Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in cancer treatment. Nevertheless, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which have been suggested to cause CAR T-cell fratricide and exhaustion. Whether CMT indeed causes CAR T-cell dysfunction and the molecular mechanisms conferring CMT remain unknown. Using a selective degrader of trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen on the CAR T-cell surface directly causes CAR T-cell fratricide and exhaustion. By performing small molecule screening using a custom high-throughput CMT screening assay, we found that the cysteine protease cathepsin B is essential for CMT and that inhibition of cathepsin B is sufficient to prevent CAR T-cell fratricide and exhaustion, leading to improved long-term in vitro and in vivo CAR T-cell persistence and in vitro antitumor activity. Our data demonstrate that it is feasible to separate CMT from cytotoxic activity, that CAR T-cell persistence, a key factor associated with clinical CAR T-cell efficacy, is directly linked to cathepsin B activity in CAR T cells, and that it is possible to improve CAR T-cell function through selective inhibition of CMT.

2561 Background: BCMA-directed chimeric antigen receptor (CAR) T-cell therapies induce deep responses in relapsed/refractory multiple myeloma (MM), but increasing potency has expanded the spectrum of treatment-related toxicities. While cytokine release syndrome and immune effector cell–associated neurotoxicity are well characterized, the consequences of extreme and prolonged CAR T-cell persistence remain poorly understood, particularly as CAR T-cell strategies are extended to solid tumors. Methods: We performed longitudinal, multi-compartment immunomonitoring in a patient with high-risk MM treated with ciltacabtagene autoleucel (cilta-cel) who developed an unusual leukemia-like clinical course. Peripheral blood (PB), bone marrow (BM), and cerebrospinal fluid (CSF) samples were analyzed by multiparameter flow cytometry, functional cytokine secretion assays, multiplex cytokine profiling, single-cell RNA sequencing with paired T-cell receptor (TCR) repertoire analysis, and lentiviral CAR integration site mapping. Results: Following cilta-cel infusion, the patient developed massive and persistent expansion of non-malignant CD8⁺ effector-memory CAR T cells, comprising >90% of circulating lymphocytes and ~95% of BM lymphocytes, with absence of detectable non-transduced T cells. Despite achieving complete remission of MM, extensive BM infiltration by CAR T cells was associated with marked marrow hypocellularity, trilineage hypoplasia, and prolonged pancytopenia. A distinct trafficking-competent CD8⁺ effector-memory subset (TEM5) was selectively enriched in CSF and associated with severe neurotoxicity and a local pro-inflammatory cytokine milieu. Single-cell transcriptomics demonstrated a highly migratory, cytotoxic effector program with suppressed proliferation, MAPK/TCR signaling, and tissue-residency signatures. TCR repertoire and integration site analyses confirmed polyclonality and excluded malignant transformation or insertional oncogenesis. Sustained CAR T-cell dominance coincided with profound hypogammaglobulinemia, failure of immune reconstitution, recurrent life-threatening infections, and ultimately fatal sepsis. Conclusions: These findings define a previously underrecognized toxicity paradigm of BCMA CAR T-cell therapy characterized by pathologic immune dominance and extreme persistence of cytotoxic CAR T cells, leading to marrow failure, neurotoxicity, and lethal immunosuppression. Extended multi-parametric immunomonitoring may identify patients at risk and inform risk-adapted management strategies, and this principle has potential implications for the design of next-generation CAR T-cell therapies, including for solid tumors.

