γδt cell targeting PD-l1, and preparation method therefor and use thereof

Abstract

Provided are a γδT cell targeting PD-L1, and a preparation method therefor and the use thereof. By means of unnatural sugar metabolic labeling, the γδT cell is modified with a first active group, and then same and a PD-L1 targeting group modified with a second active group are subjected to a bioorthogonal reaction, thereby obtaining the γδT cell targeting PD-L1. The γδT cell targeting PD-L1 has a targeted and selective killing effect on PD-L1-positive tumor cells; has the advantages of a short preparation cycle, suitability for large-scale production, uniform and stable quality, etc.; and has good application prospects.

Description

A γδT cell targeting PD-L1 and its preparation method and application Technical Field

[0001] The present invention relates to the fields of bioengineering and medical technology, and in particular to a γδT cell targeting PD-L1 and a preparation method and application thereof. Background Art

[0002] [Corrected 30.10.2024 in accordance with Regulation 91] Cancer incidence and mortality rates are increasing annually. With the continued exploration and application of human immunity, immunotherapy research has become increasingly popular. Immune cell therapy, which activates and enhances the immune system to kill tumors, has become the fourth pillar of cancer treatment. Adoptive immune cell therapy, such as chimeric antigen receptor T (CAR-T) cells, has achieved significant results in combating blood cancers, but it is associated with high production costs, lengthy preparation times, and complex processes.

[0003] [Corrected 30.10.2024 in accordance with Rule 91] PD-L1 is often highly expressed on the surface of tumor cells. As one of the most important immune checkpoints, PD-L1, upon binding to PD-1, can induce T cell apoptosis, dysfunction, and exhaustion, thereby inhibiting the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+ T cells, leading to tumor immune escape. Because normal cells also express PD-L1, conventional CAR-T cells cannot simultaneously kill PD-L1-expressing tumor cells without harming PD-L1-expressing normal cells.

[0004] Therefore, the development of a new, convenient, and low-cost immune cell PD-L1 antibody labeling technology that can selectively kill tumor cells without damaging normal cells will greatly promote immune cell therapy.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a PD-L1-targeted γδT cell and its preparation method and application. The cell has a targeted and selective killing effect on PD-L1-positive tumor cells, and has the advantages of a short preparation cycle, large-scale preparation, uniform and stable quality, and has good application prospects.

[0007] To this end, in a first aspect, the present invention provides a PD-L1-targeting γδT cell, which is obtained by connecting a γδT cell modified with a first active group and a PD-L1 targeting group modified with a second active group through a bioorthogonal reaction; the second active group is suitable for undergoing a bioorthogonal reaction with the first active group;

[0008] The γδT cells modified with the first active group are obtained by metabolically labeling the γδT cells with non-natural sugar molecules; the non-natural sugar molecules include at least one selected from Formulas 1 to 6:

[0009] Wherein, X is the first active group;

[0010] [Corrected 30.10.2024 according to Rule 91] R1, R2, R3, R4, R5, R6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxy, amino, nitro, mercapto, C 1～6 alkyl.

[0011] By applying the above-mentioned non-natural sugar molecules, γδT cells can be efficiently labeled with the first active group, which is further conducive to their connection with the PD-L1 targeting group, so that γδT cells can target PD-L1-positive tumor cells and have the function of distinguishing between tumors and normal cells, significantly promoting the killing effect on tumor cells.

[0012] In some embodiments, the non-natural sugar molecule comprises at least one selected from the group consisting of:

[0013] Wherein, X is the first active group;

[0014] [Corrected 30.10.2024 according to Rule 91] R1 and R2 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxy, amino, nitro, mercapto, C 1～6 alkyl.

[0015] In some embodiments, the non-natural sugar molecule comprises at least one selected from the group consisting of:

[0016] Wherein, X is the first active group.

[0017] In some embodiments, the bioorthogonal reaction comprises at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitization, and glycosylation.

[0018] In some embodiments, the first reactive group comprises at least one selected from the group consisting of: azide, alkynyl, trans-cyclooctene, triazine, tetrazine, hydrazide, cyclopropene, isocyanide, and cyclooctyne.

