Combined drug delivery system based on T cell adapter and adoptive T cells as well as preparation method and application of combined drug delivery system

Abstract

The invention relates to a combined drug delivery system based on a T cell adapter and adoptive T cells as well as a preparation method and application of the combined drug delivery system, and belongs to the technical field of biological medicines. The invention provides a combined drug delivery system consisting of a T cell adapter D-C-aPDL1 and an azide metabolism labeled adoptive T cell T-N3. The combined drug delivery system is prepared by the following steps: modifying cytochrome C by using DBCO to obtain DBCO-CC, connecting the DBCO-CC and aPD-L1 by using a BS3 cross-linking agent to obtain D-C-aPDL1, and then carrying out metabolism labeling on the adoptive T cell by using azide sugar to obtain T-N3 of which the surface expresses an azide group. Through intravenous injection administration, the T cell adapter is efficiently combined with T-N3 in a targeted manner in vivo, adoptive T cells and tumor cells are coupled in a tumor, and the killing effect of the adoptive T cells on the tumor cells is improved; or the adoptive T cells and the dendritic cells are coupled, activation of the dendritic cells on the adoptive T cells is improved, the anti-tumor curative effect is synergistically improved, and the problem that in the prior art, the adoptive T cell therapy is limited in solid tumor curative effect is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a combined drug delivery system based on a T cell adaptor and adoptive T cells, and a preparation method and application thereof. Background Art

[0002] Adoptive T Cell Therapy (ACT) is a biotherapeutic technique that enhances the body's anti-tumor immune response by modifying, expanding, and reinfusing T cells in vitro. Autologous or allogeneic T cells are modified and expanded in vitro and then reinfused into the patient via intravenous or local injection. T cells target the tumor microenvironment, releasing cytokines (such as IFN-γ and granzyme B) to directly kill tumor cells and form immune memory.

[0003] However, from the current perspective, the efficacy of adoptive T cell therapy for solid tumors still needs to be greatly improved, and the poor in vivo persistence of ACT cells is an important reason that restricts its efficacy. PD-L1, which is highly expressed on the surface of tumor cells and dendritic cells (DCs), can bind to PD-1 on the surface of T cells, thereby inhibiting T cell function. Immune checkpoint inhibitors (ICIs) such as PD-L1 antibodies (aPD-L1) can effectively block the PD-1 / PD-L1 signaling pathway and enhance the efficacy of T cells. The amount of systemically administered ICIs acting on ACT cells in vivo is small. On the other hand, when ICIs are used alone, the overall response rate to ICIs in patients with solid tumors is low due to the lack of tumor-specific T cells in the tumor. However, ACT can increase the number of tumor-specific T cells in the tumor microenvironment (TME), thereby improving the patient's response rate to ICIs.

[0004] As a dual-target functional molecule, T cell engager (TCE) can simultaneously bind to T cells and tumor cells, shorten the distance between T cells and tumor cells, and provide T cell activation signals, giving T cells stronger anti-tumor activity. In existing technologies, CD19×CD3 bispecific single-chain antibody adapters can efficiently bind to CD19 + B cell lymphoma cells and T cells produce a highly effective blood tumor killing effect. CLEC9A×PD-1 bispecific DC cell-T cell adapter not only blocks the immune checkpoint signaling pathway, but also achieves CD8 + The physical connection between T cells and cDC1 promotes the presentation of tumor antigens and the transmission of co-stimulatory signals, leading to more efficient T cell activation. However, TCEs currently suffer from issues such as insufficient targeting and significant toxic side effects, and there is still a lack of TCEs specifically suitable for ACT cells. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a combined drug delivery system consisting of a T cell adapter DC-aPDL1 and an azide metabolically labeled adoptive T cell T-N3, the preparation steps of which include modifying cytochrome C with DBCO to obtain DBCO-CC, and using BS 3 A crosslinker connects DBCO-CC and aPD-L1 to generate DC-aPDL1. Azidosugar is then used to metabolically label the adoptive T cells, yielding T-N3 cells expressing azide groups on their surface. This combined drug delivery system can be used to prepare tumor therapeutics and enhance the efficacy of adoptive T cell therapy against solid tumors.

[0006] The first object of the present invention is to provide a method for preparing a combined drug delivery system, wherein the combined drug delivery system is composed of a T cell adaptor DC-aPDL1 and adoptive T cells T-N3, and the preparation method comprises the following steps:

[0007] (1) using dibenzocyclooctyne-polyethylene glycol-N-hydroxysuccinimide to modify cytochrome C to obtain DBCO-CC, and using di(sulfosuccinimide) suberate as a crosslinker to link the DBCO-CC and the anti-programmed death ligand 1 antibody aPD-L1 to obtain DC-aPDL1;

[0008] (2) The adoptive T cells were metabolically labeled with azido sugars to obtain adoptive T cells T-N3 expressing azido groups on their surface.

[0009] Furthermore, the molar ratio of the dibenzocyclooctyne-polyethylene glycol-N-hydroxysuccinimide to cytochrome C is (1-11):1.

[0010] In one embodiment of the present invention, the molar ratio of dibenzocyclooctyne-polyethylene glycol-N-hydroxysuccinimide to cytochrome C is 3:1.

[0011] Furthermore, the molar ratio of aPD-L1, di(sulfosuccinimide) suberate crosslinker and DBCO-CC is 1:(10-80):(18-22).

[0012] In one embodiment of the present invention, the molar ratio of aPD-L1, di(sulfosuccinimidyl) suberate crosslinker, and DBCO-CC is 1:10:20.

[0013] Furthermore, the azido sugar is tetraacylated N-azidoacetylaminomannose, tetraacetyl-N-azidoacetylglucose or tetraacylated N-azidoacetylaminogalactose.

[0014] In one embodiment of the present invention, the azido sugar is tetraacylated N-azidoacetylaminomannose.

[0015] Furthermore, the added amount of the azido sugar is 12.5-50 μM.

[0016] In one embodiment of the present invention, the amount of the azido sugar added is 25 μM.

[0017] The second object of the present invention is to provide a combined drug delivery system prepared by the above method.

[0018] Furthermore, the number of DBCOs bound to the aPD-L1 is 0-9.

[0019] In one embodiment of the present invention, the number of DBCOs bound to aPD-L1 is 6.9.

[0020] Furthermore, the number of cytochrome C bound to the aPD-L1 is 0-4.

[0021] The third object of the present invention is to provide the use of the above-mentioned combined drug delivery system in the preparation of tumor therapeutic drugs.

[0022] The combined drug delivery system provided by the present invention works in vivo as follows Figure 1 As shown in Figure 2, DC-aPDL1 efficiently targets T-N3 in vivo through a click chemistry reaction between the DBCO and N3 groups, allowing ACT cells to "hitch a ride" on tumor-infiltrating cells. Within the tumor, DC-aPDL1 couples adoptively transferred T cells to tumor cells, effectively blocking tumor cell immunosuppression and enhancing ACT cell cytotoxicity.

[0023] At the same time, DC-aPDL1 also blocks PD-L1 on dendritic cells, significantly promoting the activation of adoptive T cells by dendritic cells through co-stimulatory signals, thereby enhancing the anti-tumor immunity of adoptive T cells.

[0024] Furthermore, the ratio of T cell adapter DC-aPDL1 and adoptive T cell T-N3 in the combined drug delivery system is (10-15): 10 6 (μg: piece).

[0025] In one embodiment of the present invention, the ratio of the T cell adapter DBCO-CC@aPD-L1 to the adoptive T cell T-N3 in the combined drug delivery system is 10:10. 6 (μg: piece).

[0026] Furthermore, the tumor treatment drug is administered by subcutaneous injection or intravenous injection.

[0027] Preferably, the tumor therapeutic drug is administered by intravenous injection.

[0028] Furthermore, the T cell engager DC-aPDL1 is administered 0-100 hours after the administration of adoptive T cells T-N3.

