Abstract A021: Spatiotemporal Regulation of Chimeric Antigen Receptor T-cells Using Focused Ultrasound Therapy to Enable Use of High-Affinity Binders

CRG-023 Is a Novel Tri-Specific CAR T Product Candidate Engineered to Prevent Antigen Escape and Sustain Durable Anti-Tumor Functionality Against B-Cell Malignancies

The acidic tumor microenvironment (TME) in solid tumors, driven by abnormal metabolism and lactic acid accumulation, suppresses chimeric antigen receptor-T (CAR-T) cell efficacy while posing safety risks from on-target, off-tumor toxicity (OTOT). This study aims to develop a novel CAR-T technology that leverages lactic acid as a tumor-specific trigger to achieve precise control of CAR activity. The objective is to enable adaptation to the acidic TME while maintaining robust anti-tumor efficacy and mitigating OTOT. We engineered a lactic acid-responsive promoter (LARP) using RNA sequencing-identified lactic acid-sensitive genes. This promoter was integrated into HER2-targeting CAR to construct LAR CAR-T cells. CAR expression dynamics under acidic vs neutral conditions were quantified via flow cytometry. Phenotypic profiling (memory markers), in vitro cytotoxicity, and cytokine secretion were assessed. In vivo OTOT was evaluated in our previously constructed humanized HER2 mice, while anti-tumor efficacy and OTOT were further tested in this mouse model bearing tumors. Our findings demonstrate that the LARP responds to lactic acid, leading to increased CAR expression in acidic conditions. The ex vivo-expanded LAR CAR-T cells exhibited an enhanced memory phenotype and superior tumor-killing capacity in vitro under acidity. In vivo, LAR CAR-T cells achieved tumor eradication comparable to conventional CAR-T cells and exhibited significantly enhanced safety profiles, characterized by the absence of acute hepatotoxicity and minimal off-target organ toxicity. Our LARP strategy exploits tumor acidity as a precise low/high switch for CAR-T cells. By restricting potent CAR expression to the acidic TME while minimizing activity in normal tissues, LAR CAR-T overcomes key barriers of efficacy and OTOT in solid tumors. This lactic acid-sensing paradigm offers a clinically translatable platform for precise immunotherapy.

Purpose: Mesothelin (MSLN) is a surface antigen associated with tumor invasion and is overexpressed in mesothelioma and lung, pancreatic, ovarian, and other cancers. Chimeric antigen receptor (CAR) T cells targeting MSLN have been tested in patients with MSLN-positive solid tumors. Despite reported patient fatalities in trials sponsored by PENN, Memorial Sloan Kettering/Atara Bio, and MD Anderson/TCR2, the design approach for MSLN CAR remains constructing high affinity CARs to maximize target cell killing. In this study, we designed MSLN CARs with two different antibodies: one targeting human MSLN specifically and another binding both human and mouse MLSN with comparable affinities. The objectives are to 1) elucidate the risk of fatal on-target off-tumor toxicity associated with high affinity MSLN CAR, and 2) demonstrate how optimizing the affinity of CARs can render CAR T cells to spare normal tissues while retaining cytotoxicity against tumors. Methods: To asses on-target off-tumor toxicity in animal models, we developed a CAR using a nanobody isolated from an immune alpaca library with nanomolar affinity for both human and mouse MSLN (labeled as hmMSLN CAR). For comparison, we constructed a CAR using a scFv specific only to human MSLN, designated hMSLN CAR. Each CAR is designed to co-express somatostatin receptor 2 to enable PET/CT imaging of CAR T cells with 18F-NOTA-Octreotide. Anti-tumor activities and toxicities were assessed in NSG mice using MSTO-211H cell line expressing human MSLN. Results: In NSG mice bearing subcutaneous MSTO-211H tumors, hMSLN CAR T cells eliminated tumors rapidly without inducing any overt toxicity. In contrast, hmMSLN CAR T cells not only failed to control tumor growth but also caused severe toxicity, resulting in significant body weight loss and necessitating euthanasia. PET/CT imaging revealed while hMSLN CAR T cells expanded exclusively within the tumor and subsequently contracted after tumor elimination, hmMSLN CAR T cells exhibited extensive infiltration and expansion in the lungs, liver, and bone marrow, indicating off-tumor, normal tissue-driven proliferation of CAR T cells. To test if affinity-tuned hmMSLN CAR T cells can selectively target tumors, we used a yeast display system to screen for affinity variants of the hmMSLN CAR. We identified one micromolar affinity CAR that binds approximately 1,000 times weaker to MSLN than the parental CAR. In stark contrast to the onset of severe toxicity soon after the administration of the parental hmMSLN CAR T cells, animals treated with micromolar affinity hmMSLN CAR T cells exhibited tumor regression without overt toxicity. Conclusions: Our findings underscore the risks of using high affinity CAR against MSLN, which may cause unpredictable on-target, off-tumor toxicities. Fine tuning the affinity of MSLN CAR offers a promising strategy for developing a potent and safe CAR for the treatment of MSLN-expressing solid tumors. Citation Format: Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Ju Y. Son, Niloufar Salehi, Irene M. Min, Moonsoo M. Jin. Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 41.

