Enzymatic self-assembly polypeptide, NK cell modified by enzymatic self-assembly polypeptide, and preparation method and application of enzymatic self-assembly polypeptide

Abstract

The invention provides an enzymatic self-assembly polypeptide, an NK cell modified by the enzymatic self-assembly polypeptide and a preparation method and application of the NK cell, and belongs to the technical field of tumor immunotherapy. The preparation method comprises the following steps: incubating NK cells and Ac4ManNAz to obtain NK-N3 cells; the NK-tumor cell connection enhancer NBD-GFFpYK (DBCO) RGD is sequentially mixed with the cancer cells and the NK-N3 cells for incubation, or the NK-tumor cell connection enhancer NBD-GFFpYK (DBCO) RGD is sequentially mixed with the NK-N3 cells and the cancer cells for incubation. According to the invention, the adhesion efficiency and targeted killing ability of NK cells and cancer cells can be significantly enhanced, and by means of a dual-targeting mechanism of enzyme response and a membrane receptor, it is ensured that the NK-tumor cell connection enhancer is only enriched at a tumor site, and the influence on normal cells is reduced; a nanofiber network formed by the self-assembled polypeptide can resist tumor microenvironment interference and stably maintain intercellular connection.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, and particularly relates to an enzyme-catalyzed self-assembling polypeptide, NK cells modified with it, preparation methods and applications. Background Technology

[0002] With the incidence of cancer remaining high, adoptive NK cell therapy has become a research hotspot due to its high safety and lack of graft-versus-host disease risk. However, it also faces challenges such as insufficient tumor targeting and low killing efficiency. Traditional CAR-NK cell therapy relies on viral transfection, which has drawbacks such as safety concerns and complex preparation. While bispecific antibodies can bridge immune cells and cancer cells, they are susceptible to the hook effect at high doses.

[0003] Cell-to-cell interactions are crucial to the efficacy of immunotherapy. While interactions between different cells (such as immune cells and cancer cells) are significant in cancer treatment, their regulation is challenging. Existing technologies for enhancing interactions between immune cells and cancer cells have limitations: nanoconjugates (such as nanoparticles modified with antibodies or aptamers) can bridge the gap between the two, but their size limitations pose challenges for deep tumor delivery; self-assembled peptides can form nanofiber networks on single-cell surfaces, providing structural support for intercellular interactions, but their application in heterocellular assembly is still immature.

[0004] Supramolecular self-assembly peptides have become a research hotspot due to their designability and biocompatibility. Introducing self-assembly modules (such as FF dipeptides) can maintain the structure of functional short peptides, while introducing enzyme-responsive units (such as phosphorylated tyrosine pY) can enable in-situ assembly of cancer cell membranes. The resulting nanofibers can exert their effects through physical disruption or signal modulation. Furthermore, peptides can mimic antibodies to block immune checkpoints or, through co-assembly, shorten the distance between immune cells and cancer cells. However, how to utilize them to establish stable and specific "cell junctions" between NK cells and cancer cells remains a problem to be solved. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an enzyme-catalyzed self-assembly polypeptide, modified NK cells thereof, preparation method and application. By constructing an NK-tumor cell linker NBD-GFFpYK(DBCO)RGD containing an enzyme response unit, a tumor cell membrane receptor targeting unit and a bioorthogonal reaction unit, and combining it with NK-N3 cells modified with metabolic sugar, a stable link is established between cancer cells and NK cells through enzyme-catalyzed self-assembly and bioorthogonal reaction. This biocompatible polypeptide-based "cell link" is used to enhance the anti-tumor effect of NK cells, providing a new approach for cell immunotherapy of solid tumors.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an NK-tumor cell linkage enhancer, NBD-GFFpYK(DBCO)RGD, wherein the structural formula of the NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD is as follows:

[0008]

[0009] This invention also provides a method for preparing the aforementioned NK-tumor cell linker NBD-GFFpYK(DBCO)RGD, comprising the following steps:

[0010] NBD-GFFpYKRGD and DBCO-NHS were reacted in the liquid phase to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD.

[0011] Preferably, the molar ratio of NBD-GFFpYKRGD to DBCO-NHS is 1.2 to 1.5:1;

[0012] The temperature of the liquid phase reaction is 20–35°C, and the time of the liquid phase reaction is 12–24 h.

[0013] This invention also provides the application of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD or the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD prepared by the aforementioned method in enhancing NK cell anti-tumor activity.

[0014] Preferably, it includes the following steps:

[0015] 1) NK cells and Ac4ManNAz were incubated in MEMα medium to obtain NK-N3 cells;

[0016] 2) NBD-GFFpYK(DBCO)RGD was incubated with cancer cells to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of cancer cells;

[0017] 3) Add NK-N3 to the cancer cell culture system linked to NBD-GFFpYK(DBCO)RGD, add growth factor IL-2, and continue co-culture to enable NK-N3 cells to kill cancer cells.

