Three-specificity adapter for regulating and controlling killing function of NK (Natural Killer) cells and application of three-specificity adapter

Abstract

The invention belongs to the technical field of biological medicine, and relates to a three-specificity adapter for regulating and controlling the killing function of NK cells and application of the three-specificity adapter. The biomarker is formed by sequentially connecting a target cell specific recognition domain, a first linking peptide, a killing activation domain, a second linking peptide and an inhibition removal domain, the target cell specific recognition domain can specifically recognize and combine target cells; the killing activation domain is a single-chain antibody targeting NKp46; the inhibition release domain is a single-chain antibody targeting NKG2A; the first linking peptide is a linear peptide with an amino acid sequence as shown in SEQ ID NO: 4; and the second linking peptide is a linear peptide with an amino acid sequence as shown in SEQ ID NO: 5. The specific killing activation domain, the inhibition release domain and the matched linker are accurately selected, so that the killing effect of the NK cells is accurately regulated and controlled, the immune response is activated, and the disease treatment effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a trispecific connector for regulating the killing function of NK cells and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Natural killer (NK) cells are key effector cells in the innate immune system, playing a central role in tumor clearance, pathogen defense, and immune regulation. The activation of their functional activity strictly depends on the dynamic balance of signals transmitted by various activating and inhibitory receptors on the cell surface. In recent years, NK cell-based immunotherapy strategies have become a research hotspot in the fields of oncology and infectious diseases. Their advantages lie not only in their ability to directly recognize and kill malignant transformed or infected cells, but also in their ability to further activate and coordinate adaptive immune responses by secreting key cytokines such as interferon-γ and tumor necrosis factor-α, thereby producing broad and durable anti-disease effects.

[0004] To achieve precise targeting of NK cell function, various multispecific molecular connectors have been developed, among which trispecific antibodies have attracted much attention due to their ability to simultaneously bind to multiple targets on both target cells and NK cells. However, existing trispecific antibodies have significant limitations in their design: First, the cytotoxic activation domains they employ often suffer from insufficient affinity or low signal activation efficiency, resulting in ineffective activation of NK cells; second, the inhibitory deactivation domains they contain have limited blocking ability against inhibitory receptors, failing to effectively relieve the inhibition of NK cell function under conditions such as the tumor microenvironment; more critically, the design of connectors between functional modules is often neglected, lacking reasonable rigidity and length regulation, resulting in the inability of the various structural domains to work synergistically in space, severely weakening the overall signal regulation efficiency.

[0005] The aforementioned deficiencies collectively make it difficult for existing connectives to effectively reverse the "inhibition stronger than activation" signaling state under physiological conditions. This not only reduces the killing efficiency of NK cells against target cells but may also lead to severe cytokine release syndrome due to unbalanced signal input, or cause premature depletion of NK cells and a significant decrease in functional durability. Furthermore, diseased cells (such as tumor cells) can achieve immune escape by dynamically downregulating activating ligands and upregulating inhibitory ligands, making the effectiveness of existing connectives with single mechanisms or imperfect structures unstable and limited in practical clinical applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a trispecific linker for regulating NK cell killing function and its application. The present invention achieves precise regulation of NK cell killing by accurately selecting specific killing activation domains, inhibition deactivation domains, and matching linkers, thereby activating the immune response and improving the therapeutic effect of diseases.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In the first aspect, a trispecific adaptor for regulating NK cell killing function is formed by sequentially connecting a target cell-specific recognition domain, a first adaptor peptide, a killing activation domain, a second adaptor peptide, and an inhibition release domain. The target cell-specific recognition domain is capable of specifically recognizing and binding to target cells; the killing activation domain is a single-chain antibody (scFv) targeting NKp46; the inhibition deactivation domain is a single-chain antibody (scFv) targeting NKG2A; the first adaptor peptide is a linear peptide with the amino acid sequence shown in SEQ ID NO: 4; the second adaptor peptide is a linear peptide with the amino acid sequence shown in SEQ ID NO: 5.

