Contributions
Abstract: S252
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
Adoptive immunotherapy with T lymphocytes genetically engineered to overexpress a tumor-specific T cell receptor represents a novel immunotherapeutic option for cancer patients. However, the lack of long-term persistence of infused tumor-reactive T cells and the immunosuppressive tumor microenvironment represent major drawbacks of ACT. Specifically, chronically stimulated tumor-specific lymphocytes become exhausted, a dysfunctional status characterized by loss of effector functions. Exhausted T cells overexpress several inhibitory receptors (IRs), such as PD-1, CTLA-4, Lag-3, Tim-3 and 2B4. The use of monoclonal antibodies targeting IRs (e.g. anti-CTLA-4 and anti-PD-1), can revert T cell exhaustion but proved effective only in a fraction of patients. Also, autoimmune-related adverse events often occur after immune checkpoint blockade, mainly due to the unleashing of autoreactive clones.
Aims
we aim to generate innovative cell therapy products, endowed with long-term persisting capacity and resistance to inhibitory signals and to investigate how the disruption of selected IRs could modulate the resistance to exhaustion in multiple myeloma models.
Methods
CRISPR/Cas9 reagents were used to simultaneously disrupt TIM-3, LAG-3 or 2B4 gene and the TCR alpha chain constant region (TRAC) gene. The frequency of NHEJ was assessed with ddPCR. TRAC-KO-IR-KO T cells were transduced with a lentiviral vector encoding for an HLA-A2-restricted NY-ESO-1-specific TCR. T cells were activated and kept in culture with a protocol to expand long-living early differentiated stem cell memory (TSCM) and central memory (TCM) cells (Cieri et al., Blood 2013). TCR-edited-IR-KO cells and TCR-edited-IR-competent (TCR-edited-IRCOMP) cells were challenged in vitro and in vivo with HLA-A2posNY-ESO-1pos multiple myeloma cell lines. HLA-A2neg NYESO-1neg cell lines were used as control.
Results
We efficiently inactivated the endogenous TCR (>98% of NHEJ) and more than 80% of gene disruption was also obtained at LAG-3, Tim-3 or 2B4 locus. TCR disrupted-IR-KO cells were efficiently transduced (> 50%) with the NY-ESO1157-165-specific TCR, thus obtaining TCR-edited-IR-KO T cells or TCR-edited-IRCOMPT cells. Importantly, TCR-edited-IR-KO T cells retain their expansion capacity and an early differentiated TSCM/TCM phenotype.
Upon chronic stimulation with multiple myeloma cells, TCR-edited-Tim-3-KO and TCR-edited-2B4-KO T cells retained a higher degranulation capacity compared to TCR-edited-LAG-3 KO and TCR-edited-IRCOMP T cells, while TCR-edited-LAG-3-KO T cells showed a limited upregulation of additional inhibitory receptors.
In a preclinical model of aggressive multiple myeloma, TCR-edited-Tim3-KO and TCR-edited-2B4-KO T cells caused higher immunological pressure and were enriched in a higher proportion of highly activated T cells compared to TCR-edited-IRCOMP-treated mice. In a similar pre-clinical multiple myeloma model, TCR-edited-LAG-3-KO, but not TCR-edited-IRCOMP, T cells showed a reduced expression of additional IRs and prevented the engraftment of the second infusion of tumor cells, thus indicating an enhanced T cell fitness and resistance to exhaustion of TCR-edited-LAG-3-KO T cells.
Conclusion
our results show that by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to different inhibitory signals.
Keyword(s): Gene therapy, Immunotherapy
Abstract: S252
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
Adoptive immunotherapy with T lymphocytes genetically engineered to overexpress a tumor-specific T cell receptor represents a novel immunotherapeutic option for cancer patients. However, the lack of long-term persistence of infused tumor-reactive T cells and the immunosuppressive tumor microenvironment represent major drawbacks of ACT. Specifically, chronically stimulated tumor-specific lymphocytes become exhausted, a dysfunctional status characterized by loss of effector functions. Exhausted T cells overexpress several inhibitory receptors (IRs), such as PD-1, CTLA-4, Lag-3, Tim-3 and 2B4. The use of monoclonal antibodies targeting IRs (e.g. anti-CTLA-4 and anti-PD-1), can revert T cell exhaustion but proved effective only in a fraction of patients. Also, autoimmune-related adverse events often occur after immune checkpoint blockade, mainly due to the unleashing of autoreactive clones.
Aims
we aim to generate innovative cell therapy products, endowed with long-term persisting capacity and resistance to inhibitory signals and to investigate how the disruption of selected IRs could modulate the resistance to exhaustion in multiple myeloma models.
Methods
CRISPR/Cas9 reagents were used to simultaneously disrupt TIM-3, LAG-3 or 2B4 gene and the TCR alpha chain constant region (TRAC) gene. The frequency of NHEJ was assessed with ddPCR. TRAC-KO-IR-KO T cells were transduced with a lentiviral vector encoding for an HLA-A2-restricted NY-ESO-1-specific TCR. T cells were activated and kept in culture with a protocol to expand long-living early differentiated stem cell memory (TSCM) and central memory (TCM) cells (Cieri et al., Blood 2013). TCR-edited-IR-KO cells and TCR-edited-IR-competent (TCR-edited-IRCOMP) cells were challenged in vitro and in vivo with HLA-A2posNY-ESO-1pos multiple myeloma cell lines. HLA-A2neg NYESO-1neg cell lines were used as control.
Results
We efficiently inactivated the endogenous TCR (>98% of NHEJ) and more than 80% of gene disruption was also obtained at LAG-3, Tim-3 or 2B4 locus. TCR disrupted-IR-KO cells were efficiently transduced (> 50%) with the NY-ESO1157-165-specific TCR, thus obtaining TCR-edited-IR-KO T cells or TCR-edited-IRCOMPT cells. Importantly, TCR-edited-IR-KO T cells retain their expansion capacity and an early differentiated TSCM/TCM phenotype.
Upon chronic stimulation with multiple myeloma cells, TCR-edited-Tim-3-KO and TCR-edited-2B4-KO T cells retained a higher degranulation capacity compared to TCR-edited-LAG-3 KO and TCR-edited-IRCOMP T cells, while TCR-edited-LAG-3-KO T cells showed a limited upregulation of additional inhibitory receptors.
In a preclinical model of aggressive multiple myeloma, TCR-edited-Tim3-KO and TCR-edited-2B4-KO T cells caused higher immunological pressure and were enriched in a higher proportion of highly activated T cells compared to TCR-edited-IRCOMP-treated mice. In a similar pre-clinical multiple myeloma model, TCR-edited-LAG-3-KO, but not TCR-edited-IRCOMP, T cells showed a reduced expression of additional IRs and prevented the engraftment of the second infusion of tumor cells, thus indicating an enhanced T cell fitness and resistance to exhaustion of TCR-edited-LAG-3-KO T cells.
Conclusion
our results show that by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to different inhibitory signals.
Keyword(s): Gene therapy, Immunotherapy