6 TH International Conference on Lymphocyte Engineering 15–17 July 2026 Milan,Italy

TRISPECIFIC TARGETING OF T CELLS ENGINEERED WITH TCR MIMIC ANTIBODIES TO LIMIT ANTIGEN ESCAPE

Tao Dao¹, Guangyan Xiong², Jeremy Meyerberg¹, Zita Aretz¹, Akihiko Shiiya¹, Tatyana Korotsvit¹, Jingbao Liu², Ziyou Cui², Chenyang Zhan¹, Hongbing Zhang², Neal Panchwagh¹, Winson Cai¹, Cheng Liu², David Scheinberg¹

¹Memorial Sloan Kettering Cancer Center, New York, United States of America, ²Eureka Therapeutics Inc, Emerywille, United States of America

AS: Academic Abstract Body: ntigen loss, tumor heterogeneity, and lack of tumor specific antigens, pose significant challenges for immunotherapies including chimeric antigen receptor T cell (CAR T) and T cell receptor (TCR) therapies, allowing tumor escape or causing toxicity, respectively. While TCR-based therapies target intracellular proteins presented by MHC molecules, many solid tumors downregulate the immunoproteasomes, resulting in failure to present certain immunogenic epitopes. To address these challenges, we previously developed a dual targeting engineered T cell format (AbTCR-CSR), combining a TCR mimic mAb (TCRm) ESK2, specific for Wilm’s tumor 1 (WT1) RMFPNAPYL/HLA-A2, with a CD33-directed costimulatory signaling receptor (CSR), that simultaneously recognize WT1 epitope RMF in the context of HLA-A2, as well as CD33 via CSR. This format allows improved specificity and potency against acute myeloid leukemia (AML). In this study, we expanded this strategy into a tri-specific T-cell construct to further mitigate antigen escape. To broaden coverage, we integrated a second TCRm, ESK3, which recognizes VLDFAPPGA—a WT1-derived epitope that is processed independently of the immunoproteasome. By tandemly integrating ESK2 and ESK3 into a single AbTCR-CSR molecule, the T cells can simultaneously recognize two distinct WT1 epitopes. Furthermore, we engineered the co-stimulatory receptor (CSR) domain to target either CD33 (for hematologic cancers) or mesothelin (for solid tumors). This triple-targeting approach—combining dual-epitope recognition with tumor-specific co-stimulation—aims to enhance functional potency, improve selectivity, and reduce the risk of relapse driven by antigen loss. This versatile platform offers a potential breakthrough for achieving durable therapeutic responses in both leukemias and solid tumors.

OP002 / #117

SUPERPOWERED LYMPHOCYTES

SYSTEMATIC DISCOVERY OF CRISPR-BOOSTED CAR T CELL IMMUNOTHERAPIES

Paul Datlinger^1,2, Eugenia Panckevich², Cosmas Arnold², Nicole Pranckevicius², Jenny Lin², Daria Romanovskaia², Moritz Schäfer^2,3, Francesco Piras^2,3, Anne-Christine Orts², Amelie Nemc², Paulina Biesaga², Michelle Chan², Theresa Neuwirth², Artem Artemov³, Wentao Li², Sabrina Ladstätter², Thomas Krausgruber^2,3, Christoph Bock^2,3

¹Arc Institute, Palo Alto, United States of America, ²CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria, ³Medical University of Vienna, Institute Of Artificial Intelligence, Center For Medical Data Science, Vienna, Austria

AS: Academic Abstract Body: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure. High-content CRISPR screening provides a method to systematically identify genetic perturbations that can enhance CAR T cell performance. Here we present CELLFIE, a platform for enhancing CAR T cells across multiple clinical objectives using high-content CRISPR screening. We developed a CROP-seq vector for the co-delivery and readout of a CAR and CRISPR library, along with a highly scalable mRNA system for delivering CRISPR editors, which extends the available tool kit for genome engineering in human T cells from loss-of-function mutations to precise base editing and CRISPR activation. In vitro and in vivo screens converged on knockouts of FAS, PRDM1, and most notably RHOG, as enhancers of CAR T cell function. We demonstrated enhanced performance of CAR T cells containing single knockouts and the RHOG-and-FAS double knockout. These boosted CAR T cells decreased tumor load and improved survival across multiple in vivo models, CAR designs, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we conducted combinatorial CRISPR screens to identify dual gene knockouts with synergistic effects on CAR ​T cell function. We further established saturation base editing screens to characterize variants of RHOG as candidates for clinical translation of CRISPR-boosted CAR T cells. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells, establishing a foundational resource for optimizing cell-based immunotherapies and providing candidate gene knockouts for clinical translation.

OP003 / #176

SUPERPOWERED LYMPHOCYTES

“FUELING” CAR T-CELLS TO IMPROVE THERAPEUTIC EFFICACY IN BRAIN TUMORS

Alina Brosque¹, Yael Engel², Dan Peer³, Michael Berger², Dinorah Friedmann-Morvinski¹

¹Tel Aviv University, Biochemistry And Molecular Biology, Tel Aviv, Israel, ²Hebre University of Jerusalem, Faculty Of Medicine, Jerusalem, Israel, ³Tel Aviv University, The Shmunis School Of Biomedicine And Cancer Researchy, Tel Aviv, Israel

AS: Academic Abstract Body: Despite therapeutic advances, conventional treatments for pediatric and adult high-grade gliomas, still result in dismal survival outcomes. This urgent unmet need has accelerated efforts to develop innovative strategies, particularly within the rapidly expanding field of CAR T-cell immunotherapy. However, applying CAR T-cell therapy to solid tumors, especially aggressive brain tumors, faces major obstacles, including profound biological heterogeneity, anatomical barriers such as the blood–brain barrier that restrict immune cell access, and a hostile, immunosuppressive tumor microenvironment (TME) that inhibits T-cell function and promotes exhaustion. Our laboratory recently designed a novel CAR T-cell therapy targeting an unexplored tumor-associated antigen, engineered for a dual mechanism of action: eliminating malignant glioma cells and tumor-derived endothelial cells. To overcome the immunosuppressive TME, we are pursuing combination strategies that pair CAR T cells with immunomodulatory agents delivered using a lipid-nanoparticle (LNP) mRNA-based platform, and we are also developing “metabolically empowered” CAR T-cells capable of using alternative energy sources to function in the nutrient-deprived GBM TME. In preclinical GBM models, treatment with our novel CAR T-cells led to a marked reduction in tumor progression, significantly prolonged overall survival, and decreased tumor vascular density. Preliminary data validate the feasibility of the LNP mRNA tool for localized immunomodulator action, while metabolically empowered CAR T-cells exhibit enhanced survival, sustained proliferation, robust cytokine secretion under glucose-restricted conditions, and superior therapeutic efficacy. Collectively, this integrated approach, combining novel CAR designs with superior metabolic fitness and advanced TME-modulating strategies, has the potential to transform outcomes for patients with high-grade gliomas.

OP004 / #86

TARGETED T CELL GENE ENGINEERING AND FUNCTIONAL OPTIMIZATION

HDR GENE CORRECTION AND SELECTION OF CD4⁺ T CELLS FOR THE TREATMENT OF HYPER IGM1: TOWARDS A PHASE I/II CLINICAL TRIAL

Claudia Asperti¹, Simona Porcellini¹, Daniele Canarutto¹, Giorgia Centonze¹, Elisa Montaldo¹, Ilaria Mei¹, Milena Perrone¹, Ilaria Visigalli¹, Francesca Cecere¹, Michela Vezzoli¹, Stefano Beretta¹, Francesca Ferrua², Valentina Vavassori¹, Samuele Ferrari¹, Francesca Sanvito³, Martina Ragazzi⁴, Sean Russell⁴, Stefano Zancan⁴, Leonard Pattenden⁴, Giuseppe Gaipa⁵, Daniela Belotti⁶, Benedetta Cabiati⁶, Marta Galuzzi⁶, Luis Gonzales-Granado⁷, Vassilios Lougaris⁸, Raffaele Badolato⁸, Andrea Finocchi⁹, Ansgar Schulz¹⁰, Wadih Abou-Chahla¹¹, Anna Shirmpton¹², Susana Lopes Da Silva¹³, Paola Albertini¹, Giuliana Ferrari¹, Anna Villa¹, Andrea Biondi¹⁴, Alessandro Aiuti², Marina Radrizzani¹, Luigi Naldini¹

¹San Raffaele Telethon Institute for Gene Therapy, Milan, Italy, ²IRCCS San Raffaele Scientific Institute, Pediatric Immunohematology Unit And Bone Marrow Transplantation Unit, Milan, Italy, ³IRCCS San Raffaele Scientific Institute, Patholgy Unit, Milan, Italy, ⁴Fondazione Telethon ETS, Rome, Italy, ⁵Fondazione IRCCS San Gerardo dei Tintori, Tettamanti Center, Monza, Italy, ⁶Fondazione IRCCS San Gerardo dei Tintori, Laboratorio Di Terapia Cellulare E Genica Stefano Verri, Monza, Italy, ⁷Research Institute Hospital 12 octubre, Immunodeficiencies Unit, Madrid, Spain, ⁸Pediatrics Clinic and Institute for Molecular Medicine A. Nocivelli, Clinical And Experimental Sciences, Brescia, Italy, ⁹Bambino Gesú Children's Hospital, Academic Department Of Pediatrics (dpuo), Rome, Italy, ¹⁰University Medical Center Ulm, Department Of Pediatrics, Ulm, Germany, ¹¹Université de Lille, Service D'hématologie Pédiatrique, Lille, France, ¹²Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom, ¹³Hospitalar Graduada de Imunoalergologia ULSSM, Lisboa, Portugal, ¹⁴Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy

AS: Academic Abstract Body: Hyper IgM1 (HIGM1) is an X-linked inborn error of immunity caused by mutations in the CD40LG gene, which impairs the interaction between activated CD4⁺T cells and effector immune cells, ultimately compromising immune response against pathogens. We developed a scalable good manufacturing practice (GMP)-compliant process using a knock-in gene editing (GE) strategy based on CRISPR/Cas9 and integrase-defective lentiviral vector (IDLV) delivery of the corrective template coupled to a LNGFR-selection marker. This process, allowing for correction, selection and expansion of edited cells, was successfully transferred to IRCCS San Gerardo GMP-facility, using healthy donor leukapheresis as surrogate starting material. GLP biodistribution study demonstrated the safety of this approach. An application for an autologous gene therapy phase I/II clinical trial has been submitted. To verify the suitability of the manufacturing process on patient cells, CD4⁺T cells derived from a cohort of patients aged 1-54 years were corrected and extensively characterized. Patient cells were edited and selected with high efficiency; product identity and purity were confirmed. As compared to the donor counterpart, patient CD4⁺T cells displayed a slightly different proliferation kinetic, which resulted in an overall slightly lower process yield, although minimal target dose was achieved. No cytokine-independent growth was observed and large deletions at target site were purged out by LNGFR-selection. Importantly, corrected patient cells displayed restoration of regulated CD40L expression, binding to CD40, and downstream signal transduction. These data provided valuable information for tailoring the process in real-world patient cells and demonstrated the feasibility of GMP-compliant manufacturing of gene corrected HIGM1 CD4⁺T cells.

