Method for constructing antibody-gamma delta T cell conjugate by chemical engineering method

Abstract

The invention discloses a method for constructing an antibody-gamma delta T cell conjugate by using a chemical engineering method. The method comprises the following steps: carrying out metabolism labeling on glycan on the surface of a gamma delta T cell by utilizing a rapid metabolism glycan labeling strategy, and coupling a functionalized modified antibody with the labeled gamma delta T cell through click chemistry to construct the antibody-gamma delta T cell conjugate. Experiments show that the conjugate constructed by the method has remarkable killing activity on PD-L1 positive tumor cells, and a new effective strategy is provided for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a method for constructing antibody-γδT cell conjugates using chemical engineering. Background Technology

[0002] γδT cells are a unique subset of T lymphocytes, possessing characteristics of both innate and adaptive immunity, and have shown significant advantages in the field of tumor immunotherapy. Unlike traditional αβT cells, γδT cells can directly recognize tumor antigens without relying on major histocompatibility complex (MHC) molecules, making them an ideal candidate for tumor immunotherapy. Furthermore, γδT cells can be infused allogeneically without requiring strict matching of donor and patient tissue types, facilitating clinical application. However, current research shows that unmodified γδT cells have low targeting efficiency against solid tumors, limiting the clinical application of current γδT cell therapy. Therefore, there is an urgent need to modify them to enhance their tumor-targeting ability and anti-tumor activity.

[0003] Conjugating antibodies to γδT cells holds promise for developing more efficient and specific immunotherapeutic strategies. It is hoped that the constructed antibody-γδT cell conjugates can precisely recognize antigens on the surface of tumor cells and effectively activate the immune-killing function of γδT cells. However, current technologies fall short of these requirements, limiting the development and clinical application of related immunotherapeutic drugs. Currently known antibody-γδT cell conjugates constructed using click chemistry still have significant room for improvement in antitumor activity. Summary of the Invention

[0004] To address the shortcomings of existing technologies and solve the aforementioned problems, this invention aims to propose a method for constructing antibody-γδT cell conjugates using chemical engineering. This invention involves simultaneously adding metabolic regulators to γδT cells during azide modification to achieve endogenous regulation, resulting in modified γδT cells. Then, a tumor-targeting antibody is precisely conjugated to the γδT cells, enabling the antibody to efficiently and stably anchor on the γδT cell surface. This not only enhances the γδT cells' ability to target and recognize tumor cells but also effectively improves their anti-tumor activity.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A γδT cell with high anti-tumor activity, wherein the method for preparing the γδT cell includes the following steps: adding γδT cells to a drug-containing culture medium for culture to obtain γδT cells with high anti-tumor activity; The drug-containing culture medium contains azide sugar and metabolic regulators, including at least one of dibutyryl cyclic adenosine monophosphate, sodium 6-Bnz-cAMP, and 8-Bromo-cAMP.

[0006] Preferably, experimental verification showed that dibutyryl cyclic adenosine monophosphate has a better regulatory effect than other compounds.

[0007] Azidose can be recognized by various glycosyltransferases, leading to non-specific labeling or low integration efficiency. By adding azidose and using low-dose metabolic regulators to slightly disrupt endogenous pathways, the effects of azidose modification and γδT cell activity can be enhanced.

[0008] Preferably, the azide sugar comprises 1,6-Pr2ManNAz. Using commercially available azide compounds for chemical engineering modification is cost-effective, and the raw materials are readily available, which helps control costs.

[0009] Preferably, the content of the metabolic regulator in the drug-containing culture medium is 10~30 μM.

[0010] Preferably, the content of the azide sugar in the drug-containing culture medium is 50~200 μM.

[0011] Preferably, the culture time is 48 hours.

[0012] A method for constructing antibody-γδT cell conjugates using chemical engineering includes the following steps: (1) Add DBCO-PEG4-NHS Ester to the antibody solution, mix gently, incubate, and then purify to obtain DBCO-antibody modified with DBCO. (2) Mix the γδT cells with the DBCO-antibody from step (1) and incubate for 45-50 minutes. Centrifuge, wash, and obtain antibody-γδT cell conjugate.

[0013] Preferably, step (2) is as follows: the highly anti-tumor active γδT cells are mixed with the DBCO-antibody and compound BAF312 from step (1) and incubated for 45-50 minutes, centrifuged, washed, and antibody-γδT cell conjugate is obtained.

[0014] Preferably, the antibody includes a PD-L1 antibody, the molar ratio of antibody to DBCO is 1:5; the incubation time is 2 hours; and the cell density in step (2) is 5 × 10⁻⁶ cells / day. 6 The cell / mL ratio was 8:2, the final concentration of DBCO-antibody was 1 μg / mL, and the final concentration of BAF312 was 10~20 μM.