[0008] Single-chain antibodies targeting NKp46 can efficiently activate the NKp46 killing activation signal receptor on the surface of NK cells, significantly enhancing the killing ability of NK cells and solving the problem of poor signal regulation performance of traditional activation modules. Single-chain antibodies targeting NKG2A can potently block the NKG2A killing inhibition signal receptor on the surface of NK cells, relieving the killing inhibition of NK cells on target cells and solving the problem of poor or no signal regulation performance of traditional blocking modules. At the same time, through the rigidity, flexibility and length design of the first and second adaptor peptides, it is ensured that the killing activation domain and the inhibition deactivation domain can act on the same NK cell simultaneously, synergistically regulating the killing activation signal and the killing inhibition signal, efficiently initiating the killing action of NK cells, and ensuring that the target cell specific recognition domain accurately binds to the target cell, further enhancing the synergy of signal regulation and the efficiency of targeted killing.

[0009] Secondly, a nucleic acid molecule encoding the three-specificity adapter described in the first aspect of the present invention.

[0010] Thirdly, a composition comprising the trispecific adapter described in the first aspect of the invention or the nucleic acid molecule described in the second aspect of the invention, and a carrier.

[0011] Fourthly, the use of a trispecific connector as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, or a composition as described in the third aspect of the present invention in the preparation of a medicament.

[0012] The beneficial effects of this invention are as follows: 1. Dual signal regulation of highly efficient activation and potent de-inhibition. This invention employs a high-affinity single-chain antibody targeting NKp46 as the cytotoxic activation domain, significantly enhancing the intensity and persistence of NK cell activation signals; simultaneously, a single-chain antibody targeting NKG2A is selected as the de-inhibition domain, effectively blocking inhibitory signal transduction. This design overcomes the overactivation risk caused by traditional "dual activation" structures, significantly reducing the probability of cytokine release syndrome while enhancing cytotoxic efficacy, avoiding premature NK cell depletion, and achieving long-term, safe immune function regulation.

[0013] 2. Spatial Coordination and Conformation Optimization of Connector Peptides. This invention introduces a rigid connector peptide (SEQ ID NO: 4) and a flexible connector peptide (SEQ ID NO: 5) to rationally regulate the relative orientation and degrees of freedom between functional domains. This connector system not only ensures that the activation domain, inhibition release domain, and targeting module do not interfere with each other during binding, but also promotes the formation of a synergistic signaling complex on the surface of NK cells, significantly enhancing signal integration efficiency and targeting accuracy.

[0014] 3. Significantly enhanced killing efficiency and broad therapeutic potential. In vitro experiments have confirmed that the trispecific binder provided by this invention can significantly enhance the killing activity of NK cells against various tumor cells, while maintaining high clearance capacity under low effector-to-target ratio conditions. This molecular design balances signal strength, safety, and biological stability, and has important clinical application value and development prospects in the fields of solid tumors, hematological malignancies, and viral infections. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is a schematic diagram of the three-specificity connector that regulates the killing function of NK cells in Embodiment 1 of the present invention; Figure 2 TrKE in Embodiment 2 of the present invention B7H3 Western blot analysis results; Figure 3 TrKE in Embodiment 3 of the present invention B7H3 Effect of NK cell killing activity (Figure); Figure 4 TrKE in Embodiment 4 of the present invention B7H3 The effect of intratumoral injection on mouse survival. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] In view of the problems of low NK cell killing efficiency, easy to cause side effects and functional exhaustion caused by insufficient killing activation signal, insufficient blocking of inhibition signal and unreasonable connection structure between modules in the NK cell connector, this invention proposes a three-specific connector for regulating NK cell killing function and its application.

[0020] A typical embodiment of the present invention provides a trispecific adaptor for regulating NK cell killing function, which is formed by sequentially connecting a target cell specific recognition domain, a first adaptor peptide, a killing activation domain, a second adaptor peptide, and an inhibition release domain. The target cell-specific recognition domain is capable of specifically recognizing and binding to target cells; the killing activation domain is a single-chain antibody targeting NKp46; the inhibition deactivation domain is a single-chain antibody targeting NKG2A; the first adaptor peptide is a linear peptide with an amino acid sequence as shown in SEQ ID NO: 4; the second adaptor peptide is a linear peptide with an amino acid sequence as shown in SEQ ID NO: 5.

