Mardiros, Armen

TPS2673 Background: The main challenge for developing CAR T therapies for solid tumors is the lack of targets that distinguish tumor from normal cells, resulting in on-target, off-tumor toxicity. Tmod logic-gated CAR T therapy addresses this challenge by incorporating 2 CARs on the same T cell: an activator targeting a marker on both tumor and normal cells, and a blocker targeting HLA-A*02 that inhibits CAR T activity against normal cells while allowing activation against tumor cells (with HLA-A*02 LOH), improving tumor selectivity and decreasing toxicity. Early safety results from 3 ongoing phase 1/2 clinical trials of logic-gated, Tmod CAR T therapy (EVEREST-1, EVEREST-2, and DENALI-1) have demonstrated manageable safety and tolerability in patients with advanced solid tumors (Grierson et al, SITC , 2024; Ward et al, SITC 2025; Specht et al, SABCS , 2025). Early efficacy results include the first ever reported complete response in a patient with non-small cell lung cancer following treatment with a CAR T-cell therapy (A2B694). A2B543 is an autologous Tmod CAR T therapy that contains the same Tmod construct as A2B694 with an added membrane-tethered IL-12 (memIL12) booster. Specifically, A2B543 is comprised of autologous Tmod cells transduced with 2 lentiviral vectors: one expressing both the HLA-A*02-targeted blocker and the mesothelin-targeted CAR activator; and a second expressing the memIL12 booster. Interleukin 12 (IL-12) is a potent, pro-inflammatory cytokine that plays a crucial role in inducing antitumor immune responses; however, systemic IL-12 can be prohibitively toxic (Jia et al, Front Immunol , 2022). In A2B543, expression of the memIL12 cassette is under the control of an NFAT promoter and is induced during antigen engagement or T cell activation. This inducible memIL12 is designed to reduce the toxicity associated with systemic IL-12 while enhancing the long-term potency and persistence of Tmod (Zhang et al, J Immunother Cancer , 2025). Methods: EVEREST-2 (NCT06051695) is a phase 1/2, open-label, nonrandomized study evaluating the safety and efficacy of A2B543 in adults with recurrent/metastatic mesothelin-expressing cancers with tumor-associated HLA-A*02 LOH, including mesothelioma, colorectal, non-small cell lung, pancreatic, or ovarian cancer. Patients are enrolled through BASECAMP-1 (NCT04981119), a master prescreening study that identifies patients with HLA LOH via next-generation sequencing and cryopreserves leukapheresis product. Upon progression, A2B543 is manufactured and then administered after lymphodepletion. The phase 1 primary objective is to evaluate the safety and tolerability of A2B543 and identify a recommended phase 2 dose (RP2D). The phase 2 primary objective is to assess overall response rate. Clinical trial information: NCT06051695 .

