Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cell therapy, where the lymphocytes of a cancer patient's tumor are harvested, expandedin vitrousing IL-2 stimulation, and then infused back into the patient Rosenberg and Restifo (2015Science34862-68), Bonini and Mondino (2015Eur. J. Immunol.452457-69). However, even with the use of TIL therapy, cancer cells can survive for various reasons, such as poor lymphocyte infiltration into tumors, chronic activation of the T cell receptor and the immunosuppressive tumor microenvironment Morganet al(1976Science1931007-8). Cytokine-inducible SH2-containing (CISH) protein is a negative regulator of T cell activation, and in a recent clinical trial was knocked out in TILs to improve TIL therapy efficacy Rosenberget al(1985J. Exp. Med.1611169-88). A mechanistic signaling pathway model was developed to theoretically evaluate the efficacy ofCISHknockout (CISHKO) in T cell activation and examine potential alternative target genes that can theoretically be targeted using multiplex gene-editing or drugs to further improve T cell activation and function Donohueet al(1984J. Immunol.1322123-8). Based on the results,CISHknockout increases the transcription of activation biomarkers IL-2 and TNF-α, but also inhibitory biomarkers such as PD1 and FasL. Using global sensitivity analysis, we also found thatGSK3B, which is responsible for the deactivation of NFAT, is also predicted to further increase T cell activation when knocked out. In addition, it was predicted thatPDCD1, FASandCTLA4can be knocked out in combination withCISHto further enhance T cell activation and prevent exhaustion and apoptosis.

Over the past decade, Immuno-Oncology has largely focused on blocking inhibitory surface receptors like PD-1 to enhance T cell anti-tumor activity. However, intracellular immune checkpoints such as CISH, which function independently of tumor-expressed ligands, offer powerful and previously untapped therapeutic potential. As a downstream regulator of TCR signaling, CISH controls T cell activation, expansion, and neoantigen reactivity. Though historically considered undruggable, recent advances in CRISPR engineering have enabled functional interrogation of these targets. We demonstrate that CISH deletion enhances T cell activation and anti-cancer functions more effectively than other emerging intracellular checkpoints. In CAR-T cells, CISH inactivation significantly increased sensitivity to tumor antigen, enabling robust recognition and killing even at low antigen levels, conditions that often lead to treatment failure with conventional T cell therapies, mirroring antigen escape scenarios seen in solid tumors. Our findings further validate CISH as a potent and druggable intracellular checkpoint capable of boosting anti-tumor T cell responses across diverse cancer types, independent of PD-L1 status. The underlying mechanisms of CISH inhibition may help explain the positive outcomes reported in recent clinical studies of this approach in solid tumor immunotherapy.

Abstract Introduction In chronic infections or cancer, stimulated CD8+ T cells progress along a trajectory towards terminal exhaustion in sites of antigen abundance. However, how these cells respond when antigen recognition is lost has not been concretely elucidated. Methods The Masopust lab generated a UBC-CreERT2 x P14 fl/fl mouse model that allows for the inducible excision of P14 TCR to precisely answer these questions, which has been used successfully in the setting of acute and chronic infections (unpublished work). Here, we combined this mouse model with the KP-NINJA tumor model (Fitzgerald et. al., 2021) that develop autochthonous lung tumors that progress slowly and express the gp33/H-2Db epitope that is recognized by the P14 TCR. Results Preliminary results showed successful infiltration and activation of these transgenic T cells in the KP-NINJA tumor microenvironment as well as accumulation within the tumor-draining lymph node following adoptive transfer. After three weeks of antigen exposure, the TCR was eliminated from 50% of transferred T cells and flow cytometry of the tumor, tumor-draining lymph node and additional tissues was performed one-week post-excision. TCR-negative T cells persisted within solid tumors and draining lymph node, although their phenotype was distinct from co-transferred TCR-positive P14 T cells. TCR-negative cells had decreased PD-1 and TOX expression in the tumor and tumor-draining lymph node and increased CXCR6 and CD127 expression within these tissues. Conclusion Ongoing studies will define tumor-specific T cell differentiation and fate after TCR excision, investigate changes to cell localization, and test functional potential. This work will add to our understanding of the development of T cell exhaustion and the plasticity of lineage commitment in cancer to better inform future anti-cancer therapeutics. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

