Author Correction: Single-cell RNA sequencing reveals the effects of chemotherapy on human pancreatic adenocarcinoma and its tumor microenvironment
POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.
Single-cell RNA sequencing reveals the effects of chemotherapy on human pancreatic adenocarcinoma and its tumor microenvironment
The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.
Prospective BASECAMP-1 experience in patients with gastrointestinal (GI) cancer: Identifying patients with human leukocyte antigen (HLA) loss of heterozygosity (LOH) for a future therapeutic trial exploiting LOH as a tumor vulnerability [Meeting Abstract]
Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal cancers are the leading causes of GI cancer-related mortality (5-y survival: 15%, 3%, and 5%-6%, respectively) (ACS 2022). HLA LOH is a recurrent mechanism of immune escape observed in 15%-20% of GI cancers (Hecht R., ASCO GI 2022). The Tmod platform is a logic-gated chimeric antigen receptor (CAR) T-cell modular system, comprising a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor. Both in vitro/in vivo, Tmod CAR T therapy kills cells with HLA-A*02 LOH (tumor) without harming cells with retained HLA-A*02 expression (normal). However, HLA-A*02 LOH can only be therapeutically exploited if patients are identifiable through a feasible and timely clinical workflow.
Method(s): We established a biobanking protocol (BASECAMP-1, NCT04981119) to determine whether HLA-A*02 LOH patients can be prospectively identified. Patients with CRC, PANC, or non-small cell lung cancer (NSCLC), and a high risk for incurable relapse, were screened first using a standard HLA assay. Heterozygous HLAA* 02 positive tumor samples were then assessed for LOH using a bioinformatic algorithm applied via the Tempus xT platform.
Result(s): As of Sep 1, 2022, 83 patients were consented at 4 institutions. HLA status was obtained from 70 patients and 28 were identified as HLA-A*02:01 heterozygous (40%; expected frequency based on USA NMDP data, 27.6%). LOH results were available for 16 patients; 4 LOH-positive patients were identified (25%, 2 PANC, 2 NSCLC). The LOH assay sensitivity declines below a tumor purity of 40% (Hecht R., ASCO GI 2022). Six patients had a tumor purity of 20% (all with PANC, a tumor known for high stromal content), limiting possible LOH detection. The impact of tumor purity on LOH sensitivity was highlighted in a patient with a low initial sample tumor purity (30%) that resulted in a 41% probability of HLA-A*02:01 LOH (below positive threshold). A second sample with a higher tumor purity (70%), obtained from formalin-fixed, paraffin-embedded sections, resulted in a 92% probability of HLA-A*02:01 LOH (positive).
Conclusion(s): BASECAMP-1 prospective identification of HLA-A*02 LOH is feasible in the real-world setting. The frequencies of the HLA-A*02 allele and of HLA-A*02 LOH in this cohort mirrored expected population frequencies. LOH results can be obtained within a clinically feasible workflow and timeframe, although samples with a,40% tumor purity have a reduced sensitivity for LOH detection, an issue recurrently observed in patients with PANC. The BASECAMP-1 strategy enables prospective identification of appropriate patients for future therapeutic clinical trials using Tmod CEA and MSLN logic-gated CAR T cells
Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies
Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.
Corrigendum: Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies
[This corrects the article DOI: 10.3389/fimmu.2023.1067352.].
