A novel cooperative therapeutic regimen using gene fusion neoantigen-specific TCR-engineered T cells mediated sustained preclinical efficacy in solid tumors independent on inherent mutation profiling
Neoantigens derived from non-synonymous somatic mutations, especially gene fusion mutation, are restricted to tumor cells and thus considered ideal targets for T-cell receptor-engineering T-cells (TCR-T) immunotherapy. Adoptive transfer of neoantigen-specific TCR-T cells preferentially exhibit potent cytotoxicity preferentially to tumor cells, and has shown promising efficacy in several preclinical human cancer models. However, most neoantigens are unique to individual patient, which hinders the application of TCR-T cell therapy. In this study, we first discovered a paired ?/? TCR repertoire, Tcr-1, that specifically targeted STY-SSX fusion neoantigen shared by almost all synovial sarcomas. Engineered T-cell expressing Tcr-1 (Tcr-T1) exhibited antigen-specific HLA-A*2402-restricted antitumoral activity against synovial sarcomas cell both in vitro and in vivo. Furthermore, to furtherly extend its application beyond synovial sarcomas, we innovatively developed a cooperative therapeutic modality, in which exogenous SYT-SSX fusion neoantigen was loaded into enzyme-responsive nanoparticles (NPs) formed by mPEG-PVGLIG-PCL copolymers (FNeo-AgNPs) for tumor targeting delivery. As expected, FNeo-AgNPs were proven of great tumor penetration, and local release. In situ modification was able to direct engineered Tcr-T1 against to other malignant gastric cancer cell line with significant antigen-specific HLA-A*2402-restricted cytotoxicity resulting in remarkable tumor regression and prolonging survival in both subcutaneous and peritoneally disseminated preclinical models, despite of their inherent mutation profiles. With these favorable data, our established cooperative therapeutic modality has a great potential for further clinical investigation, and provide a now insight for future TCR-T cell therapy development. Overall design: Preparation of single-cell suspensions: Neoantigen responsive T-cell clones were harvest and CD8+ T cells were furtherly isolated through magnetic beads (Miltenyi Biotec, CD8+ T cell Isolation Kit, Cat no. 130-096-495). Purified CD8+ T cells were subjected to single-cell analysis for TCR sequencing as reported.  Droplet-based single-cell sequencing: Briefly, using a Single Cell 5' Library and Gel Bead Kit (10X Genomics, Cat no. 1000006) and Chromium Single A Chip Kit (10X Genomics, Cat no. 120236), the single cell suspension (300-600 living cells/ml) was loaded onto a Chromium single cell controller (10X Genomics) to generate single-cell gel beads in the emulsion (GEMs) according to the manufacture's protocol. Single cells were suspended in PBS containing 0.04% BSA. Approximately 10,000 cells were added to each channel and approximately 6,000 target cells were recovered. Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53oC for 45 min, followed by 85oC for 5 min and a hold at 4oC. Complementary DNA was generated and amplified, after which, quality was assessed using an Agilent 4200 (performed by CapitalBio Technology). According to the manufacturer's introduction, scRNA-seq libraries were constructed using a Single Cell 5' Library and Gel Bead kit, Human T Cell (Cat no. 1000005). The libraries were sequenced using an Illumina Novaseq6000 sequencer with a paired-end 150-bp (PE150) reading strategy (performed by CapitalBio Technology). TCR sequencing: Full-length TCR V(D)J segments were enriched from amplified cDNA from 5' libraries via PCR amplification using a Chromium Single-Cell V(D)J Enrichment kit according to the manufacture's protocol (10X Genomics). For TCR, cells with at least one productive TCR ?-chain (TRA) and on productive TCR ?-chain (TRB) were kept for further analysis.