2563 Background: CAR-T cells targeting CD19 using scFv-based CARs have been effective and approved to treat lymphoma. Response rates are high, but a significant number of patients fail to respond or relapse. This has been linked to T cell exhaustion, immune dysregulation, and/or epitope loss. To overcome this, we developed a CAR-T cell product expressing a BAFF-ligand (LMY-920). The CAR consists of truncated human BAFF on a 3rd generation CAR backbone with CD28, OX40, and CD3z intracellular signaling domains. The BAFF-ligand domain confers ability to bind the 3 BAFF receptors (BAFFR/BR3, TACI and BCMA). These are attractive tumor-associated antigens being variably expressed in all B-lineage malignancies such as B cell NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia and multiple myeloma (MM), and are important for B-cell survival, reducing the chance of antigen escape. Additionally, they are expressed on B-lineage cells involved in antibody-mediated autoimmune diseases. BAFF ligand interactions are lower affinity than scFv interactions, potentially reducing T-cell exhaustion. The novel TcBuster transposon system is used to improve manufacturing time, efficiency, cost, and safety. Methods: Patients with refractory B cell NHL are treated in this study (NCT05312801). Autologous LMY-920 CAR-T cells are manufactured, then patients receive 3 days of fludarabine (30 mg/m 2 /d) and cyclophosphamide (500 mg/m 2 /d) lymphodepletion. LMY-920 is administered intravenously in a 3+3 dose escalation design from 1-8 x 10 6 BAFF-CAR-T cells/kg. Response is assessed using the Lugano criteria. CAR-T expansion and biologic characteristics are assessed. Results: Five patients have been treated with 1-2 x 10 6 BAFF-CAR-T cells/kg in this study, 2 patients each with mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), and one with marginal zone lymphoma (MZL). Patients had received 2 – 6 prior lines of therapy and all were refractory. One patient experienced grade 1 CRS (fever), but no ICANS was reported. All patients experienced grade 3 or higher hematologic toxicity that recovered prior to day 28, and grade 1-2 fatigue. There have been no dose limiting toxicities and dose escalation continues. Responses included 2 complete responses (CR) (DLBCL), a partial response (MZL), a mixed response (MCL) and one stable disease (MCL). Of note, one of the DLBCL patients in CR had received prior axicabtagene ciloleucel (anti-CD19) CAR-T cells as well as anti-CD20 bispecific antibodies, with lymphoma cells resulting in CD19 and CD20 antigen loss. Conclusions: The successful use of a novel transposon-engineered BAFF ligand-based CAR-T cell product demonstrates the potential of a new direction in CAR-T cell development. Safety and efficacy were seen, including patients with prior CAR-T failure and epitope loss, as hypothesized. This product is also being evaluated in patients with CLL, MM and systemic lupus erythematosus. Clinical trial information: NCT05312801 .

Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand-break-induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knockin (KI), we expand the use of base editor nickase activity to stimulate homology-directed repair (HDR) and insert clinically relevant chimeric antigen receptors (CARs) into specific loci. Through a novel single-guide RNA design strategy and a DNA template delivered by a recombinant adeno-associated virus, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19, CD33, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3E/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines and a suppressive 3D spheroid culture model. This efficient engineering process of "Iterative Nicking for Synchronous Engineered Reprogramming of T cells" (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.

Abstract Background CRISPR genome editing enables precise modification of genomic targets but may also induce unintended edits at off-target sites with similar sequences. Pooled amplicon sequencing can assess on- and off-target editing across many samples, yet analyzing, aggregating, and visualizing results from multiple pooled experiments remains challenging. Tools to simplify and standardize these analyses are needed to provide reproducible and comparable interpretation of editing data. Results We developed CRISPRessoSea, a software package that processes, compares, and visualizes genome editing rates from pooled amplicon sequencing experiments. The tool provides standardized workflows for analyzing editing across multiple targets and samples, supports both nuclease- and base-editing modalities, and generates clear, data-rich summaries suitable for downstream interpretation. Conclusions CRISPRessoSea facilitates reproducible, scalable analysis of CRISPR editing outcomes across diverse experimental designs, enabling more efficient and transparent assessment of genome editing specificity. The software is freely available at https://github.com/clementlab/CRISPRessoSea .

