Targeted T cell and Her2 positive cell bispecific antibody fusion protein connected in series with 4-1BBL and application thereof

Abstract

The invention discloses a bispecific antibody targeting T cells and Her2 positive cells and a fusion protein connected in series with 4-1BBL. Belongs to the technical field of biology. The fusion protein is of a heterotetramer structure formed by connecting two similar heavy polypeptide chains and two similar light polypeptide chains through disulfide bonds, wherein the similar heavy polypeptide chains comprise a heavy chain variable region of a Her2 targeting antibody, a human IgG1 Fc region and a human 4-1BBL extracellular region; the light polypeptide-like chain comprises a light chain variable region of a Her2 targeting antibody, a human antibody light chain constant region and a single chain variable region fragment of a CD3 targeting antibody. The fusion protein can specifically bind to Her2 positive tumor cells and CD3 molecules on the surfaces of T cells at the same time, and provides a key co-stimulation signal by using 4-1BBL connected in series, so that the killing function of the T cells is efficiently activated and enhanced in the local part of the tumor. The fusion protein provides a novel candidate strategy with stronger curative effect, more lasting effect and better safety for immunotherapy of Her2 positive tumors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a bispecific antibody targeting T cells and Her2-positive cells, fused with 4-1BBL, and its applications. Background Technology

[0002] Tumor immunotherapy is a treatment strategy that mobilizes and utilizes the body's own immune system to identify, attack, and eliminate tumor cells. Compared with traditional radiotherapy and chemotherapy, it has advantages such as high specificity, relatively fewer toxic side effects, and the potential for durable efficacy, and has become a significant breakthrough in the field of cancer treatment. Molecular targeted therapy and immunotherapy are key components of this approach.

[0003] Overexpression or amplification of human epidermal growth factor receptor 2 (Her2) is a well-established driving factor and poor prognostic indicator in various solid tumors, detected in approximately 15-20% of breast cancer, 20% of gastric cancer, approximately 27% of ovarian cancer, and a significant proportion of endometrial cancer and cholangiocarcinoma. Anti-Her2 monoclonal antibodies, such as trastuzumab and pertuzumab, have significantly improved the prognosis of patients with Her2-positive breast and gastric cancer. Studies have confirmed that the efficacy of drugs like trastuzumab partly depends on their antibody-dependent cell-mediated cytotoxicity (ADCC) effect, activating immune effector cells such as natural killer cells through their Fc fragment to kill tumor cells. However, in patients with disease relapse or progression, the ADCC effect may be weakened due to factors such as Fcγ receptor polymorphism and immunosuppression in the tumor microenvironment, limiting its long-term efficacy.

[0004] To overcome the limitations of traditional monoclonal antibodies, bispecific antibodies have emerged. Among them, T cell adapters (TCEs) are an important class of bispecific antibodies. They are designed to establish a physical connection between tumor cells and T cells by binding to tumor-associated antigens (TAAs) with one arm and to the CD3 molecule on the surface of T cells with the other arm, directly guiding T cells to specifically kill tumor cells. Compared with traditional antibodies that rely on Fc effector function, TCEs do not depend on the recruitment of endogenous immune cells, theoretically possessing a stronger potential for tumor cell lysis. Compared with antibody-drug conjugates, their cytotoxicity originates from activated T cells, making them effective against both resting and proliferating tumor cells, without the risk of payload toxicity. Currently, more than 29 bispecific antibodies targeting CD3 and Her2 have entered clinical trials, demonstrating the great potential of this strategy in the treatment of solid tumors.