Mantle cell lymphoma (MCL) remains difficult to cure, and responses to chimeric antigen receptor (CAR) T-cell therapy vary in part due to modulation of target-antigen levels. We hypothesized that enhancing Ikaros transcription factor IKZF1, either directly by lentiviral overexpression or indirectly through inhibition of casein kinase 2 alpha (CK2α), a negative regulator of IKZF1 stability, would increase CD19 transcriptional output and thereby enhance CAR T-cell recognition and clearance of MCL cells. Human MCL cell lines JeKo-1 and Mino were engineered to overexpress IKZF1 using lentiviral vectors (controls: parental, empty-vector). Parallel experiments leveraged CK2α inhibition with silmitasertib to increase endogenous IKZF1 protein stability via reduced proteasomal turnover. CD19 surface density was quantified by flow cytometry with calibrated beads, CD19 transcripts by RT-qPCR, and promoter activity using CD19-luciferase reporters. Antitumor activity of CD19-directed CAR T cells was assessed in short-term co-cultures across effector:target (E:T) ratios, with target-cell viability (plate reader and flow cytometry) and cytokines (Lumit bioluminescence-based assay) collected at defined timepoints. For in vivo validation, luciferase-RFP-tagged JeKo-1/Mino xenografts were established in NSG mice followed by CAR T-cell infusion; tumor burden (bioluminescence) and survival were monitored longitudinally. IKZF1 overexpression increased CD19 mRNA and protein in JeKo-1 and Mino cells by ∼2.5- and 2.30-fold. CK2α inhibition produced a concordant phenotype, elevating endogenous IKZF1 and yielding proportional increases in CD19 surface density and transcript abundance. Promoter-reporter assays showed increased CD19 promoter activity. In vitro, CAR T-cell killing was enhanced by IKZF1 modulation: at E:T 1:1, viable target cells at hour 4 were 41.5% (IKZF1-OE) vs 63% (control), and the survival AUC decreased by 21.5%. CAR T-cell function mirrored antigen upregulation, with elevated IFN-γ, TNF, and IL-2 secretion (all p < 0.001) and reduced exhaustion-marker induction at 24 hours. CK2α-inhibited conditions similarly enhanced CAR T activation, supporting a mechanistic link between IKZF1 stabilization, heightened antigen availability and increased CAR T-cell target recognition and killing. In conclusion, increased IKZF1, either by direct overexpression or through CK2α inhibition, upregulates CD19 transcription and surface density in MCL, resulting in more rapid and durable CAR T-cell cytotoxicity in vitro and in vivo. These findings identify CK2α–IKZF1 as a druggable regulatory axis controlling antigen availability and highlight a complementary strategy to enhance CAR T-cell responses in MCL by maintaining or boosting CD19 expression. Ongoing CHIP-seq experiments will define IKZF1 occupancy at the CD19 locus and durability and safety across additional MCL in vivo models. Citation Format: Anya K. Sondhi, Tulasigeri M. Totiger, Stanzin Idga, Jonathan Schatz, Jay Spiegel, Justin Taylor. Transcriptional regulation of CD19 by IKZF1 enhances CAR T-Cell immune targeting in mantle cell lymphoma [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C009.