[0029] Beneficial effects of the present invention:

[0030] The present invention provides a combined drug delivery system consisting of a T cell adaptor DC-aPDL1 and azide metabolically labeled adoptive T cells T-N3. When administered via intravenous injection, DC-aPDL1 specifically binds to T-N3 in vivo, "hitchhiking" on ACT cells to enrich tumors; the aPD-L1 at the other end can bind to tumor cells or dendritic cells, respectively, blocking the immunosuppression of ACT cells and promoting the killing of tumor cells by ACT cells, as well as the activation of ACT cells by dendritic cells, thereby improving the therapeutic effect on solid tumors such as melanoma, and solving the problem of limited efficacy of adoptive T cell therapy for solid tumors in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0032] Figure 1 Schematic diagram of the present invention, wherein A is a schematic diagram of azidosugar metabolic labeling of ACT cells (T-N3) and DC-aPDL1, and B is a schematic diagram of the principle of the T cell adapter DC-aPDL1 and azido T cells for the efficient treatment of solid tumors;

[0033] Figure 2 The experimental results of azidosugar metabolic labeling of T cells in Example 2 of the present invention, wherein A is a flow cytometric histogram of Cy5 fluorescence on the surface of T-N3, B is the mean fluorescence intensity on the surface of T-N3, C is the toxicity of different concentrations of Man-N3 on T cells, D is a representative flow cytometric histogram of Cy5 fluorescence on the surface of T cells, E is the mean fluorescence intensity on the surface of T cells, and F is the Cy5 fluorescence on the surface of cells observed by confocal microscopy, wherein the scale bar is 10 μm, and the data are expressed as mean ± standard deviation, and were analyzed using One-way ANOVA and Tukey's post hoc test. ns indicates no significant difference, **P<0.01, ****P<0.0001;

[0034] Figure 3 The synthesis and characterization of the T cell adapter in Example 3 of the present invention, wherein A is DC-aPDL1 Cy5Schematic diagram of the synthesis route, B is the UV absorption spectrum of the mixed solution of DPN and CC at different molar ratios, C is the linear relationship between the calculated value and the theoretical value of the DBCO and CC feed ratio, D is the UV absorption spectrum of DBCO-CC, E is the number of DBCO connected to each CC molecule at different reaction ratios, and F is DC-aPDL1 synthesized at different ratios Cy5 UV absorption spectra, G is the number of CC connections on each aPD-L1, H is the number of DBCO connections on each aPD-L1, and I is the DC-aPDL1 synthesized in different ratios Cy5 Gel electrophoresis characterization, J is the hemolysis of DC-aPDL1, K is the hemolysis of D-aPDL1 Cy5 The synthetic route, UV absorption spectra of the products at different reaction ratios, and the number of DBCOs attached to each aPD-L1;

[0035] Figure 4 The experimental results of the specific binding of T cell adapters to T-N3 and tumor cells in Example 4 of the present invention are shown in Figure 1, where A is the experimental flow chart, B is a representative flow cytometric graph of Cy5 fluorescence on the surface of T cells, C is the average fluorescence intensity of Cy5 fluorescence on the surface of T cells, and D is the average fluorescence intensity of different doses of DC-aPDL1. Cy5 Representative flow cytometry histograms of Cy5 fluorescence on the surface of T cells after co-incubation with T-N3. E is different doses of DC-aPDL1. Cy5 The average fluorescence intensity of Cy5 fluorescence on the surface of T cells after co-incubation with T-N3, F is the experimental flow chart, G is the flow cytometric representation of PD-L1 expression on the surface of tumor cells induced by different concentrations of IFN-γ, H is the average fluorescence intensity of PD-L1 expression on the surface of tumor cells induced by different concentrations of IFN-γ, I is the representative flow cytometric representation of Cy5 fluorescence on the surface of cells, J is the average fluorescence intensity of Cy5 fluorescence on the surface of cells, and K is the flow cytometric representation of different doses of aPD-L1 Cy5 、DC-aPDL1 Cy5 Mean fluorescence intensity of cell surface Cy5 after co-incubation with B16-F10 cells. Data are expressed as mean ± SD (n = 3). H and J were analyzed using one-way ANOVA and Tukey's post hoc test, K was analyzed using two-way ANOVA and Tukey's post hoc test, and C and E were analyzed using two-tailed unpaired Student's t-test. ns represents no significant difference, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0036] Figure 5 The experimental results of DC-aPDL1 coupling T cells and tumor cells in Example 5 of the present invention are shown in Figure 5, where A is the experimental flow chart and B is the CFSE + Violet +Representative flow cytometry plots of cell conjugates, C represents CFSE + Violet + Statistical graph of the proportion of cell couples in B16-F10 cells. D is the efficiency of T cell-mediated tumor cell killing. E is the level of IFN-γ secretion by T cells at different effector-target ratios. Data are expressed as mean ± standard deviation (n = 3-6). C and E were analyzed using one-way ANOVA with Tukey's post hoc test, and D was analyzed using two-way ANOVA with Tukey's post hoc test and two-tailed unpaired Student's t-test. ns indicates no significant difference. *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0037] Figure 6 These are the experimental results of ACT increasing the expression of PD-L1 on the surface of DCs in tumor-bearing mice in Example 6 of the present invention, wherein A is an experimental flow chart, B is a flow cytometric histogram of PD-L1 expression on the surface of cDC1, C is a flow cytometric histogram of PD-L1 expression on the surface of cDC2, D is the mean fluorescence intensity of PD-L1 expression on the surface of cDC1, E is the mean fluorescence intensity of PD-L1 expression on the surface of cDC2, F is an experimental flow chart of tumor-specific ACT cells increasing the expression of PD-L1 on the surface of DCs in tumor-bearing mice, G is a representative flow cytometric histogram of PD-L1 expression on the surface of cDC1, H is a representative flow cytometric histogram of PD-L1 expression on the surface of cDC2, I is the mean fluorescence intensity of PD-L1 expression on the surface of cDC1, and J is the mean fluorescence intensity of PD-L1 expression on the surface of cDC2. The data are expressed as mean ± standard deviation (n = 6) and were analyzed using one-way ANOVA with Tukey's post hoc test and two-tailed unpaired Student's t-test. ns means no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0038] Figure 7 The experimental results of DC-aPDL1 enhancing the interaction between T cells and DCs in Example 7 of the present invention are shown in Figure 1, where A is the fluorescence flow cytometry histogram of PD-L1 on the surface of BMDCs under different culture conditions, B is the average fluorescence intensity of PD-L1 on the surface of BMDCs under different culture conditions, and C is the average fluorescence intensity of aPD-L1 on the surface of BMDCs under different culture conditions. Cy5 or DC-aPDL1 Cy5 Fluorescence flow cytometry histogram of Cy5 on the surface of BMDC after co-incubation with BMDC, D is aPD-L1 Cy5 or DC-aPDL1 Cy5 The average fluorescence intensity of Cy5 on the BMDC surface after co-incubation with BMDC, E is the experimental flow chart of DC-aPDL1 promoting the formation of T-DC2.4 conjugates, F is CFSE + Violet+ Representative flow cytometry plots of T-DC2.4 conjugates. G is a statistical plot of the proportion of conjugates to DC2.4. H is the mean fluorescence intensity of CD69 expression on the surface of T cells. I is the level of IFN-γ secretion by T cells. Data are expressed as mean ± standard deviation. B, G, H, and I were analyzed using one-way ANOVA and Tukey's post hoc test, and D was analyzed using two-way ANOVA and Tukey's post hoc test. ns indicates no significant difference. ***P < 0.001, ****P < 0.0001.

[0039] Figure 8 This is DC-aPDL1 2 hours after ACT injection in Example 8 of the present invention. Cy5 Results of in vivo targeted binding adoptive T-N3 experiment, where A is the experimental flow chart and B is the Cy5 + Representative flow cytometry diagram of T cell / T-N3 ratio, C is Cy5 in peripheral blood + T cell / T-N3 ratio statistics, D is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3, E is the average fluorescence intensity of Cy5 in lymph nodes + Representative flow cytometry diagram of T cell / T-N3 ratio, F is Cy5 in lymph node + T cell / T-N3 ratio statistics, G is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3, H is the average fluorescence intensity of Cy5 in spleen + Representative flow cytometry diagram of T cell / T-N3 ratio, I is Cy5 in spleen + Statistical graph of the T cell / T-N3 ratio, J is the mean fluorescence intensity of Cy5 on the surface of T cells / T-N3, data are expressed as mean ± standard deviation (n = 6), analyzed using a two-tailed unpaired Student's t-test, ns means no significant difference, *P < 0.05, ****P < 0.0001;

[0040] Figure 9 This is DC-aPDL1 48 hours after ACT injection in Example 8 of the present invention. Cy5 Results of in vivo targeted binding adoptive T-N3 experiment, where A is the experimental flow chart and B is the Cy5 + Representative flow cytometry diagram of T cell / T-N3 ratio, C is Cy5 in peripheral blood + T cell / T-N3 ratio statistics, D is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3, E is the average fluorescence intensity of Cy5 in tumor + Representative flow cytometry diagram of T cell / T-N3 ratio, F is Cy5 in tumor + T cell / T-N3 ratio statistics, G is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3, H is the average fluorescence intensity of Cy5 in spleen +Representative flow cytometry images of the T cell / T-N3 ratio, I is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3 in the spleen, J is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3 in the lymph nodes + Representative flow cytometry plots of T cell / T-N3 ratios. K represents the mean fluorescence intensity of Cy5 on the surface of T cells / T-N3 in lymph nodes. Data are expressed as mean ± standard deviation (n = 6) and analyzed using a two-tailed unpaired Student's t-test. ***P < 0.001, ****P < 0.0001.

[0041] Figure 10 This is DC-aPDL1 96 hours after ACT injection in Example 8 of the present invention. Cy5 Results of in vivo targeted binding adoptive T-N3 experiment, where A is the experimental flow chart and B is the Cy5 + Representative flow cytometry diagram of T cell / T-N3 ratio, C is Cy5 in peripheral blood + T cell / T-N3 ratio statistics, D is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3, E is the average fluorescence intensity of Cy5 in tumor + Representative flow cytometry diagram of T cell / T-N3 ratio, F is Cy5 in tumor + T cell / T-N3 ratio statistics, G is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3, H is the average fluorescence intensity of Cy5 in spleen + Representative flow cytometry images of the T cell / T-N3 ratio, I is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3 in the spleen, J is the average fluorescence intensity of Cy5 on the surface of T cells / T-N3 in the lymph nodes + Representative flow cytometry plots of T cell / T-N3 ratios. K represents the mean fluorescence intensity of Cy5 on the surface of T cells / T-N3 in lymph nodes. Data are expressed as mean ± standard deviation (n = 6) and analyzed using a two-tailed unpaired Student's t-test. ****P < 0.0001.