[0018] Preferably, in step 1), the concentration of Ac4ManNAz is 80–120 μM; the concentration of NK cells is 800,000–1,200,000 cells / mL Ac4ManNAz; the incubation temperature is 37°C; and the incubation time is 36–60 h.

[0019] Step 2) The cancer cells are tumor cells that highly express alkaline phosphatase; the concentration of NBD-GFFpYK(DBCO)RGD is 150-250 μM, the concentration of cancer cells is 100,000-300,000 / mL NBD-GFFpYK(DBCO)RGD, the incubation temperature is 37°C, and the incubation time is 1-3 h;

[0020] Step 3) The ratio of cancer cells to NK-N3 cells connected to NBD-GFFpYK(DBCO)RGD is 1:1; the culture temperature is 37℃, the culture time is 0.5-1.5h, and the final concentration of growth factor IL-2 is 80-120U / mL; the co-culture temperature is 37℃, and the co-culture time is 6-18h.

[0021] Preferably, it includes the following steps:

[0022] 1) NK cells and Ac4ManNAz were incubated in MEMα medium to obtain NK-N3 cells;

[0023] 2) NBD-GFFpYK(DBCO)RGD and NK-N3 cells were incubated to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of NK-N3 cells;

[0024] 3) Add NK-N3 cells with NBD-GFFpYK(DBCO)RGD attached to their surface to the cancer cell culture system, add growth factor IL-2, and continue co-culture to enable NK-N3 cells to kill cancer cells.

[0025] Preferably, in step 1), the concentration of Ac4ManNAz is 80–120 μM; the concentration of NK cells is 800,000–1,200,000 cells / mL of Ac4ManNAz; the incubation temperature is 37°C; and the incubation time is 36–60 h.

[0026] Step 2) The concentration of NBD-GFFpYK(DBCO)RGD is 150-250 μM, and the concentration of NK-N3 cells is 100,000-300,000 cells / mL NBD-GFFpYK(DBCO)RGD; the incubation temperature is 37°C, and the incubation time is 1-3 hours.

[0027] Step 3) The cancer cells are tumor cells that highly express alkaline phosphatase; the ratio of NK-N3 cells with NBD-GFFpYK(DBCO)RGD on their surface to cancer cells is 1:1; the culture temperature is 37℃, the culture time is 0.5-1.5h, and the final concentration of the growth factor IL-2 is 80-120U / mL; the co-culture temperature is 37℃, and the co-culture time is 6-18h.

[0028] The present invention also provides the application of the NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD or the NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD prepared by the above preparation method in the preparation of drugs for treating solid tumors.

[0029] Preferably, the solid tumor includes cervical cancer.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD constructed in this invention exhibits several superior effects: it can significantly enhance the adhesion efficiency and targeted killing ability of NK cells and cancer cells; through the dual targeting mechanism of enzyme response and membrane receptor, it ensures that the peptides are enriched only at the tumor site, reducing the impact on normal cells; the nanofiber network formed by the self-assembled peptides can resist the interference of the tumor microenvironment and stably maintain intercellular connections; the material has good biocompatibility, no obvious toxicity, and high safety.

[0032] This invention innovatively combines enzymatic self-assembly with bioorthogonal chemistry. By constructing self-assembled peptides containing enzyme response units, membrane receptor targeting units, and bioorthogonal reaction units, a stable peptide-based "cell junction" is established between NK cells and cancer cells, effectively addressing the problem of insufficient NK cell-cancer cell interaction in existing tumor immunotherapy. This strategy achieves specific enrichment of peptides at tumor sites through a dual-targeting mechanism, enhancing the targeted killing ability of NK cells. Furthermore, the nanofiber network formed by self-assembly can resist interference from the tumor microenvironment and maintain junction stability. Simultaneously, the peptide self-assembly characteristics can optimize the interaction of the ternary system, exhibiting good biocompatibility and clinical translational potential, providing a new and effective approach for immunotherapy of solid tumors. Attached Figure Description

[0033] Figure 1 These are high-resolution mass spectrometry results from NBD-GFFpYK(DBCO)RGD;

[0034] Figure 2 These are transmission electron microscopy results of NBD-GFFpYK(DBCO)RGD self-assembly;

[0035] Figure 3 This is the result of laser confocal microscopy imaging of NBD-GFFpYK(DBCO)RGD assembled on the surface of cervical cancer cells;

[0036] Figure 4 These are laser confocal microscopy imaging results of NBD-GFFpYK(DBCO)RGD modification on the surface of NK cells;

[0037] Figure 5 This is the laser confocal microscopy imaging result of the polypeptide-based "cell junction" constructed in Example 4 (green fluorescence corresponds to NBD-GFFpYK(DBCO)RGD);

[0038] Figure 6 This is the laser confocal microscopy imaging result of the polypeptide-based "cell junction" constructed in Example 5 (green fluorescence corresponds to NBD-GFFpYK(DBCO)RGD);

[0039] Figure 7 The polypeptide-based "cell linker" constructed in Example 4 enhances the in vitro tumor cell killing effect of NK cells.

