Kirtane, Kedar

Abstract Background: Chimeric antigen receptor T-cell (CAR T) therapies have demonstrated significant clinical efficacy in hematologic malignancies, but have proven challenging in solid tumors, in part owing to a lack of tumor-specific targets and unmanageable toxicity. Mesothelin (MSLN) expression normally is limited to the mesothelium of major body cavities but can be upregulated in diverse solid tumor types (cancer.gov/tcga), making it a potential target for cancer therapy. MSLN-targeted cell therapy approaches, including CAR T and T-cell receptor fusion therapies, have shown promising clinical activity; however, on-target, off-tumor toxicity including fatal events have occurred (Beatty, et al. Gastroenterology. 2018.; Haas, et al. Mol Ther. 2023.; Hong, et al. ESMO 2021. Abstract 9590.). A2B694 is an autologous logic-gated, MSLN-targeted Tmod CAR T therapy that addresses the challenges of on-target, off-tumor toxicity by combining 2 CARs: an activating and blocking receptor. The activator recognizes MSLN present on the surface of both tumor and normal cells; the blocker inhibits the CAR T activator when it binds human leukocyte antigen (HLA)-A*02, which is present in normal cells and often lost in tumor cells. Thus, in patients with tumor-associated HLA-A*02 loss of heterozygosity (LOH), the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (Hamburger, et al. Mol Immunol. 2020). Through this unique selective mechanism, A2B694 may provide a therapeutic window to treat patients with MSLN-expressing solid tumors exhibiting HLA-A*02 LOH. Methods: EVEREST-2 (NCT06051695) is a first-in-human, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B694 in adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression, including non-small cell lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, mesothelioma, or other solid tumors with MSLN expression. Eligible patients must have progressed following standard of care therapy. Enrollment to EVEREST-2 occurs through the prescreening study BASECAMP-1 (NCT04981119), which identifies patients with tumor-associated HLA-A*02 LOH via next-generation sequencing (Tempus AI, Inc). Eligible patients enroll in the BASECAMP-1 study and undergo leukapheresis. A2B694 is manufactured from cryopreserved T cells when clinically appropriate for patients. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B694 and identify the recommended phase 2 dose (RP2D). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Phase 2 will assess overall response rate per RECIST v1.1. The first patient was enrolled on EVEREST-2 in April 2024. Dose escalation is ongoing. Citation Format: Julian R. Molina, Antonious Hazim, Oliver Dorigo, Salman R. Punekar, Monica Avila, Diane M. Simeone, Kedar Kirtane, Gottfried E. Konecny, Jeffrey Ward, Matthew Block, Jennifer Specht, Leslie Boyd, Yanyan Lou, Jong Chul Park, Jacqueline D. Xuan, Ramez N. Eskander, Gayanie Ong, Marco Davila, Wendy Langeberg, Marcela Maus, Armen Mardiros, Frederick L. Locke, Andrea Wise, David G. Maloney, John Welch, J. Randolph Hecht. EVEREST-2: A seamless phase 1/2 study of A2B694, a logic-gated Tmod CAR T-cell therapy, in patients with mesothelin-expressing solid tumors with human leukocyte antigen-A*02 loss of heterozygosity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT186.