[0042] Figure 11 This is the adoptive T-N3 enhanced DC-aPDL1 in Example 9 of the present invention. Cy5 Figure 1 shows the tumor enrichment results, where A is the experimental flow chart, B is the in vivo IVIS imaging of mice at different time points, C is the total fluorescence intensity of Cy5 in the tumor site at different time points, D is the fluorescence image of the mouse organs 72 hours after intravenous administration, and E is the total fluorescence intensity of Cy5 in the mouse organs 72 hours after intravenous administration, where He is heart, Li is liver, Sp is spleen, Lu is lung, Ki is kidney, Tu is tumor, and LN is lymph node. Data are expressed as mean ± standard deviation and analyzed using One-way ANOVA and Tukey post hoc test. ns indicates no significant difference, **P<0.01, ***P<0.001, ****P<0.0001;

[0043] Figure 12The experimental results of DC-aPDL1 in Example 10 of the present invention to enhance the number and function of adoptive T-N3 cells in the tumor, where A is the experimental flow chart, B is the CD8 + Thy1.1 + T cells account for CD45 + Representative flow cytometry graph of cell ratio, C is intratumoral CD8 + Thy1.1 + Cell number statistics, D is IFN-γ in tumor + Thy1.1 + Flow cytometry representation of the proportion of T cells in ACT, E is IFN-γ in tumor + Thy1.1 + T cell count chart, F is CD8 + Thy1.1 + T cells account for CD45 + Representative flow cytometry graph of cell ratio, G is CD8 in lymph nodes + Thy1.1 + T cells account for CD45 + Cell ratio statistics, H is CD8 in spleen + Thy1.1 + T cells account for CD45 + Representative flow cytometry chart of cell ratio, I is CD8 in spleen + Thy1.1 + T cells account for CD45 + Cell ratio statistics, data are expressed as mean ± SD (n = 6), analyzed using One-way ANOVA and Tukey post hoc test, **P < 0.01, ****P < 0.0001;

[0044] Figure 13 The anti-tumor efficacy of DC-aPDL1 in Example 11 of the present invention is enhanced by adoptive T-N3 therapy, wherein A is an experimental flow chart, B is a tumor growth curve, C is a mouse survival curve, D is a mouse body weight change curve, E is an experimental flow chart of the effect of DC-aPDL1 injected via different routes on the efficacy of ACT, F is a tumor growth curve, G is a mouse survival curve, and H is a mouse body weight change curve. Data are expressed as mean ± standard deviation (n=6). B and F were analyzed using two-way ANOVA and Tukey post hoc test, and C and G were analyzed using the Log-rank test. ns indicates no significant difference. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0046] aPD-L1(10F.9G2 TM ) and aPD-1 (RMP1-14) were purchased from BioXCell, USA. 3 Cross-linking agents were purchased from Thermo Fisher Scientific, Inc., USA. Cytochrome C was purchased from Shanghai Yuanye Biotechnology Co., Ltd., DBCO-PEG4-NHS (DPN) was purchased from BroadPharm, USA. Water-soluble Cy5-NHS was purchased from Shaanxi Xinyan Bomei Biotechnology Co., Ltd.

[0047] B16-OVA and EG7-OVA cells were purchased from ATCC, and B16-OVA-GFP cells were generated in our laboratory. C57BL / 6 mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., and all mice were 6-8 weeks old. OT-1 mice were purchased from Shanghai Model Organisms Science Co., Ltd., and Pmel-1 mice were purchased from the Jackson Laboratory in the United States. All mice were bred and maintained in an SPF animal facility under conditions of 20 ± 2°C humidity, 50 ± 5%, and 12 h of light per day. Mice were acclimated for 3 days before the start of the experiments. All animal experiments were conducted in accordance with the relevant standards for laboratory animal welfare and the Laboratory Animal Ethics Committee of Soochow University.

[0048] Example 1: T cell activation

[0049] Add 5 mL of PBS buffer containing anti-CD3 (1 μg / mL) and anti-CD28 (5 μg / mL) to each well of a 6-well plate and let it stand overnight in a refrigerator at 4°C. Grind the spleen of C57BL / 6 mice on a 70 μm cell strainer, rinse with PBS buffer, and centrifuge at 700 × g for 5 minutes. Add 1 mL of red blood cell lysis buffer to resuspend the pellet, let it stand at room temperature for 3 minutes, add excess PBS buffer to terminate the lysis, and transfer to a 40 μm cell strainer for filtration. Centrifuge at 700 × g for 5 minutes at 4°C and remove the supernatant. Add an appropriate amount of PBS buffer to resuspend the cell pellet and use EasySep TM Mouse CD8 + T cell isolation kit to isolate CD8 + T cells were resuspended in T cell culture medium containing mouse IL-2 (10 ng / mL) and IL-7 (10 ng / mL), transferred to a 6-well plate containing anti-CD3 / CD28 antibodies, and activated in a 37°C incubator for 48 hours. Fresh T cell culture medium was replaced and the cells were expanded for another 2 days before use in subsequent experiments.

[0050] Extract spleen cells from OT-1 / Pmel-1 mice. Resuspend the cells in T cell culture medium, count and adjust the density to 1×10 6 / mL, and added OVA 257-264 peptide or gp100 25-33 (1μM / mL), mouse IL-2 (10ng / mL) and IL-7 (1ng / mL). Place in a 37℃ incubator for activation for 3 days. Add Ficoll-Pague Plus monocyte separation medium for gradient centrifugation and recover CD8 + T cells were expanded for 48 h.

[0051] Example 2: Metabolic labeling of T cells with azidosugar

[0052] (1) Effect of Man-N3 on T cell bioactivity

[0053] The effect of Man-N3 on T cell activity was evaluated by CCK-8 assay. + T cells were counted and expressed as 5 × 10 3 The cells were plated in a 96-well plate at a density of 100 μL / well. A blank group (no cells, no Man-N3), a negative group (with cells, no Man-N3) and experimental groups with different proportions (with cells, 12.5, 25, 50 μM Man-N3 were added, respectively) were set up, with 6 replicates in each group. After incubation for 48 hours, centrifuge at 1200 × g for 5 minutes and discard the supernatant. 100 μL of culture medium containing 10% CCK-8 solution was added to each group and incubated at 37 ° C for 4 hours. The absorbance of the sample at 450 nm was read using an enzyme reader, and the cell survival rate of each group was calculated according to the following formula (1). Among them, OD blank is the absorbance of the blank group; OD control is the absorbance of the negative group; OD test is the absorbance of the experimental group.

[0054]

[0055] When the concentration of Man-N3 was 25 or 50 μM, the expression of azide groups on the T cell surface was slightly higher than that at 12.5 μM ( Figure 2 A, B). Compared with normal cultured T cells, 12.5 and 25 μM Man-N3 concentrations did not significantly affect the activity of T cells ( Figure 2 C), while 50 μM Man-N3 reduced T cell activity. Therefore, 25 μM Man-N3 was finally selected as the concentration for subsequent T-N3 construction.

[0056] (2) Stability of azide expression on T cell surface

[0057] After 48 hours of culture in medium supplemented with 25 μM Man-N3, T-N3 cells were recovered and resuspended in an appropriate amount of PBS buffer. Centrifuge at 700 × g for 5 minutes at 4°C to thoroughly remove the Man-N3-containing medium. Resuspend the cell pellet and add RPMI medium supplemented with murine IL-2 (10 ng / mL) and IL-7 (10 ng / mL) for another 0-5 days.

[0058] a. Flow cytometry: Cells were collected on days 0, 1, 3, and 5, and stained with 20 μM DBCO-Cy5 and analyzed by flow cytometry.

[0059] b. Confocal microscopy: On days 0, 1, 3, and 5, harvest T cells and add the nuclear dye Hoechst 33342. Incubate at 37°C (incubator) for 10 minutes. After staining, remove free dye by centrifugation. Add DBCO-Cy5 (20 μM) and incubate with T-N3 for 30 minutes. Centrifuge at 1200 × g for 5 minutes, resuspend the cells, and fix them for 30 minutes. Count the cells and resuspend them in an appropriate amount of anti-fluorescence quencher. Place 10 μL of the cell suspension on a glass slide, mount the slide, and wait for imaging.

[0060] The flow cytometry results showed that from day 0 to day 5, the azide groups on the T cell surface could be continuously expressed, and the expression intensity on days 1, 3, and 5 was 87%, 56%, and 54% of that on day 0, respectively. The Cy5 fluorescence showed a uniform ring shape on the cell surface, indicating that the azido sugar metabolic labeling strategy can enable the azide groups to be continuously expressed on the T cell surface for more than 5 days ( Figure 2 DF).