[0019] In some embodiments, the γδ T cells are human γδ T cells.

[0020] In some embodiments, the γδT cells include at least one of the following subtypes: Vγ9Vδ2T cell subtype, Vδ1T cell subtype.

[0021] In some embodiments, the second reactive group is modified to the PD-L1 targeting group by a linker compound; the linker compound has both a group that can covalently link to a thiol group and the second reactive group.

[0022] In some embodiments, the group covalently linkable to a thiol group is a succinimide active ester group, a maleimide group, or a maleic anhydride group.

[0023] In some embodiments, the linker compound comprises a molecule selected from Formula L1 and Formula L2:

[0024] Wherein, Y is the second active group;

[0025] R7 is selected from C 1～20 Alkylene or C 1～20 Alkylene ether group.

[0026] In some embodiments, R7 is selected from C 4～20 Alkylene or C 4～20 Alkylene ether group.

[0027] In some embodiments, the linker compound is DBCO-PEG4-NHS or DBCO-PEG4-MAL;

[0028] In some embodiments, the PD-L1 targeting group comprises an antibody or a fragment thereof that specifically binds to PD-L1.

[0029] In some embodiments, the PD-L1 targeting group includes at least one selected from the following group: an immunoglobulin, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single region antibody that specifically binds to PD-L1, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies.

[0030] A second aspect of the present invention provides a method for preparing PD-L1-targeting γδT cells according to the first aspect of the present invention, comprising: linking a γδT cell modified with a first active group and a PD-L1 targeting group modified with a second active group through a bioorthogonal reaction to obtain the PD-L1-targeting γδT cell; wherein the second active group is suitable for undergoing a bioorthogonal reaction with the first active group;

[0031] The γδT cells modified with the first active group are obtained by metabolically labeling the γδT cells with non-natural sugar molecules; the non-natural sugar molecules include at least one selected from Formulas 1 to 6:

[0032] Wherein, X is the first active group;

[0033] [Corrected 30.10.2024 according to Rule 91] R1, R2, R3, R4, R5, R6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxy, amino, nitro, mercapto, C 1～6 alkyl.

[0034] In some embodiments, the metabolic labeling step comprises: culturing the γδT cells using a culture medium containing the non-natural sugar molecule.

[0035] In some embodiments, the concentration of the non-natural sugar molecule in the culture medium is 10 to 200 μM; for example, it can be about 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, etc.

[0036] In some embodiments, the culture time is 2 to 48 hours; for example, it can be about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, etc.

[0037] The third aspect of the present invention provides the use of the γδT cells targeting PD-L1 in the preparation of products for treating PD-L1-positive tumors.

[0038] In some embodiments, the PD-L1 positive tumor comprises at least one of the following groups: PD-L1 positive lung cancer, lymphoma, intestinal cancer, head and neck squamous cell carcinoma, esophageal cancer, liver cancer, and ovarian cancer.

[0039] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0040] (1) The present invention provides γδT cells targeting PD-L1 and a preparation method thereof. Through the corresponding antibody coupling method, the PD-L1 targeting group can be quickly and efficiently modified on the surface of γδT cells to achieve targeted and selective killing of tumor cells expressing PD-L1.

[0041] (2) The PD-L1-targeting γδT cells provided by the present invention have been verified to be safe at the cell and animal experimental levels, and can also improve tumor targeting and enhance the anti-tumor effect of γδT cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiment below. The accompanying drawings are only for the purpose of illustrating the preferred embodiment and are not to be considered as limiting the present invention. In the accompanying drawings:

[0043] Figure 1 Schematic diagram of the process for preparing γδT cells targeting PD-L1;

[0044] Figure 2 is a flow cytometry characterization of γδT cell purity before and after PBMC culture; the left picture is before culture, and the right picture is after culture;

[0045] Figure 3 is an imaging representation of γδT modified with the first active group; scale bar is 20 μm;

[0046] Figure 4 shows the imaging characterization of γδT modified with PD-L1 targeting antibody; scale bar is 20 μm;