We performed the first in-depth, comparative and prospective biomonitoring of Multiple Myeloma (MM) patients (N = 39) receiving ciltacabtagene autoleucel (cilta-cel) or idecabtagene vicleucel (ide-cel) chimeric antigen receptor T cells (CAR T) in the real-world setting. In cilta-cel patients response rates were higher and atypical neurotoxicities/infections more frequent. Peak CAR T counts were significantly higher in cilta-cel patients, driven by CD4+ CAR expansion, correlating with clinical responses. Expansion of cilta-cel cells was associated with higher CAR and CD27 expression while, in contrast to ide-cel, there was no correlation between TIM3 expression and CAR T proliferation. Cilta-cel CAR T expansion was followed by a CAR-specific switch from proliferation-associated genes to genes/surface markers indicating effector/memory function. The longer persistence of cilta-cel CAR T was associated with increased IL-7R expression; in vitro data showed persistent antigen-independent activation and higher metabolic activity of cilta-cel vs. ide-cel CAR T. Among cilta-cel-treated patients experiencing atypical neurotoxicities, central nervous system (CNS)-infiltrating, effector-type CAR T presented a distinct inflammatory phenotypic/cytokine-expression profile. This in-depth biomonitoring report following real-world cilta-cel or ide-cel highlights intrinsic biological differences between BCMA-targeting CAR T products, potentially explaining differences in clinical activity and toxicity. Our findings may guide optimization of cellular immunotherapy strategies in MM.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in cancer treatment. Still, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which has been suggested to cause CAR T cell fratricide and exhaustion. Whether CMT directly causes CAR T cell dysfunction and the molecular mechanism conferring CMT remain unknown. Here, we developed a system for the selective degradation of trogocytosed antigen. Removal of trogocytosed antigen resulted in more CAR T cells and lower levels of TIM-3 and LAG-3, demonstrating that the presence of trogocytosed antigen on the CAR T cell surface directly causes CAR T cell fratricide and exhaustion. By performing a small molecule screening using a custom high throughput CMT-screening assay, we found that the cysteine protease cathepsin B is essential for CMT and that inhibition of cathepsin B is sufficient to prevent CAR T cell fratricide and exhaustion without compromising antitumor activity. Our data demonstrate that it is feasible to separate CMT from cytotoxic activity and that CAR T cell persistence, a key factor associated with clinical CAR T cell efficacy, is directly linked to cathepsin B activity in CAR T cells. Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Interleukin 16 (IL-16) is a cytokine that is overexpressed in autoimmune diseases and cancer. The function of IL-16 largely remains unknown although chemotaxis and induction of signaling in T cells through interaction with CD4 has been suggested. Here, we work to clarify the role of IL-16 in T cell physiology. To assess IL-16 binding to T cells, resting and activated human peripheral blood mononuclear cells were stained with fluorescently labeled IL-16 and evaluated by flow cytometry. Resting CD4 and CD8 T cells showed only minimal binding to IL-16, questioning the proposed high-affinity interaction between IL-16 and CD4. In contrast, IL-16 efficiently bound to activated CD4+ and CD8+ T cells, with T cell subsets showing differential levels of binding to IL-16 (TN > TCM > TEM > TEMRA). While T cells required prior activation to bind IL-16, the level of activation did not correlate with IL-16 binding. In mixed lymphocyte reactions using splenocytes from MHC-mismatched C57BL/6 mice, we found that addition of recombinant IL-16 significantly increased T cell activation but not IL-2 secretion. Experiments are ongoing to determine the molecular mechanism behind IL-16-mediated T cell activation and to further delineate the phenotype of IL-16-binding T cells in order to guide identification of alternative receptors for IL-16. This work will inform future therapeutic strategies targeting IL-16 in autoimmune diseases and cancer. Supported by NIAID T32AI095190; Maryland Department of Health’s Cigarette Restitution Fund Program (CH-649-CRF). Cytokines and Chemokines and Their Receptors (CCR)

Idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel) are approved chimeric antigen receptor T cell (CAR T) therapies for multiple myeloma. Unfortunately, most patients receiving these treatments will experience toxicities and/or relapse highlighting the need for optimizing CAR T strategies. We performed the first in-depth, comparative and prospective biomonitoring of patients (N=39) receiving cilta-cel or ide-cel in the real-world setting. Cilta-cel response rates were higher, although not statistically significant, and atypical neurotoxicities/infections were more frequent in the cilta-cel group. Peak circulating CAR T counts were significantly higher in cilta-cel patients, driven by a pronounced CD4+ CAR T expansion, and correlated with depth of clinical response. The pronounced expansion of cilta-cel CAR T was associated with higher CAR expression and overexpression of CD27. The cilta-cel CAR T expansion was followed by a CAR-specific switch from proliferation-associated genes to genes/surface markers associated with effector/memory (EM) function. The comparably longer persistence of cilta-cell CAR T was associated with increased resistance to exhaustion and increased IL-7R expression; in vitro data showed persistent and antigen-independent activation and higher metabolic activity of cilta-cel vs. ide-cel CAR T. Among cilta-cel-treated patients experiencing atypical neurotoxicities, central nervous system (CNS)-infiltrating, effector-type cilta-cel CAR T presented a distinct inflammatory phenotypic/cytokine-expression profile. This first report of in-depth, comparative patient biomonitoring following real-world cilta-cel or ide-cel therapy highlights intrinsic biological differences between these BCMA-targeting CAR T products, potentially explaining differences in clinical activity and toxicity. Our findings may guide the optimization of cellular immunotherapy strategies in myeloma patients. Djordje Atanackovic, Tim Luetkens, Dina Schneider, Peirong Hu, Xu Wang, Amol C. Shetty, Luke Tallon, Imari Patel, Rohan Singh, Etse Gebru, Rediet Mulatu, Destiny Omili, Daniel Yamoah, Xiaoxuan Fan, Aerielle Matsangos, Patricia Lesho, Kenneth A. Dietze, Ariel A. Fromowitz, Kim Hankey, Saurabh Dahiya, Jean A. Yared, Nancy M. Hardy, Rima Koka, Michael E. Kallen, Ashraf Badros, Aaron P. Rapoport, Mehmet Kocoglu. Distinct expansion, phenotype, function, and toxicity of cilta-cel vs. ide-cel CAR T cells in the real world [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 869.

