Bispecific bridging body and application thereof in treating malignant tumors

Abstract

The invention discloses a bispecific bridging body, the bridging body contains an xPD1 fragment capable of being specifically combined with PD-L1 molecules on the surface of a tumor cell with high affinity and a CD16A scFv fragment capable of specifically recognizing a CD16A receptor on the surface of an NK cell, a nucleotide sequence for coding the xPD1 fragment is shown as SEQ ID NO.1, and a nucleotide sequence for coding the CD16A scFv fragment is shown as SEQ ID NO.3. The invention further discloses a preparation method of the bispecific bridging body. The targeting recognition precision of the NK cells on tumor cells is remarkably improved, invalid migration of the NK cells in vivo is avoided, and the killing activity of the NK cells can be enhanced through the synergistic effect of CD16A receptor aggregation activation and PD-L1 pathway blocking.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a bispecific bridge and application thereof in treatment of malignant tumors, and the classification number is C07K16 / 46. Specifically, the application designs and constructs a bispecific bridge aiming at the PD-1 / PD-L1 pathway and the CD16A receptor on the surface of NK cells, and the killing activity of the NK cells on tumor cells can be significantly improved. BACKGROUND

[0003] In recent years, with the continuous development of immunology theory and technology, immunotherapy has become a research hotspot and core development direction in the field of tumor treatment due to its unique mechanism of action and significant therapeutic effect, and has shown excellent antitumor activity in the clinical treatment of various malignant tumors. Unlike the mechanism of traditional therapy which directly acts on tumor cells to achieve killing, immunotherapy mainly adjusts the body's own immune system, activates immune cells with tumor recognition ability in the body such as T cells, NK cells, dendritic cells and macrophages, and enhances the recognition and response ability of the body's immune system to tumor cells, thereby indirectly achieving specific removal and growth control of tumor cells, and fundamentally improving the immune function state of tumor patients, providing a new idea and strategy for cancer treatment.

[0004] Among various immune cells involved in tumor immune response, NK cells, as one of the core components of the innate immune system, can quickly recognize and kill abnormal cells in the body, including cancer cells, virus-infected cells, etc., and this killing process does not require a prior antigen sensitization process or the participation of antibodies for mediation, and is not limited by the major histocompatibility complex (MHC), so that it can still effectively play an antitumor role when the tumor occurs in the early stage or the tumor cells escape T cell immune surveillance by down-regulating MHC molecule expression. Therefore, NK cells show broad application prospects in the field of cellular immunotherapy, can play a good therapeutic effect on tumor types with down-regulated MHC-I molecule expression, and usually have lighter treatment-related side effects in clinical application.

[0005] Surface markers of NK cells have been important research objects in the field of immunotherapy, as they are the key molecules for identifying, sorting, functional research and activity evaluation of NK cells. Among them, CD16A molecule, as an important receptor on the surface of NK cells, plays a core role in mediating the antibody-dependent cell-mediated cytotoxicity (ADCC) effect of NK cells. CD16A can couple the specific IgG antibody produced by the adaptive immune system with the killing activity of NK cells, enabling NK cells to accurately recognize and kill tumor cells marked by antibodies, and becomes the core mechanism for the therapeutic effect of various therapeutic antibody drugs in clinical treatment. In addition, the structural and functional properties of CD16A molecule make it an important target for the design and optimization of current immunotherapy drugs. By modifying or regulating CD16A molecule, it is expected to further enhance the anti-tumor activity of NK cells and the therapeutic effect of antibody drugs, providing key technical support for the development of new and efficient immunotherapy programs.

[0006] In tumor immune checkpoint therapy, the PD-1 / PD-L1 pathway has become one of the most widely studied and most successfully clinically transformed targets due to its key role in tumor immune escape. The blocking strategy based on this pathway has become an important part of cancer immunotherapy. PD-L1 molecule, as the ligand of PD-1 receptor, is often expressed on the surface of tumor cells, and through its specific binding with PD-1 receptor on the membrane of activated cytotoxic T lymphocytes, it transmits an immunosuppressive signal to T cells, significantly inhibiting the proliferation, activation and cytotoxicity of T cells, and ultimately leading to the successful escape of tumor cells from the monitoring and elimination of the body's immune system, achieving immune escape.