[0021] The target cell-specific recognition domain can specifically recognize the following target cell sites: CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56, CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECLp1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptor, PRSS21, VEGFR2, PDGFR-B, SSEA-4, EGFR, NCAM, mesothelin, EBNA-1, LEMD1, phosphatidylserine, and carcinoembryonic antigen (CEA). A) B cell maturation antigen (BCMA), phosphatidylinositol polysaccharide 3 (GPC3), follicle-stimulating hormone receptor, prostaglandin, thymidine kinase (TK1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), human epidermal growth factor receptor 2 (HER2), fibroblast activator protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) ligand, disialotyl-ganglioside 2 (GD2), PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, IGLLp1 and combinations thereof. Among them, carcinoembryonic antigen (CEA) is selected from MUC16, CCAT2, CTAG1A, CTAG1B, CT45A1, CT45A2, CT45A3, CT45A5, CT45A6, CT45A8, CT45A10, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A8, CT47A9, CT47A10, CT47A11, and C. T47A12, CT47B1, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, PRAME, PCA3, MAGEC1, MAGEC2, MAGED2, AFP, MAGEA8, MAGE9, MAGEA11, MAGEA12, ILp13RA2, PLAC1, SDCCAG8, LSP1, SAGE1, CT55, and B7H3. In some embodiments, the target cell-specific recognition domain is capable of specifically recognizing the B7H3 site of the target cancer cell.

[0022] The target cell-specific recognition domain may include any portion known to specifically bind to the target. As described in more detail below, the target cell-specific recognition domain may include antibodies, affinity molecules, or other protein-based ligands (e.g., Gp2-based protein ligands) known to bind to the selected target. The term "antibody" generally refers to an immunoglobulin or a fragment thereof. Thus, as used herein, the term "antibody" includes antibody fragments capable of binding to a biomolecule (e.g., an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab′, and F(ab′)2, pFc′, Fd, single-domain antibodies (sdAb), variable fragments (Fv), single-chain variable fragments (scFv) or disulfide-linked Fv (sdFv), bivalent antibodies or bivalent-bivalent-divalent antibodies, linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments (e.g., trisomes). The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the target cell-specific recognition domain is a single-chain antibody targeting B7H3. Specifically, the amino acid sequence of the target cell-specific recognition domain is shown in SEQ ID NO: 2.

[0023] In some embodiments, the amino acid sequence of the single-chain antibody targeting NKp46 is shown in SEQ ID NO: 2.

[0024] In some embodiments, the amino acid sequence of the single-chain antibody targeting NKG2A is shown in SEQ ID NO: 3.

[0025] In some embodiments, the target cell-specific recognition domain, the first adaptor peptide, the cytotoxic activation domain, the second adaptor peptide, and the inhibition deactivation domain are sequentially connected from the N-terminus to the C-terminus.

[0026] In some embodiments, the amino acid sequence is as shown in SEQ ID NO: 6.

[0027] In another embodiment of the present invention, a nucleic acid molecule is provided that encodes the above-mentioned three specificity adapters.

[0028] In some embodiments, the nucleic acid molecule is a plasmid, messenger RNA (mRNA), circular RNA (circRNA), or self-replicating RNA (saRNA); preferably, it is a plasmid, messenger RNA (mRNA), or circular RNA (circRNA). Specifically, its nucleotide sequence is shown in SEQ ID NO: 7.

[0029] A third embodiment of the present invention provides a composition comprising the above-described three-specificity adapter or the above-described nucleic acid molecule, and a carrier.

[0030] Specifically, the three-specificity adaptor or nucleic acid molecule is encapsulated in a vector. After the composition is delivered into cells, it is translated into a functional protein by the host ribosomes.

[0031] In some embodiments, the carrier is selected from one or more of lipid nanoparticles (LNPs), inorganic nanoparticles, exosomes, adenoviruses, lentiviruses, retroviruses, oncolytic viruses, and protein-lipid carriers. Lipid nanoparticles (LNPs) are preferred.

[0032] A fourth embodiment of the present invention provides the application of the above-mentioned three-specificity connector, nucleic acid molecule or composition in the preparation of a drug.

[0033] In some embodiments, the drug has the effects of target cell killing, tumor immunity, infectious diseases, regulation of inflammatory responses and / or regulation of autoimmune diseases.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Example 1: TrKE, a three-specificity adaptor regulating NK cell killing function B7H3 The synthesis of.