OP005 / #42

TARGETING AUTOIMMUNE DISEASES

ENGINEERED FOOD ANTIGEN SPECIFIC T REGULATORY CELLS ARE A POTENTIAL CELL THERAPY FOR INFLAMMATORY BOWEL DISEASE

Peter Eggenhuizen, Marie Lee, Diana Tan, Indiana Zorkau, Kathryn Kumar, Daniel Cheong, Joshua Ooi, Rimma Goldberg

Monash University, Department Of Medicine, Clayton, Melbourne, Australia

AS: Academic Abstract Body: Background and Aim: T regulatory cells (Tregs) are defective in number and function in inflammatory bowel disease (IBD). Treg therapy for IBD was explored but did not progress past phase II trial. We propose a new approach to engineer patient induced Tregs (iTregs) with food antigen (FA)-specific T cell receptors (TCR), developing a cell therapy with a well-characterised, gut-homing receptor. Methods: IBD donor CD4+ T cells were transduced with FA-αβTCR-GFP-encoding lentivirus and induced into FA-TCR-iTregs in vitro. Humanised dextran-sodium-sulfate (DSS)-induced colitis model using NSG-MHCnull mice was established to assess in vivo efficacy. Results: FA-TCRs efficiently transduced onto induced Tregs. In vitro production led to FA-TCR-iTregs expanding 121-fold, enabling enough cell product for a potential human cell therapy. After the in vitro production process, FA-TCR-iTregs showed a stable Treg phenotype 88.5% FOXP3+ expression, similar to thymic tTregs (82.6%). FA-TCR-iTregs suppressed in a FA-specific manner showing significantly increased in vitro suppression of CD4+T effector cells compared to un-transduced iTregs. A DSS-induced colitis model in humanised NSG-MHCnull mice fed the FA showed FA-TCR-iTregs traffic to the inflamed bowel and interact with transferred autologous human dendritic cells. Mice treated with FA-TCR-iTregs showed significant improvement in clinical score compared to un-transduced iTregs and no iTregs. Conclusion: These data show FA-TCR-iTregs better suppress IBD-related inflammation and colon damage in the presence of FA and act at the site of disease, which make it a promising cell therapy candidate in future clinical trials for IBD.

OP006 / #111

TARGETING AUTOIMMUNE DISEASES

ELIMINATION OF ANTIGEN-SPECIFIC AUTOREACTIVE T CELLS USING A PHLA-BASED TARGETING

Ofri Pasternak, Eric Shifrut

Tel Aviv University and Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

AS: Academic Abstract Body: Precise targeting of pathogenic T cells remains a major unmet challenge, particularly in autoimmune diseases. Current treatments for autoimmune pathologies rely on broad immunosuppression and compromise protective immunity. To address this challenge, we developed a peptide-HLA-based chimeric antigen receptor (pHLA-CAR) designed to selectively recognize and eliminate autoreactive T cells through their antigen-specific TCR. This approach leverages the basic interaction between the TCR and the pHLA complex, to enable T cell-directed T cell cytotoxicity. Using the well-characterized NY-ESO-1 peptide and its cognate 1G4 TCR model system, we engineered primary human T cells to express a pHLA-CAR presenting the NY-ESO-1 peptide in the context of HLA-A2. T cells engineered to express this novel receptor demonstrated robust and stable surface staining following transduction. Functional assessment in co-culture assays showed highly efficient and antigen-specific cytotoxicity, with >90% elimination of cognate 1G4-expressing target cells within 20 hours. Non-cognate T cells were spared, confirming strict antigen specificity. To evaluate potential bidirectional activation, we co-cultured pHLA-CAR T cells with primary human T cells expressing the cognate TCR. While effective target cell elimination was observed, pHLA-CAR T cells maintained high viability, showing only minimal evidence of potential reciprocal cytotoxicity. These findings establish proof-of-concept for pHLA-based CAR T cells as a platform for highly specific and selective depletion of antigen-defined T cell populations. This strategy may enable selective removal of autoreactive T cell clones while preserving global immune competence, offering a new therapeutic direction for autoimmune diseases and immunotherapy-associated toxicities.

OP007 / #150

BEYOND ALPHA-BETA T-CELLS

DESIGN OF CYTOKINE AND CAR-ENGINEERED INVARIANT NATURAL KILLER T (INKT) CELLS FOR ENHANCED IMMUNOTHERAPY AGAINST METASTATIC COLON CANCER

Gloria Delfanti¹, Iris Stefanetta¹, Alessandra Perini¹, Giulia Lopatriello¹, Michela Consonni¹, Monica Casucci², Gianluca De Bellis³, Giovanni Sitia⁴, Paolo Dellabona¹, Giulia Casorati¹

¹IRCCS San Raffaele Scientific Institute, Experimental Immunology Unit, Division Of Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²IRCCS San Raffaele Scientific Institute, Innovative Immunotherapies Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ³Institute for Biomedical Technologies (ITB); National Research Council (CNR), Segrate, Italy, ⁴IRCCS San Raffaele Scientific Institute, Experimental Hepatology Unit, Division Of Immunology, Transplantation And Infectious Diseases, Milan, Italy

AS: Academic Abstract Body: Colorectal cancer (CRC) liver metastases represent a major cause of mortality and an unmet clinical need due to the limited efficacy of conventional therapies. Adoptive cell therapy (ACT) with tumor-redirected T cells is promising but often constrained by poor trafficking into solid tumors and immune suppression within the tumor microenvironment (TME). CD1d-restricted invariant natural killer T (iNKT) cells contribute to tumor immunosurveillance by limiting cancer-supporting myeloid populations and promoting immunostimulatory conditions. Our recent findings show that tumor-specific TCR-engineered iNKT cells can simultaneously target malignant and immunosuppressive myeloid cells, providing a donor-unrestricted ACT platform. Here, we evaluated iNKT cells engineered with a chimeric antigen receptor (CAR) specific for human carcinoembryonic antigen (hCEA) in CRC liver metastases. In hCEA-transgenic mice bearing orthotopic hCEA-expressing syngeneic CRC metastases, hCEA-CAR iNKT cells demonstrated robust homing to liver lesions and eradicated both early and advanced metastases, outperforming conventional CAR-T cells. Additionally, human iNKT cells engineered with hCEA-CAR are also being tested in NSG mice with human CRC liver metastases. To further enhance persistence and function, murine iNKT cells were engineered to express IL-15 (±IL-15Rα), IL-12, or IFNα. IL-15 and IFNα promoted iNKT expansion with favorable safety, whereas IL-12 induced potent TH1-skewed antitumor responses, but systemic toxicity driven by widespread IFNγ responses. A membrane-bound IL-12 construct is under development to spatially restrict cytokine activity. Collectively, these findings support iNKT-based ACT as a promising strategy for CRC liver metastases. Combining CAR redirection with optimized cytokine engineering may provide a potent and safer immunotherapeutic approach for metastatic CRC.

OP008 / #58

HSC GENETIC ENGINEERING AND EPITOPE EDITING

NOVEL ADENINE BASE EDITORS WITH TAILORED SUBSTRATE RECOGNITION ENABLE IMMUNE ENGINEERING WITH SINGLE-BASE PRECISION

Gabriele Casirati, Enrico Drago, Moises Chavez-Navarro, Jan-Phillipp Gerhards, Danilo Pellin, Pietro Genovese

Boston Children's Hospital, Hematology/oncology, Boston, United States of America

AS: Academic Abstract Body: Base editing enables nucleotide transitions without dsDNA breaks, offering unparalleled safety and efficacy for immune-cell engineering. However, BE can generate bystander deamination, compromising translatability. Critically, ∼69.7% genomic sites harbor ≥1 bystander within ±3nt. To discover deaminases with enhanced on-target discrimination, we engineered TadA-8e via directed-evolution in human cells and phenotypic selection of desired editing outcomes. We uncovered substantial functional redundancy within engineered TadAs, including divergent activity profiles and robust suppression of bystanders, achieving near-exclusive on-target deamination (32%→82%). We re-tested candidates in CD34⁺HSPCs (N=24): on-target precision correlated with cell-line data (R=0.87, p<0.0001), confirming biological robustness and translational relevance. To comprehensively chart sequence-context determinants, TadA variants were profiled against a matched sgRNA:target library (N=12,000). Clustering analyses revealed pronounced sequence-dependencies, with ±1nt flanking bases exerting the greatest effect on editing, which was further modulated by spacer position and broader motifs, offering unprecedented opportunities for tailored editing strategies and rational deaminase design. Profile-guided TadA selection enabled programmable single-base preference: in a representative multi-A substrate (GA₅A₆TA₈G), evolved variants selectively suppressed either A₅-A₆(−94%) or A₈(−57%), demonstrating selective engagement of individual adenines within the same protospacer. We applied this framework to our previously published FLT3 epitope-editing strategy (Nature, 2023) and obtained near-complete elimination of bystanders (68.7%→4.7%) while maintaining efficiency on the desired epitope mutation (75%). This work establishes that TadA can be rationally evolved to discriminate within multi-A contexts, overcoming the long-standing bottleneck of bystander mutagenesis. To our knowledge, this constitutes the first demonstration of bespoke ABEs with programmable substrate recognition, providing a blueprint for precision immune-engineering.

OP009 / #85

IN VIVO LYMPHOCYTE ENGINEERING

IN VIVO PROGRAMMING OF CAR-DENDRITIC CELLS TRIGGERS SYSTEMIC ANTIGEN SPREADING AND PERMANENT TUMOR IMMUNITY

Xiaoqian Niu

University of Singapore, Yong Loo Lin School Of Medicine, Singapore, Singapore

AS: Academic Abstract Body: Background: While CAR-T therapies have redefined cancer treatment, they are constrained by complex ex vivo manufacturing and potential antigen escape. Dendritic cells (DCs) represent a powerful yet underutilized alternative due to their unique ability to orchestrate multi-antigenic endogenous immunity. Methods: We developed a novel delivery platform using modified lnp.Targeting efficiency was evaluated in vivo against benchmark LNPs. A specialized CAR structure optimized for DC signaling was transcribed into mRNA. CT26-HER2 tumor model was used to test the in vivo anti tumor ablility. To evaluate systemic immunosurveillance and antigen spreading, treated mice were simultaneously rechallenged with CT26 cells and CT26-HER2 cells via subcutaneous injection into the bilateral axillary regions. Results: Our engineered lnp demonstrated superior DC-targeting, increasing mRNA transfection efficiency in CD11c⁺MHCII⁺cells from ∼3.5% (standard LNPs) to ∼11% in vivo. In the CT26-HER2 model, treatment led to robust tumor eradication, with mean volumes maintained below 100 mm³ (vs. 1500 mm³ in controls). Notably, in the dual-axillary rechallenge model, control group and treated mice rejected both CT26 and CT26-HER2 tumors. This systemic protection was associated with a >30-fold expansion of endogenous CD8⁺AH1⁺ tumor-specific T cells, confirming the induction of potent antigen spreading and long-term memory. Conclusion: We established a highly efficient in vivo CAR-DC induction platform. By triggering massive expansion of endogenous T cells against both primary and secondary (neo)antigens, this “all-in-one” approach effectively prevents antigen escape and tumor recurrence.