[0015] In addition to PD-L1 antibodies, other related antibodies can be conjugated to the surface of γδT cells to construct engineered cells with different target specificities. Furthermore, γδT cells conjugated with multiple different targeting antibodies can be further developed to achieve multi-target attack on tumors, thereby improving treatment efficacy and reducing the risk of tumor escape.

[0016] An antibody-γδT cell conjugate was prepared by the aforementioned method.

[0017] The application of the highly anti-tumor γδT cells or the antibody-γδT cell conjugate in the preparation of anti-tumor drugs. The tumors include solid tumors expressing PD-L1, specifically ovarian cancer, lung cancer, breast cancer, colorectal cancer, etc.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention employs a chemical modification and click chemical conjugation strategy. By modifying the surface of γδ T cells with azidosaccharide and modifying PD-L1 antibodies with DBCO, it achieves highly efficient and stable conjugation between the antibody and γδ T cells, ensuring uniform antibody exposure on the cell surface. Combining azidosaccharide with different metabolic regulators (such as dibutyrylcyclophosphamide and 6-Bnz-cAMP sodium salt) and optimizing their concentration ratios can synergistically enhance the tumor-killing activity of γδ T cells.

[0019] Further research revealed that adding BAF312 during the conjugation process can further enhance the antitumor effect of the conjugate, providing a new direction for optimizing the activity of engineered cells.

[0020] Compared to traditional CAR-T therapy, this chemical engineering strategy does not involve gene editing. The entire modification and conjugation process is achieved through chemical methods, making it simpler and more gentle, avoiding the complex preparation process and high costs of CAR-T therapy. This makes the technology easier to promote and apply, especially in resource-constrained medical settings.

[0021] The entire preparation process is relatively simple, and it only takes a few days to prepare engineered antibody-γδT cell conjugates, which greatly shortens the time patients wait for treatment.

[0022] The antibody-γδT cell conjugate prepared by this method is particularly suitable for the treatment of solid tumors expressing PD-L1, such as ovarian cancer. Attached Figure Description

[0023] Figure 1 : Validation of in vitro antitumor activity.

[0024] Figure 2 : Anti-tumor effect in vivo. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical content of this invention, the technical solution of this invention will be further described in detail below with reference to specific embodiments. The experimental methods and reagents described below are all common knowledge in the art and are conventional products on the market.

[0026] 1,6-Pr2ManNAz, CAS No: 2919936-11-9 Dibutyrylcyclic adenosine monophosphate, CAS No: 16980-89-5 6-Bnz-cAMP sodium salt, CAS No: 1135306-29-4 8-Bromo-cAMP, CAS No: 76939-46-3 BAF312 (Siponimod), CAS No.: 1230487-00-9 Example 1: Chemical Engineering Modification of γδT Cells (1) Acquisition and expansion of γδT cells Peripheral blood was collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. T cells in PBMCs were stimulated with 5 μM zoledronic acid, 1000 IU / mL IL-2, and 600 IU / mL IL-15 to induce differentiation and proliferation into γδT cells. γδT cells were cultured in medium to maintain their viability and proliferative capacity in preparation for subsequent experiments.

[0027] (2) γδT cell modification Take 1 mL of γδT cells (1×10⁻⁶) 6 Add 1 mL of the corresponding drug-containing culture medium (cells / mL) and co-culture at 37.0 ± 0.2°C, 5.0 ± 0.1% CO2 for 48 hours. Centrifuge to collect cells (400g, 3 min), and wash with PBS to obtain modified γδT cells.

[0028] Grouping of drug-containing culture media: Group A-1: ​​AIM-V medium containing 100 μM 1,6-Pr2ManNAz + 10 μM dibutyryl cyclic adenosine monophosphate. Group A-2: AIM-V medium contains 100 μM 1,6-Pr2ManNAz + 10 μM 6-Bnz-cAMP sodium salt Group A-3: AIM-V medium containing 100 μM 1,6-Pr2ManNAz + 10 μM 8-Bromo-cAMP (CAS No.: 76939-46-3) Group B-1: AIM-V medium containing 50 μM 1,6-Pr2ManNAz + 10 μM dibutyryl cyclic adenosine monophosphate. Group B-2: AIM-V medium containing 200 μM 1,6-Pr2ManNAz + 10 μM dibutyryl cyclic adenosine monophosphate. Group C-1: AIM-V medium containing 100 μM 1,6-Pr2ManNAz + 20 μM dibutyryl cyclic adenosine monophosphate. Group C-2: AIM-V medium containing 100 μM 1,6-Pr2ManNAz + 30 μM dibutyryl cyclic adenosine monophosphate. Group D: AIM-V medium contains 100 μM 1,6-Pr2ManNAz Group E: AIM-V medium contains 10 μM dibutyryl cyclic adenosine monophosphate. Group F: Blank control (AIM-V medium only) (3) PD-L1 antibody modification: DBCO-PEG4-NHS Ester was added to the antibody solution (molar ratio, antibody:DBCO = 1:5), gently mixed, and incubated at 4°C for 2 hours in the dark with gentle shaking. The solution was then purified using a 7K MWCO desalting column to obtain the DBCO-modified DBCO-antibody.