Abstract Background: Despite the success treating hematologic malignancies, chimeric antigen receptor T-cell (CAR T) therapies face challenges in breast cancer and other solid tumors due to lack of targets that distinguish tumor from normal cells. Epithelial growth factor receptor (EGFR) plays a critical role in oncogenesis across several cancers and is often upregulated [1]. While monoclonal antibodies targeting EGFR have demonstrated efficacy, these approaches are often limited by on-target, off-tumor toxicities, such as skin and gastrointestinal toxicity, which constrains dose escalation and efficacy [2]. A2B395 is an allogeneic, logic-gated, EGFR-targeted, TmodTM CAR T therapy designed to address these limitations and provide a convenient and consistent off-the-shelf option. This therapy incorporates 2 CARs: an activator targeting EGFR and a blocker targeting human leukocyte antigen (HLA)-A*02. The activator recognizes EGFR on both tumor and normal cells, whereas the blocker inhibits CAR T activity against normal cells (with preserved HLA expression) while allowing activation against tumor cells (with HLA-A*02 loss of heterozygosity [LOH]). This approach is designed to avoid on-target, off-tumor toxicity, a significant limitation to CAR T treatment of solid tumors [3]. To address potential graft-vs-host response, a short-hairpin RNA expression module targeting beta-2 microglobulin is included in the Tmod construct, which significantly reduces major histocompatibility complex class I levels and subsequent host immune response [4]. Importantly, the Tmod system is modular and adaptable to multiple targets. Initial data on autologous Tmod CAR T therapy suggest reduced off-tumor toxicity and encouraging clinical efficacy [5]. A2B395 represents a novel approach for targeting EGFR-expressing solid tumors with HLA-A*02 loss of heterozygosity. Methods: DENALI-1 is a phase 1/2, open-label, nonrandomized study evaluating the safety and efficacy of A2B395 in adults. Patients are enrolled through BASECAMP-1 (NCT04981119), a master prescreening study that identifies patients with HLA loss of heterozygosity at any time in the course of their disease via next-generation sequencing. Key inclusion criteria include histologically confirmed recurrent, unresectable, locally advanced, or metastatic cancers associated with EGFR expression, including triple-negative breast, colorectal, non-small cell lung, squamous cell head and neck, and renal cell cancers. Patients must have received ≥1 line of prior therapy, such as a checkpoint inhibitor, molecular targeted therapy, or chemotherapy. The primary objective of the phase 1 portion is to evaluate safety, tolerability, and the recommended phase 2 dose (RP2D) using a Bayesian optimal interval design for dose escalation. The dose-expansion phase will confirm the RP2D and collect and analyze biomarker data. The Phase 2 portion will assess overall response rate per RECIST v1.1. As of 03 July 2025, the first patient dosed with A2B395, who has breast cancer, completed the 28-day safety review period. Dose-escalation is ongoing. Citation Format: J. M. Specht, S. R. Punekar, M. Ulrickson, D. J. Wong, J. R. Hecht, M. Maus, P. M. Grierson, J. B. Sunwoo, J. R. Molina, D. G. Maloney, K. Spencer, H. E. Fuentes Bayne, M. Davila, S. P. Patel, B. Creelan, D. B. Zhen, J. Niu, D. C. Prasad, R. L. Marar, N. A. Bagegni, G. Palluconi, J. Lee, A. Mardiros, W. Bretzlaff, W. J. Langeberg, J. S. Welch, E. Ng, W. Y. Go, K. Kirtane, R. A. Shatsky. Denali-1: a seamless phase 1/2 study of A2B395, a logic-gated, allogeneic, Tmod chimeric antigen receptor T-cell (CAR T) therapy, in patients with triple-negative breast cancer and other EGFR-expressing solid tumors with HLA-A*02 loss of heterozygosity (LOH) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-08-03.

3040 Background: EVEREST-2 is a first-in-human, phase 1/2 trial to assess the safety and efficacy of A2B694, an autologous, logic-gated Tmod CAR T therapy targeted to MSLN, which is normally expressed in the mesothelium and can be upregulated in many solid tumors. A2B694 is designed to overcome challenges of on-target, off-tumor toxicity that have limited other MSLN-targeted approaches by combining a CAR-activating receptor targeting MSLN with a blocker CAR that recognizes HLA-A*02, to distinguish between normal and tumor cells (Tokatlian, et al. J Immunother Cancer. 2022). Methods: Adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression who have progressed after standard-of-care therapy are eligible for EVEREST-2. Enrollment to EVEREST-2 and collection of T-cells occurs through the ongoing prescreening study BASECAMP-1 (NCT04981119). When clinically appropriate, A2B694 is manufactured from cryopreserved T cells, and patients undergo lymphodepletion before A2B694 infusion. The dose-escalation phase is evaluating the safety and tolerability of A2B694 to identify the recommended phase 2 dose (RP2D). Dose escalation was started at 1x10 8 Tmod positive cells (dose level [DL] 1) and will increase up to 14x10 8 in combination with low-dose IL-2 (DL 5). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Results: As of January 15, 2025, 5 participants (median age: 60 years; range, 50-84) have enrolled on EVEREST-2 and received A2B694 at DLs 1-2; participants had ovarian cancer (n = 3), pancreatic cancer (n = 1), and non-small cell lung cancer (n = 1) and had received a median of 4 prior lines of therapy (range, 1-7). Lymphodepleting chemotherapy was well tolerated with no significant cytopenias observed. The most common adverse events were lymphopenia (7 [14.6%]) and decreased appetite (6 [12.5%]). There were no dose-limiting toxicities, cytokine release syndrome, nor related neurotoxicity. One participant was admitted to the hospital for decreased appetite. No long-term toxicities have been noted up to 7.5 months post-infusion. Of the participants who have received A2B694, 5 were efficacy evaluable at DLs 1-2. A2B694 was detected post-infusion in the peripheral blood in all patients. Additionally, A2B694 was detected in an abdominal tumor biopsy from a patient with pancreatic cancer 42 days post-infusion. Conclusions: The logic-gated approach was successful at reducing toxicity seen with prior MSLN-targeted CAR T therapies, and A2B694 showed successful CAR T expansion and tumor infiltration. The maximum tolerated dose has not been reached, and results from the dose-escalation phase continue to determine the RP2D. Clinical trial information: NCT06051695 .

