. The strand 1 and strand 2 include the combinations in the following table: Combination 1: chain 1 has the structure VL1-L1-IL15 and chain 2 has the structure VH1-L2-IL15Ra-L3-Fc; Combination 2: chain 1 has the structure IL15-L1-VL1 and chain 2 has the structure VH1-L2-IL15Ra-L3-Fc; Combination 3: chain 1 has the structure VL1-L1-IL15 and chain 2 has the structure IL15Ra-L2-VH1-L3-Fc; Combination 4: chain 1 has the structure IL15-L1-VL1 and chain 2 has the structure IL15Ra-L2-VH1-L3-Fc; Combination 5: chain 1 has the structure VH1-L1-IL15 and chain 2 has the structure VL1-L2-IL15Ra-L3-Fc; Combination 6: chain 1 has the structure IL15-L1-VH1 and chain 2 has the structure VL1-L2-IL15Ra-L3-Fc; Combination 7: chain 1 has the structure VH1-L1-IL15 and chain 2 has the structure IL15Ra-L2-VL1-L3-Fc; Combination 8: chain 1 has the structure IL15-L1-VH1 and chain 2 has the structure IL15Ra-L2-VL1-L3-Fc; Combination 9: chain 1 has the structure VL1-L1-IL15Ra and chain 2 has the structure VH1-L2-IL15-L3-Fc; Combination 10: chain 1 has the structure IL15Ra-L1-VL1 and chain 2 has the structure VH1-L2-IL15-L3-Fc; Combination 11: chain 1 has the structure VL1-L1-IL15Ra and chain 2 has the structure IL15-L2-VH1-L3-Fc; Combination 12: chain 1 has the structure IL15Ra-L1-VL1 and chain 2 has the structure IL15-L2-VH1-L3-Fc; Combination 13: chain 1 has the structure VH1-L1-IL15Ra and chain 2 has the structure VL1-L2-IL15-L3-Fc; Combination 14: chain 1 has the structure IL15Ra-L1-VH1 and chain 2 has the structure VL1-L2-IL15-L3-Fc; Combination 15: chain 1 has the structure VH1-L1-IL15Ra and chain 2 has the structure IL15-L2-VL1-L3-Fc; or Combination 16: Chain 1 has the structure IL15Ra-L1-VH1 and Chain 2 has the structure IL15-L2-VL1-L3-Fc; The Fc is an Fc-A chain or an Fc-B chain. The bispecific antibody of claim 5.

7. Its configuration from N-terminus to C-terminus has the structure shown in Formula III: 【Transformation 3】 where: VH1, VL1 represent the variable domains of the first antibody; VH2, VL2 represent the variable domains of the second antibody; CH1, CL represent the constant domains of the second antibody; "-" represents a peptide bond; L1, L2, and L3 are each independently an amino acid linker sequence with low immunogenicity; The Fc in chain 2 and chain 3 is an A chain or a B chain with different mutations, which promotes the interaction of the A chain and the B chain to form a heterodimer. The bispecific antibody of claim 5.

8. The first antibody has one or more pairs of disulfide bonds between its variable domains VH1 and VL1, and the VH1 and VL1 contain the following combinations of mutations based on EU numbering: Combination 1: 37C in VH1 and 95C in VL1; Combination 2: 44C in VH1 and 100C in VL1; Combination 3: 44C in VH1 and 105C in VL1; Combination 4: 45C in VH1 and 87C in VL1; Combination 5: 100C in VH1 and 50C in VL1; Combination 6: 100C in VH1 and 49C in VL1; Combination 7: 98C in VH1 and 46C in VL1; Combination 8: 101C in VH1 and 46C in VL1; Combination 9: 105C in VH1 and 43C in VL1; or Combination 10: 106C in VH1, 57C in VL1, The bispecific antibody according to any one of claims 1 to 7.

9. The first antigen is selected from the group consisting of CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD217, CD239, CD274, CD276, CS1, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDGFR2, FVIII, C-MET, EGFR, EGFR, SCA, ephA2, ADAM17, 17-A1, and NKG2D. ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, IL-6R, IL-2R, CCR4, VEGFR-2, CD6, CTLA-4, integrin α4, DNA / histone complex, PDGFRα, NeuGcGM3, IL-4Rα, or IL-6Rα, and the second antigen is a different epitope of the first antigen, or the above-mentioned other antigen different from the first antigen. The bispecific antibody of claim 1.

10. The first antibody and / or the second antibody is a chimeric, humanized, or fully human antibody. The bispecific antibody of claim 1.

11. The IL15 sequence is as shown in SEQ ID No. 1 or SEQ ID No. 2, and the IL15Rα sequence is as shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, or SEQ ID No.

9. The bispecific antibody of claim 1.

12. The sequence of the A chain is as shown in SEQ ID No. 18, and the sequence of the B chain is as shown in SEQ ID No.

19. The bispecific antibody according to any one of claims 4 to 6.

13. the first antigen and the second antigen are antigens that bind to two different epitopes of Her2, respectively; The bispecific antibody is obtained by fusion of the sequences SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13 and SEQ ID No. 12, wherein the sequence of strand 1 is as set forth in SEQ ID No. 10, the sequence of strand 2 is as set forth in SEQ ID No. 11, the sequence of strand 3 is as set forth in SEQ ID No. 13, and the sequence of strand 4 is as set forth in SEQ ID No.

12. The bispecific antibody of claim 1.

14. the first antigen and the second antigen are a CS1 antigen and a CD38 antigen, respectively; The bispecific antibody is obtained by fusion of the sequences SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No. 16, wherein the sequence of strand 1 is as set forth in SEQ ID No. 14, the sequence of strand 2 is as set forth in SEQ ID No. 15, the sequence of strand 3 is as set forth in SEQ ID No. 17, and the sequence of strand 4 is as set forth in SEQ ID No.

16. The bispecific antibody of claim 1.

15. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (a) a bispecific antibody according to any one of claims 1 to 14; and (b) a pharmaceutically acceptable carrier.

16. 15. Use of a bispecific antibody according to claim 13 or claim 14 in the preparation of a medicament for treating cancer or a tumor, an infectious disease or an immunomodulatory disease, or for inhibiting tumor growth.

17. The cancer or tumor comprises colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal carcinoma, testicular cancer, bone marrow cancer, lymphoma, leukemia, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

17. The bispecific antibody of claim 16.

18. 14. Use of the bispecific antibody of claim 13 in the preparation of a reagent or kit for diagnosing Her2-positive tumors, characterized in that the Her2-positive tumors include breast cancer, ovarian cancer, and gastric cancer.

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