OP010 / #174

IN VIVO LYMPHOCYTE ENGINEERING

TWO-IN-ONE COMET LV COUPLES MRNA TRANSFER AND GENOME ENGINEERING

Chiara Bresesti¹, Eleonora Carito¹, Gabriele Pedrazzani¹, Federico Rossari^1,2, Giovanna Giacca¹, Marco Notaro¹, Maristella Borghetti¹, Sara Breggion¹, Giorgio Colleoni¹, Fabrizio Benedicenti¹, Francesco Gazzo¹, Daniele Canarutto¹, Luisa Albano¹, Michela Milani¹, Alessio Cantore¹, Eugenio Montini¹, Saar Gill², Luigi Naldini¹, Attya Omer-Javed¹, Mario Squadrito³

¹San Raffaele Telethon Institute for Gene Therapy, Milan, Italy, ²University of Pennsylvania, Philadelphia, United States of America, ³San Raffaele Telethon Institute for Gene Therapy, Milano, Italy

AS: Academic Abstract Body: Lentiviral vectors (LVs) are versatile tools for genetic engineering employed in multiple FDA- and EMA-approved gene therapy products. Nevertheless, their therapeutic potential could be expanded by combining stable gene expression with transient modulation of target cell function. Here, we describe co-packaged mRNA expression technology (Comet) LVs, a novel platform enabling the simultaneous delivery of integrating and transiently expressed payload to target cells. Comet LVs incorporate RNA-binding proteins tethered to the viral envelope, which in turn bind mRNAs of interest, promoting their packaging into the viral particle. As a result, Comet LVs co-deliver mRNAs and integrating payloads in vitro to target cells, or in vivo to mice by intravenous injection. We employed Comet LVs to deliver CXCR4 mRNA to hematopoietic stem and progenitor cells (HSPCs) while introducing an integrating payload. Of note, transient CXCR4 expression enhances HSPC engraftment, enabling a chemotherapy-free, mobilization-based conditioning regiment. HSPCs transduced with Comet LVs displayed transient CXCR4 overexpression and improved engraftment potential upon transplantation. Additionally, we applied Comet LVs to in vivo antitumor vaccination by delivering tumor antigen (TA) mRNAs alongside an integrating cassette encoding IFNa, an immunostimulatory cytokine. Treated mice exhibited increased circulating IFNα levels and developed TA-specific T cells capable of eliminating TA-expressing tumor clones. Comet LVs offer a modular, customizable tool for cell engineering that couples the benefits of mRNA transient expression with LV potential for stable transduction and favorable safety profile. For these reasons, Comet LVs could significantly advance both therapeutic and research applications in genetic engineering.

OP011 / #70

ADVANCED TCR ENGINEERING

NOVEL COILED-COIL DIMERIZATION MOTIF ENFORCES OPTIMAL TCR CHAIN PAIRING, EXPRESSION, AND FUNCTION

Morteza Hafezi¹, Leo Scheller², Margaux Saillard³, Leila Hadadi³, Romain Vuillefroy De Silly³, Paris Kosti³, Greta Maria Paola Giordano Attianese³, Patrick Reichenbach³, Melanie Triboulet³, Raphael Porret⁴, Yannick Muller⁴, Philippe Guillaume³, Benita Wolf³, Vincent Zoete³, Michel Cuendet³, Bernhard Gentner³, Camilla Jandus³, Bruno Correia², Melita Irving¹, George Coukos¹

¹Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne and Lausanne University Hospital (CHUV); Lausanne, Switzerland, Lausanne, Switzerland, ²Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL); Lausanne, Switzerland, Lausanne, Switzerland, ³Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne and Lausanne University Hospital (CHUV); Lausanne, Switzerland., Lausanne, Switzerland, ⁴Division of Immunology and Allergy, University Hospital of Lausanne, Switzerland, Lausanne, Switzerland

AS: Academic Abstract Body: Abstract: T-cell receptor (TCR)-engineered T cells have shown promise against cancer and other diseases but mispairing among the introduced and native chains can curtail their therapeutic efficacy and increase the risk of toxicity. Here we demonstrate that introducing a novel coiled-coil dimerization motif into the constant regions of the α and β chains robustly enforces proper chain pairing and enhances stability, enabling optimal TCR expression to levels, comparable to CRISPR-engineered TCR T cells across diverse mouse and human TCRs. Optimal exogenous TCR assembly and expression circumvents the need for gene editing of the endogenous TCR chains as well as for TCR affinity optimization, while enhancing the efficacy and safety of TCR-engineered T cells. Overall, this novel dimerization motif holds important potential for clinical translation as it addresses key gaps in TCR-based immunotherapy.

COMBINATORIAL STRATEGY FOR IMPROVED CDH17 CAR T CELL THERAPY OF LIVER METASTASES FROM COLORECTAL CANCER

Katia Palmisano¹, Alessia Potenza², Chiara Balestrieri², Chiara Pilavi¹, Laura Falcone¹, Barbara Camisa², Oronzina Botrugno³, Jolanda Bruno³, Cristina Faccani⁴, Achille Anselmo⁵, Claudia De Lalla⁴, Francesco Fiorio⁶, Federica Ungaro⁶, Eliana Ruggiero², Fabio Ciceri⁷, Chiara Bonini², Monica Casucci¹

¹IRCCS San Raffaele Scientific Institute, Innovative Immunotherapies Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ³IRCCS San Raffaele Scientific Institute, Functional Genomics Of Cancer Unit | Division Of Experimental Oncology, Milan, Italy, ⁴IRCCS San Raffaele Scientific Institute, Experimental Immunology | Division Of Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁵IRCCS San Raffaele Scientific Institute, Flow Cytometry Resource, Advanced Cytometry Technical Applications Laboratory, Milan, Italy, ⁶IRCCS San Raffaele Scientific Institute, Experimental Gastroenterology Unit | Division Of Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁷IRCCS San Raffaele Scientific Institute, Hematology And Bone Marrow Transplantation Unit, Milan, Italy

AS: Academic Abstract Body: Colorectal cancer (CRC) is the third most diagnosed malignancy and the second leading cause of cancer death worldwide, largely due to liver metastases (CRC-LM). Standard therapies offer limited benefit in advanced disease, highlighting the need for more effective treatments. Through integrative transcriptomic and sequencing analyses, we identified cadherin-17 (CDH17) as a promising CAR-T target in CRC-LM. CDH17-CAR-T cells showed strong antitumor activity without reactivity toward healthy colon, but efficacy remained limited by tumor relapse and potential off-tumor toxicity due to intestinal CDH17 expression. To enhance the therapeutic index, we implemented a dual-antigen strategy coexpressing a CDH17-CAR with a chimeric costimulatory receptor (CCR) specific for B7-H3, upregulated in metastatic CRC and minimally expressed in normal colon. Constructs with different costimulatory domains were tested in CRC-variants engineered to express graded CDH17 and B7-H3 levels, enabling evaluation of antigen density effects. In vitro, CCR-CAR combinations enhanced transcriptional activation and functional avidity in Jurkat Reporter cells. Among CCR-CAR constructs, CDH17.28z_B7H3.BB-T cells maintained cytotoxicity under chronic antigen stimulation with significantly reduced exhaustion compared to the original CAR. In a CRC-LM mouse model, CDH17.28z_B7H3.BB-T cells demonstrated superior tumor control, greater expansion, and augmented cytokine production. However, persistent CD39 expression suggested adenosine-mediated immunosuppression. CRISPR-Cas9-mediated CD39-knockout further enhanced cytotoxicity of CCR-CAR-T cells against CRC lines and patient-derived organoids, accompanied by elevated extracellular ATP. Ongoing studies aim to validate efficacy improvements in immunodeficient and humanized CRC-LM mouse models. These results support dual-antigen targeting combined with CD39 disruption as a promising strategy to ameliorate CDH17-directed CAR-T therapy for CRC-LM.

PB002 / #52

TOP 10 POSTER PRESENTATIONS

IMPROVED CAR T-CELL EFFECTIVENESS AGAINST SOLID TUMORS BY TUMOR-RESTRICTED CYTOKINE DELIVERY VIA IMMUNOCYTOKINES

Christian Pellegrino¹, Sarah Oppolzer¹, Sarah Alberio¹, Marianna Ponzo¹, Ramon Benz¹, Roberto De Luca², Emanuele Puca², Samuele Cazzamalli², Dario Neri², Markus Manz¹, Chiara Magnani¹

¹University Hospital Zürich (USZ) and University of Zürich (UZH), Medical Oncology And Hematology, Zurich, Switzerland, ²Philochem AG, Biology, Otelfingen, Switzerland

AS: Academic Abstract Body: Chimeric antigen receptor (CAR) T-cell therapies benefit patients with blood cancers but are ineffective against solid tumors due to limited infiltration, poor persistence, and immunosuppressive environments. Cytokine boosting can enhance CAR T-cell activity, but systemic delivery is limited by toxicity. We aimed to combine targeted cytokine delivery with the immunocytokine Darleukin™ and standard CAR T therapy in renal cell carcinoma (RCC) and colorectal adenocarcinoma (COAD) models. We evaluated CAR T cells targeting CAIX or CEA in RCC (SK-RC-52) and COAD (HT29), combined with tumor-targeted immunocytokines that deliver interleukin-2 (Darleukin) to the fibronectin extra-domain B (EDB), expressed in tumor-associated neovasculature and stroma. We conducted in vitro and in vivo studies using NSG mice with tumors to analyze antitumor effects, CAR T-cell proliferation, and tumor infiltration. Combining CAR T cells with tumor-specific IL-2 immunocytokines produced superior responses compared with CAR T alone in both RCC and COAD. Importantly, co-administration of immunocytokines enabled effective treatment at lower CAR T doses without systemic toxicity. Tumor-targeted delivery, compared with the recombinant systemic cytokine, promoted CAR T expansion within tumors and improved tumor control in CAIX-positive RCC models. In conclusion, targeted cytokine delivery via immunocytokines boosts CAR T proliferation and activity in solid tumors without increased toxicity, as activity is tumor confined. This approach offers a flexible strategy to improve solid-tumor CAR T outcomes in future trials.

PB003 / #54

TOP 10 POSTER PRESENTATIONS

A MODULAR CAR T CELL PLATFORM FOR TARGETED IMMUNOTHERAPY OF ACUTE MYELOID LEUKEMIA

Kathrin Gabriel¹, Sophia Stock¹, Anja Hauptstein¹, Taddhäus Strzalkowski¹, Lisa Rohrbacher², Jana Engesser¹, Adrian Gottschlich¹, Janina Dörr¹, Vivien Menkhoff¹, Sayantan Nandi¹, Gerulf Hänel², Charlotte Carcopino¹, Donjete Simnica¹, Luisa Fertig¹, Marlena Surowka³, Peter Bruenker³, Diana Darowski³, Stefan Endres¹, Michael Von Bergwelt-Baildon⁴, Marion Subklewe², Christian Klein⁵, Sebastian Kobold¹

¹Institute of Clinical Pharmacology, LMU Klinikum, Munich, Germany, ²LMU, Gene Center, Munich, Germany, ³Roche Innovation Center, Zurich, Switzerland, ⁴LMU Klinikum, München, Germany, ⁵LMU, Department Of Biochemistry, Munich, Germany

AS: Academic Abstract Body: Chimeric antigen receptor (CAR) T cell therapy has shown limited efficacy in acute myeloid leukemia (AML) due to marked genetic heterogeneity and antigen-negative relapse. Adapter CAR T cells represent a modular strategy to address these challenges. Anti-P329G adapter CAR T cells recognize the P329G mutation within the Fc region of Fc-silenced human IgG1 antibodies (L234A/L235A), which have already been clinically validated against multiple tumor antigens and can be readily combined with matching CAR T cells. In this study, scFv-based second-generation anti-P329G CAR T cells were combined with P329G-Fc-mutated antibodies targeting CD33, CD123, and the novel AML-associated antigen CSF1R. Conventional scFv-matched CAR T cells served as positive controls, and untransduced or anti-CD19 CAR T cells as negative controls. Target antigen expression was assessed in AML cell lines and primary patient samples. T cell activation, proliferation, and cytotoxicity were evaluated using flow cytometry-, ELISA-, luciferase-, live-cell imaging-, and confocal microscopy-based assays, including antigen knockout models to simulate antigen escape. In vivo efficacy was tested in immunodeficient mice bearing THP-1 or MV4-11 tumors, including mixed wildtype/knockout models to simulate antigen heterogeneity. Anti-P329G CAR T cells mediated robust, antigen-specific effector functions comparable to conventional CAR T cells, with activity dependent on antigen expression levels. The platform enabled reversible, combinatorial, and sequential targeting, and switching adapter antibodies restored cytotoxicity in antigen-escape settings. Tumor growth was effectively controlled in vivo. These data support anti-P329G adapter CAR T cells as a flexible strategy to overcome antigen heterogeneity and escape in AML.