[0005] While CD3 / HER2 bispecific antibodies excel at recruiting T cells to tumor sites, complete T cell activation and sustained anti-tumor function typically require two key signals: a first signal (specific recognition mediated by the TCR / CD3 complex) and a second signal (i.e., co-stimulatory signaling). A lack of effective co-stimulatory signals can lead to T cell atrophy or exhaustion, thus affecting the durability of therapeutic efficacy. 4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, provides co-stimulatory signals through its ligand 4-1BBL, which are crucial for the activation, proliferation, survival, and cytokine production of CD8+ T cells. In the field of CAR-T cell therapy, CAR-T cells containing the 4-1BB co-stimulatory domain have demonstrated more durable in vivo survival and a lower incidence of neurotoxicity, proving the core value of this pathway in enhancing T cell anti-tumor immunity.

[0006] However, most current TCE drugs only provide the first signal (CD3-mediated T cell recruitment and initial activation) and fail to simultaneously provide the crucial 4-1BB co-stimulatory signal. While fusing co-stimulatory molecules (such as 4-1BB) with tumor-targeting molecules is a promising strategy to enhance local immune activation while reducing systemic toxicity, how to rationally and efficiently fuse them with bispecific antibodies (especially CD3 / HER2 bispecific antibodies) that already possess T cell recruitment capabilities to construct an integrated molecule that can simultaneously provide T cell recruitment, tumor targeting, and effective co-stimulation remains a technological gap.

[0007] Therefore, the development of a novel fusion protein in this field can not only efficiently guide T cells to Her2-positive tumor cells via bispecific antibodies, but also further enhance the activation and killing function of T cells through integrated co-stimulatory signals (such as 4-1BBL), thereby potentially generating a stronger and more durable anti-tumor immune response and providing a new solution for the treatment of Her2-positive tumors. This is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a bispecific antibody targeting T cells and Her2-positive cells, combined with a fusion protein of 4-1BBL and its applications. The fusion protein is a fusion protein of a bispecific antibody and an active co-stimulatory factor, which is formed by two amino acid sequences forming a heterodimer and by disulfide bonds of Fc forming a tetramer. This fusion protein provides a new method for the detection, prevention, or treatment of tumors, and has significant economic and social implications.

[0009] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0010] The primary objective of this application is to provide a fusion protein consisting of a bispecific antibody targeting T cells and Her2-positive cells, tandemly linked with 4-1BBL, wherein the fusion protein is a heterotetramer structure consisting of two heavy polypeptide chains and two light polypeptide chains linked by disulfide bonds. The heavy polypeptide chain includes the heavy chain variable region of the Her2 targeting antibody, the heavy chain constant region of the human IgG1 antibody, and the 4-1BBL extracellular region. The light polypeptide chain comprises a light chain variable region of the Her2-targeting antibody, a light chain constant region of the human antibody, and a single-chain variable region of the CD3-targeting antibody.