[0040] Figure 8 The polypeptide-based "cell linker" constructed in Example 5 enhances the in vitro tumor cell killing effect of NK cells.

[0041] Figure 9 Example 4 shows the effect of the polypeptide-based "cell linker" constructed to enhance the inhibitory effect of NK cells on tumor growth in vivo and the comparison of body weight of mice in different groups.

[0042] Figure 10 Example 5 shows the effect of the polypeptide-based "cell linker" constructed to enhance the inhibitory effect of NK cells on tumor growth in vivo and the comparison of body weight of mice in different groups. Detailed Implementation

[0043] This invention provides an NK-tumor cell linkage enhancer, NBD-GFFpYK(DBCO)RGD, wherein the structural formula of the NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD is as follows:

[0044]

[0045] This invention also provides a method for preparing the aforementioned NK-tumor cell linker NBD-GFFpYK(DBCO)RGD, comprising the following steps:

[0046] NBD-GFFpYKRGD and DBCO-NHS were reacted in the liquid phase to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD.

[0047] In this invention, NBD-GFFpYKRGD and DBCO-NHS are reacted in a liquid phase to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD. The molar ratio of NBD-GFFpYKRGD to DBCO-NHS is preferably 1.2–1.5:1, more preferably 1.3–1.4; the temperature of the liquid phase reaction is preferably 20–35°C, and the reaction time is preferably 12–24 h.

[0048] This invention also provides the application of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD or the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD prepared by the aforementioned method in enhancing NK cell anti-tumor activity.

[0049] The present invention includes the following steps:

[0050] 1) NK cells and Ac4ManNAz were incubated in MEMα medium to obtain NK-N3 cells;

[0051] 2) NBD-GFFpYK(DBCO)RGD was incubated with cancer cells to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of cancer cells;

[0052] 3) Add NK-N3 to the cancer cell culture system linked to NBD-GFFpYK(DBCO)RGD, add growth factor IL-2, and continue co-culture to enable NK-N3 cells to kill cancer cells.

[0053] In this invention, NK cells and Ac4ManNAz are incubated in MEMα medium to obtain NK-N3 cells. The NK cells are preferably NK-92 cells; the concentration of Ac4ManNAz is preferably 80–120 μM, more preferably 90–110 μM, and even more preferably 100 μM; the concentration of NK cells is preferably 800,000–1,200,000 cells / mL Ac4ManNAz, more preferably 900,000–1,100,000 cells / mL Ac4ManNAz, and even more preferably 1,000,000 cells / mL Ac4ManNAz; the incubation temperature is 37°C; and the incubation time is preferably 36–60 h, more preferably 42–54 h, and even more preferably 48 h.

[0054] In this invention, NBD-GFFpYK(DBCO)RGD is incubated with cancer cells to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of the cancer cells. The cancer cells are tumor cells that highly express alkaline phosphatase. The concentration of NBD-GFFpYK(DBCO)RGD is preferably 150–250 μM, more preferably 180–220 μM, and even more preferably 200 μM. The concentration of the cancer cells is preferably 100,000–300,000 cells / mL of NBD-GFFpYK(DBCO)RGD, more preferably 150,000–250,000 cells / mL of NBD-GFFpYK(DBCO)RGD, and even more preferably 200,000 cells / mL of NBD-GFFpYK(DBCO)RGD. The incubation temperature is 37°C. The incubation time is preferably 1–3 h, more preferably 1.5–2.5 h, and even more preferably 2 h.

[0055] In this invention, NK-N3 is added to a cancer cell culture system linked to NBD-GFFpYK(DBCO)RGD, and growth factor IL-2 is added. The cells are then co-cultured to enable NK-N3 cells to kill cancer cells. Before addition, cancer cells are washed with PBS to remove free NBD-GFFpYK(DBCO)RGD, preferably 2-4 times, more preferably 3 times; the ratio of cancer cells linked to NBD-GFFpYK(DBCO)RGD to NK-N3 cells is 1:1; the culture temperature is 37℃; the culture time is preferably 0.5-1.5h, more preferably 1h; after culture, free NK-N3 is removed by washing with PBS, preferably 2-4 times, more preferably 3 times; the final concentration of growth factor IL-2 is preferably 80-120U / mL, more preferably 90-110U / mL, and even more preferably 100U / mL; the co-culture temperature is 37℃; the co-culture time is preferably 6-18h, more preferably 9-15h, and even more preferably 12h.

[0056] The present invention includes the following steps:

[0057] 1) NK cells and Ac4ManNAz were incubated in MEMα medium to obtain NK-N3 cells;

[0058] 2) NBD-GFFpYK(DBCO)RGD and NK-N3 cells were incubated to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of NK-N3 cells;

[0059] 3) NK-N3 cells with NBD-GFFpYK(DBCO)RGD attached to their surface were added to the cancer cell culture system and then co-cultured with growth factor IL-2 to enable NK-N3 cells to kill cancer cells.