PB004 / #77

TOP 10 POSTER PRESENTATIONS

CAFFEINE-REGULATED MOLECULAR SWITCHES FOR FUNCTIONAL CONTROL OF CAR T CELLS

Elise Sylvander¹, Benjamin Salzer², Dominik Emminger², Hayeon Baik², Giulia D′accardio³, Markus Schäfer⁴, Konstantina Mouratidis², Michelle Buri², Álvaro Muñoz-López⁵, Fabian Engert⁵, Boris Engels⁵, Joerg Mittelstaet⁵, Antonio Rosato⁶, Johannes Zuber⁴, Eva Putz⁷, Charlotte Zajc⁷, Manfred Lehner², Michael Traxlmayr²

¹BOKU Vienna, Institute Of Biochemistry, Vienna, Austria, ²St Anna CCRI, Vienna, Austria, ³Veneto Institute of Oncology IOV –, Padua, Italy, ⁴IMP Vienna, Vienna, Austria, ⁵Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany, ⁶università degli studi di padova, padova, Italy, ⁷Medical University of Vienna, Vienna, Austria

AS: Academic Abstract Body: Chimeric Antigen Receptor (CAR) T cell therapy has shown great clinical results in the past few years. However, uncontrolled proliferation and activation of these cellular therapeutics after administration can lead to severe side effects. Systems to regulate CAR T cell activity in vivo are thus highly important to improve the safety of the therapy. In this study we developed an ON-switch that can be applied to develop CARs with caffeine-inducible function. Caffeine has an excellent safety profile, ideal pharmacological properties and is exceptionally well studied, making it an ideal drug for long term in vivo application. Our starting point was a nanobody that homodimerizes in the presence of caffeine. As most interesting applications request a heterodimeric switch, we used directed evolution to engineer two disulfide free, complementary mutants of this nanobody that heterodimerize in a caffeine dependent way. The switches were incorporated into a caffeine-dependent split CAR (CaffCAR) which achieved tumor lysis and cytokine production levels comparable to that of a traditional CD19 CAR while showing a total lack of background activity in the absence of caffeine. Importantly, the caffeine concentration necessary to fully activate CaffCARs is reached in humans after drinking 1-2 cups of coffee. CaffCAR T cell anti-tumor activity was also confirmed to be caffeine dependent in vivo, even upon overexpression of c-Jun and massive CAR T cell expansion. In summary, we present a novel caffeine dependent heterodimeric switch that can be used to tightly regulate CAR T cell function.

PB005 / #126

TOP 10 POSTER PRESENTATIONS

BREAKING THE MATRIX BARRIER: ENHANCING CAR-T CELL ACCESS TO SOLID TUMORS

Emilia Vigolo¹, Giulia D’Accardio¹, Sara Boscarato², Hieu Ngo Trong¹, Pierangela Palmerini¹, Roberta Sommaggio², Antonio Rosato^1,2

¹Veneto Institute of Oncology - IOV, Padova, Italy, ²University of Padua, Department Of Surgery, Oncology And Gastroenterology (discog), Padova, Italy

AS: Academic Abstract Body: The immunosuppressive tumor microenvironment (TME) limits Chimeric Antigen Receptor (CAR)-T cell functionality in solid tumors. In particular, tumor extracellular matrix (ECM) rich in collagen and hyaluronic acid (HA) as in prostate cancer blocks immune cell infiltration. To improve therapy efficacy, we engineered T cells to degrade ECM by co-expressing PSMA-targeting CAR molecule, and either MMP collagenases or hyaluronidase (PH20) enzyme (CAR.E-T), in both membrane-bound and soluble forms. Enzymes expression was assessed by flow cytometry and ELISA. At day 9, CAR.E-T cells displayed significantly higher cytotoxicity against PSMA-positive PC3 (PiP) (69.18±13.4%) compared to PSMA-negative cells (21±4.7%), as assessed by real-time xCELLigence assay. Moreover, CAR.E-T cells exhibited enhanced in vitro infiltration compared with CAR-T cells expressing only the CAR molecule (CAR.Mock-T), as demonstrated by Transwell invasion assay, performed in either collagen-rich Matrigel or HA-hydrogel, followed by 3D confocal imaging. By co-culture with HA-producing cells, CAR.PH20-T induced a significant reduction of pericellular HA compared with CAR.Mock-T cells, as shown by particle exclusion assay. In vivo studies with NSG mice subcutaneously injected with PiP cells expressing luciferase, showed that intravenously-administered CAR.PH20-T cells (n=10) slowed tumor progression and achieved complete tumor eradication by day 10 post-treatment, as shown by bioluminescence imaging. On the contrary, CAR.Mock-T cells (n=10) exhibited tumor growth comparable to untreated controls (n=10). Ongoing multiplexed IHC analyses are assessing CAR.E-T cell infiltration within the tumor mass. Overall, ECM-degrading enzymes expression endowed CAR-T cells with improved invasion capacity, representing a promising strategy to enhance their therapeutic efficacy in stroma-rich tumors.

PB006 / #129

TOP 10 POSTER PRESENTATIONS

ULTRA-DEEP MURINE EPITOPE-SPECIFIC TCR LIBRARIES IDENTIFY DISPARATE CONTRIBUTIONS OF T CELL RECEPTOR CHAINS AS A COMMON MODE OF FUNCTIONAL EPITOPE RECOGNITION

Kersten Heyer¹, Adrian Straub¹, Abagail Delahoussaye¹, Zahra Abedi¹, Sophia Engels¹, Matthias Warkotsch¹, Philip Bradley^2,3, Dirk Busch^1,4

¹Technical University of Munich, Institute For Medical Microbiology, Immunology, And Hygiene, Munich, Germany, ²Fred Hutchinson Cancer Center, Herbold Computational Biology Program, Division Of Public Health Sciences, Seattle, United States of America, ³University of Washington, Institute For Protein Design, Seattle, United States of America, ⁴German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany

AS: Academic Abstract Body: A central challenge in T cell receptor (TCR)-based therapy is the immense and largely unpredictable diversity generated during TCR recombination, combined with the vast landscape of potential peptide–MHC (pMHC) targets. Improved understanding of the molecular determinants that govern TCR affinity, specificity, and cross-reactivity is essential for the rational identification and design of safe and effective therapeutic TCRs. We generated ultra-deep (>1,000 unique clonotypes per specificity) TCR libraries using single-cell RNA and paired TCR sequencing from more than 650 immunized C57BL/6 mice. These libraries encompass four well-characterized CD8⁺ T cell epitopes presented by two MHC class I alleles (H-2K^b/OVA-SIINFEKL, H-2K^b/M38-SSPPMFRV, H-2D^b/GP33-KAVYNFATC, and H-2D^b/M45-HGIRNASFI). The depth of these datasets enabled comprehensive dissection of TCRα- and TCRβ-chain contributions and the relative roles of germline-encoded versus non-templated CDR3 regions in functional epitope recognition. Across all epitopes, conserved framework gene usage combined with dominant non-templated CDR3 motifs, primarily driven by TCRβ-chains, emerged as a common feature. Importantly, the contribution of associated TCRα-chains to functional epitope-recognition is distinct and often involve germline-encoded Jα-segments. Structural comparison of available TCR–pMHC crystal structures, together with large-scale AlphaFold3-guided modeling of the ultra-deep libraries, supports TCRβ-chain dominated epitope engagement as a common binding mode in both mice and humans. Consistently, identical TCRα-chains can be identified that promote functional epitope recognition to different TCRß-chains for all 4 studied epitopes. Taken togehter, these findings provide a conceptual framework for improving TCR candidate selection and rational therapeutic design

PB007 / #132

TOP 10 POSTER PRESENTATIONS

ANTI-GD2 CAR T CELLS EFFICIENTLY TARGET EWING’S SARCOMA

Chiara Chiavelli¹, Giulia Golinelli¹, Malvina Prapa², Marco Silingardi¹, Leonardo Brini¹, Giuseppe Pugliese¹, Giovanni Neri¹, Rosanna Di Tinco³, Laura Bertoni³, Giulia Rovesti¹, Riccardo Cuoghi Costantini⁴, Tiziana Petrachi⁵, Jonathan Vinet⁶, Carlotta Spano¹, Marco Bestagno⁷, Maria Cristina Baschieri¹, Federico Banchelli⁴, Giovanni Palazzi⁸, Edwin M Horwitz⁹, Bruce L Levine¹⁰, Massimo Dominici¹

¹University-Hospital of Modena and Reggio Emilia, Modena, Italy, Division Of Oncology, Department Of Medical And Surgical Sciences For Children & Adults, Modena, Italy, ²Albanian University, Faculty of Medical Sciences, Tirana, Albania, Tirana, Albania, ³University of Modena and Reggio Emilia, Department Of Surgery, Medicine, Dentistry And Morphological Sciences With Interest In Transplant, Oncology And Regenerative Medicine, Modena, Italy, ⁴University of Modena and Reggio Emilia, Center Of Medical Statistic, Department Of Medical And Surgical Sciences For Children And Adults, Modena, Italy, ⁵Technopole “Mario Veronesi”, Mirandola, Modena, Italy, Mirandola, Italy, ⁶University of Modena and Reggio Emilia, Centro Interdipartimentale Grandi Strumenti (cigs), Modena, Italy, ⁷ICGEB Trieste, Trieste, Italy, ⁸University of Modena and Reggio Emilia, Modena, Italy, Pediatric Oncology And Hematology Unit, Department Of Medical And Surgical Sciences For Mothers, Children And Adults, Modena, Italy, ⁹Department of Pediatrics, Emory University School of Medicine, Atlanta (GA), USA, Atlanta, United States of America, ¹⁰Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA, Philadelphia, United States of America

AS: Academic Abstract Body: Ewing’s Sarcoma (ES) is a highly aggressive malignancy predominantly affecting children and young adults, and effective therapeutic options for relapsed or refractory disease remain limited. Although chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in hematologic cancers, its translation to solid tumors has been hindered by tumor heterogeneity, limited trafficking, and an immunosuppressive microenvironment. We previously demonstrated the antitumor activity of anti-GD2 CAR T cells in brain tumor models and showed that a truncated GD2 CAR enhances the targeting and therapeutic efficacy of mesenchymal stromal/stem cells against metastatic ES. Here, we further investigated the therapeutic potential of anti-GD2 CAR T cells in preclinical models of Ewing’s Sarcoma. Antitumor activity was evaluated in multiple in vitro two-dimensional (2D) and three-dimensional (3D) Ewing's Sarcoma models, as well as in an in vivo ES xenotransplant model. GD2 CAR T cells mediated robust cytotoxicity in both 2D and 3D systems, including against a weakly GD2-positive cell line, indicating effective antigen recognition and tumor cell killing even at low target density. In vivo, GD2 CAR T cells efficiently targeted localized tumors, significantly controlled tumor growth, and demonstrated substantial intratumoral infiltration. CAR T cells were also detectable in peripheral blood and spleen, supporting their in vivo persistence and systemic distribution. Collectively, these findings demonstrate preclinical efficacy of anti-GD2 CAR T cells against Ewing’s Sarcoma and support their further development as a therapeutic strategy for this and other GD2-positive malignancies with high unmet clinical need. Co-first Authors: Chiavelli C, Golinelli G.

