Contributions
Abstract: EP719
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Aggressive blood malignancies can benefit from the immune pressure imposed by allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT), able to eradicate cancer cells surviving chemo-radiotherapy. However, allo-HSCT may also mediate allo-reactions with potentially fatal outcomes. In order to boost HTSC efficacy while reducing toxicities, adoptive T cell therapy with genetically engineered T cells redirected against tumor antigens (ACT) is an appealing therapeutic candidate. However, ACT applicability in blood tumors is limited by the low number of known tumor-reactive T-Cell Receptors (TCRs).
Aims
We defined an experimental pipeline to isolate tumor-specific TCRs from allo-HSCT treated patients to widen the TCR repertoire for ACT by taking advantage of either multimers or inhibitory receptors surface markers expression.
Methods
Circulating Tumor-specific T cells in blood cancer patients who underwent allo-HSCT were phenotypically characterized, then purified and in vitro expanded by means of multimers directed toward immunodominant peptides of MAGE-A2, WT1, Survivin and hTERT tumor-associated antigens, restricted for HLA-A*0201 and HLA-A*2402 alleles. Tumor-Specific T cell TCR repertoire was studied by RNA sequencing.
Results
We successfully isolated and expanded markedly oligoclonal tumor-specific T-cell cultures from N=13/20 patients. Despite documented in vitro enrichment and expansion, cell cultures failed to lyse peptide-pulsed target cell lines during in vitro assays, suggesting that T cells were hypo-functional. To circumvent hypo-functionality, the dominant TCR from N=6 of those T-cell cultures was reconstructed and transferred into fit donor T cells which endogenous TCR have been previously knocked-down by means of CRISPR/Cas9 system. Newly engineered T cells proved able to specifically recognise and eliminate peptide-pulsed cell lines with different affinities in in vitro killing assays. A high-dimensional flow cytometry analysis performed on N=33 patients strengthened the hypothesis that tumor-specific T cells are hypo-functional ex vivo. Indeed, tumor-specific but not viral-specific T cells preferentially expressed multiple inhibitory receptors and showed an impaired differentiation program, with accumulation of stem cell-like memory T cells and terminal effectors. Interestingly, such phenotype was accentuated in those patients deemed to relapse after allo-HSCT. In order to take advantage such unique exhaustion signature, we exploited inhibitory receptors to segregate from three Acute Myeloid Leukemia patients a T cell fraction positive for multiple inhibitory receptors (IR+) and putatively enriched with tumor specificities. We then serially stimulated with autologous Leukemic-Antigen Presenting Cells (L-APCs) the IR+ and the control IR- fraction. In all the three tested patients, L-APCs promoted the occurrence of a dominant TCR clonotype in the IR+ but not in the IR- group. These dominant TCRs, when inserted into TCR-knocked-down donor T cells led to the generation of engineered T cells that efficiently lysed autologous leukemia in vitro while sparing healthy bone marrow cells.
Conclusion
Our data suggest the presence of circulating, though exhausted, tumor-specific T cells in patients after allo-HSCT. By exploiting multimers or the IR signature, we here provide a pipeline to isolate novel tumor-specific TCRs with different specificity, affinity and peptide sensitivity to be employed in ACT.
Keyword(s): Adoptive immunotherapy, Gene transfer, Leukemia
Abstract: EP719
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Aggressive blood malignancies can benefit from the immune pressure imposed by allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT), able to eradicate cancer cells surviving chemo-radiotherapy. However, allo-HSCT may also mediate allo-reactions with potentially fatal outcomes. In order to boost HTSC efficacy while reducing toxicities, adoptive T cell therapy with genetically engineered T cells redirected against tumor antigens (ACT) is an appealing therapeutic candidate. However, ACT applicability in blood tumors is limited by the low number of known tumor-reactive T-Cell Receptors (TCRs).
Aims
We defined an experimental pipeline to isolate tumor-specific TCRs from allo-HSCT treated patients to widen the TCR repertoire for ACT by taking advantage of either multimers or inhibitory receptors surface markers expression.
Methods
Circulating Tumor-specific T cells in blood cancer patients who underwent allo-HSCT were phenotypically characterized, then purified and in vitro expanded by means of multimers directed toward immunodominant peptides of MAGE-A2, WT1, Survivin and hTERT tumor-associated antigens, restricted for HLA-A*0201 and HLA-A*2402 alleles. Tumor-Specific T cell TCR repertoire was studied by RNA sequencing.
Results
We successfully isolated and expanded markedly oligoclonal tumor-specific T-cell cultures from N=13/20 patients. Despite documented in vitro enrichment and expansion, cell cultures failed to lyse peptide-pulsed target cell lines during in vitro assays, suggesting that T cells were hypo-functional. To circumvent hypo-functionality, the dominant TCR from N=6 of those T-cell cultures was reconstructed and transferred into fit donor T cells which endogenous TCR have been previously knocked-down by means of CRISPR/Cas9 system. Newly engineered T cells proved able to specifically recognise and eliminate peptide-pulsed cell lines with different affinities in in vitro killing assays. A high-dimensional flow cytometry analysis performed on N=33 patients strengthened the hypothesis that tumor-specific T cells are hypo-functional ex vivo. Indeed, tumor-specific but not viral-specific T cells preferentially expressed multiple inhibitory receptors and showed an impaired differentiation program, with accumulation of stem cell-like memory T cells and terminal effectors. Interestingly, such phenotype was accentuated in those patients deemed to relapse after allo-HSCT. In order to take advantage such unique exhaustion signature, we exploited inhibitory receptors to segregate from three Acute Myeloid Leukemia patients a T cell fraction positive for multiple inhibitory receptors (IR+) and putatively enriched with tumor specificities. We then serially stimulated with autologous Leukemic-Antigen Presenting Cells (L-APCs) the IR+ and the control IR- fraction. In all the three tested patients, L-APCs promoted the occurrence of a dominant TCR clonotype in the IR+ but not in the IR- group. These dominant TCRs, when inserted into TCR-knocked-down donor T cells led to the generation of engineered T cells that efficiently lysed autologous leukemia in vitro while sparing healthy bone marrow cells.
Conclusion
Our data suggest the presence of circulating, though exhausted, tumor-specific T cells in patients after allo-HSCT. By exploiting multimers or the IR signature, we here provide a pipeline to isolate novel tumor-specific TCRs with different specificity, affinity and peptide sensitivity to be employed in ACT.
Keyword(s): Adoptive immunotherapy, Gene transfer, Leukemia