Beutner, Karl R.

209 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. Methods: 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 HLA-A*02 positive tumor samples were then assessed for LOH using a bioinformatic algorithm applied via the Tempus xT platform. Results: 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). Conclusions: 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. Clinical trial information: NCT04981119 .

TPS2676 Background: Solid tumors comprise > 90% of cancers. Metastatic colorectal cancer, non-small cell lung cancer, and pancreatic cancer are among the leading causes of cancer-related mortality (5-year overall survival: 14%, 6%, and 3%, respectively) (ACS. 2021). Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies (Neelapu S. et al. N Engl J Med. 2017). 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 (Parkhurst M, et al. Mol Ther. 2011; Tanyi J. Cellicon Valley '21). To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust NOT logic gate capable of killing tumor cells, while leaving healthy cells intact (Hamburger A, et al. Mol Immunol. 2020). 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. The frequency of HLA LOH among advanced GI solid tumor cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%-20.8% between colorectal, pancreatic, and gastroesophageal tumors (Hecht R. et al. ASCO-GI 2022. Abstract #190). As such, HLA LOH offers a definitive tumor versus normal discriminator target for CAR T-cell therapy. 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 of 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) To obtain leukapheresis and feasibility for the future EVEREST Tmod CAR T-cell trial. Methods: BASECAMP-1 (NCT04981119) patient eligibility has 2 parts: 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 xT-Onco NGS testing. 2) If the tumor demonstrates HLA-A*02 LOH and the patient is eligible after screening, the patient will undergo leukapheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. Clinical trial information: NCT04981119.

190 Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal (GE) cancers are the leading causes of GI cancer–related mortality (5-yr survival rate, 14%, 3% and ̃5-6%, respectively). T-cell immunotherapy targeting GI-associated tumor antigens has been attempted, but efficacy has been constrained by on-target off-tumor toxicity, limiting the therapeutic window. The Tmod (TM) platform is an AND-NOT logic-gated CAR T modular system, versions of which have a CEA- or MSLN-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor to protect normal cells. Tmod CAR T exploits HLA LOH, common in GI malignancies (10-33% in primary solid tumors [TCGA]) and can kill tumor cells without harming healthy cells in vitro and in vivo. However, the prevalence of HLA LOH across GI tumors is unknown in the real-world setting. We utilized the Tempus xT oncology NGS database of patients with multiple GI tumors. From a standard-of-care NGS assay, GI cancer patients can be readily identified for HLA LOH and future treatment with Tmod CAR T therapy. Methods: The occurrence of HLA LOH in GI tumors of 1439 patients was assessed using paired germline and somatic DNA sequencing using a research assay [6]. CRC, PANC and GE patients with ≥ stage 3 were then extracted, and rates of HLA LOH were identified (ie, whether loss occurred across high-frequency HLA-A alleles). In addition, mutations in KRAS and BRAF, as well as MSI status were stratified to determine any association with HLA-A LOH. Results: HLA-A LOH was detected in 830 (17.3%) of all solid tumor records, and a similar proportion when all GI cancer records were analyzed (17.0%). For GI subtypes, these values ranged from 13.5% to 23.1% (Table). No high-frequency HLA-A allele (A*01, A*02, A*03, A*11) was more likely to be lost. Clinical biomarkers ( KRAS, BRAF and MSI status) were not associated with HLA-LOH. Conclusions: The frequency of HLA LOH among advanced solid tumor cancers in this dataset is 17.3%, with a range of 13.5-23% between CRC, PANC and GE. The HLA LOH frequency observed in these GI tumors is consistent with that in primary tumors from TCGA, which also used germline-matched and tumor samples. Clinical biomarkers were not associated with HLA LOH. Tempus NGS was able to identify HLA LOH, which can be used for Tmod CAR T therapy to an enhanced therapeutic window. Identification of these patients in BASECAMP-1 (NCT04981119) will enable novel Tmod CAR T therapy. [Table: see text]