[0061] Example 3: D-aPDL1 Cy5 and DC-aPDL1 Cy5 Synthesis and characterization of

[0062] (1)DC-aPDL1 Cy5 Synthesis and characterization of

[0063] The preparation process of DC-aPDL1 is as follows Figure 3 As shown in A. DBCO-PEG4-NHS (DPN) and cytochrome C (CC) were mixed in PBS at a molar ratio of 2:1, 3:1, 5:1, and 10:1, and the pH of the reaction system was adjusted to 8.0. After the reaction was allowed to proceed overnight, the product was recovered and transferred to a 10 kDa ultrafiltration tube for purification. The DBCO-CC in the supernatant of the ultrafiltration tube was resuspended in PBS buffer and immediately subjected to UV spectroscopy ( Figure 3B). Since CC has characteristic absorption peaks at 408 nm and DBCO group at 308 nm, the concentration and molar ratio of DBCO group to CC were obtained by substituting their absorbance values into the standard curve of DPN or CC, respectively. The results showed that the calculated molar ratio showed a good linear regression relationship with the ratio at the time of addition (R 2 =0.9998), confirming the reliability of the concentrations of DBCO and CC in the verification mixture using UV absorption spectroscopy ( Figure 3 C). The formula is shown in the following formula (2):

[0064]

[0065] DPN and CC were reacted overnight at molar ratios of 0:1, 2:1, 3:1, 5:1, and 10:1, and characterized by UV absorption spectroscopy ( Figure 3 D). With the increase of DPN feed amount, the number of DBCO groups connected on CC increased significantly, and 1.2, 2.0, 3.9, and 8.5 DBCO groups could be connected to CC, respectively. Figure 3 E). Although increasing the number of DBCO groups enhances its ability to target the azide group, overreaction of the amino group on CC reduces the efficiency of subsequent DBCO-CC linkage to aPD-L1 via the amino group. Therefore, a reaction molar ratio of 3:1 (DPN:CC) was selected for subsequent product synthesis.

[0066] In order to facilitate quantification, aPD-L1 was first labeled with Cy5 fluorescence to prepare aPD-L1. Cy5 The steps include reacting Cy5-NHS with aPD-L1 at a reaction molar ratio of 3:1. After 24 hours, the product is purified using a 50kDa ultrafiltration tube and the unreacted Cy5-NHS is removed by centrifugation. Subsequently, aPD-L1 Cy5 With BS 3 The cross-linking agents were mixed at a molar ratio of 1:0, 1:10, 1:20, 1:40, and 1:80 for 3 h, and the product was purified using a 50 kDa ultrafiltration tube and centrifuged at 6500 rpm for 7 min to remove free BS. 3 Cross-linking agent to obtain the intermediate NHS-aPD-L1 Cy5 (Process as Figure 3 The product concentration was calculated by UV spectrophotometry.

[0067] NHS-aPD-L1 Cy5 React with DBCO-CC at a molar ratio of 1:20, adjust the solution pH to 8.0, and react on a four-dimensional rotator. After 24 hours of reaction, purify the product using a 50 kDa ultrafiltration tube and centrifuge at 6500 rpm for 7 minutes to remove unreacted DBCO-CC.

[0068] The number of DBCO-CC couplings on aPD-L1 was detected using an ultraviolet spectrophotometer and polyacrylamide gel electrophoresis (SDS-PAGE).

[0069] a. Ultraviolet spectrophotometer: The ultraviolet spectrophotometer detects the absorbance value A647 of Cy5 at 647nm and the absorbance value A647 of CC at 408nm. 408 The molar concentrations of aPD-L1 and CC in the product solution were calculated based on the absorbance values of NHS-Cy5 and CC, respectively. The number of CCs on each aPD-L1 was calculated according to the following formula (3), and the number of DBCO connections on each aPD-L1 was deduced.

[0070]

[0071] b. SDS-PAGE: Dilute DC-aPDL1 to 1 mg / mL and add non-reducing protein loading buffer (5×). Vortex for 10 seconds to mix thoroughly before loading. Prepare a 1.5 mm thick SDS-PAGE gel using the 6% ExpressCast PAGE Color Gel Rapid Kit. In Tris-Glycine running buffer, remove the comb from the upper gel of the gel preparation plate and load 10 μL of sample into each well. Set the voltage to 100 V and run the gel for 90 minutes. After electrophoresis, remove the lower gel and incubate with an appropriate amount of Coomassie Brilliant Blue stain. Place the gel on a shaker at room temperature for 20 minutes. After staining, decolorize with deionized water for 15 minutes, remove the gel, and record the results.

[0072] The UV absorption spectra showed that the number of CCs connected to each aPD-L1 in G1 to G5 were 0, 3.7, 2.6, 1.8, and 1.8, respectively ( Figure 3 F, G), the corresponding calculated numbers of DBCO are 0, 6.9, 4.8, 3.3, and 3.2 ( Figure 3 H) The synthesis of the product was characterized by SDS-PAGE gel electrophoresis. 3 When (G1) aPD-L1 Cy5 Unreacted, molecular weight is about 150kDa ( Figure 3 I). When aPD-L1 Cy5 With BS 3 When the reaction ratio was 1:10 (G2), the DC-aPDL1 Cy5 The molecular weight range is between 150-250 kDa, indicating that DBCO-CC is successfully connected and there is no aPD-L1 Cy5 Antibody cross-linking. 3As the proportion of cross-linking agent increases (G3-G5), the molecular weight of the synthesized product gradually decreases and the connection efficiency decreases. Select the DBCO with the largest number of DBCO connections. 6.9 -CC@aPD-L1 Cy5 Used for subsequent experiments.

[0073] The synthesized DC-aPDL1 was diluted in PBS buffer to a series of concentrations: 1000, 500, 250, 100, 50, and 25 μg / mL, with three groups for each concentration. Five mL of rabbit ear venous blood was collected and 100 μL of sodium heparin was added and mixed. An appropriate amount of PBS buffer was added and the mixture was centrifuged at 10,000 × g for 5 minutes at 4°C. The supernatant was discarded. The solution was rinsed 4-5 times and the erythrocyte pellet was resuspended in PBS buffer to 50 mL. The erythrocyte solution was incubated with the product at a volume ratio of 1:4 and allowed to stand at room temperature for 4 hours. The PBS group was designated as the negative control (Abs1), and the deionized water group was designated as the positive control (Abs2). After the incubation, the mixture was centrifuged at 10,000 × g for 5 minutes at 4°C and photographed. 100 μL of supernatant was transferred to a 96-well plate. The reference wavelength of the microplate reader was set to 655 nm, and the absorbance Abs3 at 577 nm was measured. The hemolysis rate was calculated according to the following formula (4).

[0074]

[0075] The biosafety of DC-aPDL1 was evaluated by hemolysis assay. Figure 3 J It can be seen that when the highest concentration of DC-aPDL1 is 1000 μg / mL, its hemolysis rate is only 1.9±0.2%, indicating that the product has good biosafety.

[0076] (2)D-aPDL1 Cy5 Synthesis and characterization of

[0077] According to DPN and aPD-L1 Cy5 DPN was added in a molar ratio of 3:1, 6:1, 12:1, 24:1, and the pH of the reaction system was adjusted to 8.0. The reaction was carried out on a four-dimensional rotating instrument ( Figure 3 K). After 24 h, the product was purified using a 50 kDa ultrafiltration tube to obtain D-aPDL1 Cy5 Add appropriate amount of PBS buffer to adjust the product concentration, place it in the sample cell of UV spectrophotometer and detect the absorbance value A at 647nm 647 and absorbance A at 308 nm 308 According to different concentrations of NHS-Cy5 (A 647 ) and DPN(A 308) were used to standardize the absorbance values of the samples to calculate the concentrations of DBCO and aPD-L1 in the samples, and the number of DBCO on each aPD-L1 was calculated according to the following formula (5).

[0078]

[0079] When the reaction molar ratio is 3:1, 6:1, 12:1, and 24:1, each aPD-L1 can be connected to an average of 2.3, 4.4, 7.8, and 15.0 DBCO groups.

[0080] Example 4: Ability of DC-aPDL1 to specifically bind to T-N3 and tumor cells

[0081] (1)DC-aPDL1 Cy5 Specific targeting and binding to T-N3

[0082] The product D-aPDL1 was prepared according to the method of Example 3 Cy5 and DC-aPDL1 Cy5 The specific groups are as follows: G1: PBS; G2: DBCO 2.3 -aPD-L1 Cy5 ;G3:DBCO 4.4 -aPD-L1 Cy5 ;G4:DBCO 7.8 -aPD-L1 Cy5 ;G5:DBCO 15 -aPD-L1 Cy5 ;G6:DBCO 6.9 -CC@aPD-L1 Cy5 , G7:aPD-L1 Cy5 OT-1CD8 was obtained according to the method of Example 1. + T cells and T-N3 were fluorescently labeled with CellTrace CFSE. T-N3 without fluorescent labeling was mixed with T cells labeled with CFSE at a ratio of 1:1, and 5 μg of D-aPDL1 was added to the cell suspension. Cy5 or DC-aPDL1 Cy5 After co-incubation, unbound aPD-L1 was washed away and flow cytometry was performed (process as shown in Figure 4 (as shown in A).