[0060] In this invention, NK cells and Ac4ManNAz are incubated in MEMα medium to obtain NK-N3 cells. The NK cells are preferably NK-92 cells; the concentration of Ac4ManNAz is preferably 80–120 μM, more preferably 90–110 μM, and even more preferably 100 μM; the concentration of NK cells is preferably 800,000–1,200,000 cells / mL Ac4ManNAz, more preferably 900,000–1,100,000 cells / mL Ac4ManNAz, and even more preferably 1,000,000 cells / mL Ac4ManNAz; the incubation temperature is 37°C; and the incubation time is preferably 36–60 h, more preferably 42–54 h, and even more preferably 48 h.

[0061] In this invention, NBD-GFFpYK(DBCO)RGD and NK-N3 cells are incubated to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of NK-N3 cells. The concentration of NBD-GFFpYK(DBCO)RGD is preferably 150–250 μM, more preferably 180–220 μM, and even more preferably 200 μM; the concentration of NK-N3 cells is preferably 100,000–300,000 cells / mL of NBD-GFFpYK(DBCO)RGD, more preferably 150,000–250,000 cells / mL of NBD-GFFpYK(DBCO)RGD, and even more preferably 200,000 cells / mL of NBD-GFFpYK(DBCO)RGD; the incubation temperature is 37°C; and the incubation time is preferably 1–3 h, more preferably 1.5–2.5 h, and even more preferably 2 h.

[0062] In this invention, NK-N3 cells with NBD-GFFpYK(DBCO)RGD attached to their surface are added to a cancer cell culture system and then co-cultured with growth factor IL-2 to enable NK-N3 cells to kill cancer cells. Before addition, NK-N3 cells are washed with PBS to remove free NBD-GFFpYK(DBCO)RGD, preferably 2-4 times, more preferably 3 times; the cancer cells are tumor cells that highly express alkaline phosphatase; the ratio of NK-N3 cells with NBD-GFFpYK(DBCO)RGD attached to the surface to cancer cells is 1:1; the culture temperature is 37℃; the culture time is preferably 0.5-1.5h, more preferably 1h; after culture, free cancer cells are removed by washing with PBS, preferably 2-4 times, more preferably 3 times; the final concentration of growth factor IL-2 is preferably 80-120U / mL, more preferably 90-110U / mL, and even more preferably 100U / mL; the co-culture temperature is 37℃; the co-culture time is preferably 6-18h, more preferably 9-15h, and even more preferably 12h.

[0063] This invention also provides the application of the aforementioned NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD, or the NK-tumor cell linkage enhancer NBD-GFFpYK(DBCO)RGD prepared by the aforementioned method, in the preparation of drugs for treating solid tumors. In this invention, the solid tumor includes cervical cancer.

[0064] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] Preparation of NBD-GFFpYKRGD: NBD-GFFpYKRGD was prepared by solid-phase synthesis. First, 1 mmol of 2-chlorotriphenylmethyl resin was weighed and placed in a solid-phase tube, and soaked in dichloromethane (DCM) for 10 min, then the DCM was extruded. 2 mmol of Fmoc-Asp(OtBu)-OH was weighed and dissolved in 15 mL of DCM, and 660 μL of N,N-diisopropylethylamine (DIEA) was added and mixed thoroughly. The mixture was then added to the solid-phase tube and reacted for 3 h. The resin was then washed 5 times with 10 mL of DCM for 1 min each time. The resin was then blocked with 20 mL of blocking solution (17 mL DCM + 2 mL methanol + 1 mL DIEA) for 10 min. The resin was then washed 5 times with 10 mL of DCM for 1 min each time, and then washed 5 times with 10 mL of N,N-dimethylformamide (DMF) for 1 min each time. After extruding the DMF, 20 mL of DMF was added. The resin was soaked in 20% piperidine at room temperature for 30 min, then washed with DMF 5 times, 1 min each time, using 10 mL of DMF each time. After extruding the DMF, 2 mmol of Fmoc-Gly-OH and 2 mmol of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) were weighed and dissolved in 15 mL of DMF. 660 μL of N,N-diisopropylethylamine (DIEA) was added and mixed thoroughly. The mixture was then added to a solid-phase tube and reacted for 3 h. The process of washing the resin with DMF 5 times, soaking in 20% piperidine for 30 min, and washing the resin with DMF 5 times was repeated. Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(HPO3Bzl)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and NBD-CO were then sequentially connected. OH; Finally, wash the resin 5 times with DMF for 1 min each time, using 10 mL of DMF each time, and then wash the resin 5 times with DCM for 1 min each time, using 10 mL of DCM each time; after extruding the DCM, add 20 mL of cutting solution (95% TFA + 2.5% TIS + 2.5% water) and soak at room temperature for 3 h. After removing most of the TFA by rotary evaporation, precipitate the peptide with 50 mL of cold diethyl ether, and finally purify it by high performance liquid chromatography (methanol / water). After drying with a freeze dryer, NBD-GFFpYKRGD is obtained.

