In situ antigen modification-based target-redirected universal chimeric antigen receptor T (TRUE CAR-T) cell therapy in solid tumors

Abstract

Background: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable success in the treatment of hematologic malignancies, while the success has not yet been replicated in solid tumors. To some extent, the disappointing results can be attributed to the paucity and heterogeneity of target antigens in solid tumors since adequate antigens are the cornerstone for CAR-T cells to recognize and attack tumor cells.

Methods: We established a target-redirected universal CAR-T (TRUE CAR-T) cell therapeutic modality, in which exogenous antigens are loaded onto fusogenic nanoparticles to achieve in situ modification of cell membrane in solid tumors, providing targets for subsequent CAR-T cell therapy. The modification effect was evaluated by flow cytometry and confocal microscopic imaging. The in vivo metabolism and biodistribution of fusogenic antigen loaded nanoparticles (F-AgNPs) was explored using near infrared living imaging. Then F-AgNPs mediated in situ antigen modification were cooperated with corresponding CAR-T cell therapy, and its antitumor efficacy was evaluated using immune function experiments and further investigated in different tumor models.

Results: Using F-AgNPs, exogenous antigens were selectively modified onto tumor cell membranes through membrane fusion, spread deeper into tumor tissues through intercellular lipid transfer, further activating corresponding CAR-T cells and mediating antitumor immune responses towards multiple types of tumor cells, despite of their inherent antigen profiles. The cooperative treatment of F-AgNPs and CAR-T cell therapy successfully suppressed tumor proliferation and prolonged survival in both subcutaneous and peritoneally disseminated tumor models.

Conclusion: The fusogenic nanoparticle-based in situ antigen modification overcome the limitation of target antigens paucity and heterogeneity in solid tumors, improving the efficacy and broadening the applications of CAR-T cells, thus establishing a novel TRUE CAR-T cell therapeutic modality with universal application and translational potential in immunotherapies for solid tumors.

Keywords: Antigen modification; Cancer mmunotherapies; Fusogenic nanoparticles; Solid tumors; Universal CAR-T cells.

Fig. 1

The construction of F-AgNPs and the proposed immunotherapeutic mechanism of TRUE- CAR-T therapy. a Schematic diagram of the synthesis of lipid-conjugated antigen peptides, DSPE-PEG-EvIII and the subsequent construction of F-AgNPs. b The schematic illustration demonstrating F-AgNPs modified exogenous antigens onto tumor cell membrane via membrane fusion. c In situ antigen modification of tumor cell membrane provides adequate targets for CAR-T cell recognition, enabling CAR-T cell to exert antitumor effects independent of tumor inherent antigen profiles (immunotherapeutic mechanism of TRUE- CAR-T cell therapy). d Particle size distribution of F-AgNPs. e Surface charge distribution of F-AgNPs. f The TEM image of F-AgNPs constructs, showing hollow near spherical structure of liposome. Imaged using JEOL 1200 EX TEM. Negative staining by 2% phosphotungstic acid. Scale bar represents 100 nm

Fig. 2

F-AgNPs mediated cell membrane antigen modification and transfer. a Schematic diagram of F-AgNPs mediated antigen modification onto tumor cells, and flow cytometry histograms of tumor cells without treatment (grey), incubated with FAM-F-AgNPs (green) or incubated with F-AgNPs followed by detection by PE-anti-EvIII mAb (red). b Localization of antigen peptides modified by F-AgNPs on tumor cells MGC803 (upper) and MKN45 (lower). Nucleus: DAPI (blue); antigen peptide: FAM (green); plasma membrane: DiI (red). Scale bar represents 50 μm. c Analysis of the dosage-effect relationship of F-AgNPs mediated antigen modification on tumor cells. d Flow cytometric analysis of stability of F-AgNPs mediated antigen modification over the culture periods. Data represent mean ± s.e.m., n = 3. e, f Representative images (e) and quantitative analysis (f) of F-AgNPs mediated antigen modification efficiency on gastric cancer cells, MKN45 and MGC803, and normal cells, GES-1, NIH-3T3, HUVEC and HMrV5 cells. Nucleus: DAPI (blue); antigen peptide: FAM (green). Scale bar represents 100 μm. Data are represented as mean ± s.e.m., n = 3. A one-way ANOVA was used for statistical analysis. ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001. g Confocal images of HGC27 spheroids to assess penetration capacity of DiI delivered by NF-NPs, P-NPs or F-AgNPs. Scale bars, 250 μm