TPS2086 Background: Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor. More than 300,000 new cases are diagnosed globally with over 250,000 deaths each year (Sung H, et al. CA Cancer J Clin. 2021). Patients with recurrent GBM have a poor prognosis, with limited treatment options and a median survival of less than 1 year (Gallego. Curr Oncol, 2015). While prior attempts to treat GBM with chimeric antigen receptor (CAR) T-cells have been limited by tumor heterogeneity, chlorotoxin (CLTX)-directed CAR T-cells in mice demonstrated broad anti-tumor activity and prolonged survival with no off-tumor toxicity or antigen escape (Wang, et al. Sci Transl Med, 2020). CLTX, a 36–amino acid peptide identified in scorpion venom, selectively binds to malignant glioma cells through matrix metalloproteinase-2 (MMP2) and clinical administration of CLTX-based biologics has been well tolerated in patients (Mamelak, et al. J Clin Oncol, 2005, Patil, et al. Neurosurgery, 2019). CHM 1101 is the first CAR T to utilize CLTX as its tumor targeting domain for autologous CAR T-cell therapy. A single-center first-in-human phase 1 study of CHM 1101 in patients with recurrent GBM is ongoing. Methods: Clinical Trial NCT05627323 is a phase 1b, multi-center study of CHM 1101 in adult subjects with MMP2+ recurrent or progressive GBM after standard therapy. After leukapheresis and tumor resection, pts will receive CHM 1101 at Dose Level 1 (240 × 106 CAR T cells) or Dose Level 2 (440 x 106d CAR T-cells), divided across 3 once-weekly intracranial (intracavitary and intraventricular) administrations; after disease assessment at 28 days, additional doses of CHM 1101 can be administered on a weekly schedule. Eligible subjects have a prior histologically confirmed diagnosis of a grade 4 GBM, a grade 2 or 3 malignant glioma with radiographic progression consistent with a grade 4 GBM (IDH wild type), grade 4 diffuse astrocytoma (IDH mutant), or a unifocal relapse of GBM. MMP2+ tumor expression is confirmed by IHC (≥20% moderate/high MMP2 score [2+ or 3+]). The primary endpoint is safety; key secondary endpoints include feasibility, progression-free survival, overall survival, response rate (RANO criteria), and cellular kinetics. Recruitment is ongoing. Clinical trial information: NCT05627323 .

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia

Chimeric antigen receptor (CAR) T cell therapy has proven to be highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. These efforts face multiple challenges, including off-tumor target expression and checkpoint inhibition of CAR T cell activity in the tumor microenvironment. CAR T cells are much more potent compared to antibody therapeutics, therefore there is a need for more stringent CAR T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within the context of intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. In this study we employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model, which expresses both BCMA and ROR1. The anti-ROR1 CAR T cells used in this study recognized both the mouse and human proteins whereas the anti-BCMA CAR T cells recognized only the human protein. RNA ISH revealed that BCMA was only expressed in the xenograft tumor and in no mouse organs, while ROR1 was found to be expressed in the xenograft tumor as well as at low levels in mouse lung and liver. Duplex RNA ISH assay with probes targeting the CAR 3’ UTR and either IFNG or GZMB mRNA allowed highly sensitive and specific detection of CAR T cells and their activation state in both tumor and normal tissues from vehicle, anti-ROR1 CAR T cell, or anti-BCMA CAR T cell treated mice. Activated anti-BCMA CAR T cells expressing GZMB and IFNG were found only in the xenograft tumor, where BCMA was expressed. In contrast, activated anti-ROR1 CAR T cells were found almost exclusively in mouse lung and liver with very few anti-ROR1 CAR T cells being found in the xenograft tumor, consistent with the previously observed pulmonary and hepatic toxicity of anti-ROR1 CAR T cells that was not predicted by IHC analysis of ROR1 protein presumably due to lack of antibody sensitivity. Lastly, we employed a multiplex ISH-IHC approach to confirm the presence of activated anti-BCMA CAR T cells in the xenograft tumor through simultaneous detection of the BCMA CAR 3’ UTR, IFNG, GZMB, and CD3, validating the anti-tumor activity of anti-BCMA CAR T cells. These data thus demonstrate how the RNAscope ISH assay can be utilized for CAR T efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR T cell pharmacodynamics and activation in tumor models. This technology has equal utility in understanding both CAR T and TCR-T cell activity in patient tumors. Citation Format: Helly Pimentel, Helen Jarnagin, Hailing Zong, Courtney Todorov, Kenneth Ganley, Fay Eng, Kevin Friedman, Molly Perkins, Shannon Grande, Courtney M. Anderson, Bingqing Zhang, Christopher Bunker, James B. Rottman, Xiao-Jun Ma. Preclinical safety, biodistribution, and tumor infiltration analysis of CAR T cell targets using in situ hybridization technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2324.