[0011] As a preferred technical solution, the single-chain variable region fragment of the CD3-targeting antibody in the light polypeptide chain includes a heavy chain variable region VH and a light chain variable region VL, wherein: The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region VH are shown below; GFTFNTYA, SEQ ID NO.3; IRSKYNNYAT, SEQ ID NO.4; ARHGNFGNSYVSWFAY,SEQ ID NO.5; The amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region VL are shown below; TGAVTTSNY, SEQ ID NO.6; GTN; ALWYSNLWV, SEQ ID NO.7; The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region VH of the Her2-targeting antibody in the heavy polypeptide chain are shown below. GFNIKDTY, SEQ ID NO.8; IYPTNGYT, SEQ ID NO.9; SRWGGDGFYAMDY, SEQ ID NO.10; The amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region VL of the Her2-targeting antibody in the light polypeptide chain are shown below. QDVNTA, SEQ ID NO.11; SAS; QQHYTTPPT, SEQ ID NO.12; As a preferred technical solution, the heavy chain variable region VH of the CD3-targeting antibody in the light polypeptide chain further includes FR-H1, FR-H2, FR-H3, and FR-H4 framework regions, whose amino acid sequences are shown below; EVQLVESGGGLVQPGGSLRLSCAAS, SEQ ID NO.13; MNWVRQAPGKGLEWVAR, SEQ ID NO.14; YYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC, SEQ ID NO.15; WGQGTLVTVSS, SEQ ID NO.16; The light chain variable region VL of the CD3-targeting antibody in the light polypeptide chain also includes FR-L1, FR-L2, FR-L3, and FR-L4 framework regions, whose amino acid sequences are shown below; QTVVTQEPSLTVSPGGTVTLTCRSS, SEQ ID NO.17; ANWVQQKPGQAPRGLIG, SEQ ID NO.18; KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYC, SEQ ID NO.19; FGGGTKLTVL, SEQ ID NO.20; The heavy chain variable region VH of the Her2-targeting antibody in the heavy polypeptide chain also includes FR-H1, FR-H2, FR-H3, and FR-H4 framework regions, whose amino acid sequences are shown below; EVQLVESGGGLVQPGGSLRLSCAAS, SEQ ID NO.21; IHWVRQAPGKGLEWVAR, SEQ ID NO.22; RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC, SEQ ID NO.23; WGQGTLVTVSS, SEQ ID NO.24; The light chain variable region VL of the Her2-targeting antibody in the light polypeptide chain also includes FR-L1, FR-L2, FR-L3, and FR-L4 framework regions, whose amino acid sequences are shown below; DIQMTQSPSSSLSASVGDRVTITCRAS, SEQ ID NO.25; VAWYQQKPGKAPKLLIY, SEQ ID NO.26; FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC, SEQ ID NO. 27.

[0012] FGQGTKVEIK, SEQ ID NO.28.

[0013] As a preferred technical solution, the amino acid sequence of the heavy chain variable region VH of the CD3-targeting antibody in the light polypeptide chain is shown below; EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS, SEQ ID NO.29; The amino acid sequence of the light chain variable region VL of the CD3-targeting antibody in the light polypeptide chain is shown below. QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL, SEQ ID NO.30; The amino acid sequence of the heavy chain variable region VH of the Her2-targeting antibody in the heavy polypeptide chain is shown below; EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, SEQ ID NO.31; The amino acid sequence of the light chain variable region VL of the Her2-targeting antibody in the light polypeptide chain is shown below; DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, SEQ ID NO.32.

[0014] As a preferred technical solution, the amino acid sequence of the single-chain variable region of the CD3-targeting antibody in the light polypeptide chain is shown below; EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL, SEQ ID NO.33.

[0015] The single-chain variable region of the CD3-targeting antibody in the light polypeptide chain is composed of a heavy chain variable region VH and a light chain variable region VL connected by a flexible linker peptide, the amino acid sequence of which is shown below. GGGGSGGGGSGGGGS, SEQ ID NO. 34.

[0016] The amino acid sequence of the extracellular region of human 4-1BBL in the heavy-like polypeptide chain is shown below; REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE, SEQ ID NO. 35.

[0017] The amino acid sequence of the heavy chain constant region of the human IgG1 antibody in the heavy polypeptide chain is shown below; ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV SEQ ID NO. 36.

[0018] The amino acid sequence of the light chain constant region of the human antibody in the light polypeptide chain is shown below; SEQ ID NO. 37.

[0019] As a preferred technical solution, the amino acid sequence of the heavy polypeptide chain of the fusion protein is shown in SEQ ID NO. 1, and the amino acid sequence of the light polypeptide chain is shown in SEQ ID NO. 2.

[0020] ,SEQ ID NO.1; DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL, SEQ ID NO.2.

[0021] Another point of this application is to provide: a nucleic acid molecule encoding the said fusion protein.

[0022] Another point of this application is to provide: an expression vector or host cell comprising the nucleic acid molecules as described.

[0023] Another aspect of this application is to provide: a method for preparing the fusion protein, comprising culturing the host cells under conditions suitable for expression and recovering the fusion protein from the culture.

[0024] Another aspect of this application is to provide: the use of the fusion protein in the preparation of reagents and / or medicaments for the detection and / or treatment of Her2-positive tumors.