[0047] Figure 5 shows the in vitro killing of PD-L1-positive ovarian cancer cells by γδT-αPDL1 and γδT cells; *** indicates P < 0.001, **** indicates P < 0.0001;

[0048] Figure 6 is a graph showing the evaluation of the tumor inhibition effect in the PD-L1-positive ovarian cancer mouse peritoneal metastasis model; ** indicates P < 0.01, **** indicates P < 0.0001. DETAILED DESCRIPTION

[0049] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0050] In existing technologies, immune cell therapy primarily targets T lymphocytes. The majority (approximately 95%) of mature T cells have T cell surface receptors (TCRs) composed of two heterodimeric peptide chains, α and β. A smaller subset consists of γ and δ chains, representing γδT cells. γδT cells are T cells that perform innate immune functions, capable of killing cancer cells and tumor stem cells, as well as recognizing cancer antigens, demonstrating their potent lethality.

[0051] The present invention couples PD-L1 targeting groups to γδT cells, enabling them to obtain the ability to target PD-L1 while retaining their tumor recognition and killing functions. This allows them to enrich PD-L1-positive cancer cells and significantly improve their tumor cell killing effect. In order to efficiently label PD-L1 targeting groups on γδT cells, the present invention provides non-natural sugar molecules represented by formulas 1 to 6. Through metabolic labeling, the first active group with bioorthogonal reactivity can be labeled on the surface of γδT cells, which has the advantages of high labeling efficiency, rapidity, and high stability.

[0052] In some embodiments, a non-natural sugar molecule as shown in formula 5-1-1 is used.

[0053] It can be prepared by the following method, which is divided into 2 steps:

[0054] (1) Synthesis of intermediate 1-O-Ac-ManNAz:

[0055] 460 mg of ManNAz and 297 mg of sodium carbonate were dissolved in 40 mL of water and cooled to 0 ° C in an ice-water bath. 195.8 μL of thioacetic acid and 297 mg of DMC were added in sequence (DMC was dissolved in 630 μL of water, and an aqueous solution of DMC was added during the reaction). After reacting at 0 ° C for 15 minutes, 297 mg of sodium carbonate and 195.8 μL of thioacetic acid were added again, and then 297 mg of DMC was added, and the reaction was continued at 0 ° C for 15 minutes. This step-by-step addition method was repeated three times (a total of 5 rounds of reaction). After the reaction was completed, the reaction system was diluted with 40 mL of water, the aqueous phase was washed once with DCM, and lyophilized to obtain a light yellow powder. It was purified by silica gel column (dichloromethane and methanol as eluents) to obtain 370 mg of 1-O-Ac-ManNAz with a yield of 69%. 1H NMR(600MHz,Deuterium Oxide)δ5.96(d,J=1.7Hz,1H),5.88(d,J=1.7Hz,1H),4.63(dd,J=4.6,1.8Hz,1H),4.48 (dd,J=4.8,1.8Hz,1H),4.17-4.07(m,6H),3.96(dd,J=9.5,4.5Hz,1H),3.91(dd,J=12. 4,2.2Hz,1H),3.87-3.80(m,3H),3.78(ddd,J=10.0,4.1,2.7Hz,1H),3.69(t,J=9.9Hz, 1H), 3.61 (t, J=9.7Hz, 1H), 3.56 (ddd, J=9.9, 4.7, 2.2Hz, 1H), 2.19 (s, 3H), 2.13 (s, 3H). 13 C NMR (151 MHz, D2O) δ 172.03, 171.68, 171.66, 171.03, 92.44, 91.69, 77.20, 74.39, 71.19 68.67, 66.16, 66.05, 60.05, 60.03, 52.25, 51.66, 51.60, 51.55, 20.22, 20.19. HRMS (ESI) requires C 10 H 16 N4NaO7[M+Na] + 327.09167, detected value 327.09094.