[0007] In order to effectively block the immunosuppressive effect mediated by the PD-1 / PD-L1 pathway, the PD-1 molecule was modified by genetic engineering technology, and a high-affinity PD-1 variant (xPD1) was obtained. Compared with the wild-type PD-1 molecule, xPD1 can specifically bind to the PD-L1 molecule expressed on the surface of tumor cells with higher affinity, thereby more effectively competing to block the PD-1 / PD-L1 pathway and relieving the immunosuppressive effect of tumor cells on immune effector cells such as T cells and NK cells. This blocking strategy can quickly restore the normal anti-tumor immune response function of the body's immune system, prompting immune effector cells to efficiently recognize and attack tumor cells.

[0008] Therefore, there is an urgent need for a bridge that can specifically recognize the PD-L1 molecule on the surface of tumor cells and the CD16A receptor on the surface of NK cells, which can relieve the immunosuppressive effect of tumor cells on immune effector cells such as T cells and NK cells by blocking the PD-1 / PD-L1 pathway, and at the same time, by binding with CD16A, activate NK cells, further enhance the targeting killing efficiency of NK cells on tumor cells. Summary of the Invention

[0009] This invention addresses the technical problem by overcoming the shortcomings of existing technologies and provides a bispecific bridging agent and its application in the treatment of malignant tumors. This bridging agent targets the PD-L1 molecule on the surface of tumor cells and the CD16A receptor on the surface of NK cells. By blocking the PD-1 / PD-L1 immunosuppressive pathway and simultaneously activating NK cells by binding to CD16A, it spatially shortens the distance between tumor cells and NK cells, significantly enhancing the killing efficiency of NK cells against tumor cells, thereby significantly improving the therapeutic effect of cancer.

[0010] This invention addresses the insufficient synergy between cell therapy and immune checkpoint blockade in existing cancer immunotherapies by integrating these two therapeutic strategies. A bispecific bridging complex is designed with a modular structure, comprising a CD16A-specific binding domain and a PD-L1-specific binding domain. The CD16A-specific binding domain specifically recognizes and binds to the CD16A receptor on the surface of NK cells, while the PD-L1-specific binding domain specifically targets the highly expressed PD-L1 antigen on the surface of tumor cells. This dual-target recognition establishes a stable bridge between NK cells and tumor cells, forming a ternary complex of NK cell-bridging complex-tumor cell. This invention not only significantly improves the targeting accuracy of NK cells against tumor cells and avoids ineffective migration of NK cells in vivo, but also enhances the cytotoxic activity of NK cells through the synergistic effect of CD16A receptor aggregation activation and PD-L1 pathway blockade.

[0011] One objective of this invention is to provide a bispecific bridging agent containing an xPD1 fragment that can specifically bind to PD-L1 molecules on the surface of tumor cells with high affinity and a CD16A scFv fragment that can specifically recognize the CD16A receptor on the surface of NK cells. The nucleotide sequence encoding the xPD1 fragment is shown in SEQ ID NO.1, the nucleotide sequence encoding the CD16A scFv fragment is shown in SEQ ID NO.3, and the amino acid sequence of the bispecific bridging agent is shown in SEQ ID NO.12.

[0012] The further optimized technical solution of this invention is as follows: Preferably, the bispecific bridger further contains a linker peptide that links the xPD1 fragment to the CD16A scFv fragment.

[0013] Preferably, the nucleotide sequence encoding the linker peptide is shown in SEQ ID NO.2.

[0014] Preferably, the N-terminus of the linker peptide is linked to the xPD1 fragment, and the C-terminus is linked to the CD16A scFv fragment.

[0015] The above structure can effectively maintain the activity of xPD1 and CD16A scFv proteins in the bridging body.

[0016] The second objective of this invention is to provide a method for preparing a bispecific bridging agent, comprising: Step 1: Obtain the DNA molecule encoding the xPD1 fragment and the DNA molecule encoding the CD16A scFv fragment; Step 2: The DNA molecule encoding the xPD1 fragment obtained in Step 1 is ligated with the DNA molecule encoding the CD16A scFv fragment, fused with the expression vector, and transformed into host cells; Step 3: Induce host cells containing the recombinant expression vector to express the fusion protein, and isolate and purify to obtain high-purity bridging protein.