Abstract Introduction Tissue-resident memory (Trm/TDRM) T cells populate non-lymphoid tissues and provide a first line of defense against pathogen re-exposures. KLF2, Runx3, Hobit, and Blimp1 are transcription factors (TF) that are reported to contribute to Trm/TDRM differentiation or maintenance. Yet it is unclear what the relative importance of each TF is, and how that may vary among tissue microenvironments. Methods We used CRISPR-Cas9-mediated deletion of TFs to address CD8+ Trm/TDRM development in 15 distinct tissues following lymphocytic choreomengitis virus infection (Armstrong strain). Results We found modest contributions by Hobit with few significant changes in knockout Trm/TDRM cells. Runx3 deletion had minimal effects on Trm/TDRM establishment, however integrin expression was significantly altered, including reduced CD103 expression. Blimp1 deletion increased the proportion of recirculating memory T cells that expressed CD62L but had minimal impact on resident cells. Most notably, the loss of KLF2 promoted greater accumulation of Trm/TDRM-phenotype CD8 T cells within the liver and secondary lymphoid organs (SLOs) by seven days after infection and persisted long after antigen clearance. Trm/TDRM-phenotype memory cells expressing CD69, CD49a, and P2rX7 were particularly elevated within SLOs when KLF2 was lost. Conclusion These data indicate that the loss of KLF2 has a major influence on promoting Trm/TDRM establishment, Runx3 shapes Trm/TDRM phenotype, and Hobit and Blimp1 have fewer observable roles after LCMV infection of the parameters analyzed. A better understanding of the TF-mediated regulation of Trm/TDRM will help inform manipulation or programming of T cells for adoptive cell therapies, therapeutic depletion, and other applications. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-major histocompatibility complex-associated antigens on transformed and pathogen-infected cells. In addition to their inability to mediate graft versus host disease, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene-edited chimeric antigen receptor (CAR)-γδ T cells for therapeutic application. Engineered CAR-γδ T cells demonstrate robust functionality in vitro and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo.

Abstract Introduction Antigen experienced T cell subsets are heterogenous, and can be described based on their phenotype, function, and migration properties. We wished to better understand how antigen stimulation regulates the trafficking, survival, differentiation and developmental plasticity of T cell subsets. Methods We developed a new mouse model that allows for the in vivo excision of the P14 TCR on a defined fraction of antigen-specific CD8 T cells. Specifically, we engineered a novel tamoxifen-inducible TCR-knockout transgenic mouse using a rAAV6 vector encoding for the floxed P14 TCR gene. This model allows for permanent cessation of antigen stimulation on select T cells at time points of our choosing. Results After acute infection with LCMV Armstrong, elimination of the TCR on established memory CD8 T cells maintained stable subset composition, but resulted in detectable downregulation of exhaustion markers, indicating that TCR sensing occurs among steady-state memory T cells. TCR deletion during chronic infections (LCMV Minnesota and LCMV Cl13 ± aCD4 depletion) affected both the population structure and phenotype. Terminal (Tex/TXt), as well as progenitor exhausted (Tpex/TXp) T cell subsets substantially declined after TCR deletion in persistently viremic infection with LCMV Cl13 + aCD4. However, the loss of Tpex/TXp after TCR deletion was moderated in settings of chronic infections without CD4 T cell depletion, in which most host viral load was more tightly controlled. Conclusion These results demonstrate that the antigen dependence of Tpex/TXp depends on context, and a subset may survive in the absence of further antigen stimulation, which has important implications for maintaining immunity to chronic infections and cancer. Funding Source Walter Benjamin Program, DFG Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated B cell activation, autoantibody production, and nephritis. B cell activating factor (BAFF) overexpression enhances autoreactive B-cell survival, driving autoimmunity. BAFF specific belimumab and CD20 specific rituximab antibodies are used for SLE therapy but are not curative, highlighting the need for alternative B cell depletion therapies. Here, we use BAFF ligand based chimeric antigen receptor T (CAR-T) cells targeting BAFFr, BCMA and TACI expressed on mature B cells and plasma cells. BAFF CAR-T cells efficiently killed B cells after co-culture with peripheral blood mononuclear cells (PBMCs) from SLE patients and in a patient derived SLE xenograft humanized mouse model developed by injecting patient PBMCs into immunocompromised mice. We also generated murine CD8+ T cells expressing human BAFF CAR to test their therapeutic efficacy in spontaneous (MRL/lpr) and pristane induced mouse models of SLE. In both models, BAFF CAR-T cells mediated persistent elimination of mature B cells, resulting in a decrease in the production of autoantibodies (IgM, IgG, Anti-ANA, and Anti-dsDNA IgG) and proteinuria along with prolonged survival. Adoptive transfer of B cells from control MRL/lpr lupus mice to previously BAFF CAR-T treated MRL/lpr lupus mice showed continued depletion of B cells and prolonged survival. Potential advantages of BAFF CAR-T therapy include avoiding B cell aplasia as BAFF receptors are not expressed by early B cells and preventing the escape of long-lived plasma cells post BAFF CAR-T therapy as they express receptors of BAFF. These data demonstrate the potential for a cellular immunotherapy based approach to induce remission of SLE pathogenesis using BAFF-CAR-T therapy.

