Anti-CD3 antibody variants, fusion proteins and applications

Abstract

Anti-CD3 antibody variants, fusion proteins and applications are provided. Anti-CD3 antibodies are mutated to obtain variants thereof. The fusion proteins provided include (1) anti-tumor associated antigen (TAA) / anti-CD3 bispecific antibodies, (2) anti-tumor associated antigen (TAA), anti-CD3 and IL15 / IL15Ra-containing multifunctional fusion proteins, and (3) anti-tumor associated antigen (TAA) and IL15 / IL15Ra-containing fusion proteins. Nucleic acid molecules and vectors encoding the antibody molecules are further provided, as well as pharmaceutical uses of the antibody molecules.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical technology, specifically to anti-CD3 antibody variants, fusion proteins and applications. [Background technology]

[0002] T lymphocytes, also known as T cells, are lymphoid stem cells derived from bone marrow. After differentiation and maturation in the thymus, they are distributed throughout the body to immune organs and tissues via lymphatic and blood circulation, thereby exerting their immune functions. T cells are highly efficient killers capable of rapidly eliminating virus-infected and cancer cells. The killing effect of T cells requires the formation of an immune synapse, a process that relies heavily on TCR recognition of the complex formed by the MHC molecules on the surface of antigen-presenting cells and the presented antigen peptide. An activated immune synapse releases cytotoxins and cytokines to effect killing. The immune synapse formation process is limited by the distance between the T cell and the target cell. Bispecific or multispecific antibody-directed T cells must simulate the formation of an immune synapse: one end achieves cross-linking of the T cell's TCR receptor by targeting CD3, and the other end achieves cross-linking of the target cell by targeting the target cell's surface antigen. CD3 molecules are expressed on the surface of all mature T cells and non-covalently bind to TCR to form complete TCR-CD3 complexes, jointly involved in the immune response to antigen stimulation. Currently, they are the most widely used and most successful trigger molecule on the surface of immune effector cells in bispecific antibodies. Bispecific antibodies targeting CD3 bind to CD3 on the surface of T cells and antigens on the surface of tumor cells, respectively, thereby shortening the distance between cytotoxic T cells (Tc or CTL) and tumor cells, directly activating T cells and inducing them to kill cancer cells directly, rather than relying on the dual activation signals of conventional T cells.

[0003] Among the CD3 antibodies disclosed, OKT3 is a groundbreaking antibody, pioneering the use of monoclonal antibodies in disease treatment as the first commercially available therapeutic antibody. SP34 is currently one of the few CD3 antibodies capable of reacting with monkeys. In recent years, the scientific community has made new advances in the field of biantibodies, resulting in the development of a variety of novel CD3 biantibodies (CD3-BsAbs). Currently, over 100 CD3-BsAbs with different structures have been successfully developed. Numerous forms determine many antibody characteristics, including half-life, immunogenicity, therapeutic response type, and ability to penetrate solid tumors. Amgen's blinatumomab (CD19xCD3 in BiTE format), approved by the FDA in 2014, is currently the most successful CD3-BsAb biantibody. This drug is used to treat patients with relapsed or refractory acute lymphoblastic leukemia (ALL). Compared with standard chemotherapy, up to 40% of patients treated with blinatumomab achieve a complete or partial response (CR / PR), and median overall survival can be improved by several months. In addition to blinatumomab, there are many other CD3-BsAbs currently undergoing clinical trials, including CD19, CD20, CD38, BCMA, CD33, and CD123. Furthermore, in a phase 1 / 2 clinical trial, patients with acute myeloid leukemia (AML) were treated with MacroGenics' flotetuzumab (CD123xCD3-BsAb), which showed an overall response rate (ORR) of 30%. In another Phase 1 / 2 clinical trial of Abbvie / Genmab's Epcoritamab (CD20xCD3-BsAb), patients with diffuse lymphocytic large B-cell neoplasia (DLBCL) showed a 44% complete response (CR) and an 11% partial response (PR), and patients with follicular lymphoma showed a 100% PR. Several other CD20xCD3 biantibodies have also achieved similarly impressive results. For example, in NSG mice, Regeneron's REGN1979 (CD20xCD3-BsAb) was able to control tumor growth better than Roche's Rituximab (CD20 monoclonal antibody), further demonstrating the therapeutic efficacy of CD3 biantibody drugs.Updated data on Roche's CD20xCD3 T cell-binding bispecific antibody glofitamab (CD20-TCB) in treating patients with relapsed or refractory (R / R) diffuse large B-cell lymphoma (DLBCL) show that the extension study included heavily pretreated, highly refractory DLBCL patients, with 58.3% of patients failing to respond to their initial treatment, and approximately one-third (33.1%) of patients having previously received CAR-T cell therapy. According to independent review committee (IRC) assessment, with a median follow-up of 12.6 months, 39.4% of patients (61 / 155) achieved complete response (CR), the primary outcome. Half of the patients (51.6%, 80 / 155) achieved an overall response (partial response [PR] + complete response [CR], a secondary outcome). From a safety perspective, cytokine release syndrome (CRS) was the most common adverse event, occurring in 63.0% of patients. CRS events were predictable and generally low-grade (primarily grade 1 [47.4%] or grade 2 [11.7%]), occurring during the first dose. Only one patient discontinued glofitamab treatment due to CRS. The incidence of grade 3 or higher CRS was low (3.9%), with no grade 5 events. Glofitamab has a novel "2:1" structural pattern, containing two Fab regions targeting CD20 and one Fab region targeting CD3.

[0004] Clinical trials of Amgen's blinatumomab have shown that cytokine storm (CRS) is one of the most significant toxic side effects. When CD19-expressing tumor cells coexist with a large number of healthy B and T cells, CD3-BsAb-mediated T cell activation occurs suddenly, resulting in the excessive release of proinflammatory cytokines such as IFN-γ, IL-6, and TNF-α, ultimately leading to symptoms ranging from fever to multiple organ failure. However, CRS is not a toxic side effect unique to blinatumomab. In fact, CRS frequently occurs with most CD3-BsAb and CAR-T treatments. Humanized mouse models have shown that the main mediator of CD3-BsAb-induced CRS is TNF-α produced by activated T cells, which triggers the massive secretion of proinflammatory cytokines by monocytes. Studies have shown that blocking upstream TNF-α and its downstream IL-1β or IL-6 can effectively alleviate CRS. Furthermore, several preclinical studies conducted in mouse and monkey models indicate that the severity of the cytokine storm can be reduced by reducing the affinity of CD3 (i.e., using "weaker" CD3).

[0005] Interleukin-15 (IL-15) is a 14-15 kDa cytokine crucial for the function of NK cells, NKT cells, and memory CD8+ T cells. IL-15 binds to its receptor, IL-15Rα, to generate the highly potent IL-15 superagonist (IL-15 SA), which is then transduced and transported to target cells. IL-15 SA potently activates cells that respond to and express IL-15R, particularly NK cells and T cells, thereby promoting antitumor and antiviral functions. IL-15 has a wide range of immunomodulatory effects and can be involved in regulating the survival, proliferation, and function of T cells, particularly NK cells and memory CD8+ T cells. IL-15 is structurally very similar to IL-2 and belongs to the spiral cytokine family. The heterotrimeric receptor for IL-15 shares the IL-2 receptor with the IL-2R / IL-15Rβ (CD122) and a common γc chain (CD132). Through these common receptor components and the shared JAK1 / JAK3 / STAT5 signaling pathway, IL-15 has the same functions as IL-2, including stimulating T cell proliferation, producing cytotoxic T lymphocytes, stimulating B cell immunoglobulin synthesis, and promoting the generation and sustained survival of NK cells. In many adaptive immune responses, IL-2 and IL-15 have distinct and often competing effects. There are four key points: 1. IL-2 can regulate the activation of Treg cells, whereas IL-15 cannot. 2. IL-2 inhibits T cell responses by activating the cell death of induced CD8+ effector T cells. 3. IL-15 plays an essential role in the differentiation of NK, effector CD8+, and memory CD8+ T cells. 4. Clinical studies have shown that IL-15 toxicity differs from that of IL-2, and compared with IL-2, IL-15 induces little vascular capillary leakage. These factors make IL-15 a more potent cytokine in tumor immunotherapy.Currently, several IL-15-targeting products are in clinical trials, most notably the IL-15 superagonist N-803, a fusion protein formed by mutating the IL-15 protein N72D and co-expressing and binding IL-15Ra and IgG1Fc. On May 23, 2022, ImmunityBio submitted a marketing application to the FDA for the IL-15 superagonist N-803, used in combination with BCG to treat non-muscle-invasive cancer (NMIBC) unresponsive to BCG vaccination. Clinical data published in 2021 demonstrated significant efficacy of the N-803 + BCG combination treatment, with a CR rate of 71% (59 / 83) and a mean CR duration of 24.1 months. Phase 1 clinical trials have shown that the combination of nivolumab (PD-1 antibody, Opdivo) and N-803 in the treatment of metastatic non-small cell lung cancer significantly extends patient long-term survival. Genentech, in collaboration with Xencor, has developed the IL-15 cytokine XmAb® 24306 and is currently conducting clinical studies of XmAb® 24306 in combination with Tecentriq. Hengrui's SHR-1501 (IL-15 fusion protein) was approved for clinical trials on May 14, 2019. Furthermore, SHR-1501 will be combined with SHR-1316 (a PD-L1 monoclonal antibody drug) for patients with advanced malignancies who have failed previous treatments.

[0006] Currently, there is still an unmet need for the development of therapeutic CD3 bispecific antibodies, and continuous development research is required to resolve the challenges faced in solid tumors and improve the safety of CD3-BsAb biantibodies. Summary of the Invention

[0007] [Means for solving the problem]

[0008] A first object of the present invention is to provide an anti-CD3 antibody mutant, wherein the anti-CD3 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VHCDR1, VHCDR2, and VHCDR3, and the light chain variable region comprising VLCDR1, VLCDR2, and VLCDR3, and the amino acid sequence of the anti-CD3 antibody comprises VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 set forth in SEQ ID NOs: 29 to 34, and the mutation sites of the anti-CD3 antibody mutant include any one or more of the tenth position in the sequence set forth in SEQ ID NO: 30, the second position and the seventh position in the sequence set forth in SEQ ID NO: 31, the third position and the fourth position in the sequence set forth in SEQ ID NO: 33, and the fourth position, the fifth position, the sixth position, and the seventh position in the sequence set forth in SEQ ID NO: 34.