[0017] More preferably, in step 1, obtaining the DNA molecule encoding the xPD1 fragment specifically involves using high affinity... PD- L1 Using the gene as a template, a DNA molecule encoding the xPD1 fragment is amplified using an upstream primer having the nucleotide sequence shown in SEQ ID NO. 8 and a downstream primer having the nucleotide sequence shown in SEQ ID NO. 9; specifically, obtaining the DNA molecule encoding the CD16A scFv fragment involves using the light chain variable region gene and heavy chain variable region gene of the CD16A antibody as templates, and amplifying the DNA molecule encoding the CD16A scFv fragment using an upstream primer having the nucleotide sequence shown in SEQ ID NO. 10 and a downstream primer having the nucleotide sequence shown in SEQ ID NO. 11.

[0018] More preferably, in step 2, the expression vector is the pGEX-6P-1 vector, and the host cell is competent Escherichia coli DH5α.

[0019] A third objective of this invention is to provide a biomolecule, carrier, or host cell, wherein the biomolecule, carrier, or host cell includes a nucleic acid molecule encoding the aforementioned bispecific bridging agent and its amino acid sequence.

[0020] A fourth objective of this invention is to provide the application of the bispecific bridging agent and / or the biomolecule, carrier, or host cell in the preparation of drugs for treating malignant tumors.

[0021] In the above applications, the malignant tumor is any one or more of liver cancer, lung cancer, kidney cancer, and colorectal cancer.

[0022] The beneficial effects of this invention are as follows: The bispecific bridging agent described in this invention exhibits outstanding advantages in clinical translation and industrial application, laying the foundation for its application as a cancer treatment drug, especially for solid tumors, which are more difficult to treat clinically. In terms of clinical value, this bridging agent, through the synergistic effect of NK cell targeted activation and precise blocking of the PD-L1 pathway, can specifically solve the problems of insufficient immune cell infiltration and strong tumor microenvironment inhibition in the treatment of solid tumors. At the same time, the dual-target design reduces non-specific damage to normal tissues, further improving the efficacy and safety of anti-tumor drugs. In terms of industrialization, this bridging agent adopts a modular structural design, which has the characteristics of simple preparation process, short production cycle and high product stability. It is not only easy to achieve large-scale production, but also allows for precise control of product quality through standardized processes, effectively reducing R&D investment and production costs.

[0023] The above advantages help reduce the economic burden on patients and avoid treatment interruptions due to excessive costs. Improved efficacy and reduced side effects can effectively improve patient treatment compliance, reduce subsequent risks caused by treatment intolerance or limited efficacy, and truly improve patients' quality of life and prognosis. From a socio-economic perspective, the research and development and transformation of this bridging agent can fill the technological gap in the field of solid tumor immunotherapy. On the other hand, its large-scale production and clinical application will form a complete industrial chain, drive the development of related industries in the biopharmaceutical field, and reduce the consumption of social medical resources caused by cancer treatment, thus possessing considerable economic benefits and broad market prospects. Attached Figure Description

[0024] Figure 1 This is an SDS-PAGE image of the purified protein of this invention.

[0025] Figure 2 This is a schematic diagram of the interaction results between the CD16A / PD-L1 bispecific bridger and PD-L1 detected by the GST pulldown method of the present invention.

[0026] Figure 3 This is a schematic diagram showing the results of the GST pulldown detection of the interaction between CD16A / PD-L1 bispecific bridger and CD16A according to the present invention.

[0027] Figure 4 This is a schematic diagram illustrating the effect of Western blotting on the overexpression of the PD-L1 gene in HepG2 cells according to the present invention.

[0028] Figure 5 This is a diagram illustrating the effect of the bispecific bridging agent of the present invention in mediating the killing of tumor cells by NK-92MI cells.