Two successful regional gene therapies have emerged in recent years: chimeric antigen receptor (CAR) T-cell therapy and oncolytic virus therapy. Coupled with the recent advances in the understanding of solid tumor immunology, the success of these therapies illustrates how regional therapy can be combined with efficient gene transfer and immune stimulation to treat solid tumors. CAR T cells and oncolytic viruses leverage the fundamental advantages of regional delivery to accomplish the following three objectives: 1. Enhance delivery of therapeutics to the tumor. Cancer-targeted gene therapy can be inefficient when infused intravenously, owing to the increased volume of distribution, sequestration in other organs, and poor or altered vascularity, which limit drug perfusion into tumor tissue. Regional CAR T-cell administration can bypass these limitations. Oncolytic viruses can be regionally infused or intralesionally injected. 2. Enhance targeting of cancer cells, thereby limiting normal-tissue toxicity. CAR T cells and oncolytic viruses are genetically modified to optimize cancer-specific targeting. The flexibility afforded by genetic engineering allows preferential targeting, infection of cancer cells, and sparing of normal tissues. 3. Generate both a local and a systemic immune response that can target metastatic disease and prevent tumor recurrence. Perhaps, the most impressive feature of these local therapies is their ability to generate an immune response that extends beyond the primary tumor to establish systemic immune surveillance. This immune response follows the tenets of the adaptive immune system: it establishes circulating immune memory that is capable of self-renewal and is long-lasting. Regional delivery of CAR T cells elicits a robust local immune response and establishes systemic immunity. Oncolytic viruses not only target tumors via local cancer cell destruction but also generate a local and systemic immune response. The regression of metastatic lesions following local delivery of oncolytic virus exemplifies the potential to generate systemic immune responses.

INSPIRED Symposium Part 5: Expanding the Use of CAR T Cells in Children and Young Adults

The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAP(hi) stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8(+) T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted.

<div>Abstract<p>The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAP<sup>hi</sup> stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8<sup>+</sup> T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted. <i>Cancer Immunol Res; 2(2); 154–66. ©2013 AACR</i>.</p></div>

Background: Gastric cancer ranks among the top 5 cancer types in terms of both incidence and mortality, with a 5-year overall survival rate of only 29%. CD44 variant 6 (CD44v6) is an isoform of CD44 and is identified in 70% of gastric cancer cases. CD44v6 is considered a marker of poor prognosis, and its expression varies among different types of gastric cancer. CD44v6 has a role in apoptosis resistance, metastasis, and tumor progression, and is shown to be enriched in recurrent tumors. CD44v6 expression in normal tissue is very low and is restricted to certain cell types. Therefore, CD44v6 is a promising target for targeted therapies against gastric cancer. In this study, we aim to develop a chimeric antigen receptor (CAR) T cell therapy targeting CD44v6 and evaluate its functionality, cytotoxic activity and antitumoral effect. The scFv was derived from the monoclonal antibody (mAb) known as bivatuzumab (BIWA-4), a humanized mAb that has been used in clinical studies as radiotherapy and as antibody drug conjugates. Methods: Plasmid containing the CAR sequence with the BIWA-4 scFv was synthesized, and Jurkat T cell line and primary human T cells were transduced with lentivirus containing the CAR sequence. Jurkat cell line was used to measure binding to recombinant protein and assess CD69 expression after antigen exposure. Primary T cells transduced with CAR were co-cultured at different E:T ratios with gastric cancer cell lines (AGS, SNU 638, and MKN45) with different levels of expression of CD44v6. Target cell killing was measured by flow cytometry, bioluminescence, and fluorescence microscopy. T cell exhaustion and exhaustion phenotype were evaluated prior killing assays. For in vivo experiments, NSG mice will be engrafted with gastric cancer cell lines and tumor progression will be evaluated by IVIS. Results: While high affinity variants of bivatuzumab have been used to construct CAR (BIWA-8), ours is the first study that used medium affinity variant known as BIWA-4. Jurkat cells transduced with the car construct demonstrated specific binding to the recombinant monomeric CD44v6, which triggered surface expression of early T cell activation marker CD69. With varying concentrations of monomeric CD44v6, the affinity of CAR to CD44v6 was determined to be ∼100 nM KD. Transduction efficiency of primary T cells remained stable, reaching ∼60% after 10 days of expansion. Most CAR T cells exhibited effector memory phenotype, with low levels of PD1. CD44v6 CAR T killing in vitro was correlated with the E: T ratio and the level of antigen expression, demonstrating specificity of CAR and avoiding off-target effects with CD44v6 negative cells. Conclusion: We have successfully developed CAR T cells possessing medium affinity against CD44v6 and confirmed CAR-specific killing of CD44v6 positive gastric cancer cells. Citation Format: gonzalo gonzalez, Moonsoo Jin, Yanping Yang, Elizabeth Ortiz, Yogindra Vedvyas. Cytotoxic and antitumoral effect of an anti-CD44v6 CAR-T against gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6113.