e16408 Background: A2B530 is a logic-gated CEA-targeting Tmod CAR T therapy being evaluated in the EVEREST-1 (NCT05736731) phase 1/2 study. Tmod CAR T therapy addresses challenges of on-target, off-tumor toxicity that have limited application of CAR T therapy to solid tumors (Tabernero, et al. J Clin Oncol. 2017); through use of a CEA-targeted CAR-activating receptor combined with a blocking receptor recognizing HLA-A*02, A2B530 can distinguish between tumor and normal cells (Sandberg, et al. Sci Transl Med. 2022). Methods: Patients with unresectable locally advanced or metastatic solid tumors expressing CEA and tumor-associated HLA-A*02 LOH are enrolled through the ongoing prescreening study BASECAMP-1 (NCT04981119). Eligible patients undergo leukapheresis and, when clinically appropriate, A2B530 is manufactured for the EVEREST-1 study. Patients undergo lymphodepletion before A2B530 infusion. The phase 1 dose escalation will evaluate safety, tolerability, manufacturing feasibility, and biomarkers starting at 1x10 8 Tmod positive cells (dose level [DL] 1) up to 6x10 8 cells and higher doses of lymphodepleting chemotherapy (LDC; DL 5). After the recommended phase 2 dose has been established, dose expansion will confirm safety and activity. Results: As of January 15, 2025, 14 total participants were enrolled on EVEREST-1 and had received A2B530 infusion at DLs 1-5, 4 (29%) with pancreatic (PANC) and 10 (71%) with colorectal cancer. Median age was 57 years (range, 34-76), with a median of 2 prior lines of therapy (range, 1-6). LDC was well tolerated: neutropenia nadired at days 7-10, with recovery in all patients, and no significant episodes of prolonged cytopenia. There were no dose-limiting toxicities, grade > 3 serious adverse events (AEs), or episodes of related neurotoxicity. Most gastrointestinal AEs occurred early and were grade 1 or 2. One patient in DL 2 (2x10 8 cells) experienced possible grade 2 cytokine release syndrome (CRS), which started on day 15 and resolved with supportive therapy within 2 days; later the CRS diagnosis was confounded by a positive blood culture from the participant’s central line. The low rate of CRS is consistent with the low levels of serum cytokines measured up to 28 days post-infusion. Central response assessment has been completed for the 6 participants in DLs 1-2, with a partial response observed in 1 of 3 (33%) of participants with PANC. Peak expansion shows a possible dose response. Conclusions: Treatment with A2B530 was well tolerated, showing potential clinical efficacy and a potential dose response, although these are initial results. Maximum tolerated dose has not been reached, and dose escalation continues to determine the optimal phase 2 dosing of this treatment regimen. Clinical trial information: NCT05736731 .

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.