[0083] The results of G1 to G5 groups showed that as D-aPDL1 Cy5 The number of DBCO groups attached to each aPD-L1 increases, and Cy5 + The proportion of T-N3 in T-N3 gradually increased ( Figure 4 B, C). Among them, DBCO 15-aPD-L1 Cy5 It can bind to 86.8% of T-N3 cells, while CFSE + The Cy5-positive ratio of T cells (without azide labeling) was only 18.6%, indicating that D-aPDL1 Cy5 The targeting ability to T-N3 gradually increases with the increase of DBCO amount. 6.9 -CC@aPD-L1 Cy5 In (G6), each aPD-L1 has only 6.9 DBCO groups attached, but Cy5 + The proportion of T-N3 reached 75.5% of T-N3 cells, second only to the DBCO in the G5 group with 15 DBCOs. 15 -aPD-L1 Cy5 , and was significantly higher than the G4 group (45.4%) with 7.8 DBCOs. At the same time, the Cy5 positive rate on T cells caused by nonspecific binding was <5.0%, which fully proved that DC-aPDL1 Cy5 Can target and bind T-N3 more efficiently.

[0084] CFSE-labeled T cells and T-N3 were transferred to 96-well plates, and different concentrations of DC-aPDL1 were added. Cy5 (10, 15, 30 μg / 10 6 T cells / T-N3) were co-cultured and analyzed by flow cytometry. Cy5 Able to efficiently combine with T-N3 ( Figure 4 D, E). With the increase of DC-aPDL1 Cy5 With the increase of the dose, the Cy5 fluorescence on T-N3 gradually increased, while the Cy5 fluorescence on the surface of T cells without azide modification did not change significantly, indicating that DC-aPDL1 Cy5 It binds specifically and dose-dependently to T-N3, but shows very low background binding to non-azide-modified T cells.

[0085] (2)DC-aPDL1 Cy5 Specific targeting of tumor cells

[0086] The expression of PD-L1 on the surface of normal cultured tumor cells in vitro is low, while IFN-γ can kill tumor cells and induce them to produce defense mechanisms. 0, 10, 20, and 30 ng / mL of IFN-γ were added to the culture system of B16-F10 cells (mouse skin melanoma cells) and incubated for 24 hours before flow cytometry detection. Compared with normal cultured B16-F10 cells, the expression of PD-L1 on the surface of B16-F10 cells induced by different concentrations of IFN-γ was significantly increased ( Figure 4 FH).

[0087] To evaluate DC-aPDL1 Cy5 The ability to bind to tumor cells, the synthetic product DBCO 2.3 -aPD-L1 Cy5 、DBCO 4.4 -aPD-L1 Cy5 、DBCO 7.8 -aPD-L1 Cy5 、DBCO 15 -aPD-L1 Cy5 、DBCO 6.9 -CC@aPD-L1 Cy5 and aPD-L1 Cy5 Co-incubated with B16-F10 cells at 1×10 6 125 ng of the synthetic product was added to each B16-F10 cell, and the cells were fixed after incubation for 30 minutes and then waited for flow cytometry detection.

[0088] The results of G2 to G5 groups showed that as D-aPDL1 Cy5 As the number of DBCO groups directly modified on aPD-L1 increased, the Cy5 fluorescence on B16-F10 cells gradually decreased ( Figure 4 I, J), when the number of DBCO connections is 7.8 (G4 group), compared with unmodified aPD-L1 Cy5 Compared with the G7 group, the Cy5 MFI of B16-F10 cells decreased to 71.6% of the G7 group. When the number of DBCOs was further increased to 15 (G5 group), the Cy5 MFI of B16-F10 cells decreased to 61.8% of the G7 group. This result indicates that direct DBCO conjugation affects the activity of aPD-L1, resulting in a decrease in its ability to bind to tumor cells. In the G6 group, the number of DBCO connections was 6.9 and the Cy5 MFI was 1.3. + The Cy5 MFI of B16-F10 cells was retained at 88.8%, which not only confirmed that DC-aPDL1 Cy5 It has excellent binding ability to tumor cells and also shows that indirect modification of the DBCO group on aPD-L1 through CC can effectively maintain the antibody activity.

[0089] To investigate DC-aPDL1 Cy5 To investigate the dose-dependency of the ability to bind to tumor cells, B16-F10 cells and B16-F10 cells pretreated with IFN-γ were cultured in vitro. B16-F10 cells that highly expressed PD-L1 after induction with 20 ng / mL IFN-γ and normal tumor cells that were not induced were transferred to 96-well plates. After 24 hours, 0, 31.25, 62.5, and 125 ng of DC-aPDL1 were added, respectively.Cy5 , incubate at room temperature in the dark for 30 min, then fix the cells and wait for flow cytometry detection. Figure 4 As shown in K, at the same dose, compared with the B16-F10 cells cultured normally, the Cy5 fluorescence value of B16-F10 cells induced by IFN-γ was significantly enhanced, indicating that DC-aPDL1 Cy5 The binding rate to B16-F10 cells was significantly increased. In addition, this binding showed a dose-dependent trend. Cy5 In comparison, DC-aPDL1 Cy5 There was no significant difference in the cell surface Cy5 fluorescence value after binding to B16-F10 cells, further proving that the modification strategy did not significantly affect the activity of the antibody and maintained the tumor cell targeting function.

[0090] Example 5: DC-aPDL1 Cy5 Increase the tumor-killing efficiency of T cells

[0091] Due to DC-aPDL1 Cy5 It can target and bind to the surface of T-N3 and specifically recognize and bind to PD-L1 and DC-aPDL1 on the surface of tumor cells. Cy5 To investigate the role of DC-aPDL1 Cy5 To investigate the ability of CFSE fluorescently labeled OT1 T-N3 and tumor cells, CFSE fluorescently labeled B16-F10 cells were mixed at a ratio of 20:1, and 5, 10, and 20 μg / 10 6 Incubate cells with three concentration gradients of DC-aPDL1 from T to N3 for 30 min at room temperature in the dark. Figure 5 (as shown in A).

[0092] When the number of T-N3 and B16-F10 cells remained unchanged, the DC-aPDL1 Cy5 With increasing doses, Cy5 + Violet + The proportion of double-positive cell combinations showed a significant upward trend ( Figure 5 B) When DC-aPDL1 Cy5 The dose is 5μg / 10 6 cells increased to 20 μg / 10 6 cells, Cy5 in B16-F10 cells + Violet + The proportion of cell conjugates increased from 22.7% to 44.9%, indicating that DC-aPDL1 Cy5Can effectively couple T-N3 and tumor cells ( Figure 5 C).

[0093] Take B16-OVA-GFP cells (mouse melanoma transfected with chicken ovalbumin OVA and green fluorescent protein GFP) in good growth state and adjust the cell density to 1×10 per well. 4 The cells were evenly plated in a flat-bottom 96-well plate and placed in a 37°C incubator. When the cells grew to a density of 70%, the supernatant was discarded and OT-1CD8 + T cells or T-N3 cells were incubated with aPD-L1 and DC-aPDL1 in a 37°C incubator for 24 hours. The groups were as follows: G1: T cells; G2: T-N3 cells; G3: T-N3 cells + aPD-L1 cells; and G4: T-N3 cells + DC-aPDL1 cells. After 24 hours, the supernatant from the original 96-well plate was slowly aspirated and transferred to a round-bottom 96-well plate. The suspension was centrifuged at 1200 × g for 5 minutes to remove excess cells, and the supernatant was transferred to another 96-well plate. IFN-γ secretion by T cells was assayed according to the ELISA kit protocol. After incubation, the supernatant was discarded. The remaining B16-OVA-GFP cells in the 96-well plate were lysed by adding 50 μL of RIPA lysis buffer. The GFP protein was excited at 480 nm and emitted at 530 nm, and fluorescence was measured using a microplate reader. The cell fluorescence value of the group with E / T of 0 was 100%, and the tumor cell survival rate (%) was calculated, that is, the killing rate = 1-cell survival rate (%).

[0094] The statistical curve of T cell killing rate showed that there was no significant difference in tumor killing rate between G1 group and G2 group; however, after adding aPD-L1, the tumor killing effect of T cells was improved ( Figure 5 D). Compared with the G3 group, the G4 group increased the tumor cell killing rate by 1.3, 1.4, 1.4, and 1.2 times at E / T ratios of 2.5:1, 5:1, 10:1, and 20:1, respectively, verifying that DC-aPDL1 can significantly enhance the killing ability of T-N3 cells against tumor cells. In addition, at all E / T (T:B16-OVA-GFP) ratios, there was no significant statistical difference in the IFN-γ secretion between T cells (G1 group) and T-N3 (G2 group), indicating that Man-N3's sugar metabolism labeling of T cells does not interfere with their normal killing function ( Figure 5E). In contrast, when T-N3 was combined with aPD-L1 (G3 group), IFN-γ secretion increased significantly. Furthermore, when T-N3 was combined with DC-aPDL1 (G4 group), IFN-γ levels increased by more than 1.3 times compared to the G1, G2, and G3 groups, further demonstrating that DC-aPDL1 can significantly enhance T-N3's ability to kill tumor cells.