[0066] Example 1

[0067] An NK-tumor cell linker NBD-GFFpYK(DBCO)RGD, wherein the structural formula of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD is as follows:

[0068]

[0069] Its preparation method is as follows:

[0070] NBD-GFFpYKRGD and DBCO-NHS were dissolved in DMSO at a ratio of 1.5:1, and the pH was adjusted to 7.4 with DIEA. The mixture was reacted in liquid phase at 20°C for 24 hours. After purification by high performance liquid chromatography, the mixture was freeze-dried to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD.

[0071] Example 2

[0072] An NK-tumor cell linker NBD-GFFpYK(DBCO)RGD, wherein the structural formula of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD is as follows:

[0073]

[0074] Its preparation method is as follows:

[0075] NBD-GFFpYKRGD and DBCO-NHS were dissolved in DMSO at a ratio of 1.3:1, and the pH was adjusted to 7.4 with DIEA. The mixture was reacted in liquid phase at 35°C for 12 h. After purification by high performance liquid chromatography, the mixture was freeze-dried to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD.

[0076] Example 3

[0077] An NK-tumor cell linker NBD-GFFpYK(DBCO)RGD, wherein the structural formula of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD is as follows:

[0078]

[0079] Its preparation method is as follows:

[0080] NBD-GFFpYKRGD and DBCO-NHS were dissolved in DMSO at a ratio of 1.2:1, and the pH was adjusted to 7.4 with DIEA. The mixture was reacted in liquid phase at 30°C for 16 h. After purification by high performance liquid chromatography, the mixture was freeze-dried to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD.

[0081] Example 4

[0082] NK-92 cells (1 million cells / mL Ac4ManNAz) were incubated with 100 μM Ac4ManNAz in MEMα medium for 48 h. Cells were collected and centrifuged at 800 rpm for 5 min to collect NK-92 cells. The supernatant was discarded, and the cells were resuspended in PBS and centrifuged again to remove free Ac4ManNAz, thus constructing NK-N3 cells.

[0083] Assembly of NBD-GFFpYK(DBCO)RGD prepared in Example 2 on the surface of tumor cells: HeLa cells were treated with 200 μM NBD-GFFpYK(DBCO)RGD at 37°C for 2 h (concentration of 200,000 cells / mL), and then the cells were washed with PBS to remove free NBD-GFFpYK(DBCO)RGD, so that NBD-GFFpYK(DBCO)RGD was assembled on the surface of HeLa cell membrane.

[0084] NK-N3 cells were co-cultured with HeLa cells whose surface was assembled with NBD-GFFpYK(DBCO)RGD. At a 1:1 ratio, they were co-cultured at 37°C for 1 h. After washing with PBS to remove free NK-N3, only NK-N3 interacting with HeLa cells were retained. 100 U / mL of IL-2 was added to the co-culture system, and co-culture was continued for another 12 h to induce NK-N3 cell killing of HeLa cells.

[0085] Example 5

[0086] NK-92 cells (1 million cells / mL Ac4ManNAz) were incubated with 100 μM Ac4ManNAz in MEMα medium for 48 h. Cells were collected and centrifuged at 800 rpm for 5 min to collect NK-92 cells. The supernatant was discarded, and the cells were resuspended in PBS and centrifuged again to remove free Ac4ManNAz, thus constructing NK-N3 cells.

[0087] Assembly of NBD-GFFpYK(DBCO)RGD prepared in Example 2 on the surface of NK cells: NK-N3 cells were treated with 200 μM NBD-GFFpYK(DBCO)RGD at 37 °C for 2 h (concentration of 200,000 cells / mL), the cells were collected by centrifugation, and the cells were resuspended with PBS to remove free NBD-GFFpYK(DBCO)RGD, so that NBD-GFFpYK(DBCO)RGD was modified onto the surface of NK-N3 cell membrane.

[0088] NK-N3 cells with a surface modified with NBD-GFFpYK(DBCO)RGD were co-cultured with HeLa cells at a 1:1 ratio at 37°C for 1 h. After washing with PBS to remove free NK-N3 cells, only NK-N3 cells interacting with HeLa cells were retained. 100 U / mL of IL-2 was added to the co-culture volume, and co-culturing continued for 12 h to induce NK-N3 cell killing of HeLa cells.

[0089] Experimental Example 1

[0090] Structural identification of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD prepared in Example 2.

[0091] Experimental results: such as Figure 1 As shown, the theoretically calculated molecular weight of NBD-GFFpYK(DBCO)RGD is [MH]. - =1588.5693, molecular weight determined by high-resolution mass spectrometry is [MH]. - =1588.5717, indicating that the molecule was successfully prepared.

[0092] Experiment Example 2

[0093] Transmission electron microscopy experiment

[0094] Preparation of sample solution: Weigh 2.5 mg of NBD-GFFpYK(DBCO)RGD prepared in Example 2, dissolve it in 0.5 mL of phosphate buffer (PBS, 10 mM, pH 7.4), add saturated sodium bicarbonate aqueous solution to adjust the pH of the peptide solution to 7.4, then add alkaline phosphatase (ALP, final concentration 5 U / mL), mix well, and incubate in a 37°C water bath for 10 h.