PB008 / #134

TOP 10 POSTER PRESENTATIONS

TROGOCYTOSIS AND THE PARADOXIMAL IMPACT OF GAMMA SECRETASE INHIBITION ON BCMA CAR T CELLS

Pinar Ataca Atilla¹, Sylvain Simon², Erden Atilla¹, Ali Turan², David Coffey³, Grace Bugos², Melissa Comstock², Andrew Cowan², Stanley Riddell², Damian Green¹

¹University of Miami, Division of Transplantation and Cellular Therapy, Sylvester Comprehensive Cancer Center, Miami, United States of America, ²Fred Hutchinson Cancer Center, Translational Science And Therapeutics, Seattle, United States of America, ³University of Miami, Myeloma Institute, Sylvester Comrehensive Cancer Center, Miami, United States of America

AS: Academic Abstract Body: BCMA directed CAR T cells have changed the treatment landscape for multiple myeloma, and γ secretase inhibitors (GSIs) may further strengthen this approach by increasing surface BCMA and limiting antigen shedding. Early clinical experience suggests that patients with higher BCMA levels after GSI exposure experience deeper and more durable responses. Despite these gains, relapse remains common. Trogocytosis—the transfer of antigen from target cells onto CAR T cells—has emerged as a potential contributor to antigen loss, functional decline, and fratricide among CAR T cells. To clarify how GSI exposure shapes these processes, we examined trogocytosis kinetics in vitro and using samples from two Phase I BCMA CAR T trials +/- GSI (NCT03502577; NCT03338972).

GSI treatment intensified trogocytosis, particularly in high antigen density targets and CD4⁺ CAR T cells. CAR T cells that acquired BCMA (Trogo⁺) showed reduced proliferation, diminished killing capacity, and higher expression of exhaustion markers. These Trogo⁺ cells were also vulnerable to fratricide by naïve CAR T cells, as confirmed by confocal and live cell imaging. Single cell RNA seq analyses suggest that Trogo⁺ CAR T cells adopt transcriptional features with heightened activation and emerging exhaustion. In patient samples, BCMA trogocytosis was detectable, although a relationship to CAR T persistence and clinical outcomes did not reach statistical significance.

Together, these findings point to paradoxical impacts of GSI: improved tumor targeting but increased susceptibility to trogocytosis associated dysfunction. Refining GSI timing and developing strategies to limit trogocytosis may help strengthen the durability of BCMA CAR T therapy.

PB009 / #139

TOP 10 POSTER PRESENTATIONS

LOCOREGIONAL IL-7RΑ–ENHANCED B7-H3 CAR T CELLS (CMD03) IN PEDIATRIC DIPG: PRELIMINARY RESULTS FROM A PHASE I DOSE-ESCALATION TRIAL

Koramit Suppipat^1,2, Supannikar Tawinwung^2,3

¹Chulalongkorn University, Excellence Center For Cellular Immunotherapy, Bangkok, Thailand, ²Thailand Hub of Talent in Cancer Immunotherapy, Bangkok, Thailand, ³Chulalongkorn University, Faculty of Pharmaceutical Sciences, Pharmacology And Physiology, Bangkok, Thailand

AS: Academic Abstract Body: Background

Diffuse intrinsic pontine glioma (DIPG) remains a universally fatal pediatric brain tumor with a median survival of ∼11 months. B7-H3 is highly expressed in DIPG and represents a promising immunotherapeutic target. We developed CMD03, a novel B7-H3–directed CAR T cell incorporating IL-7 receptor alpha (IL-7Rα) signaling to enhance T-cell persistence and fitness. Preclinical studies demonstrated a less differentiated memory phenotype, increased proliferation, reduced activation-induced cell death, and superior tumor control with prolonged survival in murine models. Methods

We initiated a single-center, Phase I dose-escalation trial (NCT06221553) evaluating repeated intracerebroventricular infusions of CMD03 CAR T cells without lymphodepletion in pediatric patients with progressive DIPG. Dose levels were 1 × 10⁷, 3 × 10⁷, and 1 × 10⁸ CAR T cells per infusion. Primary endpoints were safety and tolerability; secondary endpoints included preliminary efficacy and CAR T-cell biodistribution. Results

Three patients were treated at dose level 1 following radiographic progression, receiving a total of 10 infusions. No dose-limiting toxicities were observed. Common adverse events included transient fever and headache. Two patients developed grade 3 tumor inflammation–associated neurotoxicity (TIAN), resolving within 24–72 hours with dexamethasone and anakinra. CAR T cells were detected in cerebrospinal fluid (5–20%) but not in peripheral blood. One patient achieved stable disease at two months. Conclusion

Repeated locoregional delivery of IL-7Rα–enhanced B7-H3 CAR T cells is feasible and manageable at the initial dose level. Ongoing evaluation will further define safety and therapeutic potential in DIPG.

PB010 / #235

TOP 10 POSTER PRESENTATIONS

SINGLE-CELL PROFILING GUIDES A DISEASE-TAILORED TCR-BASED IMMUNOTHERAPY FOR EPITHELIAL OVARIAN CANCER

Maria Chiara Maffia¹, Anna Simioni¹, Elena Tassi¹, Alice Bergamini², Chiara Balestrieri¹, Zulma Magnani¹, Barbara Camisa¹, Danilo Abbati¹, Alessia Potenza¹, Martina Spiga¹, Luca Bocciolone², Massimo Candiani², Miriam Sant'Angelo³, Maurilio Ponzoni³, Birgit Schultes⁴, Aaron Prodeus⁴, Eliana Ruggiero¹, Chiara Bonini¹

¹IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²IRCCS San Raffaele Hospital, Milan, Italy, Department Of Obstetrics And Gynecology, MILAN, Italy, ³IRCCS San Raffaele Hospital, Milan, Italy, Department Of Surgical Pathology, MILANO, Italy, ⁴Intellia Therapeutics, Cambridge, United States, Cambridge, United States of America

LB: Academic Abstract Body: Epithelial ovarian cancer (EOC) is a highly lethal malignancy with limited benefit from current immunotherapies. Although tumor-infiltrating lymphocytes correlate with improved outcomes, the mechanisms driving T-cell dysfunction remain poorly understood. Identifying key inhibitory receptors (IRs) and mapping the immune landscape are essential to develop more effective treatments. Single-cell transcriptomics and high-dimensional flow cytometry of patient-derived tumors and ascites revealed distinct CD8⁺ T-cell populations, including tissue-resident memory-like cells with preserved effector potential in ascites and exhausted tumor-infiltrating cells co-expressing multiple IRs. Flow cytometry confirmed the predominance of exhausted CD8⁺ subsets within tumors. These insights guided the engineering of T cells expressing six tumor antigen-specific TCRs following CRISPR/Cas9-mediated disruption of the endogenous TCR repertoire. The most promising product was further optimized through knockout of IRs (TIM3, LAG3 or 2B4). Engineered T cells were evaluated in vitro using patient-derived ascites and tumor organoids and in vivo. Among engineered lymphocytes, WT1-redirected T cells showed the strongest and most selective cytotoxicity, which was further enhanced by TIM3 disruption. TIM3_KO WT1-TCR T cells demonstrated superior tumor killing, particularly against targets expressing high levels of TIM3 ligands, and improved tumor regression in vivo. In conclusion, we mapped the immunological heterogeneity of EOC across distinct anatomical sites (tumor and ascites) and identified TIM3 as a central driver of T-cell dysfunction. Overall, this study highlights how single-cell profiling of the EOC microenvironment can guide the design of disease-tailored T cell-based therapeutics and establish TIM3_KO WT1-TCR T cells as a promising approach to overcome immune suppression.

USING DIGITAL PCR TO PROFILE VECTOR COPY NUMBER, EXPANSION AND BIODISTRIBUTION OF ANTIGEN-SPECIFIC TCR-TREGS FOR SLE

Cheng Rui Peh, Joshua Ooi, Yi Tian Ting

Monash University, Medicine, Clayton, Australia

AS: Academic Abstract Body: Antigen-specific regulatory T cells (TCR-Tregs) engineered with lentiviral vectors (LVVs) are an attractive strategy for treating SLE, but we still know relatively little about their in vivo behaviour or how best to monitor them. We aimed to describe in vivo expansion and organ biodistribution of SLE targeted TCR-Tregs in a murine model using a semi-automated manufacturing process and digital PCR (dPCR), and use in vitro–expanded human Tregs and Tconvs, transduced across graded multiplicities of infection (MOIs), to compare vector copy number (VCN) and GFP expression and test our in-house dPCR assay. For in vivo studies, PBMCs from HLA-DR15–positive SLE patients were used to generate antigen-specific TCR-Tregs on a Prodigy plus Tyto platform; final purity will be reported. These cells were infused into mice, and LVV-marked cells were tracked in blood and organs by dPCR. For in vitro studies, Tregs and Tconvs from HLA-DR15 heterozygous healthy donors were transduced with SM2-1 TCR LVV at MOIs 1, 3, 5 and 10. VCN was measured by commercial qPCR and in-house dPCR, and GFP by flow cytometry on days 10 and 20. In vitro, TCR-Tregs showed higher GFP and significantly higher VCN than Tconvs (P < 0.05). VCN rose with MOI and remained stable till day 20, whereas GFP declined. In vivo, dPCR revealed a TCR-Treg expansion peak around day 30 with subsequent contraction and detectable vector genomes in multiple organs. Overall, TCR-Tregs maintain stable VCN while down-modulating GFP, and dPCR provides a practical way to track kinetics, biodistribution and VCN.

PV002 / #260

Posters Viewing

ADVANCED TCR ENGINEERING

TARGETING CATHEPSIN G WITH A DUAL HLA-RESTRICTED TCR MEDIATES ACUTE MYELOID LEUKEMIA CONTROL

Francesca Marzuttini¹, Alessia Potenza¹, Ludovica Celli¹, Anna Simioni¹, Laura Conte², Barbara Camisa¹, Zulma Magnani³, Lucia Sergi Sergi¹, Neda Mohammadi¹, Samantha Scaramuzza⁴, Rita El Khoury⁵, Ciro Improta¹, Alessia Ugolini¹, Martina Spiga¹, Erica Remiddi¹, Alice Grometto¹, Erica Carnevale¹, Vanessa Cavallaro¹, Stefania Veronese¹, Alessandra Scola¹, Cristina Toffalori⁵, Danilo Abbati¹, Francesca Sanvito⁶, Maurilio Ponzoni⁷, Maria Teresa Lupo-Stanghellini⁸, Maria Themeli⁹, Monica Casucci¹⁰, Massimo Degano², Luca Vago², Ivan Merelli¹¹, Fabio Ciceri¹², Chiara Bonini¹³, Eliana Ruggiero^1,2

¹IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²Ospedale San Raffaele, Milano, Italy, ³San Raffaele Scientific Institute, Immunology, Transplantation And Infectious Diseases, MILAN, Italy, ⁴San Raffaele-Telethon Institute for Gene Therapy (SR-TIGET), Milan, Italy, ⁵IRCCS San Raffaele Scientific Institute, Milan, Italy, ⁶IRCCS San Raffaele Scientific Institute, Patholgy Unit, Milan, Italy, ⁷IRCCS San Raffaele Hospital, Milan, Italy, Department Of Surgical Pathology, MILANO, Italy, ⁸IRCCS Ospedale San Raffaele, Milan, Italy, ⁹Vrije Universiteit Amsterdam, Amsterdam, Netherlands, ¹⁰IRCCS San Raffaele Scientific Institute, Innovative Immunotherapies Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ¹¹Institute for Biomedical Technologies, National Research Council, Segrate, Italy, ¹²IRCCS San Raffaele Scientific Institute, Hematology And Bone Marrow Transplantation Unit, Milan, Italy, ¹³Vita-Salute San Raffaele University, Milan, Italy

LB: Academic Abstract Body: Hematopoietic stem cell transplantation has unveiled the sensitivity of acute myeloid leukemia (AML) to immunotherapy and has paved the way for the development of adoptive cell therapies. T cell receptor (TCR)-based approaches represent a promising strategy, as they enable the recognition of both intracellular and extracellular antigens with high specificity and sensitivity, offering the possibility to target molecules shared across AML subtypes and essential for leukemic cell survival. However, their potential remains limited by HLA restriction. In this study, we identified and characterized a novel TCR specific for Cathepsin G (CG), a serine protease normally confined to neutrophil granules but aberrantly localized in the cytoplasm of AML blasts. The CG-specific TCR was introduced into primary T cells by lentiviral transduction, combined with CRISPR/Cas9-mediated knockout of endogenous TCRs and co-expression of the CD8 co-receptor to enhance functionality. CG-TCR engineered T cells displayed cytotoxic activity against primary AML blasts both in vitro and in vivo. Importantly, the identified TCR recognized CG-derived peptides presented by two common HLA alleles, HLA-A*24:02 and HLA-C*07:02, broadening its applicability across a large patient population. Notably, safety assessment demonstrated an optimal safety profile, with no evidence of on-target off-tumor toxicity, as no alterations in normal hematopoiesis were observed. In addition, the absence of off-target cross-reactivity was confirmed by peptide mutagenesis. These findings highlight the therapeutic potential of CG-specific, dual-restricted TCR-engineered T cells as a broadly applicable and effective strategy for AML treatment.