BASECAMP-1: LEVERAGING HLA LOSS OF HETEROZYGOSITY IN SOLID TUMORS BY NGS TO IDENTIFY PATIENTS WITH RELAPSED SOLID TUMORS FOR FUTURE CEA AND MSLN LOGIC-GATED TMODTM CAR T-CELL THERAPY [Meeting Abstract]
Background Solid tumors comprise >90% of cancers. Nonsmall cell lung cancer (NSCLC), metastatic colorectal cancer (CRC), and pancreatic cancer are the leading causes of cancerrelated mortality (5-year overall survival: 26%, 15%, and 11%, respectively).1 Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies.2,3 However, translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities.4,5 To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust logic gate capable of killing tumor cells, while leaving healthy cells intact.6,7 The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells (figure 1).8,9 Human leukocyte antigen (HLA) loss of heterozygosity (LOH) offers a definitive tumor versus normal discriminator target for CAR T-cell therapy.10 The frequency of HLA LOH among advanced NSCLC, CRC, and pancreatic cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%- 23.1%.11 LOH can be reliably detected using the Tempus xTOnco next-generation sequencing (NGS) assay.12,13 Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives: 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) Subsequent apheresis and manufacturing feasibility for the future EVEREST CEA or MSLN Tmod CAR T-cell studies. Methods BASECAMP-1 (NCT04981119) patient eligibility has 2 parts (figure 2): 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xTOnco NGS testing; 2) If the tumor demonstrates HLAA* 02:01 LOH and the patient is eligible after screening, the patient will undergo apheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. (Figure Presented)
AN NGS ASSAY TO IDENTIFY HLA LOSS OF HETEROZYGOSITY FOR FUTURE CEA AND MSLN LOGICGATED CAR-T SOLID TUMOR PROTOCOLS DESIGNED FOR REDUCED ON-TARGET, OFF-TUMOR TOXICITY [Meeting Abstract]
Background Chimeric antigen receptor (CAR) T-cell therapy has shown clinical efficacy in hematologic cancers, but success is limited in solid tumors due to a lack of tumor-specific targets that distinguish cancer from normal cells and an immunosuppressive tumor microenvironment.1 Integrating synthetic biology and comprehensive molecular profiling of tumors may provide active and tolerable approaches to CAR T-cell therapy in patients with solid tumors. Human leukocyte antigen (HLA) loss of heterozygosity (LOH) in tumors offers a definitive tumor vs normal discriminator target for CAR T-cell therapy.2 The Tmod platform3,4 is a modular logic-gated CAR T system comprising different versions including a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate blocker receptor targeting HLA-A*02 or other HLA alleles to protect normal cells. Compared with existing immunohistochemistry (IHC) tests, Tempus xT-Onco is a standard-of-care next-generation sequencing (NGS) assay5 that detects somatic alterations including HLA LOH and generates whole transcriptome RNA data (eg, CEA or MSLN expression) and a tumor immune infiltration profile, which can effectively identify patients appropriate for Tmod CAR T-cell therapy. Methods HLA LOH in solid tumors was assessed with paired germline and somatic DNA sequencing. Common driver mutations, microsatellite instability status, and tumor mutational burden were examined in HLA-A LOH or HLA-A intact cohorts. Tumor expression of CEA and MSLN was evaluated via RNA sequencing and compared with immunohistochemistry (IHC) results. Results A total of 21,053 tumor samples in the Tempus database were compared with their matched-normal samples. HLA-A LOH was detected in 16% of 10,867 advanced solid tumors (table 1) and similar LOH frequencies were observed among common HLA-A alleles. Clinical factors and molecular biomarkers were similar between HLA-A LOH and HLA-A intact cohorts. High CEA expression was seen in IHC-positive patients. Conclusions The frequency of HLA-A LOH in solid tumors in the Tempus database is similar to that reported in the Cancer Genome Atlas.6 Tempus xT-Onco reliably detects HLA LOH and quantifies CEA and MSLN expression. Based on these data, patients with solid tumors are now being prospectively screened for HLA LOH using xT-Onco in an ongoing tissue banking study (BASECAMP-1, NCT04981119), preparing for future interventional protocols
A2B530, AN AUTOLOGOUS CEA-DIRECTED TMOD TCELL THERAPY WITH AN INHIBITORY RECEPTOR GATED BY HLA-A*02 TO TARGET COLORECTAL, PANCREATIC, AND LUNG CANCER [Meeting Abstract]