[0025] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) To achieve a synergistic anti-tumor mechanism of “three-in-one” and overcome the key shortcomings of existing TCE therapies. This fusion protein is the first to integrate T cell targeting (CD3 scFv), tumor cell targeting (Her2 antibody), and a potent co-stimulatory signal (4-1BBL) into a single molecule. It addresses the problem that traditional TCEs (such as CD3 / HER2 bispecific antibodies) only provide T cell recruitment and initial activation signals (the first signal), lacking the crucial co-stimulatory signal (the second signal), which easily leads to T cell exhaustion or functional insufficiency, affecting the durability of therapeutic efficacy. The introduction of 4-1BBL can directly provide a powerful co-stimulatory signal to recruited T cells at the tumor site, promoting their complete activation, proliferation, survival, and cytokine secretion, thereby potentially generating a stronger and more durable anti-tumor immune response.

[0026] (2) Precise positioning and local activation significantly improve efficacy and potentially reduce systemic toxicity. This fusion protein, through its bispecific structure, triggers both TCR / CD3 signaling and 4-1BB co-stimulatory signals only within the local microenvironment where Her2-positive tumor cells encounter T cells. This avoids the severe hepatotoxicity and other systemic side effects that may be caused by systemic administration of free 4-1BB agonist antibodies. It achieves "conditional" immune activation, precisely limiting potent co-stimulatory activity to the tumor site, maximizing efficacy while significantly improving the safety window of treatment.

[0027] (3) It retains and may enhance multiple anti-tumor effects, providing redundant killing pathways. The fusion protein retains the Fc fragment of the anti-Her2 antibody, thus possessing the potential for antibody-dependent cell-mediated cytotoxicity (ADCC). This provides an additional killing mechanism for treatment. Even in immunosuppressive microenvironments where T cell function is temporarily limited, ADCC can still serve as an important supplementary killing pathway. This constructs a dual-protection mechanism of "direct T cell killing" and "Fc effector-mediated killing," enhancing the ability to combat tumor heterogeneity and immune escape, and potentially overcoming the problem of resistance to traditional anti-Her2 therapies in some patients.

[0028] In summary, the fusion protein of this application is not a simple functional superposition, but rather achieves a highly efficient synergistic effect of "precise recruitment → complete activation → multiple killing" through molecular design. It holds promise as a novel immunotherapy candidate with stronger efficacy, longer duration of action, and better safety for Her2-positive solid tumors (especially patients who do not respond well to existing targeted or immunotherapies), demonstrating significant clinical application value and development prospects. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 Here is a schematic diagram of the structure of the fusion protein 37 / 38 of this invention.

[0031] Figure 2 The image shows the enzyme digestion identification results of the fusion protein 37 / 38 expression plasmid in Example 1 of this invention. Lane 1 is the marker; lane 2 is the pcDNA3.4-37 band; lane 3 is the BamHI and EcoRI double digestion band of pcDNA3.4-37; lane 4 is the marker; lane 5 is the pcDNA3.4-38 band; and lane 6 is the BamHI and EcoRI double digestion band of pcDNA3.4-38.

[0032] Figure 3 Figure 1 shows the expression and purification results of the fusion proteins 37 / 38 prepared in Example 1 of this invention; where A represents the elution volume and OD. 280 The corresponding diagram for pH; B is the SDS-PAGE result, and lane 1 is the band of the denatured and reduced sample from lanes 37 / 38.

[0033] Figure 4 Figure 2 shows the ELISA results of the binding of fusion protein 37 / 38 prepared in Example 2 of this invention to recombinant human CD3 protein.

[0034] Figure 5 Figure 1 shows the ELISA results of the binding of fusion proteins 37 / 38 prepared in Example 2 of this invention to recombinant human Her2 protein.