766 Background: BASECAMP-1 (NCT04981119) is a pre-screening study to identify patients with tumor-associated human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH) for interventional studies, such as EVEREST-2 (NCT06051695), a phase 1/2 study of logic-gated chimeric antigen receptor T-cell (CAR T) therapy for MSLN-expressing cancers. MSLN is a cell surface protein expressed in several cancer types, including mesothelioma (MESO), colorectal (CRC), non-small cell lung (NSCLC), ovarian (OVCA), and pancreatic (PANC) cancer, which can be associated with poor prognosis (1). Longitudinal clinical, genomic, and biomarker data were collected from patients enrolled in BASECAMP-1, providing a large dataset for translational discovery and propensity scoring. The aim of this analysis was to determine whether MSLN expression measured by RNAseq and IHC are correlated among patients with solid tumors. Methods: In BASECAMP-1, tumor tissue from patients with germline heterozygous HLA-A*02 is tested for HLA-A*02 LOH using an investigational next generation sequencing device codeveloped with Tempus Labs (Lozac'hmeur, et al. NPJ Precis Oncol. 2024) that detects somatic alterations, including HLA LOH, and generates RNAseq data (eg, MSLN expression). Gene-level transcripts per million read (TPM) values were determined and the log2 TPM +1 was displayed in a scatter plot (the mean and standard deviation [SD] are provided in the Table). Patients with HLA-A*02 LOH also submit tissue for IHC testing for exploratory biomarkers (eg, MSLN expression using anti-MSLN clone 5B2). Statistical significance was determined with ANOVA or t-tests. Results: As of June 1, 2024, 314 patients had been screened for BASECAMP-1 and had RNAseq results; 34 patients also had MSLN IHC results. MSLN expression by RNAseq was consistently higher in patients with PANC or OVCA vs CRC or NSCLC ( P <0.001). MSLN expression by RNAseq and IHC were correlated overall ( P <0.001), particularly among patients with PANC and OVCA. Among the 8 patients with OVCA or PANC who were IHC+ for MSLN, only 1 had an RNAseq value below 6 log2 TPM+1; this patient had mesonephric-like ovarian adenocarcinoma, which may account for the low value. The one MESO sample with both RNAseq and IHC available was IHC+ and had a high RNAseq value. Conclusions: This analysis demonstrated high correlation between RNAseq and IHC MSLN expression in tumor tissue. 1. Faust, et al. Cancers. 2022. Clinical trial information: NCT04981119 . MSLN expression by RNASeq by tumor type and IHC status. Tumor Overall (N=314) IHC data available (n=34) MSLN Positive MSLN Negative n Mean TPM a (SD) n Mean TPM a (SD) n Mean TPM a (SD) All 314 5.1 (2.3) 20 6.0 (2.1) 14 2.3 (1.7) adNSCLC 33 3.8 (2.4) 2 4.7 (1.7) 7 1.5 (1.3) sqNSCLC 4 3.1 (1.8) 0 0 MESO 4 4.5 (4.2) 1 5.7 0 CRC 148 4.4 (2.0) 8 5.1 (2.1) 7 3.1 (1.6) PANC 100 6.1 (1.8) 3 7.8 (0.6) 0 OVCA 25 7.2 (2.1) 5 6.6 (2.8) 0 a MSLN log2 transcripts per million+1. ad, adenocarcinoma; sq, squamous.

TPS2699 Background: Despite the success in hematologic cancers, chimeric antigen receptor (CAR) T-cell therapies are challenging to implement in solid tumors owing to a lack of tumor-specific targets that discriminate cancer from normal cells. MSLN expression normally is limited to the mesothelium of major body cavities but can be upregulated in diverse solid tumor types (TCGA 2022), making it a potential target for cancer therapy. MSLN-targeted cell approaches, including CAR T-cell and T-cell receptor fusion therapies, have shown promising clinical activity; however, on-target, off-tumor toxicity including fatal events have occurred (1-3). A2B694 is an autologous logic-gated, MSLN-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining 2 CARs: an activating and blocking receptor. The activator recognizes MSLN present on the surface of both tumor and normal cells; the blocker binds HLA-A*02 and prevents CAR T-cell activity. Thus, in patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). Through this unique discriminatory mechanism, A2B694 may provide a therapeutic window to treat patients with MSLN-expressing solid tumors exhibiting HLA-A*02 LOH. Methods: EVEREST-2 (NCT06051695) is a first-in-human, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B694 in adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression, including non-small cell lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, mesothelioma, or other solid tumors with MSLN expression. Eligible patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. Enrollment to EVEREST-2 occurs through the prescreening study BASECAMP-1 (NCT04981119), which identifies patients with tumor-associated HLA-A*02 LOH via next-generation sequencing (Tempus AI, Inc.). Eligible patients enroll in the BASECAMP-1 study and undergo leukapheresis. A2B694 is manufactured from cryopreserved T cells when clinically appropriate for patients. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B694 and identify the recommended phase 2 dose (RP2D). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Phase 2 will assess overall response rate per RECIST v1.1. 1. Beatty, et al. Gastroenterology. 2018. 2. Haas, et al. Mol Ther. 2023. 3. Hong, et al. ESMO 2021. Abstract 9590. 4. Hamburger, et al. Mol Immunol. 2020. Clinical trial information: NCT06051695 .