[0009] Preferably, the mutation refers to an amino acid substitution. Preferably, the mutation sites of the variant include one or more of the following: A at position 10 is substituted with E in the sequence shown in SEQ ID NO: 30; G at position 2 is substituted with S and S at position 7 is substituted with G in the sequence shown in SEQ ID NO: 31; N at position 3 is substituted with W and K at position 4 is substituted with L in the sequence shown in SEQ ID NO: 33; Y at position 4 is substituted with N or R, S at position 5 is substituted with K, N at position 6 is substituted with G and L at position 7 is substituted with G in the sequence shown in SEQ ID NO: 34.

[0010] Preferably, the amino acid sequence of VHCDR2 is as set forth in SEQ ID NO:35, or the amino acid sequence of VHCDR3 is as set forth in SEQ ID NO:36, or the amino acid sequence of VLCDR2 is as set forth in SEQ ID NO:37 or 38, or the amino acid sequence of VLCDR3 is as set forth in SEQ ID NO:39, 40 or 41.

[0011] Preferably, the amino acid sequences of said VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are (1)SEQ ID NO:29, 35, 31, 32, 37, 39, (2)SEQ ID NO:29, 35, 36, 32, 37, 40, (3) As set forth in any one set of sequences of SEQ ID NOs: 29, 35, 36, 32, 38, 41.

[0012] Preferably, the heavy and / or light chain variable regions of the antibody variant are selected from or have at least 90% sequence identity with the heavy and / or light chain variable regions of the sequences shown in SEQ ID NO: 1, 2 or 3.

[0013] Preferably, there is a mutation between the heavy chain variable region and the light chain variable region to form a disulfide bond, and the mutation site includes any one or a combination of two or more of the following, where the mutation site is numbered according to EU, the heavy chain variable region is represented by VH, and the light chain variable region is represented by VL:

[0014] [Table 1]

[0015] Preferably, the anti-CD3 antibody mutant further comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4 or a mutant thereof, and a light chain constant region selected from human κ chain, λ chain, or a mutant thereof, wherein the heavy chain constant region comprises an Fc fragment or a mutant thereof.

[0016] Preferably, the antibody mutant is an scFv comprising a heavy chain variable region, a light chain variable region, and a linking fragment linking the heavy chain variable region and the light chain variable region, and the amino acid sequence of the linking fragment preferably comprises a few GGGGS repeats, more preferably three GGGGS repeats.

[0017] Another object of the present invention provides the use of said anti-CD3 antibody variants in the preparation of a medicament for inhibiting or treating cancer. Preferably, the cancer is selected from, or arises at, colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, cutaneous squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome cancer.

[0018] Another object of the present invention is to provide nucleic acid molecules encoding said anti-CD3 antibody variants. Another object of the present invention is to provide an expression vector comprising the above nucleic acid molecule.

[0019] Another object of the present invention is to provide an anti-tumor associated antigen (TAA) / anti-CD3 protein molecule, which is in a heterodimeric form comprising a first monomer and a second monomer, the first monomer comprises (a) an Fd fragment, (b) a light chain fragment, and a first Fc chain; the second monomer comprises (a) an Fd fragment, (b) a light chain fragment, an anti-CD3 antibody fragment, and a second Fc chain, wherein the light chain fragment comprises a VL domain and a CL domain, and the Fd fragment comprises a VH domain and a CH1 domain; and in the first monomer or the second monomer, the light chain fragment pairs with the Fd fragment to form an anti-TAA Fab domain, wherein the light chain fragment of the first monomer is fused to the first Fc chain, and the N-terminus of the anti-CD3 antibody fragment is fused to the light chain fragment of the second monomer, and the C-terminus is fused to the second Fc chain; Alternatively, the first monomer comprises (a) an Fd fragment, (b) a light chain fragment, a cytokine functional region, and a first Fc chain; the second monomer comprises (a) an Fd fragment, (b) a light chain fragment, an anti-CD3 antibody fragment, and a second Fc chain, and the cytokine functional region comprises IL-15 and IL-15Ra, wherein the N-terminus of the cytokine functional region is fused to the light chain fragment of the first monomer and the C-terminus is fused to the first Fc chain; and the N-terminus of the anti-CD3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain; Alternatively, the first monomer comprises an anti-TAA antibody fragment, a cytokine functional region, and a first Fc chain, and the second monomer comprises an anti-TAA antibody fragment, an anti-CD3 antibody fragment, and a second Fc chain, wherein the N-terminus of the cytokine functional region is fused to the anti-TAA antibody fragment of the first monomer and the C-terminus is fused to the first Fc chain, and the N-terminus of the anti-CD3 antibody fragment is fused to the anti-TAA antibody fragment of the second monomer and the C-terminus is fused to the second Fc chain; The first Fc chain and the second Fc chain are interchangeable.

[0020] Preferably, the anti-TAA antibody fragment is in the form of an scFv comprising a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region, and the amino acid fusion order of the anti-TAA antibody fragment from the N-terminus to the C-terminus of the peptide chain is "VH-VL" or "VL-VH", where "-" represents the linking fragment, or the anti-CD3 antibody fragment is in the form of an scFv comprising a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region. The scFv form includes a linked fragment, and the fusion order of the amino acids of the anti-CD3 antibody fragment from the N-terminus to the C-terminus of the peptide chain is "VH-VL" or "VL-VH", where "-" indicates a linked fragment, or the fusion order of the amino acid fragments of the cytokine functional domain from the N-terminus to the C-terminus of the peptide chain is "IL-15-IL-15Ra" or "IL-15Ra-IL-15", where "-" indicates a linked fragment, and preferably, the sequence of the IL-15-IL-15Ra is as set forth in SEQ ID NO:42, and the sequence of the IL-15Ra-IL-15 is as set forth in SEQ ID NO:43. Preferably, the amino acid sequence of the anti-CD3 antibody fragment is selected from a CD3-binding specific antibody, antibody fragment, single domain antibody or a humanized form thereof, preferably, the amino acid sequence of the anti-CD3 antibody fragment is selected from SP34, OKT3, UCTH1 or a derivative thereof.

[0021] Preferably, the anti-CD3 antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the anti-CD3 antibody fragment comprises VHCDR1, VHCDR2, and VHCDR3, and the light chain variable region of the anti-CD3 antibody fragment comprises VLCDR1, VLCDR2, and VLCDR3, and the amino acid sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are as shown in any one set of sequences of the anti-CD3 antibody variant described in claim 5.

[0022] Preferably, IL-15Ra and IL-15 form an IL-15 / IL-15Ra complex, IL-15 includes IL-15, mutations, truncations and various derivatives thereof that are capable of binding to IL-15Ra, and IL-15Ra includes IL-15, mutations, truncations and various derivatives thereof that are capable of binding to IL-15Ra, and preferably, said IL-15 / IL-15Ra complex includes, but is not limited to, mutant forms shown in any of the following combinations, and the counting method is based on the first amino acid of the amino acid sequence of IL-15 or IL-15Ra, counting it as number 1, and preferably, the parent sequence of said IL-15 is as shown in SEQ ID NO:44, and the parent sequence of said IL-15Ra is as shown in SEQ ID NO:45.

[0023] [Table 2]

[0024] Preferably, the IL-15 includes, but is not limited to, any of the mutant forms shown in the following combinations, and the counting method is based on the first amino acid of the amino acid sequence of IL-15, counting it as number 1, and preferably, the parent sequence of IL-15 is as shown in SEQ ID NO:44.

[0025] [Table 3]

[0026] Preferably, said TAA is selected from CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, 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, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB or Glypican-3.

[0027] Preferably, the first Fc chain and the second Fc chain polymerize to form an Fc segment, and the Fc segment is selected from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or a variant thereof, preferably selected from IgG1 Fc or human IgG4 Fc or a variant thereof, and the protein molecule is in the form of an Fc heterodimer, and preferably, the Fc heterodimer comprises, but is not limited to, the following combinations of mutations, which are counted according to EU:

[0028] [Table 4]

[0029] Preferably, the protein molecule comprises an Fc segment, and the selection of the Fc segment eliminates immune effector function, including but not limited to, combinations of the following mutations, which are counted according to EU:

[0030] [Table 5]

[0031] Preferably, the anti-tumor associated antigen (TAA) / anti-CD3 protein molecule is (1) SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:07, (2) SEQ ID NO:08, SEQ ID NO:06, SEQ ID NO:07, (3) SEQ ID NO:09, SEQ ID NO:06, SEQ ID NO:07, (4) SEQ ID NO:10, SEQ ID NO:06, SEQ ID NO:07, (5) SEQ ID NO:05, SEQ ID NO:17, SEQ ID NO:07, (6) SEQ ID NO:01, SEQ ID NO:18, SEQ ID NO:07, (7) SEQ ID NO:19, SEQ ID NO:17, SEQ ID NO:07, (8) SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:07, (9) SEQ ID NO:20, SEQ ID NO:21, (10) SEQ ID NO:22, SEQ ID NO:21, (11) SEQ ID NO:20, SEQ ID NO:23, (12) SEQ ID NO:22, SEQ ID NO:23, (13) SEQ ID NO:24, SEQ ID NO:25, (14) SEQ ID NO:26, SEQ ID NO:25, (15) SEQ ID NO:24, SEQ ID NO:27, (16) Obtained by fusing amino acid fragments shown in either one of the following pairs of sequences: SEQ ID NO: 26, SEQ ID NO: 27.

[0032] Another object of the present invention provides nucleic acid molecules encoding said anti-tumor associated antigen (TAA) / anti-CD3 protein molecules. Another object of the present invention provides the use of said anti-tumor associated antigen (TAA) / anti-CD3 protein molecule in the preparation of a medicament for inhibiting or treating cancer, comprising or arising at a site selected from colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, cutaneous squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0033] Another object of the present invention is to provide an anti-tumor-associated antigen (TAA) and IL-15 / IL-15Ra-containing fusion protein, which comprises two polypeptide chains, one of which comprises an anti-TAA antibody fragment, a cytokine functional domain, and an Fc fragment, the anti-TAA antibody fragment being in the form of an scFv, the cytokine functional domain comprising IL-15 and IL-15Ra, and the N-terminus of the cytokine functional domain being fused to the anti-TAA antibody fragment and the C-terminus being fused to the Fc fragment.

