Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.
METHODS. Two different pipelines of neoantigen identification were established in this study: (a) Clinical-grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were synthesized de novo. (b) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in 6 patients. RESULTS. Immunogenic neoepitopes were recognized by autologous T cells in 3 of 4 patients who used the de novo synthesis mode and in 6 of 13 patients who used the shared neoantigen peptide library. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining 4 patients achieved prolonged stabilization of disease with a median progression-free survival of 8.6 months. CONCLUSION. The current study provides feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate neoantigen-based translational immunotherapy research.
Human tumors, including gastric cancer, frequently express high levels of epidermal growth factor receptors (EGFRs), which are associated with a poor prognosis. Targeted delivery of anticancer drugs to cancerous tissues shows potential in sparing unaffected tissues. However, it has been a major challenge for drug penetration in solid tumor tissues due to the complicated tumor microenvironment. We have constructed a recombinant protein named anti-EGFR-iRGD consisting of an anti-EGFR VHH (the variable domain from the heavy chain of the antibody) fused to iRGD, a tumor-specific binding peptide with high permeability. Anti-EGFR-iRGD, which targets EGFR and αvβ3, spreads extensively throughout both the multicellular spheroids and the tumor mass. The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines, multicellular spheroids, and mice. Moreover, anti-EGFR-iRGD could improve anticancer drugs, such as doxorubicin (DOX), bevacizumab, nanoparticle permeability and efficacy in multicellular spheroids. This study draws attention to the importance of iRGD peptide in the therapeutic approach of anti-EGFR-iRGD. As a consequence, anti-EGFR-iRGD could be a drug candidate for cancer treatment and a useful adjunct of other anticancer drugs.
Breast cancer susceptibility gene 1 (BRCA1) has a central role in chemotherapy-induced DNA damage response. The protein inhibitor of activated STAT (PIAS) family of proteins, PIAS1 and PIAS4, are also necessary for adequate DNA damage repair. To further understand the role of BRCA1 in DNA repair, we examined the mRNA expression of these genes in 133 advanced (stage III-IV) gastric cancer patients using quantitative reverse transcription polymerase chain reaction. All P values were two-sided. The median overall survival was 12.5 months (95% confidence interval [CI] = 9.8 to 13.4 months). Among 59 patients receiving second-line docetaxel, the median overall survival was 25.8 months (95% CI = 9.2 to 42.4 months) for patients with high BRCA1 expression, 19.1 months (95% CI = 3.4 to 34.8 months) for those with intermediate expression, and 9.5 months (95% CI = 8.7 to 10.2 months) for those with low expression (P = .0062). The risk of mortality was higher in patients with low BRCA1 levels compared with high BRCA1 levels (hazard ratio of death = 2.49, 95% CI = 1.03 to 5.97, P = .037). Survival in patients receiving second-line docetaxel-based chemotherapy showed a similar trend with PIAS1 and PIAS4 mRNA expression levels, although the associations for PIAS4 were not statistically significant.
The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC. These studies reveal possibility of generating PD-1-disrupted CTL by CRISPR-Cas9 system and demonstrate enhanced immune response of these PD-1-disrupted CTLs to the EBV-LMP2A antigen and superior cytotoxicity to the EBV-positive gastric cancer cell. In addition, when combined with low-dose radiotherapy, these PD-1-disrupted CTLs mediated an impressive antitumor effect in a xenograft mouse model of EBVaGC. Taken together, these studies illustrate PD-1/PD-L1-mediated immune tolerance of EBVaGC and provide a new strategy for targeting immune checkpoints to break the tolerance for the T cell-based adoptive therapy.
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