[0029] Figure 6This is a diagram illustrating the effect of the bispecific bridging agent of the present invention in mediating the killing of tumor cells by NK cells derived from PBMCs. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to the embodiments: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0031] Materials: 1×TE buffer (pH=8.0) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; trypsin, DMEM medium, and NK-92 MI special medium were purchased from Wuhan Pronosei Life Sciences Co., Ltd.; Opti-MEM... ® I. Culture medium was purchased from Gibco; restriction endonucleases... Age I, Eco RI and Bam HI was purchased from NEB; T4 DNA ligase was purchased from Nanjing Novizan Biotechnology Co., Ltd.; Escherichia coli DH5α was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; Lenti-X293T cell line was purchased from Clontech; puromycin was purchased from Amresco; plasmids pLKO.1, pCMV-VsVg, and pCMV-deltaR8.2 were purchased from Addgene; HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0032] The other materials and reagents mentioned in this invention are available to the public through commercial channels both domestically and internationally, and will not be described in detail here. Example 1

[0033] (1) Construction of recombinant plasmids ① Construction of recombinant plasmids pET-32a-CD16A and pET-32a-PD-L1 Obtain the nucleotide sequence information of the target gene from the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank). CD16A The gene's accession number is NM_000569.8. PD-L1 The gene accession number is NM_014143.4, and the PCR primer sequences are designed as follows: CD16A The upstream primer for the gene is: 5'-GCCATGGCTGATATCGGATCCATGCGGACTGAAGATCTCCCAAAG -3'; CD16A The downstream primer for the gene is: 5'-GTGGTGGTGGTGGTGCTCGAGTCATTTGTCTTGAGGGTCCTTTCTCC-3'; PD-L1 The upstream primer for the gene is: 5'-GCTGATATCGGATCCGAATTCGCATTTACTGTCACGGTTCC -3' PD-L1 The downstream primer for the gene is: 5'-GTGGTGGTGGTGGTGCTCGAGTTAGTATGGGGCATTGACTTTCAC-3' All PCR primers used in this invention were synthesized by Genewiz Biotechnology Co., Ltd.

[0034] Using cDNA from human chronic myeloid leukemia cells K-562, preserved in the Tumor Biology Laboratory of the School of Biological Science and Technology, Yangzhou University, as a template, a PCR amplification system was established for PCR amplification of the fragment. After purification using a PCR product recovery kit, the fragment was ligated with... Xho I / Bam The HI-double-digested vector pET-32a was ligated using standard methods, and the ligation product was transformed into competent *E. coli* DH5α. Single colonies were selected and cultured overnight, and plasmids were extracted to obtain recombinant plasmids. Xho I / Bam HI double digestion and sequencing analysis were used to identify the recombinant plasmids, obtaining the recombinant plasmids pET-32a-CD16A and pET-32a-PD-L1.

[0035] ② Construction of recombinant plasmid pGEX-6P-1-xPD1-CD16A scFv The PCR primer sequences are as follows: the upstream primer for xPD1 is 5'-TTCCAGGGGCCCCTGGGATCCccaggatggttcttagactcc-3'; the downstream primer is 5'-GCTTCCTCCACCGCCAGAGCCACCTCCTCCACTTCCGCCACCTCCTCTAGAcaccagggtttggaactggc-3'; The upstream primer for CD16A_scFv is 5'-GCTCTGGCGGTGGAGGAAGCttcgaacaggtgcagctggtgcagag-3'; the downstream primer is 5'-CTCGAGTCGACCCGGGAATTCtcacagcacggtcagc-3'.

[0036] The nucleotide sequences of CD16A_scFv, the linker peptide, and the xPD1 fragment were spliced ​​and amplified using overlap extension PCR. xPD1 is located at the N-terminus of the linker peptide, and its nucleotide sequence is shown in SEQ ID NO.1. CD16A_scFv is located at the C-terminus of the linker peptide, and its nucleotide sequence is shown in SEQ ID NO.3. The nucleotide sequence of the linker peptide is shown in SEQ ID NO.2. Then, using... Bam HI and Eco The pGEX-6P-1 vector was double-digested with RI enzymes. The PCR-amplified fragment xPD1-CD16A scFv was ligated into the purified double-digested vector, transformed into competent E. coli DH5α, and single colonies were picked and cultured overnight. Extraction yielded the recombinant plasmid. Xho I / Acc The recombinant plasmid was identified by double digestion with 65I and sequencing analysis.

