Welch, John S.

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 .

Objective Identifying eligible patients for precision oncology clinical trials is challenging, particularly for rare molecular subpopulations. To address this challenge, A2 Biotherapeutics developed BASECAMP-1 ( NCT04981119 ), a non-interventional master screening study to identify patients eligible for interventional studies of logic-gated Tmod chimeric antigen receptor T-cell therapies. Eligible patients for these interventional trials have an advanced solid malignancy and are germline human leucocyte antigen (HLA)-A*02 heterozygous, with tumour-associated HLA-A loss of heterozygosity (LOH). HLA-A LOH occurs in ~16% of advanced solid malignancies; therefore, an efficient screening strategy is required. This report describes BASECAMP-1; compares the efficiency of two screening methods; and discusses the broader advantages of BASECAMP-1 beyond efficient enrolment. Methods and analysis Patients are identified for BASECAMP-1 using two approaches. In the traditional approach, common for clinical trials, investigators consent and screen all patients who might be good candidates for cell therapy trials, with no prior knowledge of patient HLA-A type or LOH status. To further optimise our approach, we co-developed with Tempus AI (Tempus) the bioinformatic programme Aware, which identifies potentially eligible patients with tumour-associated HLA-A*02 LOH within a clinico-genomic database that includes linked genomic and transcriptomic sequencing and clinical data collected during routine care. Results Over 42 months of using a traditional approach to identify eligible patients, 1918 patients at 13 study sites were consented and screened for BASECAMP-1; of these, 30 patients with tumour-associated HLA-A*02 LOH were enrolled (~0.7 participants per month). Over the last 30 months of that same period, Tempus Aware screening was implemented and 55 patients with tumour-associated HLA-A*02 LOH were enrolled (~1.8 participants per month). The bioinformatic approach identified more patients than the traditional approach and used sequencing results produced as part of the standard clinical tumour sequencing workflow, reducing resource use and study staff burden. Additional advantages of using a screening study, such as BASECAMP-1, include manufacturing efficiencies and collection of a large dataset of molecular and clinical parameters that can be used to supplement trial analyses. Conclusions The BASECAMP-1 study demonstrates a clinico-genomic screening approach can more efficiently identify patients for precision oncology trials. Furthermore, precision oncology can be enhanced through collaborative data-sharing. Trial registration number NCT04981119 .

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 .

TPS2677 Background: Despite the success in hematologic malignancies, CAR T therapies face significant challenges in solid tumors due to the lack of tumor-specific targets that distinguish cancer from normal cells. EGFR plays a critical role in oncogenesis across several cancers and is often upregulated (TCGA 2022). While monoclonal antibodies targeting EGFR have demonstrated efficacy, these approaches are often limited by on-target, off-tumor toxicities, such as skin rash, which constrains dose escalation and efficacy (Macdonald, et al. J Am Acad Dermatol . 2015). A2B395 is an allogeneic, logic-gated, EGFR-targeted Tmod 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 HLA-A*02. The activator recognizes EGFR on both tumor and normal cells, while the blocker inhibits CAR T activity against normal cells with preserved HLA expression and decreases the risk for graft-versus-host disease (Hamburger, et al. Mol Immunol . 2020). To address potential host-vs-graft response, an shRNA expression module targeting B2M is included in the Tmod construct, which significantly reduces major histocompatibility complex class I levels and subsequent host immune response (DiAndreth, et al. Clin Immunol. 2022). 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 (Grierson, et al. SITC 2024. Abstract 588). A2B395 represents a novel approach for EGFR-expressing solid tumors with HLA-A*02 LOH. Methods: DENALI-1 (NCT06682793) 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 LOH at any time in the course of their disease via next-generation sequencing (Tempus AI, Inc.). Key inclusion criteria include histologically confirmed recurrent unresectable, locally advanced, or metastatic cancers associated with EGFR expression, including colorectal, non-small cell lung, squamous cell head and neck, triple negative breast, 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 phase 1 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 RP2D and collect biomarker data. Phase 2 will assess overall response rate per RECIST v1.1. Clinical trial information: NCT06682793 .

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.

TPS788 Background: MSLN is a desirable candidate for targeted therapy given its limited expression in normal tissues and its overexpression in several malignancies, including PANC, mesothelioma, non-small cell lung, and ovarian cancers (TCGA 2022). MSLN-targeted CAR T and T-cell receptor therapies have shown promising clinical activity; however, on-target, off-tumor toxicity, including fatal events, have occurred (1-3). Autologous logic-gated Tmod CAR T therapies address the challenges of on-target, off-tumor toxicity by combining 2 CARs: activating and blocking receptors. The activator recognizes a tumor-associated antigen present on the surface of both tumor and normal cells; the blocker prevents CAR T activity when it binds HLA-A*02, which is present in normal cells and often lost in tumor cells. Thus, in patients with tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells due to retained HLA-A*02 expression (4). HLA-A*02 LOH is observed in various solid tumors, including ~20% of PANC (5). Two autologous CAR T therapies are currently under investigation: A2B530, targeting carcinoembryonic antigen, in the EVEREST-1 trial, and A2B694, targeting MSLN, in the EVEREST-2 trial, described herein. A2B694 may provide a therapeutic window to treat patients with PANC and other MSLN-expressing solid tumors exhibiting HLA-A*02 LOH due to its unique discriminatory mechanism. Methods: EVEREST-2 (NCT06051695) is a first-in-human, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B694 for recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression, including PANC. Eligible patients must have exhausted options of potentially curative therapy and have recurrent disease. Enrollment to EVEREST-2 occurs through the prescreening study BASECAMP-1 (NCT04981119), in which patients with tumor-associated HLA-A*02 LOH are identified via next-generation sequencing (Tempus AI, Inc) and leukapheresis product is collected. 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 the primary endpoint overall response rate per RECIST v1.1 and secondary endpoints progression-free survival and overall survival. The first patient was dosed on April 24, 2024 and dose escalation is ongoing. 1. Beatty, et al. Gastroenterology . 2018. 2. Haas, et al. Mol Ther . 2023. 3. Hong, et al. ESMO 2021. Abstr 9590. 4. Hamburger, et al. Mol Immunol . 2020. 5. Hecht, et al. J Clin Oncol . 2022. Clinical trial information: NCT06051695 .