[0095] Example 6: ACT enhances the expression of PD-L1 on the surface of DCs in vivo

[0096] To investigate the expression of PD-L1 on dendritic cells (DCs) in vivo, mice were subcutaneously inoculated with 1×10 6 B16-OVA cells. Three days after tumor implantation, CD8 + T cells. When the tumor grows to 100mm 3 At approximately 100 mg / kg, mice were weighed and intraperitoneally injected with 20 mg / mL cyclophosphamide solution. Mice were randomly divided into the following groups: G1: Healthy; G2: Healthy, ACT; G3: Tumor-bearing; G4: Tumor-bearing, ACT. 24 hours after lymphodepletion (LD), mice in groups G2 and G4 were injected intravenously with 5×10 6 OT-1CD8 + T cells. 48 hours after the mice were infused with T cells, the tumor, spleen, and bilateral inguinal lymph nodes of each mouse were removed, weighed, and ground. The cells were recovered into a 96-well plate and stained and analyzed by flow cytometry (process as shown in the following example). Figure 6 (as shown in A).

[0097] In B16-OVA tumor-bearing mice, OT1 CD8 + After T cells (G4 group), the expression of PD-L1 on the surface of cDC1 and cDC2 in lymph nodes and spleen increased to a certain extent, and was more significant in tumor tissues ( Figure 6 BE). Specifically, the expression of PD-L1 on the cDC1 and cDC2 surfaces in the G4 group was 3.0-fold and 2.0-fold higher, respectively, compared to the G3 group. This result suggests that PD-L1 expression on DC surfaces is closely associated with ACT cells.

[0098] 1×10 6 B16-F10 cells. T cell extraction and lymphocyte lysis were performed as above. The random groups were as follows: G1: Tumor-bearing; G2: Tumor-bearing, OT-1ACT;

[0099] G3: Tumor-bearing, Pmel-1 ACT. 24 hours after the washout, mice in groups G2 and G4 were injected with 5×10 6 OT-1 or Pmel-1 CD8 + T cells. 48 hours after T cell infusion, bilateral inguinal lymph nodes, spleen, and tumor were collected, weighed, and ground for staining and flow cytometry analysis (process as shown in the following example). Figure 6 F).

[0100] Compared with untreated B16-F10 tumor-bearing mice (G1 group), mice bearing tumors and injected with Pmel-1CD8 + The expression of PD-L1 on the surface of cDC1 and cDC2 in the lymph nodes and spleen of mice with T cells (G3 group) was increased, and the expression of PD-L1 on the surface of cDC1 and cDC2 in tumor tissue increased by 2.9 times and 1.9 times, respectively ( Figure 6 GJ), which fully demonstrated that the expression of PD-L1 on the surface of DCs could be increased after injection of tumor-specific T cells into tumor-bearing mice. + Compared with the G1 group, the expression of PD-L1 on the surface of cDC2 in the lymph nodes, spleen and tumor did not change significantly. The expression of PD-L1 on the surface of cDC1 in the lymph nodes increased by only 10.5%, and increased by 54.2% in the tumor. These results indicate that after the injection of T cells that can specifically recognize tumor antigens into tumor-bearing mice, the expression of PD-L1 on the surface of DC increased significantly. This phenomenon may be through Pmel-1 CD8 + T cells to B16-F10 cells (or OT-1CD8 + The specific recognition of B16-OVA cells by T cells leads to the activation of T cells by tumor cells and the secretion of large amounts of IFN-γ, thereby inducing DCs to increase PD-L1 expression.

[0101] Example 7: DC-aPDL1 enhances the interaction between T cells and bone marrow-derived dendritic cells

[0102] Femurs and tibias were removed from C57BL / 6 mice, and the epiphyses were cut open at both ends. The bone marrow cavity was flushed with PBS buffer, and the cell suspension was recovered. After centrifugation at 350×g at 4°C for 5 minutes, 1 mL of ACK red blood cell lysis buffer was added, and the cells were allowed to lyse for 3 minutes. The reaction was terminated by adding an appropriate amount of PBS. After filtration through a cell strainer, the cells were centrifuged at 350×g at 4°C for 5 minutes, and the supernatant was discarded. RPMI 1640 complete medium containing recombinant mouse GM-CSF at a concentration of 20 ng / mL was added, and the cells were transferred to a non-TC-treated (tissue culture-treated) cell culture flask and cultured in a 37°C incubator. Three days later, the flask was supplemented with an equal volume of RPMI 1640 complete medium containing 20 ng / mL GM-CSF and cultured at 37°C. On the seventh day, the suspended cells in the flask were recovered for subsequent experiments.

[0103] BMDC (bone marrow-derived dendritic cells) were divided into a normal culture group and an IFN-γ (20 ng / mL) induced culture group (pre-treated 24 h in advance). aPD-L1 was synthesized according to the experimental method in Example 3. Cy5 and DC-aPDL1 Cy5 The treated BMDCs were transferred to 96-well plates and different concentrations of aPD-L1 were added. Cy5 and DC-aPDL1 Cy5 (31.25, 62.5, 125, 250, 500ng / 10 6 BMDCs) were co-incubated for 30 minutes and fixed for flow cytometry analysis. Compared with untreated BMDCs (G1 group), the expression of PD-L1 on the surface of BMDCs increased significantly after IFN-γ (G2 group) and CpG (G3 group) stimulation for 24 hours ( Figure 7 A, B).

[0104] To investigate DC-aPDL1 Cy5 To determine whether it can effectively bind to the surface of BMDCs, BMDCs were pretreated with IFN-γ for 24 h and then incubated with different doses of aPD-L1 Cy5 and DC-aPDL1 Cy5 Co-incubation, flow cytometry was used to detect the Cy5 fluorescence level of BMDC. Compared with the normal cultured BMDC (G1 group and G2 group), the surface Cy5 fluorescence of BMDC (G3 group and G4 group) after IFN-γ pretreatment was significantly enhanced and very close to ( Figure 7 C, D). This result not only verifies that IFN-γ can enhance the expression of PD-L1 on the surface of BMDC, but also proves that DC-aPDL1 Cy5 Able to interact with aPD-L1 Cy5 It also efficiently binds to PD-L1 expressed on BMDCs.

[0105] T-N3 was labeled with CFSE, and DC2.4 (mouse dendritic cell line) was labeled with CellTrace Violet dye. T-N3:DC2.4 was mixed at a ratio of 5:1. 5, 10, and 20 μg / 10 were added to the mixed cell suspension. 6 DC-aPDL1 was incubated at three concentration gradients of T-N3 for 30 min at room temperature in the dark. After cell fixation, the cells were analyzed by flow cytometry (process as shown in Figure 7 E). With the increase of the dose of DC-aPDL1, CFSE + Violet + The proportion of double-positive cells showed an upward trend. In the G2, G3, and G4 groups, CFSE + Violet + The cells accounted for 34.3%, 41.0% and 45.9% of the total DC2.4, respectively, indicating that DC-aPDL1 could simultaneously bind to T-N3 and DC2.4, effectively forming cell coupling bodies ( Figure 7 F, G).

[0106] In activated OT-1CD8 + OVA was added to T-N3 according to the group 257-264 (50 μg / mL) (ovalbumin class I restricted epitope peptide), CpG (2 μg / mL), aPD-L1 (10 μg / mL) 6 T-N3), DC-aPDL1 (10 μg / 10 6 T-N3), BMDC (number of T cells is 10:1). Specific groups are as follows: G1: CpG+OVA 257-264 ; G2: BMDC+CpG+OVA 257-264 ; G3: BMDC+CpG+OVA 257-264 +aPD-L1;G4:BMDC+CpG+OVA 257-264 After incubation at 37°C for 48 h, the cell supernatant was collected and the secretion of IFN-γ in the supernatant was detected according to the ELISA kit instructions. The cells were then recovered and stained for flow cytometry analysis.

[0107] CD69 is an activation marker on the surface of T cells, and its expression level is positively correlated with the activation state of T cells. Compared with the group without BMDC (G1 group), BMDC in the G2 group processed the antigen peptide OVA under the promotion of CpG. 257-264 , significantly improving T cell activation, and the expression of CD69 on the surface of T-N3 increased by 1.7 times ( Figure 7H). Compared with the G2 group, when aPD-L1 was added to the culture conditions of the G3 group, the expression of CD69 on the surface of T-N3 was only slightly increased. However, in the G4 group, DC-aPDL1 significantly increased the expression of CD69 on the surface of T-N3, which was 1.3 times higher than that of the G2 group, further enhancing the activation of T-N3. In addition, compared with the G2 group, the IFN-γ secretion of T-N3 in the G3 and G4 groups increased by 1.2 and 1.5 times, respectively, demonstrating that DC-aPDL1 can more strongly promote T-N3 activation ( Figure 7 I).