There are currently no approved therapeutic approaches for the targeting of intracellular disease processes using biologics, such as monoclonal antibodies. A major hurdle to this approach is the efficient delivery of biologics across the plasma membrane of the respective target cells. Here, we develop a novel cell therapy allowing the efficient delivery of such payloads using naturally occurring fusogens causing transient membrane-membrane fusion between the engineered cells and target cells. Following identification of fusogens that are amenable to immune cell engineering, by not showing syncytia formation or altering viability, we demonstrate that stable expression of these fusogens can be combined with the co-expression of various payloads, such as DNA-modifying enzymes, single-chain variable fragments, or enzymes. We show that expression of the fusogen is sufficient to allow efficient payload transfer to different target cell types, including epithelial cells and lymphocytes. Using quantitative RT-PCR, we show that mRNA encoding the respective payload is efficiently transferred from the engineered cell to the target cells. Using various model systems, we demonstrate that the transferred payloads are functional in target cells, by demonstrating targeted gene editing using recombinases and base editors, protein degradation, and luciferase activity. This new cell therapy renders acute and chronic intracellular disease processes druggable by genetically encoded therapeutics. Department of Defense CDMRP W81XWH-22-ALSRP-TIA; Maryland Department of Health’s Cigarette Restitution Fund Program CH-649-CRF; NIH P30CA134274 Technological Innovations in Immunology (TECH)

Interleukin-16 (IL-16) is a pleiotropic cytokine secreted by lymphocytes upon stimulation. IL-16 is implicated in the pathogenesis of autoimmune diseases and cancer and preclinical models suggest that blocking IL-16 signaling has therapeutic potential. In this project, we aimed to target IL-16 using engineered antibodies. Sweeping antibodies (swAbs) contain two modifications, pH-dependent antigen binding and enhanced neonatal Fc receptor (FcRn) binding, resulting in 50-1,000-fold increased clearance of target antigen compared to wildtype (WT) antibodies. Here, we attempted to convert two WT anti-IL-16 antibodies into swAbs: 1) a novel, fully-human, high affinity antibody developed in our lab (3B6) and 2) a previously established mouse antibody (14.1). We used histidine-scanning mutagenesis of the variable regions to introduce pH-dependent binding, which was quantified by custom ELISAs/TRFIAs and bio-layer interferometry. Mutagenesis of 14.1 produced three highly pH-dependent binders and, in vitro assays using these IL-16 swAbs showed enhanced cellular uptake and degradation of IL-16 compared to WT antibodies. We are currently determining biodistribution and therapeutic activity of these antibodies in vivo. In contrast, successive rounds of engineering pH dependency in 3B6 were unsuccessful. We are currently determining the epitope of 3B6 to better understand its resistance to swAb engineering. This project informs swAb engineering and the therapeutic targeting of IL-16. Supported by NIAID T32AI095190; Maryland Department of Health’s Cigarette Restitution Fund Program (CH-649-CRF). Therapeutic Approaches to Autoimmunity (THER)

Targeting soluble antigens using conventional monoclonal antibodies is challenging due to high levels of antigen and limited antigen clearance per antibody molecule. Sweeping antibodies are engineered monoclonal antibodies that more efficiently clear soluble antigens than conventional antibodies. Sweeping antibodies contain two modifications: (1) pH-dependent antigen binding to facilitate lysosomal degradation of the targeted antigen while allowing antibody recycling and (2) enhanced neonatal Fc receptor (FcRn) engagement, resulting in 50-1,000-fold increased clearance of target antigen compared to conventional antibodies. The pleiotropic cytokine interleukin 16 (IL-16) has been proposed as a promising therapeutic target for monoclonal antibody therapy, due to its high expression and potential disease-promoting function in autoimmune diseases and cancer. Here, we develop the first fully human antibody as well as multiple sweeping antibodies targeting IL-16. We demonstrate that amenability to the introduction of pH-dependent binding into anti-IL-16 antibodies is correlated with epitope size and proximity to a positively charged IL-16 residue, informing future sweeping antibody development. We demonstrate that anti-IL-16 sweeping antibodies exhibit significantly increased antibody recycling and IL-16 degradation indicating that these molecules are a superior approach for the therapeutic targeting of IL-16 compared to conventional antibodies.