PV003 / #118

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ADVANCED TCR ENGINEERING

SELECTION OF FUNCTIONAL MHC-II RESTRICTED, HBV-SPECIFIC MURINE T CELL RECEPTORS TO STUDY THE ROLE OF CD4+ T CELLS IN PRECLINICAL CHRONIC INFECTION MODELS

Tobias Sixt^1,2,3, Jinpeng Su^1,2,3, Romina Bester¹, Ioanna Gemünd¹, Nikolas Müller¹, Edanur Ates Öz^1,2, Luis Olguin-Contreras¹, Ulrike Protzer^1,2,3

¹Technical University of Munich, Institute Of Virology, Munich, Germany, ²Helmholtz Center Munich, Institute Of Virology, Munich, Germany, ³German Center for Infection Research (DZIF), Munich, Germany

AS: Academic Abstract Body: Chronic hepatitis B virus (HBV) infection remains a global health burden, leading to liver cirrhosis and hepatocellular carcinoma with a death toll of 1.1 million humans per year. T-cell-based therapies such as therapeutic vaccination, adoptive T cell therapy, and T-cell-engager antibodies represent a promising option for curing HBV infection. We and others have shown that HBV-specific CD4⁺ T cell help is crucial for viral clearance. Further in-depth analysis of the role of CD4⁺ T cells in chronic HBV infection is required to obtain functional immunological insights, but a transgenic mouse model is currently lacking. We therefore aimed at identifying HBV-specific CD4⁺ T-cell receptors from C57BL/6 mice. First, murine HBsAg- and HBcAg-specific CD4⁺ T cell epitopes were identified by screening cytokine responses of splenocytes from TherVacB-immunized mice following restimulation with overlapping HBV peptide libraries. Identified immunodominant epitopes were used to generate peptide-MHC-II-tetramers that allowed the isolation of HBsAg- and HBcAg-specific, MHC-II-restricted T cells from C57BL/6 mice after three immunizations using recombinant HBsAg and HBcAg formulated with the Th1-inducing adjuvant LMQ. Following single-cell-RNA-sequencing of isolated T cells, a total of 4 HBsAg- and 7 HBcAg-specific clonotypes were identified within different phenotypical clusters. The TCRs of identified clonotypes were fully reconstructed and cloned into pMP71 retroviral vectors. In vitro functional validation using transduced murine splenocytes and Jurkat triple-reporter (TPR) cells is used to identify the most suitable MHC-II-restricted, HBV-specific TCR candidates, which will serve as experimental tools for studying chronic HBV infection and for the future development of a novel transgenic mouse model.

PV004 / #156

Posters Viewing

BEYOND ALPHA-BETA T CELLS

OPTIMIZING ADOPTIVE IMMUNOTHERAPY OF LEUKEMIA WITH CD1C-REDIRECTED T CELLS

Michela Consonni¹, Ivan Civettini², Claudio Garavaglia², Giulia Mondardini², Alessandra Mancino², Leonie Bourgeois³, Daniel Häussinger³, Paolo Dellabona¹, Giulia Casorati¹

¹IRCCS San Raffaele Scientific Institute, Experimental Immunology Unit, Division Of Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²Irccs Ospedale San Raffaele, Experimental Immunology Unit, Division Of Immunology, Transplantation And Infectious Diseases, Milano, Italy, ³Universität Basel, Departement Chemie, Basel, Switzerland

AS: Academic Abstract Body: Adoptive cell therapy (ACT) with genetically redirected T cells represents a promising strategy to reduce the high relapse rate of acute leukemias following frontline treatment. We previously demonstrated that AML and B-ALL blasts express CD1c and can be targeted by T cells recognizing a leukemia-associated lipid antigen presented by CD1c. We identified a lead leukemia-specific TCR (DN4.99) that effectively redirects T cells against CD1c-expressing leukemias, mediating tumor killing in vitro and delaying disease progression in immunodeficient mice. This define a viable ACT strategy for any CD1c-expressing leukemia that works across MHC-barriers. To further enhance the efficacy of DN4.99 TCR-engineered T cells, we are exploring three complementary strategies: (i) increasing the functional avidity without altering antigen specificity by removing an N-glycosylation site in the TCR Cα region (DN4.99 Gly–) and by overexpressing CD3ζ to maximize the TCR surface expression; (ii) co-engineering DN4.99 Gly–TCR-T cells with chimeric costimulatory receptors (CCR) containing intracellular costimulatory domains, but lacking CD3ζ signaling; (iii) co-expressing a membrane-bound IL-2 (tmIL-2) to support long-term leukemia control, while balancing IL-2 efficacy and toxicity. We found that removal of the glycosylation site significantly improved leukemia control in vitro and in vivo, while CD3ζ overexpression further enhanced TCR surface expression. CCRs targeting CD38 and CD33 lowered the activation threshold and sustained cytotoxicity in vitro, while tmIL-2 supported T-cell expansion in vitro, indicating functional cytokine activity. Our results delineate effective improvements for our CD1c-redirected ACT strategy for acute leukemia. We are currently gaining further insight into the relative efficacy and safety of the described approaches.

PV005 / #43

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BEYOND ALPHA-BETA T CELLS

TO ELUCIDATE THE IMMUNOMODULATORY AND ANTI-TUMOR ROLE OF M1 MACROPHAGE ON BONE MARROW DERIVED MESENCHYMAL STEM CELLS IN BREAST CANCER

Pramod Gautam

AIIMS, New Delhi, Biochemistry, New Delhi, India

AS: Academic Abstract Body: Breast cancer is the most prevalent malignancy among women globally and continues to provide a significant treatment challenge owing to tumor heterogeneity and microenvironment-driven development. In the tumor microenvironment, MSCs can transform into a type that helps tumors and becomes cancer-associated fibroblasts (CAFs), which assist tumor growth. This study aimed to understand how M1 macrophages can change tumor-associated MSCs (T-MSCs) into a normal phenotype and reduce their tumor-promoting activities. The high-purity murine peritoneal macrophages M1 polarization through the stimulation of IFN-7 and LPS. Macrophages were highly polarized (M1, 86%percent) with up-regulation of CD11c, CD86, TLR2/4, TNF-a, and IL-6 and low levels of M2 markers after 48 hours of stimulation. BM-MSCs were successfully isolated, initially exhibiting a rounded morphology that progressively transitioned into a spindle-shaped fibroblastic configuration by days 3 to 5, reaching 60 to 70% confluence by day 7. Flow cytometry analysis revealed a progressive enrichment of MSCs over passages, with CD44⁺/CD105⁺ cells increasing from 0.059% at P-1 to 71.9% at P-2 and ultimately reaching 92.0% at P-3, while remaining negative for CD31 and CD45. M1-CM treatment significantly reduced CAF-associated markers (CD29, PDPN, and FSP) and downregulated EMT-related features in T-MSCs. Cytokine analysis revealed decreased TNF-α, IL-10, VEGF, and IL-4 expression, indicating a shift toward a less tumor-supportive microenvironment. Functional assays demonstrated reduced proliferation, migration, and invasion of T-MSCs following M1-CM exposure. Collectively, these findings suggest that M1 macrophages can reverse the pro-tumorigenic programming of MSCs, highlighting the therapeutic potential of targeting the macrophage–MSC axis as a novel cell-based immunotherapeutic strategy in breast cancer.

PV006 / #92

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BEYOND ALPHA-BETA T CELLS

ENGINEERING LENTIVIRAL VECTORS FOR IN VIVO MACROPHAGE-TARGETED IFNΑ GENE THERAPY IN METASTATIC LIVER DISEASE

Giovanna Giacca, Giulia Buccarello, Sara Breggion, Chiara Bresesti, Stefano Beretta, Thomas Kerzel, Marco Notaro, Alessandro Vallorini, Attya Omer-Javed, Elisa Montaldo, Marina Radrizzani, Anna Rudnitski-Kajaste, Luigi Naldini, Mario Squadrito

SR-TIGET, Milan, Italy

AS: Academic Abstract Body: The liver’s inherently immunosuppressive microenvironment facilitates the development of liver metastases (LMS), contributing to the failure of many systemic therapies and resulting in poor clinical outcomes. We previously established an in vivo gene therapy platform using lentiviral vectors (LVs) to selectively reprogram liver macrophages to secrete type I interferon (IFNa) within metastatic lesions. Selective IFNa expression was achieved by a macrophage-specific promoter and microRNA target sequences that, upon systemic delivery of the LV to mice, led to selective transgene expression in macrophages. Gene-based IFNα delivery reduced metastatic volume of both colorectal and pancreatic ductal adenocarcinoma LMS in mice, thus offering a promising therapeutic option for patients. To drive the IFNa expression in human macrophages, we identified and employed a promoter sequence derived from the human MRC1 gene, which is robustly expressed in tumor-associated macrophages. To support clinical translation, we addressed the barrier posed by human-specific restriction mechanisms by engineering LV particles to incorporate accessory proteins. Engineered LVs enhanced transduction efficiency of cultured human macrophages by more than tenfold compared to standard LVs. In summary, engineering of LVs to incorporate accessory proteins and the identification of the novel human MRC1 promoter sequence, provide powerful tools for advancing LV-based gene therapies. These innovations enable in vivo engineering of liver macrophages and represent a critical step toward the translation of in vivo gene therapy for metastatic liver disease.