[0095] Electron microscopy sample preparation: The sample solution was dropped onto a copper grid and stained with uranium acetate dye.

[0096] Electron microscopy: The microstructure of the sample was observed using a transmission electron microscope (Hitachi H-7500).

[0097] Experimental results: such as Figure 2 As shown in the figure, in the presence of alkaline phosphatase, molecules assemble into long, fibrous structures.

[0098] Experimental Example 3

[0099] Assembly experiment of NBD-GFFpYK(DBCO)RGD prepared in Example 2 on the surface of cancer cell membrane

[0100] 3T3 cells (control) that do not secrete alkaline phosphatase (ALP) and HeLa cells that secrete ALP (example) were cultured in confocal microscopy dishes. 3T3 cells were stained with 50 μM Cell-Tracker Blue for 30 min, followed by washing away the free dye with PBS. HeLa cells were stained with 2 μM Cell-Tracker Red for 30 min, followed by washing away the free dye with PBS. Both cell types were treated with 200 μM of NBD-GFFpYK(DBCO)RGD prepared in Example 2 at 37°C for 2 h, followed by washing the cells with PBS to remove free NBD-GFFpYK(DBCO)RGD. The assembly of NBD-GFFpYK(DBCO)RGD on the surface of both cell types was observed using laser confocal microscopy.

[0101] Experimental results: such as Figure 3 As shown in the figure, NBD-GFFpYK(DBCO)RGD does not assemble on the surface of 3T3 cells that do not secrete ALP, but it does assemble on the surface of HeLa cells that secrete ALP. Blue represents 3T3 cells, red represents HeLa cells, and green represents NBD-GFFpYK(DBCO)RGD.

[0102] Experiment Example 4

[0103] Example 2: Modification experiment on the surface of NBD-GFFpYK(DBCO)RGD prepared in NK cells.

[0104] NK-92 cells were incubated with 100 μM Ac4ManNAz in MEMα medium for 48 h. Cells were collected, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in PBS and centrifuged again to remove free Ac4ManNAz, thus constructing NK-N3 cells. NK cells and NK-N3 cells were stained with 50 μM Cell-Tracker Blue for 30 min, respectively. After centrifugation, NK cells (control) and NK-N3 cells (example) were collected and resuspended in PBS to remove excess stain. NK cells and NK-N3 cells were treated with 200 μM NBD-GFFpYK(DBCO)RGD prepared in Example 2 at 37 °C for 2 h. Cells were collected by centrifugation and resuspended in PBS to remove free NBD-GFFpYK(DBCO)RGD. The modification of NK cell membranes by NBD-GFFpYK(DBCO)RGD was observed using laser confocal microscopy.

[0105] Experimental results: such as Figure 4As shown in the figure, NBD-GFFpYK(DBCO)RGD can modify the surface of NK-N3 cells, but cannot modify the surface of NK cells.

[0106] Experimental Example 5

[0107] Example 4 describes a polypeptide-based "cell linker" that enhances the interaction between NK cells and HeLa cancer cells.

[0108] NK-92 cells were incubated with 100 μM Ac4ManNAz in MEMα medium for 48 h. Cells were collected, centrifuged at 800 rpm for 5 min to collect NK-92 cells, and the supernatant was discarded. The cells were resuspended in PBS and centrifuged again to remove free Ac4ManNAz, thus constructing NK-N3 cells. NK cells and NK-N3 cells were stained with 50 μM Cell-Tracker Blue for 30 min, respectively. After centrifugation, NK cells and NK-N3 cells were collected and resuspended in PBS to remove excess stain. HeLa cells were treated with 200 μM NBD-GFFpYK(DBCO)RGD prepared in Example 2 at 37 °C for 2 h, and washed with PBS to remove free NBD-GFFpYK(DBCO)RGD. NK cells (control) and NK-N3 cells (example) were co-cultured with HeLa cells conjugated with NBD-GFFpYK(DBCO)RGD at 37 °C for 1 h. Free NK cells were washed with PBS to remove PBS. The modification of NBD-GFFpYK(DBCO)RGD on the surface of NK cell membranes was observed using laser confocal microscopy.

[0109] Experimental results: such as Figure 5 As shown in the figure, the interaction between NK-N3 cells and HeLa cells linked to NBD-GFFpYK(DBCO)RGD is significantly higher than that between NK cells and HeLa cells linked to NBD-GFFpYK(DBCO)RGD, demonstrating that the constructed polypeptide-based "cell linker" can significantly enhance the interaction between NK cells and cancer cells HeLa.

[0110] Experimental Example 6

[0111] Example 5 describes a polypeptide-based "cell linker" that enhances the interaction between NK cells and HeLa cancer cells.