[0034] Preferably, in the anti-tumor associated antigen (TAA) and IL-15 / IL-15Ra-containing fusion protein, the IL-15Ra and IL-15 form an IL-15 / IL-15Ra complex, IL-15 includes IL-15, its mutations, truncations and various derivatives capable of binding to IL-15Ra, and IL-15Ra includes IL-15, its mutations, truncations and various derivatives capable of binding to IL-15Ra, and preferably, the IL-15 / IL-15Ra complex includes, but is not limited to, mutant forms shown in any of the following combinations, and the counting method is based on the first amino acid of the amino acid sequence of IL-15 or IL-15Ra, counting it as number 1, and preferably, the parent sequence of IL-15 is as shown in SEQ ID NO:44, and the parent sequence of IL-15Ra is as shown in SEQ ID NO:45.

[0035] [Table 6]

[0036] Preferably, in the anti-tumor-associated antigen (TAA) and IL-15 / IL-15Ra-containing fusion protein, the IL-15 includes, but is not limited to, any of the mutant forms shown in the following combinations, and the counting method is based on the first amino acid of the amino acid sequence of IL-15, counting it as number 1, and preferably, the parent sequence of IL-15 is as shown in SEQ ID NO:44.

[0037] [Table 7]

[0038] Preferably, in said anti-tumor associated antigen (TAA) and IL-15 / IL-15Ra containing fusion protein, it comprises an Fc segment, and the selection of the Fc segment eliminates immune effector function, including but not limited to, combinations of the following mutations, which are counted according to EU:

[0039] [Table 8]

[0040] Preferably, the anti-tumor-associated antigen (TAA) and IL-15 / IL-15Ra-containing fusion protein has an amino acid sequence of any one of its polypeptide chains as set forth in SEQ ID NO:28 or has at least 90% sequence identity thereto.

[0041] Another object of the present invention is to provide nucleic acid molecules encoding said anti-tumor associated antigens (TAA) and IL-15 / IL-15Ra-containing fusion proteins. Another object of the present invention provides the use of said anti-tumor associated antigen (TAA) and IL-15 / IL-15Ra-containing fusion protein in the preparation of a medicament for inhibiting or treating cancer, wherein said cancer comprises or occurs at a site selected from colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloid cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, cutaneous squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. [Effects of the Invention]

[0042] Compared with existing technologies, the present invention has the following beneficial effects: (1) The present invention involves mutating an existing anti-CD3 antibody to obtain a new anti-CD3 antibody variant.

[0043] (2) The present invention provides antibody molecules with novel structures that have superior biological activity. [Brief explanation of the drawings]

[0044] [Figure 1] 1 is a graph showing FACS detection of binding of the CD3 antibody SP34 and its mutants to Jurkat cells. [Figure 2] FIG. 1 is a schematic diagram of the molecular configuration of a bispecific antibody against anti-TAA (tumor-associated antigen) and anti-CD3. [Figure 3] Molecular design diagram of QP372337241461. [Figure 4] Molecular design diagram of QP374437481461. [Figure 5] This figure shows the results of FACS detection of the binding of molecules such as CLDN18.2 / CD3 double antibody to CLDN18.2. [Figure 6] This figure shows the results of FACS detection of the binding of molecules such as CLDN18.2 / CD3 double antibody to CLDN18.2. [Figure 7]FIG. 1 shows the results of FACS detection of the binding of molecules such as the CLDN18.2 / CD3 double antibody to CD3. [Figure 8] FIG. 1 shows the results of FACS detection of the binding of molecules such as the CLDN18.2 / CD3 double antibody to CD3. [Figure 9] This shows the results of killing of human gastric cancer cells NUGC4-CLDN18.2 cells by PBMCs mediated by molecules such as CLDN18.2 / CD3 antibodies. [Figure 10] This shows the results of killing of human gastric cancer cells NUGC4-CLDN18.2 cells by PBMCs mediated by molecules such as CLDN18.2 / CD3 antibodies. [Figure 11] This shows the results of killing of human lung cancer cells HCC827-CLDN18.2 cells by PBMCs mediated by molecules such as CLDN18.2 / CD3 antibodies. [Figure 12] FIG. 1 shows a curve diagram of tumor growth inhibition by the CLDN18.2 / CD3 bibody molecule in an in vivo animal model of subcutaneously implanted colorectal cancer cells MC38-hCLDN18.2 in CD3EGD HuGEMM mice. [Figure 13] FIG. 10 is a curve diagram showing changes in mouse body weight in each group after administration in an in vivo animal model of subcutaneously implanting colorectal cancer cells MC38-hCLDN18.2 into CD3EGD HuGEMM mice. [Figure 14] FIG. 10 is a curve diagram of tumor growth inhibition by CLDN18.2 / CD3 bibody molecule in a PBMC-humanized NOG mouse X-CLDN18.2 / MIA PaCa-2 subcutaneously transplanted tumor model. [Figure 15] FIG. 10 is a curve diagram of tumor growth inhibition by CLDN18.2 / CD3 bibody molecule in a PBMC-humanized NOG mouse X-CLDN18.2 / MIA PaCa-2 subcutaneously transplanted tumor model. [Figure 16] FIG. 1 is a design diagram of the structural form of one type of multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 17] FIG. 1 is a design diagram of the structural form of one type of multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 18] FIG. 1 is a design diagram of the structural form of one type of multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 19] FIG. 1 is a design diagram of the structural form of one type of multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 20] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 21] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 22] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 23] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 24] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 25] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 26] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 27] FIG. 1 is a design diagram of another type of structural form of a multifunctional fusion protein containing anti-TAA / CD3 and IL15 / IL15Ra. [Figure 28] FIG. 10 is a structural morphology diagram of a TAA / IL15 bifunctional fusion protein for tumor-targeting T cell / NK cell activation. [Figure 29] FIG. 1 shows the results of FACS detection of the binding of the CLDN18.2 / CD3 / IL15 multifunctional fusion protein to CLDN18.2. [Figure 30]FIG. 1 shows the results of FACS detection of the binding of CLDN18.2 / CD3 / IL15 and CLDN18.2 / IL15 fusion proteins to CLDN18.2. [Figure 31] FIG. 1 shows the results of FACS detection of the binding of the CLDN18.2 / CD3 / IL15 multifunctional fusion protein to CD3. [Figure 32] FIG. 1 shows the results of FACS detection of the binding of CLDN18.2 / CD3 / IL15 and the CLDN18.2 / IL15 fusion protein to CD3. [Figure 33] FIG. 1 shows the results of an Mo7e cell proliferation experiment to detect the activity of the multifunctional fusion protein IL15. [Figure 34] FIG. 1 shows the results of an Mo7e cell proliferation experiment to detect the activity of the multifunctional fusion protein IL15. [Figure 35] FIG. 1 shows the results of an Mo7e cell proliferation experiment to detect the activity of the multifunctional fusion protein IL15. [Figure 36] FIG. 1 shows the results of CLDN18.2 / CD3 / IL15-mediated killing of human gastric cancer cells NUGC4-CLDN18.2 by PBMC. [Figure 37] FIG. 1 shows the results of CLDN18.2 / CD3 / IL15-mediated killing of human gastric cancer cells NUGC4-CLDN18.2 by PBMC. [Figure 38] FIG. 1 shows the results of CLDN18.2 / CD3 / IL15-mediated killing of human lung cancer cells HCC827-CLDN18.2 by PBMC. DETAILED DESCRIPTION OF THE INVENTION

[0045] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and examples. For experimental methods for which no specific conditions are specified in this study, we generally follow conventional conditions or those provided by the raw material or product manufacturers. Reagents for which no specific source is given are conventional reagents purchased on the market.

[0046] As used herein, " / " refers to "and." For example, an anti-tumor-associated antigen (TAA) / anti-CD3 bispecific antibody refers to an antibody that simultaneously targets a TAA and CD3.

[0047] The term "fusion" refers to direct linkage of components via peptide bonds, linkage of components via linking fragments, or fusion via intermolecular interactions. In a single peptide chain, fusion refers to direct linkage via peptide bonds or linkage via linking fragments. A "multifunctional fusion protein" refers to a protein containing two or more antigen-binding domains capable of binding to two or more different epitopes (e.g., two, three, or more different epitopes); a multifunctional fusion protein may also contain a cytokine (e.g., IL-15, IL-15Ra), etc. The "fusion position" refers to the position of a functional region or domain in a peptide chain and indicates the order in which each functional fragment is linked on the peptide chain.

[0048] The term "polypeptide" refers to an amino acid chain of any length, including proteins and fragments thereof. The present invention discloses polypeptides as sequences of amino acid residues. These sequences are written from left to right in the direction from amino terminus to carboxy terminus. According to standard nomenclature, amino acid residue sequences are named by three-letter or one-letter codes, such as alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0049] The term "single chain" refers to a molecule comprising amino acids linearly linked by peptide bonds. The term "variant" or "mutant" refers to a polypeptide or polynucleotide that contains different amino acids or nucleotides but retains essential properties. Typically, the differences between variants or between a variant and a parent antibody are limited, and the amino acid sequences are generally very similar. Herein, the antibody or antibody fragment before mutation is referred to as the parent antibody, and the antibody or antibody fragment after mutation is referred to as the variant. The variant still has antigen-binding activity.

[0050] The term "antibody" (Ab) refers to an immunoglobulin molecule (Ig) that contains at least one antigen-binding site and is capable of specifically binding to an antigen.

[0051] The term "antigen" refers to a substance that can induce an immune response in the body and specifically bind to an antibody. The binding of an antibody to an antigen is mediated by interactions formed between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the surface of an antigen that binds to an antibody is called an "antigenic determinant" or "epitope," and typically each antigen has multiple determinants.