[0108] Example 8: DC-aPDL1 Cy5 Investigation of the ability of T-N3 to be adopted for targeted transfer in vivo

[0109] (1) Injection of DC-aPDL1 2 h after injection of ACT cells Cy5

[0110] Pmel-1 CD8 was obtained according to the method of Example 1 + T or T-N3. The mice were weighed one day in advance and injected intraperitoneally with cyclophosphamide at a dose of 100 mg / kg. They were then randomly divided into groups of 6 mice each, as follows: G1: PBS; G2: T+DC-aPDL1 Cy5 ;G3:T-N3+DC-aPDL1 Cy5 24 hours after the injection of cyclophosphamide, 5×10 6 Pmel-1 CD8 + T cells or T-N3, 2 h later, each mouse in the G2 and G3 groups was injected with 50 μg DC-aPDL1 into the tail vein Cy5 After 48 hours, peripheral blood, bilateral inguinal lymph nodes and spleen of each mouse were collected for staining and flow cytometry analysis (process as shown in Figure 8 (as shown in A).

[0111] In peripheral blood, DC-aPDL1 Cy5 The targeting of adoptive T-N3 is significantly stronger than that of endogenous CD8 + T cells, Cy5 + The average proportion of T-N3 cells was as high as 87.9%, while the endogenous CD8 + The Cy5 positive ratio of T cells was only 0.6%, and the average Cy5 fluorescence value of adoptive T-N3 was 1.5477 / 1.3347 / 1.37 ... + 6.6 times of T ( Figure 8 BD). However, only 0.8% of the non-azide-labeled adoptive T cells showed Cy5 fluorescence. The average Cy5 fluorescence value on the adoptive T-N3 in the G3 group was 4.3 times that of the non-azide-labeled adoptive T cells (G2 group) ( Figure 8 BD).

[0112] In lymph nodes, Cy5 + The average proportion of adoptive T-N3 was 69.2%, while the endogenous CD8 + The average Cy5 fluorescence value of adoptive T-N3 is also the same as that of endogenous CD8 + T cells, which is 5.5 times higher than the fluorescence value of non-azide labeled adoptive T cells (3.5 times Figure 8 EG). In the spleen, endogenous CD8 + Cy5 on T cells + The ratio was 0.6%, while DC-aPDL1 Cy5 The average target binding rate on adoptive T-N3 reached 81.5%, and the average Cy5 fluorescence value was the same as that of endogenous CD8 + The T cells were 9.6 times higher than those in the G2 group and 5.0 times higher than those in the T-N3 group ( Figure 8 HJ). These results showed that DC-aPDL1 Cy5 It can specifically target adoptive T-N3 in vivo, but not endogenous CD8 + The binding ability of T cells to non-azide-labeled T cells was weak.

[0113] (2) Injection of DC-aPDL1 48 hours after ACT cell injection Cy5

[0114] To further validate DC-aPDL1 Cy5 To investigate the in vivo targeting of T-N3 at different time points after T cell injection, we injected Pmel-1 CD8 + After 48 hours, T cells / T-N3 were intravenously injected with PBS, aPD-L1 Cy5 or DC-aPDL1 Cy5 After 2 days, peripheral blood, lymph nodes, spleen and tumor of mice were collected, ground and stained and analyzed by flow cytometry (process as shown in Figure 9 (As shown in A). In the peripheral blood and tumors of mice, DC-PDL1 Cy5 It can efficiently target and bind to the surface of adoptive T-N3. In the G4 group, DC-PDL1 in peripheral blood Cy5 The average binding rate with adoptive T-N3 was 79.6%, which was significantly higher than that of endogenous CD8 + T cells, and the average fluorescence value of Cy5 on adoptive T-N3 is endogenous CD8 + In addition, the aPD-L1 Cy5 with adoptive T-N3 or endogenous CD8 + T cells only have background binding, and in the G3 group, adoptive T cells and endogenous CD8 +The Cy5 positive ratio on T cells also showed a basically consistent trend ( Figure 9 BD). In tumors, DC-aPDL1 Cy5 The average targeting ratio of adoptive T-N3 reached 76.0%, which was significantly higher than that of Cy5. + Endogenous CD8 + The ratio of T cells; and the Cy5 fluorescence value on adoptive T-N3 is the endogenous CD8 + 4.7 times of T cells ( Figure 9 EF). In addition, adoptive T cells / T-N3 or endogenous CD8 + The average Cy5 ratio on T cells was below 0.9%, indicating that aPD-L1 Cy5 Adoptive T-N3 and DC-aPDL1 Cy5 Similarly, in the G4 group, Cy5-positive endogenous CD8 + The proportion of T cells is much lower than that of adoptive T-N3 in the same group. Among them, the average fluorescence value of Cy5 on adoptive T-N3 in the spleen is the endogenous CD8 + 11.6 times of T cells ( Figure 9 H, I). The average fluorescence value of Cy5 on adoptive T-N3 in lymphocytes is the value of endogenous CD8 + In G2 and G3 groups, aPD-L1 Cy5 Adoptive transfer of T-N3 or DC-aPDL1 Cy5 Almost no specific binding ability to adoptive T cells ( Figure 9 J, K). Therefore, after adoptive T-N3 proliferation in vivo for 48 hours, DC-aPDL1 Cy5 It can still efficiently target and bind to T-N3.

[0115] (3) Injection of DC-aPDL1 96 hours after ACT cell injection Cy5

[0116] Then, we administered aPD-L1 intravenously 96 hours after adoptive transfer to T-N3. Cy5 or DC-aPDL1 Cy5 ( Figure 10 A) DC-aPDL1 in peripheral blood and tumors of mice adoptively transferred to T-N3 Cy5 The average binding rates were 91.7% and 69.0%, respectively, and their endogenous CD8 + The binding rate on T cells was low; and the average fluorescence values of Cy5 on peripheral blood and adoptive T-N3 in tumor were +The results of G2 and G3 groups showed that aPD-L1 was 8.5 times and 7.9 times higher than that of T cells. Cy5 Unable to effectively bind to adoptive T-N3 and endogenous CD8 + T cell surface and DC-aPDL1 Cy5 Adoptive T cells and endogenous CD8 + T cells only have nonspecific binding ( Figure 10 BG). In the spleen and lymph nodes, DC-aPD-L1 Cy5 The targeting ability of adoptive T-N3 was basically the same. The data of G4 group showed that endogenous CD8 + Cy5 in T + The cell ratio is much lower than that of the adoptive T-N3 in the same group. The average fluorescence value of Cy5 on the adoptive T-N3 in the spleen is the endogenous CD8 + 17.1 times of T cells ( Figure 10 H, I). In lymph nodes, the average fluorescence value of Cy5 on adoptive T-N3 cells was significantly higher than that on endogenous CD8 + T cells ( Figure 10 J, K). In G2 and G3 groups, DC-aPDL1 Cy5 with adoptive T cells or endogenous CD8 + T cells have only a certain background binding rate, and aPD-L1 Cy5 It does not specifically bind to adoptive T-N3 and endogenous CD8 + T cells.

[0117] These results indicate that although azide expression on the surface of adoptively transferred T-N3 cells gradually decreased over time in vivo, it did not significantly affect the expression of DC-aPDL1. Cy5 The specific binding ability of DC-aPDL1 to adoptive T-N3 was detected 4 days after T-N3 was injected into the body. Cy5 It can still target and bind to these T-N3s.

[0118] Example 9: Adoptive T-N3 Enhancement of DC-aPDL1 Cy5 Enriched in tumors

[0119] DC-aPDL1 Cy5 After targeted binding to adoptive T-N3, it can "hitch a ride" on T-N3 in vivo, thereby changing DC-aPDL1 Cy5 Biodistribution in vivo. C57BL / 6 mice were subcutaneously inoculated with 1.5×10 6 EG7-OVA cells. When the tumor grows to 150mm 3 At 4 hr, the mice were weighed and intraperitoneally injected with 100 mg / kg of cyclophosphamide. The mice were randomly divided into the following groups: G1: PBS; G2: aPD-L1Cy5 ;G3:T+DC-aPDL1 Cy5 ;G4:T-N3+DC-aPDL1 Cy5 After 24 hours, 5×10 6 OT-1CD8 + T cells or T-N3, and 2 h later, each mouse in the G2-G4 groups was intravenously injected with 50 μg aPD-L1 Cy5 or DC-aPDL1 Cy5 IVIS imaging was performed at 2, 9, 24, 48, and 72 hours after administration. After 72 hours, the mice were euthanized and the heart, liver, spleen, lung, kidney, and tumor were removed for in vitro imaging (process as shown in the following example). Figure 11 (as shown in A).

