T Cells Directed against the Metastatic Driver Chondromodulin-1 in Ewing Sarcoma: Comparative Engineering with CRISPR/Cas9 vs. Retroviral Gene Transfer for Adoptive Transfer.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.

Immunogenic T cell epitopes of SARS-CoV-2 are recognized by circulating memory and naïve CD8 T cells of unexposed individuals.

BACKGROUND: Recent studies have provided evidence of T cell reactivity to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in significant numbers of non-infected individuals, which has been attributed to cross-reactive CD4 memory T cells from previous exposure to seasonal coronaviruses. Less evidence of cross-reactive memory CD8 T cells has been documented to date. METHODS: We used the NetCTLPan neural network of the Epitope Database and Analysis Resource to select a series of 27 HLA-A*02:01 epitopes derived from the proteome of SARS-CoV-2. Their binding capacity was assessed by a HLA-A*02:01 stabilization assay and by quantifying their binding to HLA-A*02:01 monomers for the generation of tetramers. Their ability to stimulate and induce expansion of SARS-CoV-2 reactive CD8 T cells was measured by flow cytometry. The TCR repertoire of COVID convalescent and healthy unexposed donors was analysed using the MIRA database. FINDINGS: The HLA-A*02:01 epitopes tested were able to stabilise HLA molecules and induce activation of CD8 T cells of healthy unexposed donors. Our results, based on specific tetramer binding, provide evidence supporting the presence of frequent cross-reactive CD8 T cells to SARS-CoV-2 antigens in non-exposed individuals. Interestingly, the reactive cells were distributed into naïve, memory and effector subsets. INTERPRETATION: Our data are consistent with a significant proportion of the reactive CD8 T clones belonging to the public shared repertoire, readily available in absence of previous contact with closely related coronaviruses. Furthermore, we demonstrate the immunogenic capacity of long peptides carrying T cell epitopes, which can serve to isolate virus-specific T cell receptors among the ample repertoire of healthy unexposed subjects and could have application in COVID-19 immunotherapy. Limitations of our study are that it concentrated on one MHC I allele (HLA-A*02:01) and the low numbers of samples and epitopes tested. FUNDING: See the Acknowledgements section.