SNF5/INI1 Deficiency Redefines Chromatin Remodeling Complex Composition during Tumor Development

Malignant Rhabdoid Tumors (MRTs), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, re-expression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 re-expression indicated the recruitment of different components into the complex along with the expulsion of others. IP-Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth promoting SWI/SNF complexes to differentiation inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of malignant rhabdoid tumors

Efficacy and Safety of Autologous Tumor-Infiltrating Lymphocytes in Recurrent or Refractory Ovarian Cancer, Colorectal Cancer, and Pancreatic Ductal Adenocarcinoma

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy has shown efficacy in metastatic melanoma, non-small cell lung cancer, and other solid tumors. Our preclinical work demonstrated more robust CD8 predominant TIL production when agonistic anti-4-1BB and CD3 antibodies were used in early ex vivo TIL culture. METHODS: Patients with treatment-refractory metastatic colorectal (CRC), pancreatic (PDAC) and ovarian (OVCA) cancers were eligible. Lymphodepleting chemotherapy was followed by infusion of ex vivo expanded TIL, manufactured at MD Anderson Cancer Center with IL-2 and agonistic stimulation of CD3 and 4-1BB (urelumab). Patients received up to six doses of high-dose IL-2 after TIL infusion. Primary endpoint was evaluation of objective response rate at 12 weeks using Response Evaluation Criteria in Solid Tumors version 1.1 with secondary endpoints including disease control rate (DCR), duration of response, progression-free survival (PFS), overall survival (OS), and safety. RESULTS: 17 patients underwent TIL harvest and 16 were treated on protocol (NCT03610490), including 8 CRC, 5 PDAC, and 3 OVCA patients. Median age was 57.5 (range 33-70) and 50% were females. Median number of lines of prior therapy was 2 (range 1-8). No responses were observed at 12 weeks. Ten subjects achieved at least one stable disease (SD) assessment for a DCR of 62.5% (95% CI 35.4% to 84.8%). Best response included prolonged SD in a patient with PDAC lasting 17 months. Median PFS and OS across cohorts were 2.53 months (95% CI 1.54 to 4.11) and 18.86 months (95% CI 4.86 to NR), respectively. Grade 3 or higher toxicities attributable to therapy were seen in 14 subjects (87.5%; 95% CI 61.7% to 98.4%). Infusion product analysis showed the presence of effector memory cells with high expression of CD39 irrespective of tumor type and low expression of checkpoint markers. CONCLUSIONS: TIL manufactured with assistance of 4-1BB and CD3 agonism is feasible and treatment is associated with no new safety signals. While no responses were observed, a significant portion of patients achieved SD suggesting early/partial immunological effect. Further research is required to identify factors associated with resistance and functionally enhance T cells for a more effective therapy

Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC

Evaluation of the Combination of HDACi and IL-21 in TIL Initial Phase of Expansion

https://openworks.mdanderson.org/sumexp21/1009/thumbnail.jp

SNF5/INI1 Deficiency Redefines Chromatin Remodeling Complex Composition during Tumor Development