[0052] The term "antibody" as used herein is understood in the broadest sense and includes monoclonal antibodies (full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two distinct antigen-binding domains. Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies of other origins. The antibodies of the present invention can be derived from any animal, including, but not limited to, immunoglobulins from humans, non-human primates, mice, rats, bovine, equine, chicken, camel, and alpaca. Antibodies can also contain other modifications, such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of the antibody, and immunoglobulin molecules comprising any other modifications to the antigen recognition site, so long as the antibody exhibits the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules comprising at least one antigen-binding domain.

[0053] The basic structure of an antibody is a Y-shaped monomer consisting of two identical heavy chains (H) and two identical light chains (L) linked by disulfide bonds. Each chain contains two to five domains (also called functional regions) of approximately 110 amino acids, each with similar sequences but different functions. In antibody molecules, the amino acid sequences near the N-terminus of the light and heavy chains vary greatly, and the resulting domains are called variable regions (V regions), while the region with a relatively constant amino acid sequence near the C-terminus is called constant region (C region).

[0054] The V regions of the heavy and light chains are called VH and VL, respectively. Each VH and VL has three distinct regions of amino acid composition, the sequence of which is highly variable, called hypervariable regions (HVRs). These regions form a spatial structure complementary to antigen epitopes and are also called complementarity-determining regions (CDRs). The three CDRs of VH are designated VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL are designated VLCDR1, VLCDR2, and VLCDR3, respectively. The six CDRs of VH and VL together form the antigen-binding site. Amino acid diversity in the CDR regions provides the molecular basis for antibodies to specifically bind to a wide variety of antigens. The amino acid composition and sequence outside the CDRs of the V regions are relatively constant and are called framework regions (FRs). VH and VL each have four framework regions (referred to as framework regions), designated FR1, FR2, FR3, and FR4. VH and VL each consist of three CDRs and four FRs, and the sequence from the amino end to the carboxyl end is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0055] According to the amino acid sequence of the constant region of the antibody heavy chain, human immunoglobulins can be classified into five categories: IgM, IgG, IgA, IgD, and IgE. They can also be divided into different subtypes (isotypes). For example, human IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. No subtypes of IgM, IgD, or IgE have been identified. Based on the light chain amino acid sequence, light chains can be classified into kappa chains and lambda chains. The antibodies of the present invention can be of any type (e.g., IgM, IgG, IgA, IgD, or IgE) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2).

[0056] The heavy and light chain constant regions are called CH and CL, respectively. The heavy chain constant regions of IgG, IgA, and IgD have three domains: CH1, CH2, and CH3, while the heavy chain constant regions of IgM and IgE have four domains: CH1, CH2, CH3, and CH4.

[0057] The hinge region, located between CH1 and CH2, contains abundant prolines, making it flexible and flexible, allowing the distance between the two Y-shaped arms to change, which is advantageous for both arms to bind to the antigen epitope simultaneously.

[0058] The term "Fab fragment" refers to an antigen-binding fragment (Fab) of an antibody composed of the VL, VH, CL, and CH1 domains, which binds to a single antigen epitope (monovalent). Those skilled in the art know that under certain conditions, specific portions of antibody molecule chains can be easily hydrolyzed into various fragments by proteolytic enzymes. Papain hydrolyzes antibody molecules from the N-terminus of the hinge region into two identical antigen-binding fragments (Fab) and a crystallizable fragment (Fc).

[0059] The term "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains. The terms "Fc," "Fc segment," "Fc fragment," and "Fc domain" refer to a crystallizable fragment of an antibody that does not have antigen-binding activity and is the site where an antibody interacts with effector molecules or cell surface Fc receptors (FcRs). Fc fragments bind to cells that have the corresponding Fc receptors on their surface, producing different biological effects. In ADCC (antibody-dependent cell-mediated cytotoxicity), the Fab segment of an antibody binds to an antigen epitope on a virus-infected or tumor cell, and the Fc segment binds to the FcR on the surface of killer cells (NK cells, giant cells, etc.), mediating the killer cells to directly kill the target cells. The Fc fragment contains antibody constant region polypeptides excluding the heavy chain constant region CH1, i.e., the two carboxy-terminal constant region domains CH2 and CH3 of the heavy chain constant region of human immunoglobulin IgA, IgD, and IgG, and the three carboxy-terminal constant region domains CH2, CH3, and CH4 of the heavy chain constant region of human immunoglobulin IgE and IgM. The Fc fragment is often selected from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, or a variant thereof, and preferably selected from IgG1 Fc or human IgG4 Fc, or a variant thereof.

[0060] The Fc fragment can be composed of two chains, and the two chains of the Fc fragment are referred to herein as the first Fc chain and the second Fc chain. The first Fc chain and the second Fc chain can each be mutated, and the present invention is not particularly limited thereto. The Fc fragment can also refer to a single polypeptide chain within the Fc domain. The Fc segment of an antibody can be selected to eliminate immune effector functions, including, but not limited to, the following combinations of mutations (as counted by EU):

[0061] [Table 9]

[0062] Mutationally engineered Fc variants can form space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, etc. The interactions between Fc variants favor the formation of stable heterodimers. A preferred mutation design is a "knob-in-hole" type mutation design.

[0063] Single-chain antibody fragments (scFvs), or single-chain antibodies, consist of antibody heavy and light chain variable regions linked via a linker. The "linking fragment" can link IL-15 to IL-15Ra and the VH to VL of the CD3 antibody, ensuring correct protein folding and peptide stability. The "linking fragment" is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5, or more. If the linking fragment sequence is too short, it may affect the conformational folding of the two proteins and cause mutual interference. If the linking peptide sequence is too long, the linking peptide sequence itself may become a new antigen, resulting in immunogenicity problems.

[0064] Tumor-associated antigens (TAA) refer to antigenic molecules present in tumor cells or normal cells, including embryonic proteins, glycoprotein antigens, squamous epithelial antigens, etc., and are commonly used in clinical tumor diagnosis. Tumor-associated antigens are not inherent to tumor cells and can be synthesized in trace amounts by normal cells, but are highly expressed during tumor cell proliferation, hence the term "associated antigens." The tumor-associated antigen can be, for example, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, 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, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, or Glypican-3.

[0065] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA ring, into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell, and thereby be replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Expression vectors that are useful in recombinant DNA techniques are usually in the form of plasmids. The terms "IL-15", "IL-15Ra" may be mutants or fragments thereof.

[0066] The terms "first" and "second" are used for descriptive purposes only and do not denote or imply any degree of importance. The present invention uses the existing CD3 antibody SP34 as a parent antibody and mutates its variable region to obtain CD3 antibody variants. The present invention also provides several protein molecules that target different targets and have different structures.

[0067] The following sequences can be used for "IL-15 to IL15Ra". SEQ ID NO:42

[0068] [ka]

[0069] The following sequences can be used for "IL15Ra to IL-15". SEQ ID NO:43

[0070] [ka]

[0071] 1. Obtaining anti-CD3 antibody mutants and designing anti-TAA (tumor-associated antigen) / CD3 bispecific antibody forms Example 1: Obtaining anti-CD3 antibody mutants, molecular cloning, transient expression, and protein purification 1. Molecular cloning: Using the humanized anti-CD3 antibody SP34 (Wileman et al., 1990; US 8236308) as the parent antibody, its CDRs were mutated, and highly stable antibodies were screened to obtain three variants. The light and heavy chain variable regions of the CD3 parent antibody SP34 and its variants were constructed into a single-chain antibody VH-VL via a linker sequence, and the Fc segment of human IgG1 was fused to its C-terminus. The eukaryotic expression vector pQD was constructed and loaded by molecular cloning. The clone numbers and protein sequence numbers are as shown in the following table.

[0072] [Table 10]

[0073] The amino acid sequence is as shown below. SEQ ID NO:01 QD3685

[0074] [ka]

[0075] SEQ ID NO:02 QD3689

[0076] [ka]

[0077] SEQ ID NO:03 QD3690

[0078] [ka]

[0079] SEQ ID NO:04 QD3679(hSP34 VH-VL-FC)

[0080] [ka]

[0081] Note: The underlined amino acids represent the heavy chain variable region of the antibody, the wavy line represents the light chain variable region, and the italicized part between the heavy and light chain variable regions represents the linking sequence. The amino acid order of the variable regions is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the bolded and underlined parts represent HCDR1, HCDR3, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. A comparison of the CDR sequences of the parent antibody and its mutants is shown in the following table.

[0082] [Table 11]

[0083] 2. Protein expression: 293E cells at a cell density of 1 × 10 6 Prepare the plasmid and transfection reagent PEI. The amount of plasmid required for transfection is 100 μg / 100 ml cells, with a PEI to plasmid mass ratio of 2:1. Mix the plasmid and PEI evenly and leave for 15 minutes. Slowly add the plasmid and PEI mixture to the 293E cells and culture them in a shaker at 8% CO2, 120 rpm, and 37°C. On the 5th or 6th day after transfection, centrifuge the cells at 4700 rpm for 20 minutes in a horizontal centrifuge, and collect and purify the cell supernatant.

[0084] 3. Protein purification: Protein affinity chromatography: After high-speed centrifugation of the cell culture medium, the supernatant was collected and affinity chromatography was performed using a GE Protein A chromatography column. The equilibration buffer used for chromatography was 1x PBS (pH 7.4). After loading and binding the cell supernatant, the column was washed with PBS until the UV intensity returned to baseline. The target protein was then eluted using 0.1 M glycine (pH 3.0) elution buffer, adjusted to neutral pH using Tris, and stored. Protein volume exclusion chromatography: The product obtained by ion exchange was concentrated by ultrafiltration, followed by volume exclusion chromatography. Separation was performed using, for example, GE Superdex 200 gel to remove possible aggregates and other components, yielding a highly purified target product. The purity of the resulting protein was analyzed by SDS-PAGE and SEC-HPLC detection. Protein concentration was measured by UV spectrophotometry.

[0085] The yield and purity of the cell-expressed proteins are shown in the following table. The yield of QP3679, i.e., the parent humanized anti-CD3 antibody SP34, was 68.64 mg / L. The anti-CD3 antibody mutants all showed higher expression yields than the parent antibody QP3679, indicating that the mutants had higher yields and the proteins may be more stable. At the same time, the protein purity determined by SEC-HPLC was all very good.