[0035] Figure 6 The image shows the ELISA results of the binding of the fusion protein 37 / 38 prepared in Example 2 of this invention to recombinant human 4-1BB protein.

[0036] Figure 7 Figure 1 shows the binding FC results of the fusion protein 37 / 38 prepared in Example 3 of this invention with the Jurkat cell line and the human breast cancer cell line SK-BR-3; where A is the result of Jurkat cells; and B is the result of SK-BR-3 cells.

[0037] Figure 8 The image shows the ADCC results of primary PBMC-derived NK cells killing SK-BR-3 cells mediated by the fusion protein 37 / 38 prepared in Example 4 of this invention.

[0038] Figure 9 The image shows the ADCC results of NK92 cells killing SK-BR-3 mediated by the fusion protein 37 / 38 prepared in Example 4 of this invention.

[0039] Figure 10 Figure 4 shows the results of primary PBMC-derived T cells killing SK-BR-3 mediated by the fusion protein 37 / 38 prepared in Example 4 of this invention.

[0040] Figure 11 Figure 4 shows the results of Jurkat killing SK-BR-3 mediated by the fusion protein 37 / 38 prepared in Example 4 of this invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1 Plasmid construction, protein expression and purification (the structural diagram is shown in Figure 1) Figure 1 (As shown) (1) The aforementioned SEQ ID No.1 (numbered 37) and SEQ ID No.2 (numbered 38) sequences were synthesized by gene (with restriction endonucleases BamHI and EcoRI restriction sites added to both ends of the sequences), and then constructed into pcDNA3.4 plasmids expressing fusion proteins using pcDNA3.4-TOPOTAcloning kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.), i.e., constructed as pcDNA3.4-37 and pcDNA3.4-38.

[0043] (2) The pcDNA3.4-37 and pcDNA3.4-38 plasmids from the previous step were transformed into E. coli TOP10, amplified by shaking in LB medium, extracted using a plasmid large-scale extraction kit (Beijing Polymer Biotechnology Co., Ltd.), and then digested with restriction endonucleases BamHI and EcoRI for identification. Figure 2In this diagram, lane 1 is the marker; lane 2 is the band of plasmid pcDNA3.4-37; lane 3 is the band of plasmid pcDNA3.4-37 after double digestion with BamHI and EcoRI; lane 4 is the marker; lane 5 is the band of plasmid pcDNA3.4-38; and lane 6 is the band of plasmid pcDNA3.4-38 after double digestion with BamHI and EcoRI. The upper side of the double digestion band is the linearized band of the empty plasmid, and the lower side is the target band, which is consistent with the designed size and position.

[0044] (3) The expression plasmid was transfected into 293F cells using PEI transfection reagent for protein expression.

[0045] (4) 120 h after transfection, the supernatant was harvested, centrifuged and filtered, and the protein A affinity column (purchased from Cytiva) was treated with 5 column volumes of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2). The supernatant was loaded onto the column. After the supernatant was loaded, the column was washed with buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2) to bring the contaminating protein to baseline. The protein was then eluted with 50 mM citrate / sodium citrate buffer (pH 3.2), and samples with a concentration of 100 mAu or more were collected. The pH was then adjusted to 7.0 with 1 M Tris-Cl (pH 8.0), concentrated in a concentration tube, filtered and sterilized, and stored at 4 °C. 3 μg of the sample was then used for SDS-PAGE staining. Figure 3 ): Figure 3 A in the diagram is a chromatography chromatogram, showing the elution volume versus OD. 280 Correspondence diagram of pH; Figure 3 Image B shows the diagram below illustrating the marker band size specification and the SDS-PAGE results. Lane M represents the protein marker band, and lane 1 represents the 37 / 38 denatured / reduced sample band. The positions of the two protein chains are consistent with the designed size (after expression in cells, 37 / 38 will form a Y-shaped antibody structure, becoming a single protein, which forms two bands under denaturing / reducing conditions). Figure 3 The protein was shown in Figure B (where the upper band represents the heavy chain and the lower band represents the light chain). The protein purified by chromatography is the fusion protein 37 / 38.