PV007 / #245

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BEYOND ALPHA-BETA T CELLS

PLURIPOTENT STEM CELL-DERIVED TYPE 2 INNATE LYMPHOID CELLS EXHIBIT POTENT ANTITUMOR ACTIVITY AGAINST PANCREATIC CANCER CELLS

Grace I-Chih Kuo^1,2, Jiyoung Yun¹, Thristan Taberna¹, Julyanne Brassard¹, Ross Jones¹, Ayaz Damji¹, Si Xuan Chen¹, Peter Zandstra^1,3, Kelly Mcnagny^1,4,5

¹University of British Columbia, School Of Biomedical Engineering, Vancouver, Canada, ²University of British Columbia, Experimental Medicine, Faculty Of Medicine, Vancouver, Canada, ³University of British Columbia, Michael Smith Laboratories, Vancouver, Canada, ⁴University of British Columbia, Department Of Medical Genetics, Vancouver, Canada, ⁵Centre for Heart and Lung Innovation (HLI), St Paul’s Hospital, Vancouver, Canada

LB: Academic Abstract Body: Introduction: Innate lymphoid cells (ILCs) are tissue-resident immune cells that share developmental and transcriptional programs with T cells but lack antigen-specific receptors. They function as frontline effectors, initiating immune responses prior to antigen-specific T cell expansion and combating infections. Like T cells, they also exhibit anti-tumor functions through cytotoxicity, cytokine secretion, and immune cell recruitment. Leveraging these properties could offer a novel avenue for cancer immunotherapy, potentially overcoming some limitations of conventional T cell-based approaches in treating solid tumors. Methodology: We developed a differentiation workflow to generate ILC2-like cells from human pluripotent stem cells (hPSCs). Cytokine supplementation, ligand coating, and small molecule supplementation were optimized to promote the development of functional, cytokine-secreting ILC2s under feeder-free and animal-component-free conditions. Results: Flow cytometry revealed that ∼50% of CD45+Lineage-cells are mature ILC2-like cells secreting type 2 cytokines (IL-5 and IL-13). These cells display metabolic and immunophenotypic characteristics comparable to peripheral blood-derived ILC2s. Upon IL-33 stimulation, iPSC-ILC2s induce apoptosis in pancreatic cancer cells through the DNAM1-CD112/CD155 axis and significantly inhibit tumor cell proliferation. These effects were confirmed using co-culture imaging and MTT assays. Transwell experiments and inflammatory-mediator profiling identified both direct cytotoxic interactions and soluble factors contributing to cancer cell killing. Preliminary xenografting studies in NSG mice confirmed in vivo antitumor activity and persistence. Conclusion: This work establishes a scalable platform for generating functional ILC2s from PSCs, enabling detailed studies of human ILC2 biology. These findings support the development of ILC-based immunotherapies and suggest broader therapeutic applications in chronic inflammatory and autoimmune diseases.

PV008 / #238

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BEYOND ALPHA-BETA T CELLS

DEVELOPMENT OF NOVEL MONOCLONAL ANTIBODIES AGAINST HUMAΝ VDELTA1 T CELL RECEPTOR

Fanying Li^1,2, Leon Separautzki^1,2, Alper Yilmaz^1,2, Chantal Pelzer^1,2, Jean-Pierre Dangy^1,2, Erica Lana^1,2, Catherine Mkindi³, Maximillian Mpina³, Craig Fenwick^4,5, Said Jongo³, Claudia Daubenberger^1,2, Mathias Schmaler^1,2

¹Swiss Tropical and Public Health Institute (Swiss TPH), Department Of Medical Parasitology And Infection Biology, Allschwil, Switzerland, ²University of Basel, Basel, Switzerland, ³Ifakara Health Institute, Bagamoyo, Tanzania, ⁴Lausanne University Hospital (Centre Hospitalier Universitaire Vaudois), Service Of Immunology And Allergy, Department Of Medicine, Lausanne, Switzerland, ⁵University of Lausanne, Lausanne, Switzerland

LB: Academic Abstract Body: Human Vδ1 T cells are predominantly tissue-resident lymphocytes that play a crucial role in maintaining tissue homeostasis and providing immune protection against infections and malignancies. A highly limited range of commercially available monoclonal antibodies can specifically bind Vδ1+ T cells—irrespective of whether they are fresh or fixed—and support their selective in vitro activation or proliferation. In this report, we detail how we generated and studied two new monoclonal antibodies that recognize the Vδ1 T cell receptor chain. NMRI mice were immunized with an engineered, secreted VγVδ1 heterodimer produced in Expi293F cells. Subsequently, hybridoma technology was employed to generate antibody-producing clones. Several clones were found to produce antibodies that specifically recognized both soluble and cell-surface VγVδ1 heterodimers, as demonstrated by indirect ELISA and flow cytometry. Of these, two antibodies with IgG1 heavy and κ-light chains were selected, demonstrating high affinity (0.266 nM) and avidity for binding soluble VγVδ1 TCR. One antibody triggered an increase in CD69 expression on SKW-3 cells carrying the Vδ1 TCR, when paired with various γ-chains. Both specifically stained Vδ1+ T cells in fresh and paraformaldehyde-fixed peripheral blood mononuclear cells (PBMC), making them well suited for studying tissue residency of Vδ1 T cells in fixed or bio-banked tissues. Furthermore, the antibodies facilitated in vitro activation and proliferation of Vδ1+ T cells in PBMC of a range of human donors. In summary, we demonstrate that two novel antibodies have been generated that will allow for in-depth investigation of γδ T cells in health and disease.

PV009 / #23

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BEYOND ALPHA-BETA T CELLS

CHIMERIC ANTIGEN RECEPTOR-ENGINEERED NK92 CELLS FOR ADOPTIVE IMMUNOTHERAPY OF PANCREATIC CANCER

Gaia Zuccolotto¹, Alessandro Penna², Giulio Fracasso², Anna Tosi¹, Antonio Rosato^1,2

¹Istituto Oncologico Veneto, Padova, Italy, ²università degli studi di padova, padova, Italy

AS: Academic Abstract Body: Pancreatic cancer (PC) is one of the leading causes of malignancy-related death, with almost half-million new diagnosis every year. Aberrant over-expression of the human prostate specific membrane antigen (hPSCA) has been detected in the vast majority of patients diagnosed with PC. This molecule can be used as potential target for treatments, in particular for ready-to-use immunotherapy and adoptive cell therapy (ACT). NK92 cells are a new strategy among ACTs, without most of the inhibitory killer-cell immunoglobulin-like receptors (KIRs), with well-defined expansion kinetics. Furthermore, this cell line is already approved for the clinical use towards different tumor types and shows high cytotoxic activity, even after irradiation required by authorities. We designed a new CAR anti-hPSCA to transduce NK92 cells, in order to create a ready-to-use therapy and after 72h from the transduction, the CAR and the eGFP, as reporter gene, under the control of a bi-directional promoter, were already detectable. The phenotypic profile of activated NK cells was stable before and after the transduction. CAR-NK92 cells have shown a high and specific lysis of the antigen expressing tumor cells, as well as specific cytokines release ability and the activation of degranulation after antigen engagement. In vivo, in a disseminated and orthotopic tumor mouse models, the CAR-NK-92 controlled the tumor growth and improved survival of the mice treated. This CAR NK-92 therapy paves the way of a new therapeutic approach for PC, based on the development of an Off-the-Shelf, renewable, effective and low-cost product that can be advanced as a ready-to-use drug.

PV010 / #84

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CAR/TCR MRNA THERAPIES

DEVELOPMENT OF FAST AND COST-EFFECTIVE MRNA-CAR-T SPECIFIC TO CA125

Kamilla Aminulla, Artem Pilunov, Vladimir Bozhenko

Russian Scientific Center of Roentgenology and Radiology, Ministry of Health of the Russian Federation, Moscow, Russian Federation

AS: Academic Abstract Body: mRNA-based CAR-T therapy is an innovative approach to immunotherapy for solid tumors. Compared with viral vectors, mRNA transfection offers a superior safety profile by minimizing the risks of cytokine release syndrome and off-tumor toxicity. Furthermore, this method is characterized by rapid manufacturing, reduced costs, and the ability to control the duration of the therapeutic effect, which is critical when targeting tumor-associated antigens. One such target is the CA125 antigen (a derivative of the MUC16 protein), the overexpression of which is characteristic of ovarian cancer. We engineered third-generation CA125-specific CAR-T lymphocytes and developed a cost-effective manufacturing protocol for them. To optimize production time and cost-effectiveness, three distinct T-cell culture strategies followed by mRNA electroporation were evaluated. These strategies included the use of non-activated T-cells (<24 h), activation with CD3/CD28 magnetic beads (3–4 days), and activation using Phytohemagglutinin (PHA) combined with IL-2 (1–2 days), the latter of which significantly reduces production costs. Assessment of cytotoxic activity via Real-Time Cell Analysis (RTCA xCELLigence) confirmed the high specificity of the generated CAR-T cells toward the OVCAR3 cell line (high CA125 expression), with no activity observed against cells with low antigen levels (OAW-42). Comparative efficacy analysis revealed that non-activated CAR-T cells demonstrated cytotoxicity only at a high effector-to-target (E:T) ratio (5:1). Conversely, activated cells were effective at a 1:1 ratio, while bead-activated cells retained cytotoxic potential even at an E:T ratio of 0.25:1. Thus, the PHA-based protocol provides an optimal balance between manufacturing speed, cost-efficiency, and the functional activity of CAR-T cells.

PV011 / #167

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CAR/TCR MRNA THERAPIES

OVERCOMING TGF-Β-MEDIATED CAR T CELL DYSFUNCTION IN NEUROBLASTOMA USING DOMINANT-NEGATIVE TGF-Β RECEPTOR EXPRESSION OR TGF-Β RECEPTOR INHIBITION

Tabea Biereder¹, Laura Grunewald¹, Catharina Delebinski¹, Judith Feucht², Michael Hudecek³, Sebastian Kobold⁴, Annette Künkele¹

¹Charité Universitätsmedizin Berlin, Oncology And Hematology, Berlin, Germany, ²Universitätsklinikum Tübingen, Pediatric Oncology And Hematology, Tübingen, Germany, ³Universitätsklinikum Würzburg, Würzburg, Germany, ⁴Institute of Clinical Pharmacology, LMU Klinikum, Munich, Germany

AS: Academic Abstract Body: Background: The efficacy of CAR T cell therapy in solid tumors is severely limited by the immunosuppressive tumor microenvironment (TME). A central driver of this resistance is TGF-β, a cytokine frequently cited for its role in immune evasion, impairing T cell effector functions and long-term persistence. Methods: To counteract TGF-β-mediated suppression, we engineered L1CAM-, B7H3-, or ROR2-CAR T cells by co-expressing a dominant-negative TGF-β receptor (dnTGFβRII). In parallel, pharmacological inhibition was tested using LY2109761, a TGFβRII inhibitor. TGF-β levels and CAR T cell cytokine production were measured by ELISA. Functional assays included cytotoxicity assessed via Incucyte live-cell imaging. Flow cytometry was used to quantify activation and exhaustion markers, proliferation capacity, and intracellular pSMAD2/3 levels. Finally, metabolic fitness was evaluated using Seahorse XF assays. Results: High TGF-β levels exerted strong suppressive effects on CAR T cells, impairing cytotoxicity, proliferation, and cytokine secretion. Both co-expression of dnTGFβRII and pharmacological inhibition via LY2109761 effectively restored CAR T cell function. This was further validated by significantly reduced intracellular pSMAD signaling. Distinct functional differences were observed among CAR constructs targeting different antigens. Metabolic profiling confirmed restored mitochondrial spare respiratory capacity, while FACS data showed a significantly reduced exhaustion phenotype in protected CAR T cells. Conclusions: Our findings identify TGF-β as a critical mediator of CAR T cell dysfunction in neuroblastoma. Disruption of TGF-β signaling restores effector function, improves metabolic fitness, and mitigates T cell exhaustion. This combinatorial strategy represents a promising approach to enhance CAR T cell efficacy in solid tumors.