[0086] [Table 12]

[0087] Example 2: FACS detection of anti-CD3 antibody variants binding to Jurkat cells naturally expressing CD3 FACS detection of anti-CD3 antibody binding activity: Human lymphocytic leukemia cells (Jurkat cells) express native CD3. The binding of anti-CD3 antibodies and their mutants to Jurkat cells was detected by FACS (fluorescence-activated cell sorting). 1E5 Jurkat cells per well were seeded into a U-shaped 96-well plate, washed once with ice-cold PBS, and centrifuged at 1200 rpm for 3 minutes. After washing, 200 μL of 3% FBS / PBS blocking solution was added per well and incubated on ice for 1 hour. After blocking, the cells were centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and samples of various concentrations were added. The cells were incubated on ice for 2 hours, washed three times with ice-cold PBS, and PE-anti-human Fc antibody was added at a 1:200 dilution (50 μL per well). The cells were mixed thoroughly, incubated on ice for 1 hour, and washed three times with ice-cold PBS. Resuspend the cells in 200 μl / well of PBS, read the mean fluorescence values ​​on a FACS instrument, and analyze the results using graphpad prism software.

[0088] As shown in Figure 1, the results demonstrate that the anti-CD3 antibody SP34 and its mutants QP3685, QP3689, QP3690 all maintain jurkat binding activity.

[0089] Example 3. Design of a bispecific antibody format containing anti-TAA (tumor-associated antigen) and anti-CD3 CD3 bivalent antibodies exert their therapeutic effects primarily by simultaneously binding to tumor-associated antigens (TAA) expressed on tumor cells and CD3 targets on T cells, bridging the two cells to form an immune synapse, activating the T cells and killing the tumor cells. Multivalent TAA antibodies can improve target selectivity, especially when target specificity is insufficient. The main approach to improving the safety of CD3 bivalent antibodies is to improve TAA selectivity while simultaneously selecting appropriate CD3 antibody affinity, which can maintain efficacy while reducing side effects, thereby improving tumor selectivity and reducing the targeting of healthy cells.

[0090] The molecular design of the anti-TAA (tumor-associated antigen) and anti-CD3 bispecific antibody provided by the present invention is as shown in FIG. The anti-TAA and anti-CD3 bispecific antibody molecule shown in Figure 2 comprises a first monomer (left part of Figure 2) and a second monomer (right part of Figure 2). The first monomer comprises two chains: chain 1 having an Fd fragment (including VH and CH1), and chain 2 having a light chain fragment (including VL and CL) and a first Fc chain. The second monomer also comprises two chains: chain 1 having an Fd fragment (including VH and CH1), and chain 2 having a light chain fragment (including VL and CL), an anti-CD3 antibody fragment (the CD3 antibody in Figure 2 is in the form of an scFv), and a second Fc chain. In the first or second monomer, the light chain fragment and the Fd fragment each pair to form an anti-TAA Fab domain (to realize the function of the TAA antibody in Figure 2). Unlike conventional antibodies, a first monomeric light chain fragment is fused to a first Fc chain, and simultaneously, the N-terminus of the anti-CD3 antibody fragment is fused to a second monomeric light chain fragment and the C-terminus of the anti-CD3 antibody fragment is fused to a second Fc chain. The first Fc chain and the second Fc chain polymerize to form an Fc domain. The Fc domain can form a human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, or a variant thereof.

[0091] Molecular cloning, transient expression, and protein purification of bispecific antibodies: 1. Molecular cloning: As shown in Figure 2, the TAA selected CLDN18.2 to design an anti-CLDN18.2 / CD3 bispecific antibody. The CLDN18.2 antibody sequence can be found in the sequence of antibody QP14611463 in patent document CN202010344676.8. The anti-CD3 antibody uses the SP34 sequence and the antibody light and heavy chain sequences of its variants QP3685, QP3689, and QP3690, respectively. The anti-CD3 antibody sequence of QP374537493746 is selected from SP34, and the anti-CD3 antibody sequences of QP090837493746, QP090937493746, and QP091037493746 are selected from QP3685, QP3689, and QP3690. The protein expression and sequence numbers are as shown in the following table.

[0092] [Table 13]

[0093] For comparison, two other CLDN18.2 / CD3 bibody molecules, QP372337241461 and QP374437481461, were constructed. Based on patent US20200055932A1, Amgen's CLDN18.2 / CD3 bibody molecule AMG910 was constructed, and the AMG910 protein is designated QP3693. The protein expression and sequence number are as shown in the following table:

[0094] [Table 14]

[0095] The molecular design of QP372337241461 is as shown in Figure 3. The molecular design of QP374437481461 is as shown in Figure 4. 2. Clone construction method: Design clones as shown in Tables 4 and 5, and construct full-length expression vectors. Primer design: Design multiple primers to synthesize the gene fragments required for recombination using the online software DNAWorks (v3.2.4) (http: / / helixweb.nih.gov / dnaworks / ). Fragment splicing: Obtain the gene fragments required for recombination by PCR amplification using the multiple primers designed above, according to the instructions for TaKaRa PrimerSTAR GXL DNA polymerase. First-stage PCR: A 50 μL PCR reaction system contains 10 μL of PrimerSTAR GXL Buffer (5x), 4 μL of dNTP mixture (2.5 mmol L), 1 μL of each primer listed above, and 1 μL of PrimeSTAR GXL DNA polymerase. The PCR reaction conditions are 98°C for 2 minutes, 30 cycles of 98°C for 20 seconds, 55°C for 15 seconds, and 68°C for 30 seconds, followed by 68°C for 5 minutes. Second-stage PCR: Using the first-stage PCR product as a template, PCR amplification is performed using the first and last primers under the same conditions as in the first stage. PCR is then performed to amplify the target fragment. Construction and enzyme digestion of expression vector pQD: The expression vector pQD (with a signal peptide) is designed and constructed using several special restriction endonucleases, such as BsmBI, which have different characteristics in their identifier sequence and enzyme digestion site. The vector is digested with BsmBI enzyme and the gel is collected for storage. Recombinant construction of expression vector: The recombinant target gene fragment and the BsmBI-digested expression vector pQD (with a signal peptide fragment) are recovered and added to DH5α-sensitive cells at a 3:1 ratio. The cells are incubated at 0°C for 30 minutes on ice, heat-shocked at 42°C for 90 seconds, and then 5x the volume of LB medium is added and incubated at 37°C for 45 minutes. The cells are then plated onto LB-Amp plates and cultured overnight at 37°C. Single clones are then selected and the target clones are identified by sequencing.

[0096] 3. Protein Expression: The cell density of 293E cells was 1 x 10 6Prepare the plasmid and transfection reagent PEI. The amount of plasmid required for transfection is 100 μg / 100 ml cells, with a PEI to plasmid mass ratio of 2:1. Mix the plasmid and PEI evenly and leave for 15 minutes. Slowly add the plasmid and PEI mixture to the 293E cells and culture them in a shaker at 8% CO2, 120 rpm, and 37°C. On the 5th or 6th day after transfection, centrifuge the cells at 4700 rpm for 20 minutes in a horizontal centrifuge to collect and purify the cell supernatant.

[0097] 4. Protein purification: Protein affinity chromatography: After high-speed centrifugation of the cell culture medium, the supernatant was collected and affinity chromatography was performed using a GE Protein A chromatography column. The equilibration buffer used for chromatography was 1x PBS (pH 7.4). After loading and binding the cell supernatant, the column was washed with PBS until the UV intensity returned to baseline. The target protein was then eluted using 0.1 M glycine (pH 3.0) elution buffer, adjusted to neutral pH using Tris, and stored. Protein volume exclusion chromatography: The product obtained by ion exchange was concentrated by ultrafiltration, followed by volume exclusion chromatography. Separation was performed using, for example, GE Superdex 200 gel to remove possible aggregates and other components, yielding a highly purified target product. The purity of the resulting protein was analyzed by SDS-PAGE and SEC-HPLC detection. Protein concentration was measured by UV spectrophotometry. Endotoxin was strictly controlled throughout the purification process, and the endotoxin content of the purified protein was less than 1 EU / mg.

[0098] The results show that the CLDN18.2 / CD3 bispecific antibodies QP374537493746, QP090837493746, QP090937493746, and QP091037493746 have high cell expression yields and HPLC-SEC purities exceeding 95%, demonstrating their feasibility.

[0099] Example 4: FACS detection of binding activity of bispecific antibodies to CLDN18.2 Experimental Phase: The cell line CHOS-CLDN18.2, stably expressing CLDN18.2, was harvested and seeded at 1E5 cells / well into a U-shaped 96-well plate. The plate was washed once with ice-cold PBS and centrifuged at 1200 rpm for 3 minutes. After washing, 200 μL of 3% FBS / PBS blocking solution was added per well and incubated on ice for 1 hour. After blocking, the plate was centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and various concentrations of the sample were added. The plate was then incubated on ice for 2 hours, washed three times with ice-cold PBS, and PE-anti-human FC antibody was added at a dilution of 1:200, 50 μL per well. The plate was mixed thoroughly, incubated on ice for 1 hour, and washed three times with ice-cold PBS. The cells were resuspended in 200 μL of PBS per well, and the mean fluorescence intensity was measured using a FACS instrument. The results were analyzed using GraphPad Prism software.

[0100] As shown in Figures 5 and 6, the results demonstrate that the CLDN18.2 / CD3 bispecific antibodies QP374537493746, QP090837493746, QP090937493746, and QP091037493746 all bind to CLDN18.2. As shown in Figure 5, the binding affinity of the bispecific antibodies QP374537493746, QP090837493746, QP090937493746, and QP091037493746 of Figure 2 of the present invention to the TAA target CLDN18.2 is comparable to that of the anti-CLDN18.2 antibody IgG form QP14611463 and is significantly more potent than the control antibody AMG910 (1:1 form) QP3693, as expected by design.