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is an effective treatment for cancer patients. While CAR T cells have shown remarkable efficacy, most patients receiving CAR T cell therapy eventually relapse. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism involving the transfer of cell surface proteins from tumor cells to CAR T cells. CMT results in antigen-negative tumor cells and correlates with reduced CAR T cell persistence, possibly due to increased CAR T cell fratricide or exhaustion. To date it has not been conclusively demonstrated that antigens transferred by CMT are the cause of fratricide or exhaustion. To answer this question, we developed a system to rapidly degrade trogocytosed protein in CAR T cells. Using this system, we show that the transfer of CD19 to CAR T cells directly causes CAR T cell fratricide and exhaustion. We hypothesize that reducing CMT will increase CAR T cell persistence. To identify molecular mechanisms driving CMT as potential therapeutic targets, we inhibited various proteins essential for cell adhesion, endocytosis, actin polymerization, or antigen processing using small molecule inhibitors. We found that inhibition of the cysteine protease Cathepsin B (CTSB), with a membrane-permeable (Ca-074) or membrane-impermeable (Ca-074-Me) inhibitor, significantly reduced CMT without affecting CAR T cell cytotoxicity. We hypothesize that CTSB inhibition could be a potential therapeutic approach to limit CMT.CTSB activity is regulated by the protein Cystatin A (CSTA). Both Ca-074-Me and CSTA sterically block access to the active site cysteine of CTSB. We found that overexpression of CSTA in CAR T cells significantly reduced CTSB activity and reduced antigen transfer. CSTA overexpression minimized antigen loss on tumor cells, indicating that CTSB inhibition blocks CMT early during the trogocytic process. CSTA overexpression reduced CAR T cell exhaustion but did not alter their expansion, tumor cell killing, or phenotype, indicating that this approach selectively inhibits CMT. Next, we assessed the effect of CSTA overexpression on CAR T cell persistence in vivo. NALM6 tumor-bearing NSG mice were injected with conventional or CSTA-overexpressing CD19 CAR T cells. Four weeks after CAR T cell injection, we observed substantially increased CAR T cell numbers in the blood (10.6x, p=0.0317), bone marrow (7.3x, p=0.0159), and spleens (5.6x, p=0.0556) of mice treated with CSTA-overexpressing CAR T cells. Taken together, we provide the first experimental evidence that CMT directly causes fratricide and exhaustion, reducing CAR T cell persistence. We demonstrate that CMT can be targeted efficiently by overexpressing the CTSB inhibitor CSTA in CAR T cells, resulting in substantially increased CAR T cell persistence. This represents a promising approach to improve CAR T cell efficacy and limit the occurrence of relapse in patients receiving CAR T cell therapy. Citation Format: Kenneth A. Dietze, Michael L. Olson, Etse Gebru, Djordje Atanackovic, Aaron P. Rapoport, Tim Luetkens. Targeting trogocytosis through cathepsin B inhibition enhances CAR T cell persistence [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 43.

BACKGROUND AIMS:Chimeric antigen receptor (CAR) T-cell (CAR-T) therapies have revolutionized the treatment of B-cell lymphomas. Unfortunately, relapses after CD19-targeted CAR-T are relatively common and, therefore, there is a critical need for assays able to assess the function and potency of CAR-T products pre-infusion, which will hopefully help to optimize CAR-T therapies. We developed a novel multicolor fluorescent spot assay (MFSA) for the functional assessment of CAR-T products on a single-cell level, combining the numerical assessment of CAR-T products with their functional characterization.METHODS:We first used a standard single-cell interferon (IFN)-γ enzyme-linked immune absorbent spot assay to measure CD19-targeted CAR-T responses to CD19-coated beads. We then developed, optimized and validated an MFSA that simultaneously measures the secretion of combinations of different cytokines on a single CAR-T level.RESULTS:We identified IFN-γ/tumor necrosis factor-α/granzyme B as the most relevant cytokine combination, and we used our novel MFSA to functionally and numerically characterize two clinical-grade CAR-T products.CONCLUSIONS:In conclusion, we have developed a novel assay for the quantitative and functional potency assessment of CAR-T products. Our optimized MFSA is cost-effective, easy to perform, reliable, can be performed overnight, allowing for a fast delivery of the product to the patient, and requires relatively minimal maintenance and training. The clinical value of our novel assay will be assessed in studies correlating the pre-infusion assessment of CAR-T products with the patients' outcome in a prospective fashion.