[0029] (4) γδT cell-antibody click conjugation The modified γδ T cells were adjusted to a cell density of 5 × 10⁶. 6 Take 800 μL of cell culture and add 200 μL of purified DBCO-antibody to a final concentration of 1 μg / mL. Gently pipette to mix, place in a rotary mixer, and mix at 4°C for 45–50 minutes. Centrifuge at 300×g, 4°C for 5 minutes, carefully discard the supernatant, add PBS and wash three times. For the last wash, resuspend in 500 μL of cold PBS to obtain the antibody-γδT cell conjugate.

[0030] Experiment: Functional verification of engineered cells The expression level and distribution of antibodies on the surface of γδT cells were detected by confocal microscopy and flow cytometry to confirm that the antibodies were uniformly and stably distributed and exposed on the cell surface. Then, the killing ability of αPD-L1-γδT cells against PD-L1 positive tumor cells was evaluated by in vitro cytotoxicity assays and in vivo antitumor assays.

[0031] 1. In vitro antitumor activity assessment At an effector-to-target ratio of 2:1, PD-L1-γδT cells or γδT cells were co-cultured with PD-L1-positive ovarian cancer cell line (OVCAR-8) for 2 hours. Cytotoxicity (%) was detected using the LDH release assay (direct method) according to the LDH cytotoxicity assay kit instructions. Results are shown in Table 1 and [Table data missing]. Figure 1 .

[0032] Table 1 Cytotoxicity (mean ± standard deviation)

[0033] The experimental results showed that αPD-L1-γδT cells in each group exhibited significant killing activity against PD-L1 positive ovarian cancer cell lines in vitro.

[0034] The cytotoxicity of γδ T cells alone against PD-L1 positive ovarian cancer cells was 20.24±1.63%, while the cytotoxicity of the blank control and groups D and E containing only a single component was low (23.63%~63.50%). The cytotoxicity of each group (A-1, A-2, A-3, B-1, B-2, C-1, C-2) modified with combinations of azide sugar and different metabolic regulators was significantly increased. Among them, group C-1 (94.88±2.17%), group C-2 (94.63±1.51%), and group B-2 (93.72±1.78%) showed the highest cytotoxicity, followed by group A-1 (85.54±1.70%).

[0035] 2. Verification of in vivo anti-tumor effects PD-L1-positive ovarian cancer cells OVCAR8-Luc were injected intraperitoneally into NSG mice (female, 6-8 weeks old) at a dose of 3 × 10⁻⁶ cells per day. 5 300 cells were injected into tumor-bearing mice on days 1-3. On days 7 and 10, αPD-L1-γδT cells were reinfused into the mice via intraperitoneal injection (3 × 10⁻⁶ cells). 6 The experiment included αPD-L1-γδT cells (100 cells), with a control group (γδT cells, PBS). On day 24, bioluminescence imaging was used to assess the inhibitory effect of αPD-L1-γδT cells on tumor growth. Results are shown in Table 2. Figure 2 .

[0036] Table 2. In vivo tumor suppression effect (mean ± standard deviation)

[0037] Animal experiments showed that the PBS control group had the highest total flux value for tumors (2.23±0.18), while the γδ T cell group showed a certain inhibitory effect (1.25±0.08). The engineered groups all exhibited stronger tumor-inhibiting effects, with groups C-1 (0.34±0.05), C-2 (0.33±0.04), and B-2 (0.41±0.08) showing the lowest total flux values ​​and the most significant tumor growth inhibition, consistent with the trend observed in in vitro toxicity results.

[0038] In addition, during the click conjugation process of γδT cells and antibodies, we unexpectedly discovered that the compound BAF312 could enhance the antitumor activity of γδT cells and antibodies. Specific examples are as follows: Example 2 The B-2 modified γδ T cells from Example 1 were adjusted to a cell density of 5 × 10⁻⁶. 6 Cells / mL: Take 800 μL of cell culture, add 200 μL of purified DBCO-antibody to a final concentration of 1 μg / mL and 50 μL of LAF312 solution to a final concentration of 10 μM. Gently pipette to mix, place in a rotary mixer, and mix at 4°C for 45–50 minutes. Centrifuge at 300 × g, 4°C for 5 minutes, carefully discard the supernatant, add PBS and wash three times repeatedly, finally resuspending in 500 μL of cold PBS to obtain the antibody-γδT cell conjugate.