[0101] Example 5: FACS detection of binding activity of bispecific antibodies to CD3 Human lymphocytic leukemia cells (Jurkat cells) express native CD3, and the binding of anti-CD3 antibodies and their mutants to Jurkat cells was detected by FACS detection. 1E5 Jurkat cells per well were seeded into a U-shaped 96-well plate, washed once with ice-cold PBS, and centrifuged at 1200 rpm for 3 minutes. After washing, 200 μL of 3% FBS / PBS blocking solution was added per well and incubated on ice for 1 hour. After blocking, the cells were centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and samples of various concentrations were added. The mixture was incubated on ice for 2 hours, washed three times with ice-cold PBS, and PE-anti-human Fc antibody was added at a 1:200 dilution, 50 μL per well. The mixture was mixed thoroughly, incubated on ice for 1 hour, and washed three times with ice-cold PBS. The cells were resuspended in 200 μL of PBS per well, and the mean fluorescence values ​​were read using a FACS instrument. The results were analyzed using GraphPad Prism software.

[0102] As shown in Figures 7 and 8, the results indicate that CLDN18.2 / CD3 bispecific antibodies QP374537493746, QP090837493746, QP090937493746 and QP091037493746 all bind to CD3. As shown in Figure 7, the biantibodies QP374537493746, QP090837493746, QP090937493746, and QP091037493746 of Figure 2 of the present invention are classified into two categories based on their binding affinity to CD3, where QP374537493746 and QP090837493746 have weak binding affinity to CD3, while QP090937493746 and QP091037493746 have strong affinity. However, the binding affinity of these two molecules to CD3 is significantly weaker than that of the control antibody AMG910 (1:1 form) and QP3693, which is consistent with our expectation that the CD3 antibody affinity should be reduced to improve the safety of the CD3 biantibody.

[0103] Example 6: T cell killing of human gastric cancer cells mediated by CLDN18.2 / CD3 bispecific antibodies Experimental Method: The target cells selected were the human gastric cancer cell line NUGC4-CLDN18.2, which stably expresses CLDN18.2. The effector cells were human PBMCs. Various concentrations of antibody were added at an E:T ratio of 10:1 and cultured at 37°C and 5% CO2 for 48 hours. The percentage of LDH was measured in the cell culture supernatant using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays). The maximum target cell lysis rate (100%) was 1%, and the target cells were treated with Triton X-100 to lyse the cells and release all LDH. Control wells were set up, including target cell spontaneous lysis, effector cell spontaneous lysis, and target cell + effector cell spontaneous lysis. Data are analyzed according to the formula % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] x 100.

[0104] As shown in Figures 9 and 10, the results show that QP374537493746, QP090837493746, QP090937493746, and QP091037493746 can all mediate PBMC-mediated killing of human gastric cancer cells NUGC4-CLDN18.2 cells. As shown in Figure 9, the PBMC-killing activities of human gastric cancer cells NUGC4-CLDN18.2 cells mediated by the dual antibodies QP374537493746, QP090837493746, QP090937493746, and QP091037493746 of Figure 2 of the present invention are all stronger than those of the control molecule QP3693 (AMG910). The binding affinities of the CD3 antibodies selected in the present invention to CD3 are all weaker than that of the CD3 antibody of the AMG910 molecule (see Figure 7), but this does not affect their killing ability against target cells.

[0105] Example 7: T cell killing of human lung cancer cells mediated by CLDN18.2 / CD3 bispecific antibodies Experimental Method: The target cells selected were the human lung cancer cell line HCC827-CLDN18.2, which stably expresses CLDN18.2. The effector cells were human PBMCs. Various concentrations of antibody were added at an E:T ratio of 10:1 and cultured at 37°C and 5% CO2 for 48 hours. LDH was detected in the cell culture supernatant using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) to quantify % cytotoxicity. The maximum target cell lysis rate (100%) was 1%, and target cells were treated with Triton X-100 to lyse the cells and release all LDH. Control wells were set up, including target cell spontaneous lysis, effector cell spontaneous lysis, and target cell + effector cell spontaneous lysis. Data are analyzed according to the formula % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] x 100.

[0106] As shown in Figure 11, the results indicate that the CD3 dual antibody molecules QP374537493746, QP090837493746, QP090937493746, and QP091037493746 can kill human lung cancer cells HCC827-CLDN18.2 cells via PBMCs. The PBMC-killing activities of human lung cancer cells HCC827-CLDN18.2 cells mediated by the dual antibodies QP374537493746, QP090837493746, QP090937493746, and QP091037493746 of Figure 2 of the present invention are all stronger than those of the control molecule QP3693 (AMG910). The results of NUGC4-CLDN18.2 in HCC827-CLDN18.2 cells are reproduced (see Figure 9).

[0107] Example 8: Animal efficacy of CD3 humanized mouse model The effect of the anti-CLDN18.2 / CD3 dual antibody molecule on tumor growth was evaluated in a CD3-humanized mouse (CD3EDG HuGEMM) model. Exponentially growing colorectal cancer cells, MC38-hCLDN18.2 cells, were harvested and resuspended in PBS to a concentration suitable for seeding. 5 × 10 cells were implanted in the right back of each experimental mouse. 6 MC38-hCLDN18.2 cells were subcutaneously inoculated, and tumor growth was monitored periodically. Tumors grew to an average volume of approximately 100 mm. 3 Once the tumors have grown to 100%, the mice are randomly assigned to groups based on tumor size and body weight and administered the following treatment regimen:

[0108] [Table 15]

[0109] According to the regimen shown in the table above, six doses were administered and tumors were measured twice a week. The tumor inhibition rates are shown in Table 7, and the tumor inhibition curves are shown in Figure 12. The results show that 21 days after administration, the tumor inhibition rate (TGI) of QP374537493746 in the low-dose group of 0.6mpk reached 89.29%, which was superior to or equivalent to the tumor inhibition rate (TGI = 86.5%) of the control antibody AMG910 in the 0.7mpk group (the molar concentration of the AMG910 group of 0.7mpk is equivalent to 1.2mpk of QP374537493746). This indicates that QP374537493746 can achieve the same tumor inhibition effect as the control antibody AMG910 at half the dose. Figure 13 shows the weight changes of mice in each group after administration. The results show that the weight of mice in the administration group remained basically unchanged, and they grew well, confirming the safety of the drug.

[0110] [Table 16]

[0111] Example 9: Animal efficacy of humanized NOG mouse model Experimental Objective: To evaluate the in vivo efficacy of test substances in a PBMC-humanized NOG mouse X-CLDN18.2 / MIA PaCa-2 subcutaneous tumor model. Cell Culture: X-CLDN18.2 / MIA PaCa-2 cells were cultured in vitro at 37°C and 5% CO2 in medium supplemented with 10% fetal bovine serum. Subculture was performed twice weekly. When cells reached exponential growth phase and cell viability exceeded 95%, they were harvested, the percentage of viable cells was counted, and inoculated. Animals: NOG mice, female, 6-8 weeks old, weighing 18-20 g. A total of 36 animals (24 plus 50%) were required for the experiment. Animals were provided by a qualified supplier. Tumor inoculation: 5 x 10 6 X-CLDN18.2 / MIA PaCa-2 and 2*10 6 Each mouse was inoculated with a mixture of PBMC cells into the right neck and back. At the same time, the animals were ear-tagged, which served as the sole reference for subsequent experiments. Tumors were allowed to grow until the average tumor volume reached approximately 60–100 mm. 3 The subjects will be randomly assigned to groups and administered the drugs when the dose reaches 100 mg / kg. The experimental group assignment and administration method are shown in Table 8.

[0112] [Table 17]

[0113] According to the regimen shown in the table above, five doses were administered, with tumors measured twice weekly. The tumor inhibition rates are shown in Table 9, and the tumor inhibition curves are shown in Figure 14. Results from day 15 after administration showed that the tumor inhibition rate (TGI) of QP374537493746 in the low-dose group (4 mpk) reached 92.3%, while the tumor inhibition rate (TGI) of QP374537493746 in the high-dose group (8 mpk) reached 99.8%, both of which were significantly better than the tumor inhibition rate (TGI = 69.3%) of the control antibody AMG910 in the 5 mpk group (the molar concentration of the AMG910 5 mpk group corresponds to the 8 mpk molar concentration of the QP374537493746 high-dose group). Figure 15 shows the weight changes of mice in each group after administration. The results show that the weight of mice in the treatment groups remained almost unchanged and grew well, demonstrating good drug safety.

[0114] [Table 18]

[0115] 2. Design of multifunctional fusion proteins containing anti-TAA (tumor-associated antigen), anti-CD3 antibody, and cytokine IL15 / IL15Ra Although CD3 biantibodies have shown great potential in hematological malignancies, they still face several challenges in the treatment of solid tumors. CD3 biantibody therapy is associated with T cell infiltration and immune suppression in solid tumor tissues, and low levels of infiltrating T cells can easily lead to resistance to CD3 biantibody therapy. IL15 is a soluble cytokine that activates T cells and NK cells and mediates their proliferation and survival. The present invention designs a TAA / CD3 / IL15 multifunctional fusion protein, whose cytokine IL15 component is expected to stimulate the proliferation of immune cells, including T cells, and alter the tumor immune microenvironment.

[0116] Example 10: Design of a multifunctional fusion protein containing anti-TAA (tumor-associated antigen), anti-CD3, and IL15 / IL15Ra The present invention provides a variety of anti-TAA (tumor associated antigen), anti-CD3, and IL15 / IL15Ra-containing multifunctional fusion proteins.

[0117] 1. The first type of multifunctional fusion protein can be seen in Figures 16 to 19, where the fusion protein molecule includes a first monomer (left part of the figure) and a second monomer (right part of the figure). The first monomer includes two chains: chain 1 having an Fd fragment (including VH and CH1), and chain 2 having a light chain fragment (including VL and CL), a cytokine functional region (including IL15 and IL15Ra), and a first Fc chain. The second monomer includes two chains: chain 1 having an Fd fragment (including VH and CH1), and chain 2 having a light chain fragment (including VL and CL), an anti-CD3 antibody fragment (CD3 antibody in the figure, which is in the form of an scFv), and a second Fc chain. In the first or second monomer, the light chain fragment and the Fd fragment each pair to form an anti-TAA Fab domain (to realize the function of the anti-TAA antibody in the figure). The N-terminus of the cytokine functional region is fused to a first monomeric light chain fragment and its C-terminus is fused to a first Fc chain, the N-terminus of the anti-CD3 antibody fragment is fused to a second monomeric light chain fragment and its C-terminus is fused to a second Fc chain, and the first and second Fc chains polymerize to form an Fc domain.