[0056] (2) Synthesis of formula 5-1-1:

[0057] 260 mg of 1-O-Ac-ManNAz was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 mol / L). Three equivalents of Me-SATE phosphamide (synthesis method, see J. Med. Chem. 1995, 38, 3941-3950) were added to this solution under nitrogen. Subsequently, four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile to a concentration of approximately 0.45 mol / L) were added via syringe. The reaction was allowed to proceed overnight at zero degrees Celsius. TLC indicated the reaction was complete. A 70% solution of tert-butyl hydrogen peroxide was added at zero degrees Celsius. The reaction system was stirred for 2 hours to oxidize the trivalent phosphine product to a pentavalent phosphine product. After removing the solvent by rotary evaporation, the product was purified on a silica gel column using dichloromethane and methanol as eluents to yield 267 mg of 1-O-Ac-ManNAz-6-bis-(Me-SATE)-P, a two-step yield of 53%. The compounds were characterized by NMR and high-resolution mass spectrometry as follows. 1H NMR (600MHz, DMSO-d6) δ8.20(d,J=7.9Hz,1.3H),7.94(d,J=9.6Hz,1.0H),5.97(d,J=1.9Hz,1.3H,5.85(d,J=1.8Hz,1.0H),5.40(m,4 .6H),4.54(ddd,J=9.7,4.4,1.8Hz,1.0H),4.42(ddd,J=11.3,5.9,1.8Hz,1.0H),4.38(ddd,J=11.4,6.2,1.8Hz,1.3H),4.28-4.21(m, 3.6H),4.21-4.16(d,J=5.6Hz,9.2H),4.05-3.99(m,4.6H),3.96(dd,J=9.4,4.8Hz,1.3H),3.84(ddd,J=8.8,6.6,1.9Hz,1.3H),3.80( dd,J=9.2,4.4Hz,1.0H),3.69-3.61(m,2.3H),3.49(t,J=9.3Hz,1.0H,3.28(m,9.2H),2.50(s,13.8H),2.24(s,3.9H),2.14(s,3.0H). 13 C NMR(151MHz,DMSO-d6)δ194.83,194.81,168.78,168.55,168.39,168.36,92.00,91.71,76.22,76.18,73.60,73.56,70.61,68.06,67.46,67.42 ,67.14,67.10,66.29,66.07,65.76,65.71,65.67,65.66,65.63,65.62 ,65.60,51.91,51.43,50.77,50.56,30.52,28.82,28.77,20.77,20.69. 31 P NMR, -1.86. HRMS (ESI) calculated value C 18 H 30 N4O 12 PS2[M+H] + 589.10393, detected value 589.10428.

[0058] Unless otherwise stated, the reagents used in the examples of the present invention can be obtained through conventional commercial routes.

[0059] Example 1 Preparation and characterization of γδT cells

[0060] 1. Extraction of peripheral blood mononuclear cells (PBMC)

[0061] Human peripheral blood was drawn and human PBMCs were separated using lymphocyte separation medium.

[0062] 2. PBMC-Initiated Expansion of γδT Cells

[0063] The culture medium described in Table 1 was used, and the 6 γδT cells were resuspended at 100 cells / mL and cultured in a 37°C, 5% CO2 incubator for the first three days. After three days, the cells were cultured and passaged normally. Flow cytometric characterization of PBMCs before and after culture is shown in Figure 2. These results indicate that the purity of γδT cells can reach over 95% by day nine of culture.

[0064] Table 1. γδ T cell activation medium

[0065] Example 2 Preparation and Characterization of γδT-αPDL1 Cells

[0066] 1. γδT cell labeling with azide groups

[0067] Azide was used as the first active group to modify γδT cells. The compound shown in formula 5-1-1 was used as a non-natural sugar molecule to modify the azide group. The specific steps are as follows:

[0068] The purity-identified γδT cells from Example 1 were taken and 100 μM of non-natural sugar molecules were added and cultured for 24 h. The cells were then collected by centrifugation at 400 g for 3 min and washed once with PBS to obtain γδT cells labeled with azide groups, namely γδT-N3. 5×10 4 The validation was performed on 100 μM DBCO-PEG4-Biotin cells: cells were incubated with 100 μM DBCO-PEG4-Biotin for 10 minutes at room temperature, followed by a single wash with PBS. The cells were then incubated with 2 μg / ml Streptavidin-Alexa Fluor 488 for 15 minutes at 4°C, followed by a single wash with PBS. Laser confocal imaging was then used to detect AF488 fluorescence to characterize the azide labeling of the cells. As shown in Figure 3, after labeling γδ T cells with the non-natural sugar molecule, azide groups were present on the cell surface, demonstrating extremely high labeling efficiency.