[0039] Example 3 The B-2 modified γδ T cells from Example 1 were adjusted to a cell density of 5 × 10⁻⁶. 6 Take 800 μL of cell culture and add 200 μL of purified DBCO-antibody to a final concentration of 1 μg / mL and 50 μL of LAF312 solution to a final concentration of 15 μM. Gently pipette to mix, place in a rotary mixer, and mix at 4°C for 45–50 minutes. Centrifuge at 300 × g, 4°C for 5 minutes, carefully discard the supernatant, add PBS and wash three times repeatedly, finally resuspending in 500 μL of cold PBS to obtain the antibody-γδT cell conjugate.

[0040] Example 4 The B-2 modified γδ T cells from Example 1 were adjusted to a cell density of 5 × 10⁻⁶. 6Take 800 μL of cell culture, add 200 μL of purified DBCO-antibody to a final concentration of 1 μg / mL, and 50 μL of BAF312 solution to a final concentration of 20 μM. Gently pipette to mix, place in a rotary mixer, and mix at 4°C for 45–50 minutes. Centrifuge at 300 × g, 4°C for 5 minutes, carefully discard the supernatant, add PBS and wash three times repeatedly, finally resuspending in 500 μL of cold PBS to obtain the antibody-γδT cell conjugate.

[0041] The antitumor activity of Examples 2-4 was tested respectively, and the results are shown in Table 3.

[0042] Table 3

[0043] The results showed that the addition of BAF312 further enhanced cytotoxicity to over 98%, with Example 4 (20 μMBAF312) reaching 99.67 ± 1.89%; the total flux value of tumor in vivo was significantly reduced, with Example 4 showing only 0.02 ± 0.09, which was lower than group B-2 without BAF312, indicating that BAF312 can significantly enhance the antitumor activity of engineered cells.

[0044] Based on the above in vitro cytotoxicity and in vivo tumor suppression effects, Example 4 (adding 20 μM BAF312 to Group B-2) is the optimal regimen. Under this regimen, the engineered αPD-L1-γδ T cells achieved 99.67% in vitro cytotoxicity and the most significant in vivo tumor growth inhibition effect, demonstrating the strongest targeted anti-tumor activity.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A γδ T cell with high anti-tumor activity, characterized in that, The preparation method of the γδT cells comprises the following steps: adding γδT cells into a drug-containing culture medium for culture to obtain γδT cells with high anti-tumor activity. The drug-containing culture medium contains azidosugar and a metabolic regulator, and the metabolic regulator comprises at least one of dibutyryl cyclic adenosine phosphate, 6-Bnz-cAMP sodium salt and 8-bromo-cAMP.

2. The γδ T cells with high anti-tumor activity according to claim 1, characterized in that, The azidosugar comprises 1,6-Pr2ManNAz.

3. The γδ T cells of claim 1, wherein the γδ T cells have high anti-tumor activity. The content of the metabolic regulator in the drug-containing culture medium is 10-30 μM.

4. The γδ T cells of claim 1, wherein the γδ T cells have high anti-tumor activity. The content of the azidosugar in the drug-containing culture medium is 50-200 μM.

5. The γδ T cells of claim 1, wherein the γδ T cells have high anti-tumor activity. The culture time is 48 hours.

6. A method for constructing an antibody-γδ T cell conjugate by chemical engineering, characterized in that, The method comprises the following steps: (1) adding DBCO-PEG4-NHS Ester into an antibody solution, mixing gently, incubating, purifying and obtaining DBCO-modified DBCO-antibody; (2) mixing the γδT cells with high anti-tumor activity according to any one of claims 1-5 and the DBCO-antibody of step (1) and incubating for 45-50 minutes, centrifuging, washing and obtaining antibody-γδT cell conjugate.

7. The method of claim 6, wherein, Step (2) is: mixing the γδT cells with high anti-tumor activity according to any one of claims 1-5, the DBCO-antibody of step (1) and compound BAF312 and incubating for 45-50 minutes, centrifuging, washing and obtaining antibody-γδT cell conjugate.

8. The method of claim 6, wherein, The antibody includes a PD-L1 antibody, the molar ratio of the antibody to DBCO is 1:5; the incubation time is 2 hours; the cell density of step (2) is 5x10 6 cells / mL, the volume ratio of cell liquid to DBCO-antibody liquid is 8:2, the final concentration of DBCO-antibody is 1 pg / mL, and the final concentration of BAF312 is 10~20 pM.

9. An antibody-γδ T cell conjugate, characterized in that The antibody-γδT cell conjugate is prepared by the method of claim 6.

10. Use of the γδT cells with high anti-tumor activity of claim 1 or the antibody-γδT cell conjugate of claim 9 in the preparation of an anti-tumor drug.

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