[0118] 16 to 19, the scFv-form anti-CD3 antibody fragment can comprise a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region. The fusion order of amino acids in the anti-CD3 antibody fragment from the N-terminus to the C-terminus of the peptide chain can be "VH(CD3) to VL(CD3)" or "VL(CD3) to VH(CD3)," with "~" representing a linking fragment. The fusion order of amino acid fragments of cytokine functional regions can be "IL-15 to IL-15Ra" or "IL-15Ra to IL-15."

[0119] [ka]

[0120] [ka]

[0121] [ka]

[0122] [ka]

[0123] 2. The second type of multifunctional fusion protein can be seen in Figures 20 to 27, where the protein molecule comprises a first monomer (left part of the figure) and a second monomer (right part of the figure). The first monomer comprises an anti-TAA antibody fragment (in the form of an scFv comprising VH and VL domains), a cytokine functional region (comprising IL15 and IL15Ra), and a first Fc chain. The second monomer comprises an anti-TAA antibody fragment (in the form of an scFv comprising VH and VL domains), an anti-CD3 antibody fragment (the CD3 antibody in the figure is in the form of an scFv), and a second Fc chain. The N-terminus of the cytokine functional region is fused to the anti-TAA antibody fragment of the first monomer, and the C-terminus is fused to the first Fc chain. The N-terminus of the anti-CD3 antibody fragment is fused to the anti-TAA antibody fragment of the second monomer, and the C-terminus is fused to the second Fc chain. The first Fc chain and the second Fc chain polymerize to form an Fc domain.

[0124] Similar to the first type of multifunctional fusion protein, the anti-CD3 antibody fragment in scFv form can comprise a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region. The fusion order of amino acids in the anti-CD3 antibody fragment, from the N-terminus to the C-terminus of the peptide chain, can be "VH(CD3) to VL(CD3)" or "VL(CD3) to VH(CD3)," with "~" representing a linking fragment. The fusion order of amino acid fragments of cytokine functional regions can be "IL-15 to IL-15Ra" or "IL-15Ra to IL-15." Furthermore, the anti-TAA antibody fragment in scFv form comprises a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region, and the fusion order of the amino acids of the anti-TAA antibody fragment from the N-terminus to the C-terminus of the peptide chain can be "VH(TAA) to VL(TAA)" or "VL(TAA) to VH(TAA)", where "~" represents the linking fragment.

[0125] Each domain of the antibody molecule shown in Figures 20 to 27 uses the same amino acid sequence; the difference lies in the fusion order or configuration. Using Figure 20 as an example, the two polypeptide chains for the fusion protein are illustrated as follows: (1) first polypeptide chain: VL(TAA)-VH(TAA)-IL15-IL15Ra-first Fc chain; (2) second polypeptide chain: VL(TAA)-VH(TAA)-VH(CD3)-VL(CD3)-second Fc chain.

[0126] 3. The present invention provides a TAA / IL15 bifunctional fusion protein for tumor-targeting T cell / NK cell activation, the structural form of which can be seen in Figure 28. Compared with the second type of multifunctional fusion protein, the difference is that the anti-CD3 antibody fragment is replaced with a cytokine functional domain (including IL15 and IL15Ra). In Figure 28, the protein molecule has a symmetrical structural form.

[0127] Example 11: Molecular cloning, transient expression, and protein purification of CLDN18.2 / CD3 / IL15 multifunctional fusion protein 1. Molecular cloning: According to Example 10, a CLDN18.2 / CD3 / IL15 or CLDN18.2 / IL15 multifunctional fusion protein was designed, where the CLDN18.2 antibody can refer to the antibody QP14611463 sequence in patent document CN202010344676.8, and the anti-CD3 antibody uses the SP34 light chain and heavy chain sequences. The protein expression and sequence number are as shown in the following table.

[0128] [Table 19]

[0129] At the same time, IL15 / IL15Ra-FC from Hengrui company was constructed as a control, and the IL15 / IL15Ra-FC protein was numbered QP33123313.

[0130] The clones were designed and full-length expression vectors were constructed as shown in Table 10. For clone construction, protein expression and protein purification methods, see Example 3.

[0131] Example 12: FACS detection of binding activity between CLDN18.2 / CD3 / IL15 multifunctional fusion protein and CLDN18.2 Experimental step: The CHOS-CLDN18.2 cell line, which stably expresses CLDN18.2, was harvested and seeded at 1E5 cells / well into a U-shaped 96-well plate. The plate was washed once with ice-cold PBS and centrifuged at 1200 rpm for 3 minutes. After washing, 200 μL of 3% FBS / PBS blocking solution was added per well and incubated on ice for 1 hour. After blocking, the plate was centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and various concentrations of the sample were added. The plate was then incubated on ice for 2 hours, washed three times with ice-cold PBS, and PE-anti-human Fc antibody was added at a 1:200 dilution (50 μL / well). The plate was mixed thoroughly, incubated on ice for 1 hour, and washed three times with ice-cold PBS. The cells were resuspended in 200 μL of PBS per well, and the mean fluorescence intensity was measured using a FACS instrument. The results were analyzed using GraphPad Prism software. As shown in Figures 29 and 30, the results show that the CLDN18.2 / CD3 / IL15 and CLDN18.2 / IL15 multifunctional fusion proteins all bind to the TAA target CLDN18.2 with stronger affinity than the AMG910 analog (1:1 form) QP3693, which is consistent with expectations.

[0132] Example 13: FACS detection of binding activity of CLDN18.2 / CD3 / IL15 multifunctional fusion protein to CD3 Lymphocytic leukemia cells (Jurkat cells) express native CD3, and the binding of anti-CD3 antibodies and their mutants to Jurkat cells was detected by FACS detection. 1E5 Jurkat cells per well were seeded into a U-shaped 96-well plate, washed once with ice-cold PBS, and centrifuged at 1200 rpm for 3 minutes. After washing, 200 μL of 3% FBS / PBS blocking solution was added per well and incubated on ice for 1 hour. After blocking, the cells were centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and samples of various concentrations were added. The mixture was incubated on ice for 2 hours, washed three times with ice-cold PBS, and PE-anti-human Fc antibody was added at a 1:200 dilution, 50 μL per well. The mixture was mixed thoroughly, incubated on ice for 1 hour, and washed three times with ice-cold PBS. The cells were resuspended in 200 μL of PBS per well, and the mean fluorescence values ​​were read using a FACS instrument. The results were analyzed using GraphPad Prism software. As shown in Figures 31 and 32, the results showed that all CLDN18.2 / CD3 / IL15 multifunctional fusion proteins bound to CD3 with weaker affinities than the control molecule AMG910 (QP3693), which is consistent with our expectation that reduced CD3 antibody affinity would improve the safety of CD3 biantibodies.

[0133] Example 14: Mo7e cell proliferation experiments to evaluate the activity of the multifunctional fusion protein IL15 Mo7e (human giant cell leukemia cell line) cells express IL-15Rβγ and are cytokine-dependent. Research has shown that resting NK cells and naive T cells express the intermediate affinity IL-15Rβγ phenotype, and the results of Mo7e (IL-15Rβγ) cell proliferation experiments using the cytokine IL15 / IL15Ra are consistent with the results of unstimulated PBMC proliferation experiments (Mol Cancer Ther, 11(6) June 2012). The present invention uses Mo7e cell proliferation experiments to evaluate the activity of the multifunctional fusion protein IL15. The method and results are as follows:

[0134] Experimental reagents: Mo7e cells (human giant cell leukemia cell line) were purchased from the Reob Resource Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, Cell Proliferation and Toxicity Detection Kit (CCK-8) was purchased from MeilunBio, product number is MA0218, recombinant human GM-CSF was purchased from perprotech, product number is 300-03, human IgG was purchased from Sigma, product number is I4506, and other antibodies were prepared in-house.

[0135] Experimental method: Mo7e cells were cultured in RPMI1640 medium containing 10% FBS, 2 mM L-glutamine, and 8 ng / ml GM-CSF in a 37°C, 5% CO2 incubator. 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. 2 × 10 cells were collected in 80 μl / well. 4 The cells were seeded into a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 1 hour. Each drug medium to be tested was diluted 4-fold, and 20 μl per well was mixed evenly with the cell suspension and incubated in a 37°C, 5% CO2 incubator for 3 days. 10 μl per well of CCK-8 reagent was added to the 96-well plate to be tested and incubated in a 37°C, 5% CO2 incubator for 4 hours. The 96-well plate was then removed and the absorbance at a wavelength of 450 nm was detected using a microplate reader.

[0136] As shown in Figures 33, 34, and 35, the experimental results indicate that the differently designed CLDN18.2 / CD3 / IL15 and CLDN18.2 / IL15 multifunctional fusion proteins all possess IL15 activity, although the activity varies depending on the molecular design. Different constructions result in molecules with different IL15 activity, providing an opportunity to select the optimal therapeutic window.

[0137] Example 15: T cell killing of human gastric cancer cells mediated by CLDN18.2 / CD3 / IL15 multifunctional fusion protein Experimental Method: The target cells selected were the human gastric cancer cell line NUGC4-CLDN18.2, which stably expresses CLDN18.2. The effector cells were human PBMCs. Various concentrations of antibody were added at an E:T ratio of 10:1 and cultured at 37°C and 5% CO2 for 48 hours. The percentage of LDH was measured in the cell culture supernatant using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays). The maximum target cell lysis rate (100%) was 1%, and the target cells were treated with Triton X-100 to lyse the cells and release all LDH. Control wells were set up, including target cell spontaneous lysis, effector cell spontaneous lysis, and target cell + effector cell spontaneous lysis. The data is analyzed according to the formula % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] x 100. As shown in Figures 36 and 37, the results show that QP374508993746, QP090109003746, QP09023688, QP36670903, QP09040905, QP09060905, QP09040907 and QP09060907 can all PBMC-mediated killing of human gastric cancer NUGC4-CLDN18.2 cells. Here, the PBMC-mediated killing of human lung cancer cells NUGC4-CLDN18.2 by QP374508993746 and QP090109003746 was significantly superior to that of the control molecule AMG910, while the PBMC-killing of human lung cancer cells NUGC4-CLDN18.2 mediated by other molecules was comparable to that of the control molecule AMG910.