[0046] Example 2 Antigen-antibody binding ELISA assay (1) Commercial recombinant human CD3, Her2, 4-1BB proteins were diluted with pH 9.6 NaHCO3 coating buffer, plated on ELISA plates, 100 ng / well, and incubated overnight at 4°C.

[0047] (2) Wash the ELISA plate coated yesterday three times with PBS.

[0048] (3) Block with PBST containing 3% BSA, at room temperature for 30 min.

[0049] (4) Dilute the purified fusion protein 37 / 38 with PBST containing 3% BSA. The initial concentration was 100 μg / mL. The protein was then serially diluted 10 times to 0.001 μg / mL for a total of 6 concentrations. The diluted protein was added to an ELISA plate and incubated at room temperature for 30 min.

[0050] (5) Wash 3 times with PBST, add 100 μL of mouse anti-human HRP secondary antibody (1:5000) to each well, and incubate at room temperature for 30 min.

[0051] (6) Wash 3 times with PBST, add 50 μL of TMB colorimetric solution to each well, and incubate for 5 min.

[0052] (7) Add 50 μL of 1 M HCl to each well and detect OD using a microplate reader. 450 Absorbance. Results are as follows: Figure 4 , Figure 5 , Figure 6 As shown.

[0053] The results show that the fusion protein 37 / 38 has binding activity with the three target proteins CD3, Her2, and 4-1BB in a dose-dependent manner; however, it does not have binding activity with the control protein (Ctrl Ab: a protein that does not have binding activity with CD3, Her2, and 4-1BB).

[0054] Example 3 Combined with cell flow cytometry experiments (1) Take 1×10⁻⁶ Jurkat (human T lymphocytes) and SK-BR-3 (human breast cancer cells) in the logarithmic growth phase, respectively. 6 Centrifuge at 300 g for 5 min and discard the supernatant.

[0055] (2) Resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0056] (3) Add 50 μL of fusion protein 37 / 38 with a concentration of 40 μg / mL and incubate at room temperature for 30 min.

[0057] (4) Centrifuge and discard the supernatant, resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0058] (5) Add 50 μL of diluted goat anti-human AF488 secondary antibody (2 μL / tube) (goat anti-human secondary antibody coupled with fluorescent dye AF488, which can specifically bind to fusion protein 37 / 38, purchased from Startech Biotechnology Co., Ltd.), and incubate at room temperature for 30 min.

[0059] (6) Resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0060] (7) Resuspend in 500 μL of physiological saline and analyze by flow cytometry. Results are as follows: Figure 7 As shown.

[0061] Results analysis: Figure 7 In the figure, A represents the results for Jurkat cells, and B represents the results for SK-BR-3 cells. Ctrl in the figure indicates the control signal peak (negative control: the antibody expressed does not have the ability to bind to Jurkat and SK-BR-3 cells). 37 / 38 represents the fusion protein 37 / 38 signal peak; a rightward shift indicates a positive result, indicating binding. The results show that this fusion protein 37 / 38 can bind to Jurkat and SK-BR-3 cells expressing the corresponding antigens in situ, exhibiting binding activity under physiological conditions.

[0062] Example 4 Antibody-dependent cell-mediated cytotoxicity (ADCC) activity assay (1) Take SK-BR-3 (target cells) in the logarithmic growth phase, digest and count them, and adjust the density to 1×10⁻⁶. 5 Cells / mL, seeded into 96-well plates, 50 μL / well, i.e. 5000 cells / well, and incubated overnight in an incubator.

[0063] (2) Dilute the purified fusion protein 37 / 38 and control protein 145 (expressed control antibody that mediates the ADCC effect of NK cells against SK-BR-3 cells) with culture medium. The initial concentration is 10 μg / mL, and the concentration is serially diluted 10 times to 0.1 μg / mL, for a total of 3 concentrations, namely 10, 1 and 0.1 μg / mL.

