Periodic Reporting for period 1 - THAT IS HUNT (Triggering Haematological Adoptive T-cell Immunotherapy Strategies by HUnting Novel T-cellreceptors)
Reporting period: 2018-01-01 to 2019-12-31
Concerning the first aim, we have taken advantage of multi-parametric flow cytometry to immunoprofile and longitudinally characterize the frequency and functional phenotype of tumor-specific T cells in patients after HSCT. T cells from peripheral blood (PB) and bone marrow (BM) were analyzed in terms of expression of activation and exhaustion markers, T cell memory phenotype and for reactivity against known tumor peptides. Results of our study highlighted the existence of a profound exhaustion signature associated with tumor-specific T cells both in BM as well as in PB samples, with the expression of multiple inhibitory receptors.
We identified 8 TCRs specific for 5 epitopes belonging to 4 tumor antigens and restricted to 2 HLA alleles highly frequent in the Caucasian population.
Coming to the second aim, we focused on the identification of tumor epitopes, and tumor-specific T cells, by employing different experimental strategies.
• Starting from peripheral blood mononuclear cells of healthy donors (HDs) we designed and implemented an innovative protocol for the rapid isolation of tumor-specific T cells and for the characterization of a library of tumor-specific TCRs restricted to different HLA alleles. We selected as target antigens Cyclin A1 and Cathepsin G (CG), 2 proteins overexpressed in AML and relevant also for additional tumor entities. We achieved successful expansion of tumor-specific T cells from 15 consecutive HDs. TCR αβ sequencing led to the identification of 10 tumor TCRs specific for 10 epitopes presented by 6 HLA haplotypes.
• Starting from patient material, we enlarged our study to the identification of TCRs directed towards novel peptides of tumor antigens, independently of their HLA restriction. We exploited the exhaustion phenotype of tumor-specific T cells in AML patients and the possibility to instruct AML blasts to become potent leukemic antigen presenting cells. We identified 3 tumor-specific TCRs.
To identify novel leukemia epitopes, we employed a ligandome-based strategy.
Additionally, by interrogating the great amount of TCR sequences retrieved in our study, we sought for common molecular features. We have observed the existence of specific biases in the anti-tumor immune repertoire: the preferential usage of defined variable genes in the rearrangement process occurring during the generation of the TCRs and the presence of public clones generated mainly by convergent recombination events.
Overall, we have generated a library of 21 TCRs able to recognize 6 different tumor antigens (for a total of 15 peptides) and restricted to 7 HLA alleles, including the most frequent ones in the Caucasian population. These TCRs can be now exploited in the TCR gene editing strategy for the treatment of leukemia.
The results of this study have been presented at several national and international meetings.
Mostly relevant for the immunotherapy field, we have identified novel immunogenic tumor epitopes and consequently novel TCRs restricted to a variety of HLA alleles highly frequent in the Caucasian population. Furthermore, the comprehensive characterization of tumor-reactive T cell features and the establishment of a variety of approaches for the enrichment of tumor-specific T cells will streamline TCR hunting strategies in other tumor settings, including solid tumors. Altogether, these results will foster TCR-based strategies in the immunotherapy arena. Establishment of T cell-based approaches as a standard-of-care treatment would also have an economic relevance. In fact, it would reduce the financial expenses and resources required for cancer therapy by moving from the one-pill a day for a life-time paradigm, to the concept of a living drug which provides long-term protection thanks to the ability of T cell to persist long term in the patients and patrol for tumor recurrence.