[0138] Example 16: T cell killing of human lung cancer cells mediated by CLDN18.2 / CD3 / IL15 multifunctional fusion protein Experimental Method: The target cells selected were the human lung cancer cell line HCC827-CLDN18.2, which stably expresses CLDN18.2. The effector cells were human PBMCs. Various concentrations of antibody were added at an E:T ratio of 10:1 and cultured at 37°C and 5% CO2 for 48 hours. LDH was detected in the cell culture supernatant using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) to quantify % cytotoxicity. The maximum target cell lysis rate (100%) was 1%, and target cells were treated with Triton X-100 to lyse the cells and release all LDH. Control wells were set up, including target cell spontaneous lysis, effector cell spontaneous lysis, and target cell + effector cell spontaneous lysis. The data were analyzed according to the formula: % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] × 100. As shown in Figure 38, the results show that QP090109003746, QP36670903, QP09040905, QP09060905, and QP09040907 can all mediate PBMC-killing activity of human lung cancer cells HCC827-CLDN18.2 cells. Here, the PBMC-killing activity of human lung cancer cells HCC827-CLDN18.2 cells mediated by QP090109003746 is significantly superior to that of the control molecule AMG910. The PBMC killing activity of human lung cancer cells HCC827-CLDN18.2 cells mediated by other molecules is comparable to the control molecule AMG910.

[0139] Furthermore, in some other examples, anti-CD3 antibody fragments are selected from the antibody light chain and heavy chain sequences of SP34 variants QP3685, QP3689, and QP3690, respectively, and clones are designed, and proteins are expressed and purified as shown in Table 10 of Example 11. The results show that molecules constructed using the antibody light chain and heavy chain sequences of QP3685, QP3689, and QP3690 also have CLDN18.2-binding activity and IL15 activity, and that the anti-CD3 molecules not only have CD3-binding activity but also have T cell-mediated killing of human gastric cancer cells or lung cancer cells.

[0140] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the present invention is not limited to the above description. Various modifications and variations to the present invention will be apparent to those skilled in the art upon reading the above content. Therefore, the scope of protection of the present invention should be limited by the appended claims.

Claims

1. an anti-CD3 antibody variant, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VHCDR1, VHCDR2, and VHCDR3, and the light chain variable region comprises VLCDR1, VLCDR2, and VLCDR3; The amino acid sequences of the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are (1) SEQ ID NO: 29, 35, 31, 32, 37, 39, (2) SEQ ID NO: 29, 35, 36, 32, 37, 40, (3) SEQ ID NO: 29, 35, 36, 32, 38, 41, characterized in that it is as set forth in any one set of sequences; Anti-CD3 antibody variants.

2. The heavy and / or light chain variable regions of the antibody variant are selected from the heavy and / or light chain variable regions of the sequences shown in SEQ ID NO: 1, 2 or 3, or are characterized in that they share at least 90% sequence identity therewith. The anti-CD3 antibody mutant of claim 1.

3. There is a mutation between the heavy chain variable region and the light chain variable region to form a disulfide bond, and the mutation site includes any one or a combination of two or more of the following, and the mutation site is numbered according to EU, and the heavy chain variable region is represented by VH, and the light chain variable region is represented by VL. The anti-CD3 antibody mutant of claim 1. 【Table 1】

4. The anti-CD3 antibody mutant further comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4 or a mutant thereof, and a light chain constant region selected from human κ chain, λ chain, or a mutant thereof, wherein the heavy chain constant region comprises an Fc fragment or a mutant thereof. The anti-CD3 antibody mutant of claim 1.

5. The antibody mutant is an scFv comprising a heavy chain variable region, a light chain variable region, and a linking fragment linking the heavy chain variable region and the light chain variable region, wherein the amino acid sequence of the linking fragment is three GGGGS repeats. The anti-CD3 antibody mutant of claim 1.

6. 10. Use of the anti-CD3 antibody variant of claim 1 in the preparation of a medicament for inhibiting or treating cancer, comprising: The cancer is selected from, or arises at, colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, cutaneous squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. Application of anti-CD3 antibody variants.

7. A nucleic acid molecule comprising: A nucleic acid molecule encoding the anti-CD3 antibody mutant of claim 1.

8. An anti-tumor associated antigen (TAA) / anti-CD3 protein molecule, a heterodimeric form comprising a first monomer and a second monomer; the first monomer comprises (a) an Fd fragment, (b) a light chain fragment and a first Fc chain; the second monomer comprises (a) an Fd fragment, (b) a light chain fragment, an anti-CD3 antibody fragment, and a second Fc chain; the light chain fragment comprises a VL domain and a CL domain, the Fd fragment comprises a VH domain and a CH1 domain, and in the first monomer or the second monomer, the light chain fragment pairs with the Fd fragment to form an anti-TAA Fab domain, wherein the light chain fragment of the first monomer is fused to the first Fc chain, and the N-terminus of the anti-CD3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain; or the first monomer comprises (a) an Fd fragment, (b) a light chain fragment, a cytokine functional domain, and a first Fc chain; the second monomer comprises (a) an Fd fragment, (b) a light chain fragment, an anti-CD3 antibody fragment, and a second Fc chain; the cytokine functional region comprises IL-15 and IL-15Ra, wherein the N-terminus of the cytokine functional region is fused to the light chain fragment of the first monomer and the C-terminus is fused to the first Fc chain, and the N-terminus of the anti-CD3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain; or the first monomer comprises an anti-TAA antibody fragment, a cytokine functional domain, and a first Fc chain; The second monomer comprises an anti-TAA antibody fragment, an anti-CD3 antibody fragment, and a second Fc chain, wherein: the N-terminus of the cytokine functional domain is fused to the first monomeric anti-TAA antibody fragment and the C-terminus is fused to the first Fc chain, and the N-terminus of the anti-CD3 antibody fragment is fused to the second monomeric anti-TAA antibody fragment and the C-terminus is fused to the second Fc chain, and the first Fc chain and the second Fc chain are interchangeable; The anti-CD3 antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the anti-CD3 antibody fragment comprises VHCDR1, VHCDR2, and VHCDR3, and the light chain variable region of the anti-CD3 antibody fragment comprises VLCDR1, VLCDR2, and VLCDR3, and the amino acid sequences of the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are as set forth in any one set of sequences: (1) SEQ ID NOs: 29, 35, 31, 32, 37, and 39; (2) SEQ ID NOs: 29, 35, 36, 32, 37, and 40; and (3) SEQ ID NOs: 29, 35, 36, 32, 38, and 41. The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule.

9. The anti-TAA antibody fragment is in the form of an scFv comprising a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region, and the amino acid fusion order of the anti-TAA antibody fragment from the N-terminus to the C-terminus of the peptide chain is "VH-VL" or "VL-VH", where "-" represents the linking fragment, or The anti-CD3 antibody fragment is in the form of an scFv comprising a heavy chain variable region (VH), a light chain variable region (VL), and a linking fragment linking the heavy chain variable region and the light chain variable region, and the amino acid fusion order of the anti-CD3 antibody fragment from the N-terminus to the C-terminus of the peptide chain is "VH-VL" or "VL-VH", where "-" represents the linking fragment, or The fusion order of the amino acid fragments of the cytokine functional domain from the N-terminus to the C-terminus of the peptide chain is "IL-15 to IL-15Ra" or "IL-15Ra to IL-15", where "to" represents a linked fragment, and the sequences of IL-15 to IL-15Ra are as shown in SEQ ID NO: 42, and the sequences of IL-15Ra to IL-15 are as shown in SEQ ID NO:

43. The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8.

10. IL-15Ra and IL-15 form an IL-15 / IL-15Ra complex, IL-15 includes its mutations, truncations and various derivatives capable of binding to IL-15 and IL-15Ra, IL-15Ra includes its mutations, truncations and various derivatives capable of binding to IL-15Ra and IL-15, and said IL-15 includes mutant forms shown in any of the following combinations, and the counting method is based on the first amino acid of the amino acid sequence of IL-15, counting it as number 1, and the parent sequence of said IL-15 is as shown in SEQ ID NO:

44. 【Table 2】 The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8.

11. The IL-15 / IL-15Ra complex comprises a mutant form shown in any of the following combinations, wherein the counting method is based on the first amino acid of the amino acid sequence of IL-15 or IL-15Ra, with this being counted as number 1, and the parent sequence of IL-15 is as shown in SEQ ID NO: 44, and the parent sequence of IL-15Ra is as shown in SEQ ID NO:

45. The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 10. 【Table 3】

12. The TAA is selected from CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, 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, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, and Glypican-3. The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8.

13. the first Fc chain and the second Fc chain polymerize to form an Fc segment, and the Fc segment is selected from a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, or a variant thereof, and the protein molecule is an Fc heterodimer form; The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8.

14. The Fc heterodimer comprising the following combination of mutations, wherein the following mutations are counted according to EU: 【Table 4】 The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 13.

15. The protein molecule comprises an Fc segment, and the selection of the Fc segment eliminates immune effector functions comprising a combination of the following mutations, the following mutations being counted according to EU: The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8. 【Table 5】

16. (1) SEQ ID NO: 08, SEQ ID NO: 06, SEQ ID NO: 07, (2) SEQ ID NO: 09, SEQ ID NO: 06, SEQ ID NO: 07, (3) A polypeptide obtained by fusion of amino acid fragments shown in any one of the sequences of SEQ ID NO: 10, SEQ ID NO: 06, and SEQ ID NO:

07. The anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8.

17. A nucleic acid molecule comprising: A nucleic acid molecule encoding an anti-tumor associated antigen (TAA) / anti-CD3 protein molecule according to claim 8.

18. 10. The use of the anti-tumor associated antigen (TAA) / anti-CD3 protein molecule of claim 8 in the preparation of a drug for inhibiting or treating cancer, comprising: The cancer comprises or occurs at a site selected from colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, cutaneous squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. Application of anti-tumor associated antigen (TAA) / anti-CD3 protein molecules.

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