[0064] (3) Discard the culture medium in the 96-well plate, and add 50 μL of the antibody diluted in the previous step, blank culture medium, etc. according to the sample well settings (see Table 1-Table 2 for details). Incubate in an incubator for 40 min to form a protein-target cell complex.

[0065] (4) Dilute effector cells (PBMC-NK, NK92, PBMC-T, Jurkat) to 1×10⁻⁶. 6 and / or 5×10 5cells / mL (PBMC-NK and NK92 cell experiments were set with an effector-to-target ratio of 10:1; PBMC-T and Jurkat cell experiments were set with two effector-to-target ratios, namely 10:1 and 5:1). 50 μL of effector cells and blank culture medium were added according to the sample well settings, and incubated in an incubator for 5 h.

[0066] Table 1. Experimental layout of 96-well plate for SK-BR-3 using PBMC-NK (or NK92).

[0067] Note: Background blank: Contains culture medium; used to subtract low background absorbance values ​​from control and sample wells. Spontaneous effect: contains culture medium and effector cells; Lysis buffer control: containing culture medium, add 10 μL Lysis Solution to each well; used to subtract high background absorbance values ​​from the control. Spontaneous target cell release: Contains target cells and culture medium, without lysis treatment; used to measure spontaneous LDH release from untreated normal cells. Maximum target cell concentration: 10 μL Lysis Solution is added to each well containing target cells and culture medium; this is used to determine the maximum release of LDH from cells. The same applies below.

[0068] Table 2. Experimental layout of 96-well plate for SK-BR-3 using PBMC-T (or Jurkat).

[0069] Note: In the table, (10:1) and (5:1) represent the number of effector cells added to the corresponding sample wells as 50,000 and 25,000, respectively.

[0070] (5) After culturing for 4.5 h, add 10 μL of LysisSolution from the LDH detection kit according to the sample well settings, and continue culturing for 5 h.

[0071] (6) Take out the 96-well plate, aspirate 50 μL of culture medium from each well, and then add 50 μL of Working Solution from the LDH detection kit to each well. Incubate in the dark on a shaker for 30 min.

[0072] (7) Add Stop Solution from the LDH detection kit to each well of the aforementioned 96-well plate, and use a microplate reader to detect ABS: OD. 490 .

[0073] (8) Calculate cytotoxicity according to the cytotoxicity calculation formula.

[0074] ADCC experimental group = ABS (experimental group) - ABS (background blank); Effector cell spontaneous release = ABS (spontaneous effector release) - ABS (background blank); Spontaneous release from target cells = ABS (spontaneous release from target cells) - ABS (background blank); Maximum release from target cells = ABS (maximum target cell release) - ABS (lysate control); Cytotoxicity (%) = (ADCC experimental group - spontaneous release from effector cells - spontaneous release from target cells) / (maximum release from target cells - spontaneous release from target cells) × 100%.

[0075] The results are as follows Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown.

[0076] The results show that the fusion proteins 37 and 38 can induce antibody-dependent NK cell (PBMC-NK and NK92) and T cell (PBMC-T and Jurkat) mediated cytotoxicity, respectively, i.e., they have ADCC activity.

[0077] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0078] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fusion protein consisting of a bispecific antibody targeting T cells and Her2-positive cells, tandemly linked to 4-1BBL, characterized in that... The fusion protein is a heterotetramer structure consisting of two heavy polypeptide chains and two light polypeptide chains linked by disulfide bonds. The heavy polypeptide chain includes the heavy chain variable region of the Her2 targeting antibody, the heavy chain constant region of the human IgG1 antibody, and the 4-1BBL extracellular region. The light polypeptide chain comprises a light chain variable region of the Her2-targeting antibody, a light chain constant region of the human antibody, and a single-chain variable region of the CD3-targeting antibody.