Introduction: Harnessing the potent cytotoxicity of chimeric antigen receptor T (CAR-T) cells has revolutionized cancer immunotherapy. A key factor influencing CAR-T cell efficacy is target antigen density on cancer cells. Human epidermal growth factor receptor 2 (HER2), overexpressed in various cancers like ovarian and lung carcinomas, is a promising target for CAR-T cell therapy. Traditional cytotoxicity assays generally provide only static endpoint measurements and often rely on 2D cell culture models, which fail to capture the structural complexity of tumors in vivo—a factor crucial to CAR-T cell efficacy. Live-cell imaging offers a solution by enabling non-invasive, real-time monitoring of cytotoxicity. Therefore, this study aims to explore the dynamics of CAR-T cell-mediated cytotoxicity against cancer cells with varying HER2 expression levels, comparing outcomes between 2D and 3D cell culture models using the Omni platform. Methods: Monolayers and spheroids of SKOV3 (ovarian carcinoma) and A549 (lung adenocarcinoma) cells, tagged with GFP, were exposed to HER2 CAR-T cells at different Effector : Target (E:T) ratios. Using the Omni platform, hourly brightfield and fluorescent images were captured over 72 hours assess cancer cell growth and cytotoxicity and quantify the cytolysis of the fluorescent target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent intensity of treated wells to no treatment control wells. Results: The study demonstrated a dose-dependent cytotoxic effect of HER2 CAR-T cells that correlated with increasing amounts of CAR-T cells. Comparing SKOV3 cells with A549 cells showed higher sensitivity and earlier response for all E:T ratios. At 72 hours post HER-2 CAR T-cell addition, the 5:1 group demonstrated approximately 82.4% cytolysis of SKOV3 cells, while it demonstrated 40.3% cytolysis of A549 cells. In comparing the cytotoxic effect of HER2 CAR-T cells on SKOV3 spheroids versus monolayers, the 3D spheroid model exhibited a more rapid and efficient dose-dependent response. At 72 hours post HER-2 CAR T-cell addition, the 5:1 group demonstrated approximately 82.4% cytolysis of SKOV3 cells in spheroid, while it demonstrated 61.1% cytolysis of SKOV3 cells in monolayer. Conclusion: This study underscores the importance of target antigen density in determining CAR-T cell efficacy, as SKOV3 cells, with higher HER2 expression, were more prone to CAR-T cell killing compared to A549 cells. Additionally, it also highlights the critical role of the assay model type, 2D versus 3D, in accurately assessing cytotoxicity. The Omni platform provided dynamic, real-time insights into cell viability and cytotoxicity, highlighting its potential to optimize immunotherapy strategies. Citation Format: Skylar Dewees, Nathalie Opdam - van de Laar, Stacie Chvatal, Danielle Califano, Ben Streeter. Exploring CAR-T cell killing dynamics through live-cell fluorescence microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3908.

A multiparametric CAR T cell potency assay that employs real-time cellular impedance and live cell imaging