Liechty, Kirstin B.

TPS649 Background: While advancements in early-stage breast cancer (ESBC) treatment have improved survival, disease recurrence and therapeutic resistance remain significant challenges. Current biomarkers guide initial treatment, yet novel diagnostics are required to improve risk stratification and recurrence prediction. Circulating tumor DNA (ctDNA) offers a non-invasive method for detecting minimal residual disease (MRD), potentially enabling the identification of molecular relapse before radiographic progression. However, deeper validation of ctDNA kinetics across the treatment continuum is needed to inform precision oncology strategies in ESBC. Additionally, the logistical complexity of tissue-dependent assays presents a barrier to scalability. In order to address these gaps, the purpose of the GEMINI-BREAST study is to evaluate a methylation-based, tissue-free ctDNA assay (Tempus xM) and integrate this with comprehensive clinical and multi-omic data to determine prognostic utility for invasive disease-free survival (iDFS) and long term outcomes in patients with ESBC. Methods: GEMINI-BREAST (NCT07211178) is a prospective, non-interventional, multi-center study enrolling up to 900 participants with ESBC treated with curative intent. Eligible patients enroll into one of three analytical cohorts (n ~ 300 each): Cohort 1 enrolls pre-neoadjuvant therapy (high-risk HR+/HER2- [Stage II-III], HER2+ [Stage II-III], or TNBC [Stage I-III]); Cohort 2 enrolls post-surgery/pre-adjuvant therapy (same subtypes, no evidence of disease); and Cohort 3 enrolls long-term survivors (high-risk HR+/HER2- [Stage II-III], ≥ 5 years since diagnosis). Longitudinal blood samples for ctDNA analysis and archival tissue for NGS are collected at standard-of-care intervals, including, as applicable: pre-neoadjuvant, neoadjuvant on-treatment (weeks 3, 6, 12) and post-treatment, post-surgical/pre-adjuvant, adjuvant on-treatment (every 3 months), end of definitive therapy, surveillance for up to 5 years (every 3-6 months for Cohorts 1/2; every 6-12 months for Cohort 3), and at progression/recurrence. The primary objective is to evaluate the association between ctDNA dynamics (Cohort 1) or MRD status (Cohort 2/3) and iDFS, with secondary endpoints assessing overall survival, lead time to recurrence, and the predictive value of ctDNA clearance for pathological complete response. Recruitment is currently ongoing at U.S. sites. Clinical trial information: NCT07211178 .

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 .

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 .

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]

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 .