BACKGROUND:Aggressive B cell lymphoma with secondary central nervous system (CNS) involvement (SCNSL) carries a dismal prognosis. Chimeric antigen receptor (CAR) T cells (CAR-T) targeting CD19 have revolutionized the treatment for B cell lymphomas; however, only single cases with CNS manifestations successfully treated with CD19 CAR-T have been reported.METHODS:We prospectively enrolled 4 patients with SCNSL into our study to assess clinical responses and monitor T cell immunity.RESULTS:Two of four SNCSL patients responded to the CD19-targeted CAR-T. Only one patient showed a substantial expansion of peripheral (PB) CAR-T cells with an almost 100-fold increase within the first week after CAR-T. The same patient also showed marked neurotoxicity and progression of the SNCSL despite continuous surface expression of CD19 on the lymphoma cells and an accumulation of CD4+ central memory-type CAR-T cells in the CNS. Our studies indicate that the local production of chemokine IP-10, possibly through its receptor CXCR3 expressed on our patient's CAR-T, could potentially have mediated the local accumulation of functionally suboptimal anti-tumor T cells.CONCLUSIONS:Our results demonstrate expansion and homing of CAR-T cells into the CNS in SNCSL patients. Local production of chemokines such as IP-10 may support CNS infiltration by CAR-T cells but also carry the potential of amplifying local toxicity. Future studies investigating numbers, phenotype, and function of CAR-T in the different body compartments of SNSCL patients receiving CAR-T will help to improve local delivery of "fit" and highly tumor-reactive CAR-T with low off-target reactivity into the CNS.

Abstract Soluble, tumor-derived, pro-tumorigenic (STP) cytokines have been shown to contribute to cancer cell proliferation and drug resistance. But therapeutic targeting of STP cytokines by monoclonal antibodies is limited by high concentrations and persistent production of antigen. We hypothesize that the ability of sweeping antibodies (swAbs) to actively clear soluble antigens will overcome these limitations. SwAbs differ from conventional antibodies via (1) pH-dependent binding of antigen, and (2) increased binding to the neonatal Fc receptor (FcRn). SwAbs facilitate clearance of soluble antigens by releasing antigen in the low pH environment of FcRn-expressing cells. SwAbs are then recycled via FcRn for repeated clearance of antigen.We are developing swAbs against four STP cytokines contributing to cancer cell proliferation, survival, migration or intra-tumoral angiogenesis: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and interleukin 16 (IL-16). We selected four existing neutralizing antibodies against these cytokines with available crystal structures and generated a second antibody targeting interleukin 16 with high affinity (clone 3B6; KD = 13 nM) de novo using phage display. We are using bio-layer interferometry and custom recycling/clearance assays for antibody characterization and pre-clinical validation. To introduce pH dependency into the four antibodies with crystal structures, we performed targeted histidine mutagenesis of residues at the interface with the respective target antigens. For the de novo-generated interleukin-16 antibody, we performed an unbiased histidine scanning approach by mutating residues across all six complementarity-determining regions (CDRs), which resulted in slight increases in pH sensitivity. We then performed a second round of mutagenesis of the most pH-sensitive 3B6 variants by replacing additional residues considered developability liabilities with histidine residues. We found that this approach significantly increased expression and affinity at pH 7 of the resulting 3B6 variants but only modestly increased pH sensitivity. In contrast, replacing a single residue in the heavy CDR1 in clone 14.1, a pre-clinical anti-IL-16 antibody, resulted in substantially increased pH sensitivity (KD(pH=7) = 2 nM; KD(pH<6) = n.b.). We are currently performing pH-sensitivity screenings for the three additional STP-specific antibodies. Our findings indicate that the ability to introduce pH sensitivity is largely dependent on the parental antibody and that some antibodies may not be amenable to this process. In addition, we show that histidine mutagenesis can be an effective approach to enhance expressibility and binding, while also increasing pH sensitivity. This project will answer the question whether anti-STP cytokine swAbs are an effective approach to treat patients with cancer. Citation Format: Jillian M. Baker, Nevil J. Singh, Tim Luetkens. Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines [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 2720.