Running Title: SNF5/INI1 regulates SWI/SNF complex composition in MRT

CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-β signaling

Background The correlation between elevated T-cell infiltration and improved survival of ovarian cancer (OvCa) patients suggests that endogenous tumor-infiltrating lymphocytes (TIL) possess some degree of antitumor activity that can be harnessed for OvCa immunotherapy. We previously optimized a protocol for ex vivo OvCa TIL expansion for adoptive cell therapy, which is now being tested in a clinical trial at our institution (NCT03610490). Building on this success, we embarked on genetic modification of OvCa TIL to overcome key immunosuppressive factors present in the tumor microenvironment. Here, we present the preclinical optimization of CRISPR/Cas9-mediated knockout of the TGF-β receptor 2 (TGFBR2) in patient-derived OvCa TIL.Methods OvCa TILs were generated from four patients’ tumor samples obtained at surgical resection and subjected to CRISPR/Cas9-mediated knockout of TGFBR2 before undergoing a rapid expansion protocol. TGFBR2-directed gRNAs were comprehensively evaluated for their TGFBR2 knockout efficiency and off-target activity. Furthermore, the impact of TGFBR2 knockout on TIL expansion, function, and downstream signaling was assayed.Results TGFBR2 knockout efficiencies ranging from 59±6% to 100%±0% were achieved using 5 gRNAs tested in four independent OvCa TIL samples. TGFBR2 knockout TIL were resistant to immunosuppressive TGF-β signaling as evidenced by a lack of SMAD phosphorylation, a lack of global transcriptional changes in response to TGF-β stimulation, equally strong secretion of proinflammatory cytokines in the presence and absence of TGF-β, and improved cytotoxicity in the presence of TGF-β. CRISPR-modification itself did not alter the ex vivo expansion efficiency, immunophenotype, nor the TCR clonal diversity of OvCa TIL. Importantly for clinical translation, comprehensive analysis of CRISPR off-target effects revealed no evidence of off-target activity for our top two TGFBR2-targeting gRNAs.Conclusions CRISPR/Cas9-mediated gene knockout is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods. We achieved efficient and specific TGFBR2 knockout, yielding an expanded OvCa TIL product that was resistant to the immunosuppressive effects of TGF-β. This study lays the groundwork for clinical translation of CRISPR-modified TIL, providing opportunities for engineering more potent TIL therapies not only for OvCa treatment, but for the treatment of other solid cancers as well

Efficacy and safety of autologous tumor-infiltrating lymphocytes in recurrent or refractory ovarian cancer, colorectal cancer, and pancreatic ductal adenocarcinoma

Background Tumor-infiltrating lymphocyte (TIL) therapy has shown efficacy in metastatic melanoma, non-small cell lung cancer, and other solid tumors. Our preclinical work demonstrated more robust CD8 predominant TIL production when agonistic anti-4-1BB and CD3 antibodies were used in early ex vivo TIL culture.Methods Patients with treatment-refractory metastatic colorectal (CRC), pancreatic (PDAC) and ovarian (OVCA) cancers were eligible. Lymphodepleting chemotherapy was followed by infusion of ex vivo expanded TIL, manufactured at MD Anderson Cancer Center with IL-2 and agonistic stimulation of CD3 and 4-1BB (urelumab). Patients received up to six doses of high-dose IL-2 after TIL infusion. Primary endpoint was evaluation of objective response rate at 12 weeks using Response Evaluation Criteria in Solid Tumors version 1.1 with secondary endpoints including disease control rate (DCR), duration of response, progression-free survival (PFS), overall survival (OS), and safety.Results 17 patients underwent TIL harvest and 16 were treated on protocol (NCT03610490), including 8 CRC, 5 PDAC, and 3 OVCA patients. Median age was 57.5 (range 33–70) and 50% were females. Median number of lines of prior therapy was 2 (range 1–8). No responses were observed at 12 weeks. Ten subjects achieved at least one stable disease (SD) assessment for a DCR of 62.5% (95% CI 35.4% to 84.8%). Best response included prolonged SD in a patient with PDAC lasting 17 months. Median PFS and OS across cohorts were 2.53 months (95% CI 1.54 to 4.11) and 18.86 months (95% CI 4.86 to NR), respectively. Grade 3 or higher toxicities attributable to therapy were seen in 14 subjects (87.5%; 95% CI 61.7% to 98.4%). Infusion product analysis showed the presence of effector memory cells with high expression of CD39 irrespective of tumor type and low expression of checkpoint markers.Conclusions TIL manufactured with assistance of 4-1BB and CD3 agonism is feasible and treatment is associated with no new safety signals. While no responses were observed, a significant portion of patients achieved SD suggesting early/partial immunological effect. Further research is required to identify factors associated with resistance and functionally enhance T cells for a more effective therapy