[0036] Step ①: Recombinant protein design and expression The single-chain antibody (scFv) sequences targeting human B7H3, NKG2A, and NKp46 were obtained from publicly available patent databases US2013149236A1, US2010247526A1, and WO2024251884A1, respectively. The final trispecific intercalator TrKE... B7H3 Form as Figure 1 As shown, from the N-terminus to the C-terminus, the sequence consists of a single-chain antibody against B7H3 (anti-B7H3 single-chain variable region fragment), a first adaptor peptide, a single-chain antibody activating NKp46 (activating NKp46 single-chain variable region fragment), a second adaptor peptide, and a single-chain antibody against NKG2A (anti-NKG2A single-chain variable region fragment). The protein sequence was reverse-translated and codon-optimized into a DNA sequence, which was then chemically synthesized and sequenced using GENEWIZ.

[0037] The sequence of the single-chain antibody (scFv) targeting human B7H3 is as follows: DVQLVESGGGLVQPGGGGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSDSSAIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCGRGRENIYYGGGGSRLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIAMTQSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGGGGSGGGGSGTDFTLTINNVQSEDLAEYFCQQYNNYPFTFGGGGSGTKLEIK, as shown in SEQ ID NO: 1.

[0038] The single-chain antibody (scFv) sequence targeting human NKp46 is: QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGGGGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGGGGSGGGGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIK, as shown in SEQ ID NO: 2.

[0039] The single-chain antibody (scFv) sequence targeting human NKG2A is: MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQFLVYNAKTLAEGVPSRFSGGGGSGGGGSGTQFSLKINSLQPEDFGGGGSYYCQHHYGTPRTFGGGTKLEIKGGGGSGGGGSGGGGSMGWSYILFLLATATCVHSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPEQGLQWIGRIDPYDSETHYSQKFKDKAILTVDKSSSTAYMRLSSLTSEDSAVYYCARGGYDFDVGTLYWFFDVWGAGTTVTVSS, as shown in SEQ ID NO: 3.

[0040] The amino acid sequence of the first linker peptide is: GSEAAKEAAKGS, as shown in SEQ ID NO: 4.

[0041] The amino acid sequence of the second linker peptide is EAAKEAAKGS, as shown in SEQ ID NO: 5.

[0042] The constructed three-specificity connector TrKE B7H3 The amino acid sequence is as follows: DVQLVESGGGLVQPGGGGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSDSSAIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCGRGRENIYYGGGGSRLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIAMTQSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGGGGSGGGGSGTDFTLTINNVQSEDLAEYFCQQYNNYPFTFGGGGSGTKLEIKGSEAAKEAAKGSQVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGGGGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGGGGSGGGGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIKEAAKEAAKGSMSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQFLVYNAKTLAEGVPSRFSGGGGSGGGGSGTQFSLKINSLQPEDFGGGGSYYCQHHYGTPRTFGGGTKLEIKGGGGSGGGGSGGGGSMGWSYILFLLATATCVHSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPEQGLQWIGRIDPYDSETHYSQKFKDKAILTVDKSSSTAYMRLSSLTSEDSAVYYCARGGYDFDVGTLYWFFDVWGAGTTVTVSS, as shown in SEQ ID NO: 6.

[0043] TrKE, a trispecific adaptor B7H3 The DNA sequence of

[0044] Step 2: Establishment of stable cell lines and protein expression Recombinant virus (titer 1.5 × 10⁻⁶) was packaged in Expi293F cells using a third-generation lentivirus system. 8 ZsGreen cells were infected with TU / mL at MOI=5 and ZsGreen was obtained by flow cytometry sorting. + High-expression monoclonal lines were developed. Cells were expanded in a suspension culture system, and the supernatant was collected for subsequent purification.

[0045] Step 3: Protein purification The target protein was purified by nickel ion affinity chromatography: the cell supernatant was equilibrated with binding buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and loaded onto the plate. The target peak was collected by gradient elution (500 mM imidazole). The buffer was replaced with PBS using a desalting column. The sample was then concentrated to a final concentration of ≥5 mg / mL by ultrafiltration centrifugation.

[0046] Example 2: Western blot verification of the specific connector TrKE B7H3 Successfully built.

[0047] Take purified TrKE B7H3 Proteins were dissolved in 4×Bolt™ LDS buffer and separated by electrophoresis on a 4-20% Bis-Tris gradient gel (200 V, 35 min). After transfer to a PVDF membrane, the following steps were performed: blocking at room temperature for 1 h, overnight incubation with primary antibody (anti-His tag: 1:2000) at 4°C, and incubation with HRP-conjugated secondary antibody (1:5000) at room temperature for 1 h. Immunoblot signal was then generated using an enhanced chemiluminescence reagent. Figure 2 The results showed a distinct band at approximately 81 kD, proving that TrKE B7H3 Successfully expressed.