[0069] 2. PD-L1 antibody labeled DBCO

[0070] DBCO (dibenzocyclooctene) can be used in azide-alkyne cycloaddition reactions that do not require copper ion catalysis. It can be used as a second reactive group to modify PD-L1 antibodies. The specific steps are as follows:

[0071] The PD-L1 antibody (Corning Oncology, KN035) was labeled with DBCO-PEG4-NHS at a concentration ratio of 1:5 and reacted at 30°C for 1 hour. The reaction system was then passed through a desalting column to remove unreacted DBCO-PEG4-NHS, obtaining the DBCO-labeled PD-L1 antibody, i.e., DBCO-PD-L1.

[0072] 3. γδ T cells targeting PD-L1

[0073] The γδT-N3 cells prepared in step 1 were co-incubated with the DBCO-PD-L1 prepared in step 2. The DBCO concentration on the antibody surface was 20 μM and the cell density did not exceed 1×10 7 After labeling, the cells were centrifuged at 300 g for 5 min to collect the cells, and the cells were washed once with PBS to obtain γδT cells that can target PD-L1, namely γδT-αPDL1.

[0074] Take 5×10 4 For verification, 2 μg / mL of recombinant biotinylated PD-L1 protein extracellular domain was added and incubated with the cells at room temperature for 15 minutes. After incubation, the cells were washed once with PBS. The cells were then incubated with 2 μg / mL of Streptavidin-Alexa Fluor 488 at 4°C for 15 minutes. After incubation, the cells were washed once with PBS. AF488 fluorescence was then detected by laser confocal imaging to characterize cellular PD-L1 antibody labeling. As shown in Figure 4, anti-PD-L1 antibodies were present on the cell surface, indicating high labeling efficiency.

[0075] Example 3 Evaluation of in vitro killing effect

[0076] Ovarian cancer cells OVCAR8 were digested and plated, with 5,000 cells per well added to a 96-well plate and incubated overnight at 37°C in 5% CO2. γδT-αPDL1 cells and γδT cells were then co-incubated with the above-mentioned ovarian cancer cells at effector-target ratios of 0.5:1, 1:1, and 2:1, respectively, and incubated at 37°C for 3 hours. After incubation, the cell killing effect was detected using an LDH detection kit. As shown in Figure 5, the tumor cell killing effect of γδT-αPDL1 cells was significantly better than that of γδT cells, with a significantly improved killing effect and PD-L1 target specificity.

[0077] Example 4 Evaluation of the Effect of Treating the Ovarian Cancer Peritoneal Metastasis Model

[0078] Twenty-one 6-8 week old NSG mice were intraperitoneally injected with 1×10 6OVCAR8-LUC ovarian cancer cells. On the second day after the injection of ovarian cancer cells, 100 μL of the substrate D-luciferin potassium salt 1 mg / mL was injected intraperitoneally, and imaging and fluorescence value detection were performed. On the third day after the injection of ovarian cancer cells, the mice were randomly divided into groups (7 / group) for drug administration. The γδT group was injected with γδT cells; the γδT-αPDL1 group was injected with γδT-αPDL1 cells; and the control group (Vehicle) was injected with PBS as a control. The drug was administered once every 4 days by intraperitoneal injection at a dose of 5×10 6 cells / times.

[0079] Taking the first dose as day 0, imaging and fluorescence value detection and statistics were performed to detect changes in tumor volume in mice after administration. The statistical results of fluorescence values ​​are shown in Figure 6. The tumor inhibition effect was significantly improved by the administration of γδT-αPDL1 cells.