[0037] (2) Induction and purification of recombinant proteins Take 0.5 µL of recombinant plasmid and add it to 10 µL of BL21(DE3) competent cells or Rosetta(DE3) competent cells, and incubate on ice for 5 min; heat shock at 42℃ for 90 s, and incubate on ice for 2 min; add 200 µL of antibiotic-free LB medium, and incubate at 37℃ and 250 rpm for 10 min; spread on plates and incubate overnight at 37℃. The next day, pick several colonies and add them to 6 mL of LB medium containing ampicillin, and incubate overnight at 37℃ and 250 rpm. Then, add 6 mL of the overnight culture to 300 mL of LB medium containing ampicillin, and incubate at 37℃ and 250 rpm until OD. 600nm The concentration reached 0.4-0.6; 1 mM IPTG was added, and the mixture was incubated at 37℃ and 250 rpm for 3 h, or 0.2 mM IPTG was added, and the mixture was incubated at 20℃ and 250 rpm for 4 h; the bacteria were collected by centrifugation, resuspended with lysis buffer, and sonicated; centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant was collected; the recombinant protein in the supernatant was purified by agarose affinity chromatography, and the purification procedure was performed according to the Glutathione Sepharose 4B / Ni-NTA agarose instructions (GST fusion protein was purified according to the GE Glutathione Sepharose 4B agarose instructions, and His fusion protein was purified according to the Beyotime Ni-NTA agarose instructions). The induction and purification methods for GST-tagged and His-tagged proteins were the same as those described above. The purification effect of the target protein was identified by SDS-PAGE electrophoresis, and the results are as follows. Figure 1As shown. The amino acid sequence of the purified protein is SEQ ID NO.12.

[0038] (3) GST pulldown Take glutathione agarose resin (GST beads) into a 1.5 mL centrifuge tube, add 1 mL of BLB150 washing buffer, invert 6-8 times, centrifuge at 1000 g at 4℃, and discard the supernatant. Then, wash the GST beads sequentially with BLB500 and BLB150. Add 300 µL of the supernatant containing bait protein lysis buffer to the pretreated beads, incubate at 4℃ for 1 h, and then wash sequentially with BLB150, BLB500, and BLB150. Add 1 µg of protein to the bait protein-bound beads, incubate at 4℃ for 1 h, then wash 3-5 times with PBS buffer containing 0.1% Triton X-100, add 30 µL of 1× sample preparation buffer, heat at 95℃ for 5 min, and analyze the experimental results using Western blotting (see...). Figure 2 and Figure 3 BLB150 is a buffer solution containing 50 mM Tris-HCl, 0.01 M EDTA, 0.1% Triton X-100, 10% glycerol, and 150 mM NaCl. BLB500 is a buffer solution containing 50 mM Tris-HCl, 0.01 M EDTA, 0.1% Triton X-100, 10% glycerol, and 500 mM NaCl.

[0039] (4) Packaging of lentiviruses Lentiviral cells were packaged using the pLVX-IRES-puro-PD-L1 recombinant plasmid stored in the Tumor Biology Laboratory of the School of Biological Science and Technology, Yangzhou University: The lentiviral recombinant plasmid was prepared, and the lentivirus was packaged according to the instructions for Lipofectamine 2000 reagent. The method for packaging lentivirus in a 6-well cell culture plate is as follows: ①Inoculate Lenti-X 293T cells into 6-well cell culture plates and incubate overnight at 37°C in a 5% CO2 incubator. Start the experiment when the cell confluence reaches 90%-95%. ② Take a 1.5 mL centrifuge tube 1 and add 195 μL of Opti-MEM. ® In medium I, add the three plasmids (total plasmid amount 1.17 μg) to centrifuge tubes according to the plasmid mass ratio pLVX-IRES-puro-PD-L1∶pCMV-VsVg∶pCMV-delta-R8.2 = 3∶1∶2, mix well, and let stand at room temperature until ready for use. ③ Take a new 1.5 mL centrifuge tube 2 and add 195 μL of Opti-MEM. ® Mix culture medium I with 4.68 μL of Lipofectamin 2000 by inverting the container and let stand at room temperature for 5 min. ④ Add all the mixture from centrifuge tube 2 to centrifuge tube 1, gently invert to mix, and let stand at room temperature for 20 minutes; ⑤ Transfer the mixture from step ④ to the wells of a 6-well cell culture plate inoculated with Lenti-X 293T cells, mix gently, and incubate at 37°C in a 5% CO2 incubator. After 11 h, change the medium with fresh DMEM. ⑥ Continue culturing for 48 h, collect the supernatant into a 15 mL centrifuge tube and temporarily store it at 4℃. At the same time, add fresh DMEM medium to the 6-well cell culture plate and continue culturing for 24 h at 37℃ and 5% CO2. ⑦ Collect the cell culture supernatant again into a 15 mL centrifuge tube, and gently mix the two supernatants by inverting the tube. ⑧ Centrifuge the mixed supernatant at 3000 rpm for 10 min at room temperature, then aliquot the supernatant into 1.5 mL centrifuge tubes, 1 mL / tube, and store at -80℃ for later use or directly for cell infection.