Background Natural killer (NK) cells’ unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials.Methods Advanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.Results We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function in vitro and in vivo. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt’s lymphoma both in vitro and in vivo.Conclusions The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

Background and Significance: Despite major therapeutic advances including BTK and BCL2 inhibitors, patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) face limited treatment options after targeted therapy failure. Anti-CD19 CAR T cells show modest activity in CLL, with lisocabtagene maraleucel demonstrating only 57% overall response and 18% complete response rates in BTK/BCL2 inhibitor-treated patients. CLL cells express BAFF receptors (BAFF-R, TACI, BCMA) universally, and BAFF signaling promotes CLL cell survival through BTK-independent pathways. Additionally, CLL-associated T cell dysfunction limits CAR-T efficacy which may be overcome by pre-apheresis B cell depletion. We are conducting a trial with LMY-920, a BAFF ligand-based chimeric antigen receptor (CAR)-T cell therapy targeting all three BAFF receptors, manufactured using the TcBuster transposon system for improved manufacturing efficiency and safety with obinutuzumab intended to improve T cell quality and enhance CAR-T product function. Study Design and Methods: This is an open-label, dose escalation study (NCT 06916767) conducted at Cleveland Clinic, University Hospitals of Cleveland and The Ohio State University. Dose escalation of LMY-920 (2 - 8 x 106 cells/kg) is done using a 3+3 design to determine the maximum tolerated dose and recommended phase 2 dose. Major inclusion criteria include histologically confirmed CLL/SLL relapsed after ≥2 prior therapies including both BTK and BCL2 inhibitors (i.e. “double refractory”), active disease per iwCLL criteria, ECOG performance status ≤2, and adequate organ function. Key exclusion criteria include CNS involvement, active malignancy, cardiovascular instability, active infection, and autoimmune disease requiring immunosuppression. The treatment protocol involves: (1) pre-apheresis B cell depletion with obinutuzumab (100mg day 1, 900mg day 2) starting 14-21 days before leukapheresis; (2) standard leukapheresis and LMY-920 manufacturing over 8-11 days using transposon technology; (3) lymphodepletion with fludarabine (30 mg/m²/day) and cyclophosphamide (500 mg/m²/day) for 3 days beginning on day -5; and (4) LMY-920 infusion on day 0 at escalating doses from 2×10⁶ to 8×10⁶ BAFF CAR-T cells/kg. Up to 18 patients will be enrolled across dose escalation and expansion cohorts. Primary endpoints include determination of recommended phase 2 dose and safety profile. Secondary endpoints assess objective response rate, complete response rate, duration of response, progression-free survival, and overall survival per iwCLL criteria. Correlative studies will evaluate BAFF CAR-T persistence, cytokine profiles, T cell functionality, receptor expression, circulating tumor DNA, and the impact of pre-apheresis obinutuzumab on T cell populations and CAR-T product characteristics. Long-term safety follow-up continues for 15 years per gene therapy guidelines. Conclusion: Targeting ubiquitously expressed BAFF receptors with optimization of the starting T cell material through pre-apheresis B cell depletion for patients with double refractory disease is designed to overcome current limitations of CAR-T therapy for CLL/SLL

Naïve or memory T cells reprogram their metabolism upon antigenic stimulation. They increase their glucose uptake, relying on aerobic glycolysis for generating biomass while switching to glutamine to fuel energy production. Here we have identified a requirement for human Bcl-2 family, Noxa, in the metabolic switch to glutamine dependence in activated CD8 + T cells, that is independent of its canonical role in apoptosis at the end of the immune response. Using an in vitro co-stimulation model, we demonstrate that Noxa is induced in CD8 + T cells and remains elevated during the proliferative and differentiation phases of the response and through the onset of apoptosis. Noxa protein induction requires glutamine, is mediated via mTOR, and is independent of glutaminolysis. Glutamine, in turn, requires Noxa to facilitate its conversion to glutamate. CD8 + T cells lacking Noxa showed reduced levels of intracellular glutamate but no impairment of mitochondrial or effector function, and decreased dependence on glutamine for both respiration and growth during the proliferative phase. NOXA knockout CD8⁺ T cells also displayed significantly higher viability in the apoptotic phase of the immune response. CD8 + T cells from a human NOXA gene-replacement mouse responded normally to in vitro stimulation and in vivo acute infection. However, human Noxa-expressing murine CD8 + T cells displayed a distinctly proliferative gene signature in their transcriptome following activation, supporting an early growth-promoting role for this BH3-only protein. Our studies suggest that knocking out NOXA in human CD8 + T cells to increase their lifespan as well as their ability to survive and function in glutamine-poor microenvironments could be a promising immunotherapeutic strategy.