[0112] HeLa cells secreting ALP were cultured in confocal microarrays. NK cells and NK-N3 cells were stained with 50 μM Cell-Tracker Blue for 30 min. After centrifugation, NK cells and NK-N3 cells were collected and resuspended in PBS to remove excess stain. NK cells (control) and NK-N3 cells (example) were treated with 200 μM NBD-GFFpYK(DBCO)RGD prepared in Example 2 at 37°C for 2 h. The cells were then washed with PBS to remove free NBD-GFFpYK(DBCO)RGD. NK cells and NK-N3 cells treated with NBD-GFFpYK(DBCO)RGD were added to the HeLa cell confocal microarrays at a 1:1 ratio and co-cultured at 37°C for 1 h. The confocal microarrays were washed with PBS to remove free NK or NK-N3 cells. The interaction between NK or NK-N3 cells and HeLa cells was observed using a laser confocal microscope.

[0113] Experimental results: such as Figure 6 As shown in the figure, the interaction between NK-N3 cells linked to NBD-GFFpYK(DBCO)RGD and HeLa cells (Example) was significantly higher than the interaction between NK cells treated with NBD-GFFpYK(DBCO)RGD and HeLa cells (Control), demonstrating that the constructed polypeptide-based "cell linker" can significantly enhance the interaction between NK cells and cancer cells HeLa.

[0114] Experimental Example 7

[0115] Example 4 describes the enhancement of the in vitro tumor cell killing effect of NK cells by the constructed polypeptide-based "cell linker".

[0116] The supernatant from co-culturing with IL-2 in Example 4 was collected and centrifuged at 12000 rpm and 4°C for 5 min, and the supernatant was collected again. Using NK cells as a control, the lactate dehydrogenase (LDH) level in the supernatant was measured using a lactate dehydrogenase cytotoxicity assay kit to determine the killing effect of NK cells on HeLa cells.

[0117] Experimental results: such as Figure 7 As shown in the figure, the LDH level in the NK-N3 group (example) was significantly higher than that in the NK group (control example), proving that the polypeptide-based "cell linker" constructed in Example 4 can significantly improve the killing effect of NK cells on tumor cells in vitro.

[0118] Experimental Example 8

[0119] Example 5 describes the enhancement of the in vitro tumor cell killing effect of NK cells by the constructed polypeptide-based "cell linker".

[0120] The supernatant from co-culturing with IL-2 in Example 5 was collected and centrifuged at 12000 rpm and 4°C for 5 min, and the supernatant was collected again. Using NK cells as a control, the lactate dehydrogenase (LDH) level in the supernatant was measured using a lactate dehydrogenase cytotoxicity assay kit to determine the killing effect of NK cells on HeLa cells.

[0121] Experimental results: such as Figure 8 As shown in the figure, the LDH level in the NK-N3 group (example) was significantly higher than that in the NK group (control example), proving that the polypeptide-based "cell linker" constructed in Example 5 can significantly improve the killing effect of NK cells on tumor cells in vitro.

[0122] Experimental Example 9

[0123] Example 4 describes a polypeptide-based "cell linker" that enhances the inhibitory effect of NK cells on tumor growth in vivo.

[0124] A female BALB / c nude mouse model of human cervical cancer was constructed until the tumor volume reached approximately 50 mm. 3 The mice were divided into three groups: PBS, control, and example, with five mice in each group. The PBS group received a tail vein injection of PBS, the control group received a tail vein injection of 5 million NK-N3 cells, and the example group received a tail vein injection of 1 mM NBD-GFFpYK(DBCO)RGD to allow it to assemble on the surface of tumor cells in the mice. Twelve hours later, 5 million NK-N3 cells were injected via the tail vein. This process was repeated twice, for a total of two injections. Tumor size was measured every two days to plot tumor growth curves.

[0125] Experimental results: such as Figure 9 As shown in the figure, the tumor growth in the embodiment was the slowest, demonstrating that the polypeptide-based "cell linker" constructed in Example 4 can significantly enhance the inhibition of tumor growth by NK cells in vivo. Simultaneously, the body weight of mice in each group was monitored, and there was no significant difference in body weight between the groups, indicating that the embodiment has good in vivo biosafety.

[0126] Experimental Example 10

[0127] Example 5 describes a polypeptide-based "cell linker" that enhances the inhibitory effect of NK cells on tumor growth in vivo.

[0128] A female BALB / c nude mouse model of human cervical cancer was constructed until the tumor volume reached approximately 50 mm. 3The cells were divided into three groups: PBS, control, and example, with five cells in each group. The PBS group received a tail vein injection of PBS, the control group received a tail vein injection of 5 million NK-N3 cells, and the example group received a tail vein injection of 5 million NBD-GFFpYK(DBCO)RGD-modified NK-N3 cells prepared in Example 5. The procedure was repeated twice after three days. Tumor size was measured every two days to plot tumor growth curves.

[0129] Experimental results: such as Figure 10 As shown in the figure, the tumor growth in the embodiment was the slowest, demonstrating that the polypeptide-based "cell linker" constructed in Example 5 can significantly enhance the inhibition of tumor growth by NK cells in vivo. Simultaneously, the body weight of mice in each group was monitored, and there was no significant difference in body weight between the groups, indicating that the embodiment has good in vivo biosafety.