[0040] (5) Lentiviral infection of HepG2 cells and evaluation of overexpression effect Lentiviral cells packaged with the recombinant plasmid pLVX-IRES-puro-PD-L1 were used to infect HepG2 human liver cancer cells in logarithmic growth phase; then, the intracellular PD-L1 protein level was detected by Western blotting to evaluate the overexpression effect of PD-L1. The specific methods are as follows: Human hepatocellular carcinoma cells HepG2 were seeded in 6 cm cell culture plates at a density of 4 × 10⁴ cells per well. 5 Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. Lentiviral virus was added to the cell culture plate, followed by polybrene at a final concentration of 8 μg / mL. After 16-18 hours, the medium was replaced with fresh DMEM containing serum. Two days later, puromycin was added for selection to obtain stable cell lines. After another 5 days of culture, cells were collected, and total protein was extracted. The relative levels of intracellular PD-L1 protein were detected by Western blotting. Results are shown below. Figure 4 As shown, the target gene PD-L1 Overexpression was evident in HepG2 cells.

[0041] (6) Bispecific bridging agents mediate the killing effect of NK cells on tumor cells HepG2 cells overexpressing PD-L1 were seeded into 96-well cell culture plates at 5000 cells per well. After 24 h of culture, 5000 NK cells and purified bispecific bridging agents were added, and the cells were cultured in a cell culture incubator until the set killing time was reached. The OD was measured using a CCK assay kit. 450nm To evaluate the killing effect, GST-tagged protein and protein purification eluent were set as negative control groups. The specific operating steps are as follows; ① HepG2 cells overexpressing PD-L1 were seeded in 96-well cell culture plates at 5 × 10⁶ cells / well. 3 / well, after incubation at 37℃ and 5% CO2 for 24 h, the culture medium was discarded and fresh DMEM culture medium was added, 100 µL per well.

[0042] ② Collect NK cells in 15 mL centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in NK-specific culture medium, count the cells, and then divide them into groups of 5 × 10⁻⁶ cells. 3 80 µL / well was seeded into a 96-well cell culture plate.

[0043] ③ According to the experimental design, the bispecific bridging protein and GST-tagged protein were diluted to 0.035 µg / µL with DMEM medium, and 20 µL (0.7 µg) was added to each well. Protein elution buffer was set up as a control. The mixture was then incubated at 37℃ in a 5% CO2 incubator.

[0044] ④ After the set killing time, discard the culture medium in the cell culture plate, add fresh culture medium containing 10% CCK solution, and continue culturing in a 37℃, 5% CO2 incubator for 4 h. Measure the OD. 450nm .

[0045] Using the NK-92MI cell line, the killing time was set to 4 hours, based on OD... 450nm To evaluate whether bispecific bridging agents can enhance the cytotoxic effect of NK cells. Results are as follows: Figure 5 As shown, there was no significant difference in cell activity among the HepG2 group, HepG2+GST group, and HepG2+Engager group, suggesting that the bridging agent (Engager) itself has no significant killing effect on liver cancer cells. Compared with the above three groups, the cell survival rate of the HepG2+NK-92MI group was reduced, indicating that NK-92MI cells have the activity of killing tumor cells. Compared with the HepG2+NK-92MI group and the HepG2+NK-92MI+GST group, the cell survival rate of the HepG2+NK-92MI+Engager group was significantly reduced, suggesting that the bridging agent can significantly enhance the killing activity of NK cells against HepG2 cells.