[0080] The foregoing description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed herein are intended to be encompassed within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. [Corrected 30.10.2024 in accordance with Rule 91] A γδT cell targeting PD-L1, characterized in that: The γδT cells targeting PD-L1 are obtained by connecting a γδT cell modified with a first active group and a PD-L1 targeting group modified with a second active group through a bioorthogonal reaction; the second active group is suitable for bioorthogonal reaction with the first active group; Wherein, the γδT cells are metabolically labeled by non-natural sugar molecules to obtain the γδT cells modified with the first active group; the non-natural sugar molecules include at least one selected from Formula 1 to Formula 6: Wherein, X is the first active group; R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 1～6 alkyl.

2. [Corrected 30.10.2024 according to Rule 91] The γδT cell targeting PD-L1 according to claim 1, characterized in that The non-natural sugar molecule comprises at least one selected from the following group: Wherein, X is the first active group; R1 and R2 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 1～6 alkyl; Preferably, the non-natural sugar molecule comprises at least one selected from the following group: Wherein, X is the first active group.

3. The γδT cell targeting PD-L1 according to claim 1, characterized in that The bioorthogonal reaction comprises at least one selected from the group consisting of click chemistry, photocrosslinking, photosensitization, and glycosylation; Preferably, the first reactive group comprises at least one selected from the group consisting of azide, alkynyl, trans-cyclooctene, triazine, tetrazine, hydrazide, cyclopropene, isocyanate, and cyclooctyne.

4. The γδT cell targeting PD-L1 according to any one of claims 1 to 3, characterized in that The γδT cells are human γδT cells; Preferably, the γδT cells include at least one of the following subtypes: Vγ9Vδ2T cell subtype and Vδ1T cell subtype.

5. The γδT cell targeting PD-L1 according to claim 1, characterized in that Modifying the second active group to the PD-L1 targeting group by connecting a compound; the connecting compound simultaneously has a group that can be covalently connected to a thiol group and the second active group; Preferably, the group that can be covalently linked to the thiol group is a succinimide active ester group, a maleimide group or a maleic anhydride group; Preferably, the linking compound comprises a molecule selected from Formula L1 and Formula L2: Wherein, Y is the second active group; R7 is selected from C 1～20 Alkylene or C 1～20 Alkylene ether group; Preferably, the linking compound is DBCO-PEG4-NHS or DBCO-PEG4-MAL.

6. The γδT cell targeting PD-L1 according to any one of claims 1 to 5, characterized in that The PD-L1 targeting group includes an antibody or a fragment thereof that specifically binds to PD-L1; Preferably, the PD-L1 targeting group includes at least one selected from the following group: an immunoglobulin, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single region antibody that specifically binds to PD-L1, or a monoclonal antibody or polyclonal antibody prepared from the above antibodies.

7. [Corrected 30.10.2024 according to Rule 91] The method for preparing γδT cells targeting PD-L1 according to any one of claims 1 to 6, characterized in that: include: The γδT cells modified with a first active group and the PD-L1 targeting group modified with a second active group are connected through a bioorthogonal reaction to obtain the γδT cells targeting PD-L1; the second active group is suitable for bioorthogonal reaction with the first active group; Wherein, the γδT cells are metabolically labeled by non-natural sugar molecules to obtain the γδT cells modified with the first active group; the non-natural sugar molecules include at least one selected from Formula 1 to Formula 6: Wherein, X is the first active group; R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, halogen, substituted or unsubstituted C 1～6 Alkyl, C 1～6 Alkylthio, C 1～6 Alkylcarbonyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Alkoxy; the substitution refers to being substituted by a group selected from the following: halogen, hydroxyl, amino, nitro, mercapto, C 1～6 alkyl.

8. The preparation method according to claim 7, characterized in that: The metabolic labeling step comprises: culturing the γδT cells using a culture medium containing the non-natural sugar molecule; Preferably, in the culture medium, the concentration of the non-natural sugar molecule is 10 to 200 μM; Preferably, the culturing time is 2 to 48 hours.

9. Use of the PD-L1-targeting γδT cells according to any one of claims 1 to 6 in the preparation of a product for treating PD-L1-positive tumors.

10. The use according to claim 9, characterized in that The PD-L1 positive tumor includes at least one of the following groups: PD-L1 positive lung cancer, lymphoma, intestinal cancer, head and neck squamous cell carcinoma, esophageal cancer, liver cancer, and ovarian cancer.

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