Background Nearly all colorectal and most pancreatic and lung cancers express carcinoembryonic antigen (CEA). However, due to its expression in normal gut epithelial cells, CEAtargeted therapies have resulted in on-target, off-tumor toxicity. To overcome this, we have developed TmodTM, a logicgated T-cell therapy platform. Tmod constructs are composed of an activating CAR or T-cell receptor that targets a tumor antigen and an inhibitory receptor recognizing an antigen expressed on normal healthy tissues, but not on tumor cells due to loss of heterozygosity (LOH).1,2 A2B530 is a CEAdirected Tmod construct utilizing an LIR-1-based inhibitory receptor (blocker) targeting human leukocyte antigen A*02 (HLA-A*02). Methods To generate CEA Tmod, T cells from HLA-A*02(+) donors were transduced with a single lentivirus to express i) the CAR, ii) the blocker, and iii) an shRNA targeting b2M. Cytotoxicity was measured by culturing CEA(+) target cell line pairs (A*02[-] and A*02[+]), expressing either GFP or RFP, with engineered T cells and quantifying live target cells over time. In vivo activity was examined using NSG mice subcutaneously implanted with normal (CEA[+]A*02[+]) and tumor cells (CEA[+]A*02[-]), in the right and left flanks. Mice were treated intravenously with CEA Tmod cells or control T cells. Results Control CEA CAR T cells killed CEA(+) target cell lines in vitro irrespective of HLA-A*02 expression. In contrast, CEA Tmod cells selectively killed tumor cells (CEA[+]A*02[-]) while sparing normal cells (CEA[+]A*02[+]). In mixed target cell cultures, CEA Tmod cells killed only the A*02(-) target cells, whereas the CEA CAR T cells killed both the A*02(- ) and A*02(+) cell lines. Further, CEA Tmod cells exhibited bidirectional control between the activated and blocked states. While mice treated with control CEA CAR T cells experienced a reduction in volume and bioluminescence of both normal and tumor grafts, CEA Tmod cells specifically cleared A*02(-) tumors in mice (table 1). Finally, although expansion of Tmod cells in peripheral blood trended lower than CAR and TCR controls, anti-tumor activity was comparable in these groups. Conclusions A2B530 is an autologous CEA Tmod cell product that exploits common LOH at the HLA locus in cancer cells, enabling these engineered T cells to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study identifying patients with somatic HLA LOH, is recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B530 EVEREST-1 interventional study
A2B694, AN AUTOLOGOUS LOGIC-GATED CELL THERAPY TARGETING MESOTHELIN [Meeting Abstract]
Background Mesothelin (MSLN) is expressed on a variety of solid tumors, including mesothelioma and ovarian, uterine, gastric, pancreatic, and lung cancers.1 However, efforts to target MSLN using cellular therapies have been hampered by severe on-target, off-tumor toxicities associated with damage to normal tissues expressing MSLN.2 To avoid these toxicities, we have developed a logic-gated engineered cell therapy, TmodTM, which is composed of two chimeric antigen receptors (CARs): an activator that targets a tumor-associated antigen and an inhibitory receptor (blocker) gated by an antigen expressed on normal tissue but lost in tumor cells due to loss of heterozygosity (LOH). A2B694 is an MSLN-specific Tmod construct combining a third-generation MSLN CAR with an LIR-1-based inhibitory receptor specific for human leukocyte antigen A*02 (HLA-A*02). Methods Lentivirus encoding i) the CAR, ii) the blocker, and iii) an shRNA targeting b2M was used to transduce T cells from HLA-A*02 donors and generate MSLN Tmod cells. In vitro cytotoxicity measurements were performed using fluorescence-based imaging and luciferase readouts. In vivo assessments were performed in NSG mice subcutaneously implanted with normal cells (MSLN[+]A*02[+]), or tumor cells (MSLN[+]A*02[-]), in the left and right flanks, respectively. Following engraftment, mice were randomized and treated intravenously with MSLN Tmod cells or controls. Grafts were measured via caliper. Results MSLN Tmod cells preferentially killed tumor cells (MSLN[+]A*02[-]) over normal cells (MSLN[+]A*02[+]) in vitro, unlike clinically active comparator M5 CAR T cells, which indiscriminately killed both target cell types (figure 1A). Soluble MSLN, tested across a 0-2 mg/mL range, did not impact MSLN Tmod function. Additionally, in mixed cell cultures where T cells and tumor and normal cells were simultaneously cultured (1:1:1 ratio), MSLN Tmod cells selectively killed tumor targets while sparing normal cells. Further, MSLN Tmod cells cycled between activated and blocked states in vitro when repeatedly challenged with tumor or normal target cells. Finally, while MSLN CAR T cells killed both normal and tumor grafts in vivo, MSLN Tmod cells selectively killed tumor grafts while sparing normal grafts (figure 1B, C). Conclusions A2B694 is an autologous MSLN Tmod cell product that leverages LOH at the HLA locus in cancer cells, providing a mechanism to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study that will identify patients with somatic HLA LOH, is currently recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B694 interventional study (EVEREST-2)