[0046] Using human NK cells isolated from PBMCs, the killing time was set to 24 h, and the results were as follows: Figure 6 As shown, there was no significant difference in cell activity among the HepG2 group, HepG2+GST group, and HepG2+Engager group, suggesting that the bridging agent itself has no significant killing effect on liver cancer cells. Compared with the above three groups, the cell survival rate of the HepG2+NK group was reduced, indicating that the human NK cells isolated from PBMCs have the activity of killing tumor cells. However, compared with the HepG2+NK group or the HepG2+NK+GST group, the cell survival rate of the HepG2+NK+Engager group was significantly reduced, suggesting that the bridging agent can significantly enhance the killing effect of human NK cells on HepG2 cells.

[0047] The bridging agent of this invention clones xPD1 and CD16A scFv fragments into the prokaryotic expression vector pGEX-6P-1, and obtains high-purity protein via glutathione agarose resin affinity chromatography. In vitro pull-down experiments confirmed that this bridging agent can effectively mediate efficient and specific binding between NK cells and tumor cells. Further cell killing experiments showed that the bridging agent can significantly enhance the cytotoxic activity of NK cells against tumor cells. Therefore, the bispecific bridging agent of this invention can be developed into a drug molecule that enhances the therapeutic effect of NK cells for cancer treatment.

[0048] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A dual-specificity bridge, characterized in that, The bispecific bridging agent contains an xPD1 fragment that can specifically bind to the PD-L1 molecule on the surface of tumor cells with high affinity and a CD16A scFv fragment that can specifically recognize the CD16A receptor on the surface of NK cells. The nucleotide sequence encoding the xPD1 fragment is shown in SEQ ID NO.1, the nucleotide sequence encoding the CD16A scFv fragment is shown in SEQ ID NO.3, and the amino acid sequence of the bispecific bridging agent is shown in SEQ ID NO.

12.

2. The dual-specificity bridge according to claim 1, characterized in that, The bispecific bridger also contains a linker peptide that links the xPD1 fragment to the CD16A scFv fragment.

3. The dual-specificity bridge according to claim 2, characterized in that, The nucleotide sequence encoding the linker peptide is shown in SEQ ID NO.

2.

4. The dual-specificity bridge according to claim 3, characterized in that, The linker peptide is linked to the xPD1 fragment at its N-terminus and to the CD16A scFv fragment at its C-terminus.

5. A method for preparing a bispecific bridging agent, characterized in that, include: Step 1: Obtain the DNA molecule encoding the xPD1 fragment and the DNA molecule encoding the CD16A scFv fragment; Step 2: The DNA molecule encoding the xPD1 fragment obtained in Step 1 is ligated with the DNA molecule encoding the CD16A scFv fragment, fused with the expression vector, and transformed into host cells; Step 3: Induce host cells containing the recombinant expression vector to express the fusion protein, and isolate and purify to obtain high-purity bridging protein.

6. The method for preparing a bispecific bridging agent according to claim 5, characterized in that, In step 1, obtaining the DNA molecule encoding the xPD1 fragment specifically involves using high affinity... PD-L1 Using the gene as a template, a DNA molecule encoding the xPD1 fragment is amplified using an upstream primer having the nucleotide sequence shown in SEQ ID NO. 8 and a downstream primer having the nucleotide sequence shown in SEQ ID NO. 9; specifically, obtaining the DNA molecule encoding the CD16A scFv fragment involves using the light chain variable region gene and heavy chain variable region gene of the CD16A antibody as templates, and amplifying the DNA molecule encoding the CD16A scFv fragment using an upstream primer having the nucleotide sequence shown in SEQ ID NO. 10 and a downstream primer having the nucleotide sequence shown in SEQ ID NO.

11.

7. The method for preparing a bispecific bridging agent according to claim 5, characterized in that, In step 2, the expression vector is pGEX-6P-1 vector, and the host cell is competent Escherichia coli DH5α.

8. A biomolecule, carrier, or host cell, characterized in that, The biomolecule, vector, or host cell includes a nucleic acid molecule and its amino acid sequence encoding the bispecific bridging agent of any one of claims 1 to 7.

9. The use of the bispecific bridging agent according to any one of claims 1 to 7 and / or the biomolecule, carrier or host cell according to claim 8 in the preparation of a drug for treating malignant tumors.

10. The application according to claim 9, characterized in that, The malignant tumor is any one or more of liver cancer, lung cancer, kidney cancer, and colorectal cancer.

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