[0120] Due to DC-aPDL1 Cy5 It cannot efficiently bind to ACT cells without azide labeling, so there is no statistical difference in the tumor Cy5 fluorescence intensity between the G3 group and the G2 group at different time points ( Figure 11 B, C) In the G4 group, when T-N3 and DC-aPDL1 were adopted Cy5 After combined use, the Cy5 fluorescence value in mouse tumors was significantly enhanced and reached a peak fluorescence intensity at 48 hours. At 24 hours after administration, the Cy5 fluorescence value of the tumors in the G4 group of mice reached 2 times that of the G2 and G3 groups, and increased to 3 times at 48 hours, and still maintained a high fluorescence intensity at 72 hours. The results showed that DC-aPDL1 Cy5 It can specifically carry adoptive T-N3 in vivo and prolong aPD-L1 Cy5 Longer retention time in the body and efficient accumulation in tumors. Figure 11 As shown in D and E, there was no significant difference in Cy5 fluorescence values in the spleen, lymph nodes and tumors of the G2 and G3 groups, while the Cy5 fluorescence values in the spleen, tumor and lymph nodes of the G4 group were significantly increased, indicating that DC-aPDL1 Cy5 Through specific binding to adoptive T-N3 in vivo, it is enriched in tumors and secondary lymphoid organs.

[0121] Example 10: In vivo immune effects of DC-aPDL1 combined with adoptive T-N3

[0122] C57BL / 6 mice were subcutaneously inoculated with 1.2 × 10 6 B16-F10 tumor cells were injected intraperitoneally with cyclophosphamide (100 mg / kg) when the tumors grew to 150 mm3. The mice were randomly divided into the following groups: G1: PBS; G2: T-N3; G3: T-N3 + aPD-L1; G4: T-N3 + DC-aPDL1. 24 hours later, 5×106 Pmel-1 CD8 + T-N3, 2 hours later, each mouse in the G3-G4 group was injected with 50 μg DC-aPDL1 via the tail vein. 48 hours later, the bilateral inguinal lymph nodes, spleen, and tumor of each mouse were collected for staining and flow cytometry analysis (procedure as shown in the following). Figure 12 (as shown in A).

[0123] Compared with T-N3 alone (G2 group), aPD-L1 (G3 group) increased the number of adoptive T-N3 per mg of tumor by 2.5 times ( Figure 12 B, C). DC-aPDL1 (G4 group) further increased the number of adoptive T-N3 per mg of tumor, which was increased by 13.6 times and 5.5 times compared with G2 and G3 groups, respectively. In addition, the proportion of adoptive T-N3 in the total CD45 + The proportion of cells increased significantly ( Figure 12 B, C). Compared with G2 and G3 groups, the combination of aPD-L1 and DC-aPDL1 in G4 group significantly increased IFN-γ per mg of tumor. + The number of T-N3 was 55.5 times and 5.7 times that of G2 and G3 groups, respectively ( Figure 12 D, E). This result shows that DC-aPDL1 combined with adoptive T-N3 therapy can significantly increase the number of ACT cells in the tumor and maintain its efficacy. Similar trends were also shown in the lymph nodes and spleen. Compared with the G2 group and the G3 group, DC-aPDL1 can significantly increase the proportion of adoptive T-N3 in the lymph nodes to 3.7 times and 1.8 times ( Figure 12 F, G). In the spleen, DC-aPDL1 was able to increase the proportion of adoptive T-N3 to 2.3%, reaching 2.9 times and 2.1 times of the G2 and G3 groups ( Figure 12 H, I).

[0124] Example 11: Anti-tumor effect of DC-aPDL1 combined with adoptive T-N3

[0125] The backs of C57BL / 6 mice were shaved and each mouse was subcutaneously inoculated with 8×10 5 B16-F10 tumor cells were grown. When the tumors grew to approximately 50 mm3, the mice were weighed and intraperitoneally injected with 100 mg / kg of cyclophosphamide. The mice were randomly divided into the following groups: G1: PBS; G2: T-N3; G3: T-N3 + aPD-L1; G4: T-N3 + aPD-1; G5: T-N3 + DC-aPDL1. One day after lympholysis, 5×10 cells were injected into the tail vein of each mouse in groups G2-G4. 6 Pmel-1 CD8 +T or T-N3 cells, and 2 hours later, 50 μg of aPD-L1, aPD-1, or DC-aPDL1 was injected into the tail vein of each mouse in the G3-G4 group. Six days later, 50 μg of immune checkpoint inhibitors were injected into each mouse in the G3-G4 group again. The tumor growth and body weight changes of each group of mice were monitored every 2 days, and the survival time of each group of mice was recorded. When the experimental period ended or the tumor volume of the mouse exceeded 1500 mm 3 When the mice were euthanized (procedure as shown in Figure 13 (as shown in A).

[0126] The tumor volume change curve of mice showed that adoptive T-N3 (G2 group) could delay tumor growth. The application of aPD-L1 in G3 group and aPD-1 in G4 group enhanced the effect of adoptive T-N3. Combining DC-aPDL1 and ACT cells could significantly inhibit tumor growth ( Figure 13 B) In all mice, the tumor volume did not exceed 1500 mm 3 On the 14th day, the G2 group was able to inhibit 27.8% of tumor growth; when combined with aPD-L1 or aPD-1, the tumor growth inhibition rate increased to 48.8% and 60%, respectively. In the G4 group, DC-aPDL1 combined with adoptive T-N3 delayed 92.6% of tumor growth. The G2, G3, and G4 groups were able to prolong the survival of mice to a certain extent, but the DC-aPDL1 combined with adoptive T-N3 therapy in the G5 group significantly prolonged the survival of mice ( Figure 13 C). During the treatment period, there was no significant change in the body weight of mice in each group ( Figure 13 D), indicating that these therapies had no obvious toxicity to mice and had good safety.

[0127] The effect of DC-aPDL1 injection route on the therapeutic effect was further compared. The specific groups were as follows: G1: PBS; G2: T-N3; G3: T-N3 + aPD-L1 (iv); G4: T-N3 + aPD-L1 (sc); G5: T-N3 + DC-aPDL1 (iv); G6: T-N3 + DC-PDL1 (sc). 24 hours after the urethral cleansing, 5×10 6 Pmel-1 CD8 + T cells or T-N3, 2 hours later, the antibody drug was injected. Three days later, each mouse in the G3-G6 group was injected again with 50 μg aPD-L1 or DC-aPDL1 in the same way. During the experiment, the tumor growth and weight changes of each group of mice were monitored every 2 days, and the survival time of each group of mice was recorded. When the experimental period ended or the tumor volume of the mouse exceeded 1500 mm 3 When the mice were euthanized (procedure as shown in Figure 13 E).

[0128] Compared with the subcutaneous injection of DC-aPDL1 in the G6 group, the intravenous injection of DC-aPDL1 (G5 group) can further enhance the tumor suppression ability of adoptive T-N3, and the overall tumor volume growth level of mice is lower than that of the G6 group ( Figure 13 F). On day 16, intravenous injection of DC-aPDL1 combined with adoptive T-N3 (G5) delayed tumor growth by 87.0%, while the G6 group only inhibited tumor growth by 79.3%. The survival curve further confirmed the advantage of intravenous administration ( Figure 13 G). These results indicate that intravenous administration of DC-aPDL1 is superior to subcutaneous administration. During this period, there was no significant change in the body weight of the mice ( Figure 13 H), indicating that the therapy has good biosafety in mice and no obvious toxicity.

[0129] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing a combined drug delivery system, characterized in that: The combined drug delivery system consists of a T cell adapter DC-aPDL1 and adoptive T cells T-N3, and the preparation method comprises the following steps: (1) using dibenzocyclooctyne-polyethylene glycol-N-hydroxysuccinimide to modify cytochrome C to obtain DBCO-CC, and using di(sulfosuccinimide) suberate as a crosslinker to link the DBCO-CC and the anti-programmed death ligand 1 antibody aPD-L1 to obtain DC-aPDL1; (2) The adoptive T cells were metabolically labeled with azido sugars to obtain adoptive T cells T-N3 expressing azido groups on their surface.

2. The preparation method according to claim 1, wherein: The molar ratio of the dibenzocyclooctyne-polyethylene glycol-N-hydroxysuccinimide to cytochrome C is (1-11):

1.

3. The preparation method according to claim 1, wherein: The molar ratio of aPD-L1, di(sulfosuccinimide) suberate crosslinker and DBCO-CC is 1:(10-80):(18-22).

4. The preparation method according to claim 1, wherein: The azido sugar is tetraacylated N-azidoacetylaminomannose, tetraacetyl-N-azidoacetylglucose or tetraacylated N-azidoacetylaminogalactose.

5. The combined drug delivery system prepared by the method according to any one of claims 1 to 4.

6. The combined drug delivery system according to claim 5, characterized in that: The number of DBCOs bound to the aPD-L1 is 0-9, and the number of cytochrome Cs bound to the aPD-L1 is 0-4.

7. Use of the combined drug delivery system according to claim 5 in the preparation of tumor therapeutic drugs.

8. The use according to claim 7, characterized in that: The ratio of T cell adapter DBCO-CC@aPD-L1 and adoptive T cell T-N3 in the combined drug delivery system is (10-15): 10 6 (μg: piece).

9. The use according to claim 7, characterized in that: The tumor treatment drug is administered by subcutaneous injection or intravenous injection.

10. The use according to claim 7, characterized in that: The administration time of the T cell engager DC-aPDL1 is 0-100 hours after the administration of adoptive T cells T-N3.

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