[0048] Example 3: Verification of TrKE B7H3 Effects on NK cell killing activity.

[0049] U87-MG and U87-MG B7H3- Cells were labeled with CFSE and incubated at 37°C for 15 minutes. 1×10⁶ cells were then added. 5 U87-MG and U87-MG B7H3- Cells were seeded in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow them to adhere. The next day, 2×10⁶ cells were added to each well. 5 NK cells, using PBS or TrKE B7H3The treatment lasted 6 hours. After terminating the culture, cells from the co-culture system were collected, washed with PBS, stained with 7AAD, and the live / dead cell ratio of the target cells was detected using flow cytometry. Figure 3 The results are shown in TrKE B7H3 In the NK cell / U87-MG cell co-incubation system, NK cells showed a significantly enhanced killing effect on U87-MG.

[0050] Example 4 Intratumoral injection of TrKE B7H3 Protein assessment of in vivo efficacy.

[0051] U87-MG-Fluc cells (1×10) 5 Each mouse contained one cell line dissolved in 7 μL of 70% PBS and 30% Matrigel. The cells were stereotactically injected into the brains of C57BL / 6 mice (1.8 mm lateral, 0.6 mm anterior; 2 mm depth) to establish an orthotopic glioblastoma model. Mice began receiving intratumoral injections of TrKE on day 7 post-tumor inoculation. B7H3 Protein (2.5 mg / kg) or an equivalent volume of physiological saline was injected every 5 days for a total of 3 injections. On day 22 post-inoculation, 6 mice from each group were used for survival monitoring. Figure 4 The results show TrKE B7H3 The treatment group significantly prolonged the survival of mice with glioblastoma in situ, demonstrating superior tumor treatment efficacy.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A trispecific adaptor for regulating NK cell killing function, characterized in that, It is formed by the sequential connection of a target cell-specific recognition domain, a first adaptor peptide, a kill activation domain, a second adaptor peptide, and an inhibition deactivation domain; The target cell-specific recognition domain is capable of specifically recognizing and binding to target cells; the killing activation domain is a single-chain antibody targeting NKp46; the inhibition deactivation domain is a single-chain antibody targeting NKG2A; the first adaptor peptide is a linear peptide with an amino acid sequence as shown in SEQ ID NO: 4; the second adaptor peptide is a linear peptide with an amino acid sequence as shown in SEQ ID NO:

5.

2. The three-specificity connector as described in claim 1, characterized in that, The target cell-specific recognition domain can specifically recognize the B7H3 site of the target cancer cell. Alternatively, the target cell-specific recognition domain may be a single-chain antibody targeting B7H3; Alternatively, the amino acid sequence of the target cell-specific recognition domain is shown in SEQ ID NO:

2.

3. The three-specificity connector as described in claim 1, characterized in that, The amino acid sequence of the single-chain antibody targeting NKp46 is shown in SEQ ID NO:

2.

4. The three-specificity connector as described in claim 1, characterized in that, The amino acid sequence of the single-chain antibody targeting NKG2A is shown in SEQ ID NO:

3.

5. The three-specificity connector as described in claim 1, characterized in that, The target cell-specific recognition domain, the first adaptor peptide, the cytotoxic activation domain, the second adaptor peptide, and the inhibition deactivation domain are sequentially linked from the N-terminus to the C-terminus.

6. A nucleic acid molecule characterized by, The three-specific connector as described in any one of claims 1 to 5 is encoded.

7. The nucleic acid molecule as described in claim 6, characterized in that, Nucleic acid molecules can be plasmids, messenger RNA, circular RNA, or self-replicating RNA.

8. A composition characterized in that, It includes the trispecific adapter as described in any one of claims 1 to 5 or the nucleic acid molecule as described in claim 6 or 7, and a vector.

9. The composition of claim 8, characterized in that, The carrier is selected from one or more of the following: lipid nanoparticles, inorganic nanoparticles, exosomes, adenoviruses, lentiviruses, retroviruses, oncolytic viruses, and protein-lipid carriers.

10. The use of a trispecific connector according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6 or 7, or a composition according to claim 8 or 9 in the preparation of a medicament.

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