TPS2698 Background: Chimeric antigen receptor (CAR) T-cell therapies for treating solid tumors are challenging due to a lack of tumor-specific targets that discriminate cancer from normal cells. Previous studies using CEA T-cell receptors and T-cell engagers have resulted in dose-limiting, on-target, off-tumor toxicities (1,2). A2B530 is an autologous logic-gated, CEA-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining a CAR-activating receptor with a blocking receptor to discriminate tumor from normal cells (3). The activator recognizes CEA on the surface of both tumor and normal cells while specificity for tumor cells is provided by a blocker that binds HLA-A*02. In patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). HLA-A*02 LOH can be detected using next-generation sequencing (Tempus AI, Inc.). With this definitive discriminator target, A2B530 can potentially provide a therapeutic window to treat patients with CEA-expressing solid tumors exhibiting HLA LOH. Methods: EVEREST-1 (NCT05736731) is a seamless, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B530 in adult patients. Patients are enrolled through BASECAMP-1 (NCT04981119), a master prescreening study that identifies patients with HLA LOH at any time in the course of their disease. BASECAMP-1–eligible patients undergo leukapheresis and, when clinically appropriate, their banked T cells are manufactured for the EVEREST-1 study. The key inclusion criteria include histologically confirmed recurrent unresectable, locally advanced, or metastatic cancers associated with CEA expression: non-small cell lung, colorectal, or pancreatic cancers. Patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B530 and determine the recommended phase 2 dose (RP2D). The dose-escalation portion is based on the Bayesian optimal interval design. The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B530. Phase 2 will assess overall response rate per RECIST v1.1. As of January 29, 2024, 8 patients have been enrolled on EVEREST-1. A2B530 was successfully manufactured for all patients, and all patients have received A2B530 infusion, with the first patient dosed in May 2023. Dose escalation is ongoing. 1. Parkhurst, et al. Mol Ther. 2022. 2. Tabernero, et al. J Clin Oncol. 2017. 3. Hamburger, et al. Mol Immunol. 2020. 4. Sandberg, et al. Sci Transl Med. 2022. Clinical trial information: NCT05736731 .

1604 Background: Diversity in clinical studies is essential to address health disparities, but racial and ethnic minorities are often underrepresented in clinical studies, especially in precision medicine studies using genetic data (1). BASECAMP-1 (NCT04981119) is an ongoing prescreening study to identify patients with unresectable advanced or metastatic solid tumors and tumor-associated HLA-A*02 LOH for the EVEREST-1 and -2 Tmod chimeric antigen receptor T-cell therapy trials. When enrollment began in 2021, eligibility for Part 1 was restricted to patients with germline HLA-A*02:01 based on preclinical data. Additional analysis revealed the blocker in the Tmod construct had activity across HLA-A*02 subtypes (Mock, et al. Mol Ther Oncolytics.2022). Eligibility for Part 1 was then expanded to all HLA-A*02 subtypes, allowing enrollment of more diverse patient populations. HLA-A*02 allele subtypes vary by ethnicity and race; the frequency of HLA-A*02:01 is 96% in non-Hispanic Whites, but ranges from 53% to 73% in other ethnicities and races. The frequency of all other HLA-A*02 allele subtypes (HLA-A*02:XX) is <5% in non-Hispanic Whites but up to 66% in other ethnicities and races (2,3). Methods: Patients eligible for BASECAMP-1 were identified at United States study sites. All patients screened before January 16, 2024, for Part 1 were included for demographic and central lab HLA-A typing. The % increase in eligibility and % of heterozygous HLA-A*02 patients with HLA-A*02:XX were calculated by ethnicity and race. Results: A total of 1124 patients were screened for germline HLA-A*02 typing; 431 patients were HLA-A*02:01 heterozygous, of whom 37 (9%) were Hispanic, 14 (3%) African American (AA), 2 (<1%) American Indian or Alaska Native, 17 (4%) Asian or Pacific Islander (API), and 324 (75%) non-Hispanic White (Table). The eligibility expansion identified 62 patients with HLA-A*02:XX heterozygosity, of whom 6 (10%) were Hispanic, 6 (10%) AA, 19 (31%) API, and 25 (40%) non-Hispanic White. Conclusions: By expanding enrollment to include HLA-A*02:XX, 16% more Hispanic, 43% more AA, and 112% more API patients were identified, improving the diversity in this trial compared with that of initial enrollment. 1. Aldrighetti, et al. JAMA Netw Open 2021. 2. Ellis, et al. Human Immunol. 2000. 3. Gragert, et al. Human Immunol.2013. Clinical trial information: NCT04981119 . [Table: see text]