2. The fusion protein according to claim 1, characterized in that, The single-chain variable region of the CD3-targeting antibody in the light polypeptide chain includes a heavy chain variable region VH and a light chain variable region VL, wherein: The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region VH are shown in SEQ ID NO. 3-5, respectively. The amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region VL are shown in SEQ ID NO. 6, GTN, and SEQ ID NO. 7, respectively. The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region VH of the Her2-targeting antibody in the aforementioned heavy polypeptide chain are shown in SEQ ID NO. 8-10, respectively. The amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region VL of the Her2-targeting antibody in the light polypeptide chain are shown in SEQ ID NO. 11, SAS, and SEQ ID NO. 12, respectively.

3. The fusion protein according to claim 2, characterized in that, The heavy chain variable region VH of the CD3-targeting antibody in the light polypeptide chain also includes FR-H1, FR-H2, FR-H3, and FR-H4 framework regions, the amino acid sequences of which are shown in SEQ ID NO. 13-16; The light chain variable region VL of the CD3-targeting antibody in the light polypeptide chain also includes FR-L1, FR-L2, FR-L3, and FR-L4 framework regions, the amino acid sequences of which are shown in SEQ ID NO. 17-20; The heavy chain variable region VH of the Her2-targeting antibody in the aforementioned heavy polypeptide chain also includes FR-H1, FR-H2, FR-H3, and FR-H4 framework regions, the amino acid sequences of which are shown in SEQ ID NO. 21-24; The light chain variable region VL of the Her2-targeting antibody in the light polypeptide chain also includes FR-L1, FR-L2, FR-L3, and FR-L4 framework regions, the amino acid sequences of which are shown in SEQ ID NO. 25-28.

4. The fusion protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region VH of the CD3-targeting antibody in the light polypeptide chain is shown in SEQ ID NO.

29. The amino acid sequence of the variable region VL of the CD3-targeting antibody in the light polypeptide chain is shown in SEQ ID NO. 30; The amino acid sequence of the VH variable region of the heavy chain of the Her2-targeting antibody in the aforementioned heavy polypeptide chain is shown in SEQ ID NO. 31; The amino acid sequence of the variable region VL of the light chain of the Her2-targeting antibody in the light polypeptide chain is shown in SEQ ID NO.

32.

5. The fusion protein according to claim 4, characterized in that, The amino acid sequence of the single-chain variable region of the CD3-targeting antibody in the light polypeptide chain is shown in SEQ ID NO. 33; The single-chain variable region of the CD3-targeting antibody in the light polypeptide chain is composed of a heavy chain variable region VH and a light chain variable region VL connected by a flexible linker peptide, the amino acid sequence of which is shown in SEQ ID NO.

34. The amino acid sequence of the extracellular region of human 4-1BBL in the heavy polypeptide chain is shown in SEQ ID NO. 35; The amino acid sequence of the heavy chain constant region of the human IgG1 antibody in the aforementioned heavy polypeptide chain is shown in SEQ ID NO. 36; The amino acid sequence of the light chain constant region of the human antibody in the light polypeptide chain is shown in SEQ ID NO.

37.

6. The fusion protein according to any one of claims 1-5, characterized in that, The amino acid sequence of the heavy polypeptide chain is shown in SEQ ID NO. 1, and the amino acid sequence of the light polypeptide chain is shown in SEQ ID NO.

2.

7. A nucleic acid molecule encoding the fusion protein as described in any one of claims 1-6.

8. An expression vector or host cell comprising the nucleic acid molecule as described in claim 7.

9. A method for preparing the fusion protein according to any one of claims 1-6, comprising culturing the host cell as described in claim 8 under conditions suitable for expression, and recovering the fusion protein from the culture.

10. Use of the fusion protein according to any one of claims 1-6 in the preparation of reagents and / or medicaments for detecting and / or treating Her2-positive tumors.

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