Fig. 3

F-AgNPs mediated antigen modification cooperates with CAR-T cell therapy in vitro. a Schematic illustration of action mode of TRUE-CAR-T therapy and evaluation of in vitro antitumor response. b IFN-γ secretion of EvIII CAR-T cells after 24 h incubation with tumor cells (MKN45) treated by F-AgNPs of various concentration. Data are represented as mean ± s.e.m., n = 3. Student’s t test was used for statistical analysis. ns not significant; ns not significant; *p < 0.05; **p < 0.01. c Th1- and Th2-type cytokines secretion of EvIII CAR-T cells after 24 h incubation with F-AgNPs treated tumor cells. Data represent mean ± s.e.m., n = 4–5. Student’s t test was used for statistical analysis. ns not significant; *p < 0.05; **p < 0.01. d The expression of CD137 and CD69 on CAR negative- and positive-subpopulation after 24 h incubation with F-AgNPs treated tumor cells. Data represent mean ± s.e.m., n = 3–4. Student’s t test was used for statistical analysis. e Evaluating cytotoxicity of EvIII CAR-T cells and control T cells (mock transfection) towards F-AgNPs treated tumor cells (MKN45) and exploring the dose–effect relationships using CFSE/PI assay. E:T = 10:1. Data are represented as mean ± s.e.m., n = 3. Student’s t test was used for statistical analysis. ns not significant; **p < 0.01; ***p < 0.001. f Representative confocal images of HGC27 spheroids untreated, treated with F-AgNPs, CAR-T cells and F-AgNPs + CAR-T cells. E:T = 20:1 Dead cells:EthD-1 (red); live cells: calcein AM (green). Scale bar, 250 μm. Data represent mean ± s.e.m., n = 3. A one-way ANOVA was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Biodistribution and tumor targeting of F-AgNPs in vivo. a, b The dynamics of DiR signals over time within tumor tissue after systemically delivering DiR-labeled F-AgNPs. Data are represented as mean ± s.e.m. n = 3. Student’s t test was used for statistical analysis. ns not significant. c Evaluation of in vivo antigen modification effects through observing the fluorescence images of MKN45 subcutaneous tumors sections after intravenous injection of F-AgNPs or P-NPs (FAM-labeled). Tumors were collected 2 h, 8 h and 24 h after delivery. Nucleus (DAPI, blue); antigen peptide (FAM, green); vessal (Cy5, red). Scale bar, 100 μm. d, e The dynamics of DiR signals over time in tumor and organs after intraperitoneal injection of DiR-labeled F-AgNPs. Data are represented as mean ± s.e.m. n = 4. Student’s t test was used for statistical analysis. ns not significant; *p < 0.05

Fig. 5

F-AgNPs mediated antigen modification cooperates with CAR-T cell therapy in vivo. a Schematic illustration of treatment process in subcutaneous tumor model. b, c Enhanced antitumor effect of F-AgNPs mediated antigen modification cooperative with CAR-T cell therapy in a subcutaneous MKN45 tumor model. Tumor growth profiles (b) and survival curve (c) of mice treated with PBS, F-AgNPs, EvIII CAR-T, F-AgNPs + EvIII CAR-T respectively. Tumor volume were analyzed with one-way ANOVA. Data are represented as mean ± s.e.m. n = 6. Survival curves were analyzed with log-rank test. n = 6. *p < 0.05; **p < 0.01; ***p < 0.001. d Schematic illustration of treatment process in peritoneal metastasis tumor model. e–g Enhanced antitumor effect of F-AgNPs mediated antigen modification cooperative with CAR-T cell therapy in a disseminated peritoneal MGC803 tumor model. Tumor growth profiles (e, f) and survival curve (g) of mice treated with PBS, F-AgNPs, EvIII CAR-T, F-AgNP s + EvIII CAR-T respectively. Data are represented as mean ± s.e.m.., n = 6. A one-way ANOVA was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001