[0130] As demonstrated by the above embodiments and experimental examples, this invention, based on the principles of supramolecular self-assembly and bioorthogonal chemistry, designs self-assembled peptides containing enzyme-responsive, membrane receptor-targeting, and bioorthogonal reaction units. Two strategies are employed to construct peptide-based "cell-cell connections" between NK cells and cancer cells: first, the peptide, after enzymatic self-assembly, anchors to cancer cells and then reacts with azide-modified NK cells; second, the peptide-modified NK cells, after enzymatic self-assembly, target cancer cells. This invention addresses the problem of insufficient NK cell-cancer cell interaction in existing tumor immunotherapy. Through a dual-targeting mechanism, it achieves tumor-specific enrichment, enhances the NK cell killing ability, and the self-assembled nanofiber network can resist interference from the tumor microenvironment, possessing good biocompatibility and clinical translational potential, providing a new strategy for solid tumor immunotherapy.

[0131] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An NK-tumor cell linker NBD-GFFpYK(DBCO)RGD, characterized in that, The structural formula of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD is as follows:

2. The method for preparing the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD according to claim 1, characterized in that, Includes the following steps: NBD-GFFpYKRGD and DBCO-NHS were reacted in a liquid phase to obtain the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD.

3. The preparation method according to claim 2, characterized in that, The molar ratio of NBD-GFFpYKRGD and DBCO-NHS is 1.2 to 1.5:1; The temperature of the liquid phase reaction is 20–35°C, and the time of the liquid phase reaction is 12–24 h.

4. The application of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD as described in claim 1 or the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD prepared by the preparation method described in any one of claims 2 to 3 in enhancing NK cell anti-tumor activity.

5. The application according to claim 4, characterized in that, Includes the following steps: 1) NK cells and Ac4ManNAz were incubated in MEMα medium to obtain NK-N3 cells; 2) NBD-GFFpYK(DBCO)RGD was incubated with cancer cells to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of cancer cells; 3) Add NK-N3 to the cancer cell culture system linked to NBD-GFFpYK(DBCO)RGD, add growth factor IL-2, and continue co-culture to enable NK-N3 cells to kill cancer cells.

6. The application according to claim 5, characterized in that, Step 1) The concentration of Ac4ManNAz is 80-120 μM; the concentration of NK cells is 800,000-1,200,000 cells / mL Ac4ManNAz; the incubation temperature is 37°C; and the incubation time is 36-60 h. Step 2) The cancer cells are tumor cells that highly express alkaline phosphatase; the concentration of NBD-GFFpYK(DBCO)RGD is 150-250 μM, the concentration of cancer cells is 100,000-300,000 / mL NBD-GFFpYK(DBCO)RGD, the incubation temperature is 37°C, and the incubation time is 1-3 h; Step 3) The ratio of cancer cells to NK-N3 cells connected to NBD-GFFpYK(DBCO)RGD is 1:1; the culture temperature is 37℃, the culture time is 0.5-1.5h, and the final concentration of growth factor IL-2 is 80-120U / mL; the co-culture temperature is 37℃, and the co-culture time is 6-18h.

7. The application according to claim 4, characterized in that, Includes the following steps: 1) NK cells and Ac4ManNAz were incubated in MEMα medium to obtain NK-N3 cells; 2) NBD-GFFpYK(DBCO)RGD and NK-N3 cells were incubated to allow NBD-GFFpYK(DBCO)RGD to attach to the surface of NK-N3 cells; 3) Add NK-N3 cells with NBD-GFFpYK(DBCO)RGD attached to their surface to the cancer cell culture system, add growth factor IL-2, and continue co-culture to enable NK-N3 cells to kill cancer cells.

8. The application according to claim 7, characterized in that, Step 1) The concentration of Ac4ManNAz is 80-120 μM; the concentration of NK cells is 800,000-1,200,000 Ac4ManNAz cells / mL; the incubation temperature is 37°C; and the incubation time is 36-60 h. Step 2) The concentration of NBD-GFFpYK(DBCO)RGD is 150-250 μM, and the concentration of NK-N3 cells is 100,000-300,000 cells / mL NBD-GFFpYK(DBCO)RGD; the incubation temperature is 37°C, and the incubation time is 1-3 hours. Step 3) The cancer cells are tumor cells that highly express alkaline phosphatase; the ratio of NK-N3 cells with NBD-GFFpYK(DBCO)RGD on their surface to cancer cells is 1:1; the culture temperature is 37℃, the culture time is 0.5-1.5h, and the final concentration of the growth factor IL-2 is 80-120U / mL; the co-culture temperature is 37℃, and the co-culture time is 6-18h.

9. The use of the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD as described in claim 1 or the NK-tumor cell linker NBD-GFFpYK(DBCO)RGD prepared by the preparation method described in any one of claims 2 to 3 in the preparation of drugs for treating solid tumors.

10. The application according to claim 9, characterized in that, The solid tumors include cervical cancer.

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