PV012 / #170

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CAR/TCR MRNA THERAPIES

CONTEXT-DEPENDENT REDIRECTION OF REGULATORY T CELLS FOR PRECISION THERAPY IN TYPE 1 DIABETES

Susanna Cesarano¹, Eliana Ruggiero¹, Giulia Moriggia¹, Matteo Doglio¹, Paolo Monti², Chiara Bonini^1,3

¹IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²IRCCS San Raffaele Scientific Institute, Transplantation Immunology Unit, Milan, Italy, ³Vita-Salute San Raffaele University, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy

AS: Academic Abstract Body: CD4⁺CD25^bright^CD127^low^FOXP3⁺ regulatory T cells (Tregs) are central mediators of immune tolerance and represent a promising cellular therapy for type 1 diabetes (T1D). However, polyclonal Treg approaches have shown limited persistence and efficacy, highlighting the need for antigen-specific redirection strategies. Our project develops two complementary but conceptually distinct approaches, each tailored to a different clinical setting of T1D. The first strategy focuses on combinatorial CAR-Tregs designed for iPSC-derived beta cell transplantation. These Tregs target engineered beta cells expressing two defined surface molecules, enabling dual-antigen recognition and spatially restricted activation within the graft microenvironment. We are implementing a split-signaling design in which one CAR provides only the CD3ζ activation domain, while the second CAR delivers exclusively the CD28 costimulatory signal. Full activation therefore requires simultaneous engagement of both targets, increasing specificity and limiting off-target immunosuppression. Additional regulatory domains are incorporated to enhance Treg stability, reinforce suppressive function, and preserve FOXP3 expression. Because CAR signaling is independent of HLA restriction and co-receptor requirements, this platform enables controlled and robust activation in CD4⁺ Tregs. Expansion protocols using IL-7 and IL-15 are being optimized to improve persistence and lineage stability. In contrast, the second strategy employs TCR-engineered Tregs directed against naturally occurring beta cell autoantigens to intervene in the natural history of autoimmune diabetes. This approach aims to modulate ongoing autoimmune responses during early or preclinical disease stages. Together, these platforms address two distinct goals: transplant-specific immune protection and antigen-specific regulation of disease progression.

PV013 / #21

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CAR/TCR MRNA THERAPIES

INNOVATIVE LIPID-BASED DELIVERY SOLUTION: UNLOCKING NEW OPPORTUNITIES IN MANUFACTURING AUTOLOGOUS TRANSIENT CD19 CAR-T/NK OR ALLOGENEIC CAR-T

Claire Gueguen, Mélodie Seiler, Marine Fenat, Patrick Erbacher, Julie Chevrier

Sartorius Polyplus, ILLKIRCH, France

AS: Industry Abstract Body: Gene-modified cell therapies, such as CAR-expressing T cells and Natural Killer (NK) cells, are among the most promising treatments for cancer and autoimmune diseases. Their manufacture currently relies mainly on lentiviral vectors, which involve long production timelines, high costs, and safety concerns that limit scalability. Here, we present an innovative non-viral, lipid-based delivery solution as a practical alternative for generating both autologous CAR-T/NK cells and allogeneic CAR-T cells. We developed an innovative lipid-based platform to efficiently deliver CD19 mRNA into T and NK cells, generating transient CD19 CAR-T and CAR-NK cells. In parallel, the same platform was used to co-deliver Cas9 mRNA and TRAC-targeting sgRNA for CRISPR–Cas9–mediated TCR knockout, and to support CAR knock-in by combining CRISPR components with lentiviral or adeno-associated viral vectors. This strategy achieved efficient TRAC knockout (up to 85%), high CD19 CAR expression (up to 90%), and stable genomic integration of the CAR cassette, enabling the production of functional CD19 CAR-T and CAR-NK cells that eliminate CD19+ tumor cells, as well as “universal” CAR-T cells. Overall, this ready-to-use, non-viral lipid delivery platform emerges as a practical, scalable alternative to lentiviral vectors for manufacturing next-generation CAR-engineered immune cell therapies. By enabling both transient mRNA expression and precise CRISPR-mediated CAR knock-in, the approach achieves uniform, stable CAR expression in T cells. Its ease of use, versatility across payloads and cell types, and potential to shorten timelines and reduce costs highlight a compelling path toward safer, more efficient production of next-generation cell therapies.

PV014 / #62

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CAR/TCR MRNA THERAPIES

ANTIGEN-SPECIFIC TCR DISCOVERY USING GENE SIGNATURES THAT DISTINGUISH TCR- FROM BYSTANDER-ACTIVATED CD8+ T CELLS

Martin Daniel Qui, Yirong Dai, Anthony Tan, Antonio Bertoletti

Duke-NUS Medical School, Singapore, Singapore

AS: Academic Abstract Body: Responses to antigen stimulus by CD8+ T cells can be driven by either TCR-activated or bystander T cells, with the latter having been shown to predominate in tumors. This poses a challenge for the identification of TCRs from tumor repertoires for designing TCR-redirected T cells; we posited that gene signatures specific to TCR activation may circumvent this problem, even with low frequencies of antigen-specific cells. As a proof of concept, we leveraged scRNAseq with paired TCR sequences to track the transcriptomic responses of low frequency virus-specific memory T cells, to their cognate and non-cognate antigens ex vivo. The antigen-specificity of clones were determined through the use of barcoded pMHC-peptide complexes. Here, we have shown that a public TCR:bystander gene signature adequately identifies TCR-activated cells. We further developed a TCR:bystander gene signature to better distinguish TCR-activated cells. We show that while classical expression of GZMB and IFNG is enriched in TCR-activated cells, LAG3 and IER3 better discriminates TCR-dependent activation. These findings may improve the workflow for identification of TCR candidates for TCR-redirected T cell therapy.

PV015 / #55

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HSC GENETIC ENGINEERING AND EPITOPE EDITING

IL1RAP-HYPOMORPHIC HEMATOPOIETIC CELL TRANSPLANTATION PAIRED WITH EPITOPE-SELECTIVE CAR-T CELLS AS A COMBINATORIAL CELL THERAPY AGAINST AML

Simon Garaude^1,2, Martin Pacesa³, Michelle Buri^1,2, Julien Capin⁴, Morgane Lecointre^2,5, Jerome Bonnet⁴, Bruno Correia⁶, Douglas Hanahan^2,5, Caroline Arber^1,2, Bernhard Gentner^1,2

¹Lausanne University Hospital (CHUV), Oncology, Lausanne, Switzerland, ²Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne and Lausanne University Hospital (CHUV); Lausanne, Switzerland., Lausanne, Switzerland, ³Swiss Federal Institute of Technology Lausanne (EPFL), Laboratory Of Protein Design And Immunoengineering, Institute Of Bioengineering, Lausanne, Switzerland, ⁴University of Montpellier, INSERM U1054, CNRS UMR5048, Structural Biology Center, Montpellier, France, ⁵Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland, ⁶Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL); Lausanne, Switzerland, Lausanne, Switzerland

AS: Academic Abstract Body: IL1RAP is a signaling adaptor required for IL1 superfamily receptor activity (IL1, IL33 and IL36). IL1RAP is overexpressed on leukemic stem cells (LSC) in acute myeloid leukemia (AML), where it associates with aggressive disease. More broadly, IL1RAP has emerged as a central hub mediating systemic immunosuppression (SIS) in cancer patients (Lecointre, Guillot et al, Cancer Discov 2025). Our goal is to develop highly potent CAR-T cells against IL1RAP and, at the same time, attenuate immune hyperactivation and SIS by co-engineering the non-malignant hematopoiesis. First, we studied the phenotype of IL1RAP-knockout (KO) in human hematopoietic stem and progenitor cells (HSPCs) by disrupting a splice site through adenine base editing. IL1RAP-KO HSPC showed reduced myeloid colony formation and IL1-driven differentiation towards MDSC phenotypes validating IL1RAP as an HSPC target to block SIS. Next, we performed in silico structural analysis of the IL1/33/36Rs:IL1RAP interfaces to guide a semi-empirical base-editing screen which led to the identification of a stably expressed hypomorphic IL1RAP variant with reduced IL1 dose-sensitivity, while preserving responsiveness at higher cytokine concentrations, as may occur during infection. By establishing an in vivo chimerism with IL1RAP-hypomorphic HSPCs, we expect to attenuate both SIS and immune effector cell toxicities, which have limited CAR-T treatments in AML. In parallel, we have developed an AI-designed anti-IL1RAP binder with selectivity for wild-type over hypomorphic IL1RAP. Incorporated into CAR constructs, preliminary data support selective killing of IL1RAP–wild-type malignant cells while sparing cells carrying hypomorph IL1RAP, supporting a combined strategy of immune reprogramming and precision tumor targeting.

PV016 / #96

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IN VIVO LYMPHOCYTE ENGINEERING

TOWARDS B CELL ENGINEERING IN VIVO - A LENTIVIRAL VECTOR THAT CAN SPECIFICALLY TARGET HUMAN AND MOUSE B CELLS

Dennis Doorduijn^1,2, Ata Ul Wakeel Ahmad^1,2, Kathrin De La Rosa^1,2

¹Max Delbrück Center for Molecular Medicine, BERLIN, Germany, ²Centre for Individualised Infection Medicine (CiiM), a joint venture between the Helmholtz-Centre for Infection Research (HZI) and the Hannover Medical School (MHH), Hannover, Germany, Department Of Personalised Immunotherapy, Hannover, Germany

AS: Academic Abstract Body: Genetic engineering of the B cell receptor (BCR) would enable the design of B cells that are predisposed to produce broadly neutralizing antibodies against rapidly evolving pathogens, such as HIV. However, B cells are notoriously difficult to genetically engineer and target specifically in vivo. Here, we aimed to a develop lentiviral vector that can specifically target human and mouse B cells. Lentivirus encoding eGFP was pseudotyped with a Sindbis virus spike protein that 1) has mutations that abrogate cell attachment without affecting fusion-potential 2) contains a protein A domain (ZZ) that is an anchor for cell-targeting antibodies (m168-ZZ). m168-ZZ lentiviral particles were coated with B cell-specific antibodies (anti-CD19, anti-CD21) or an aspecific antibody (anti-MERS-CoV S), and incubated with primary human or mouse B cells to read-out transduction efficiency by flow cytometry. m168-ZZ lentivirus coated with B cell-specific antibodies targeting CD19 or CD21 transduced up to 40-80% of human or mouse B cells ex vivo. Only B cell-specific antibodies increased transduction efficiency approximately 20-fold compared to lentivirus without antibody, whereas lentivirus coated with aspecific antibody did not. Additionally, B cell-specific antibodies did not increase transduction efficiency of non-B-cell lines, suggesting B cell-specific enhancement of transduction. Altogether, we have successfully developed a lentiviral vector that can target and transduce human and mouse B cells. Further experiments will be performed to assess specific targeting of B cells and vectors with cargo to engineer BCRs in more complex ex vivo models and in vivo models.

PV017 / #142

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IN VIVO LYMPHOCYTE ENGINEERING

TRANSIENT IN VITRO AND IN VIVO CAR-T CELL GENERATION AND TUMOR TARGETING VIA MRNA-LOADED LIPID NANOPARTICLES

Judith Kemming¹, Nayad Kutlu², Marvin Müller¹, Semjon Willier¹, Ulrike Burk¹, Tobias Feuchtinger¹

¹University Medical Center Freiburg, Pediatric Hematology, Oncology And Stem Cell Transplantation, Freiburg, Germany, ²Dr. von Hauner Children's Hospital, University Hospital LMU Munich, Pediatric Hematology, Oncology, Hemostaseology And Stem Cell Transplantation, Munich, Germany

AS: Academic Abstract Body: Introduction: While CAR-T cell therapies are highly effective in hematologic malignancies, responses in pediatric solid tumors remain limited, largely due to poor T cell infiltration into the immunosuppressive tumor microenvironment (TME). Lipid nanoparticles (LNPs) offer a non-viral approach for in vivo delivery of mRNA, potentially bypassing ex vivo CAR-T cell production and improving efficiency. We aim to generate anti-B7H3 CAR-T cells targeting solid tumors using LNPs and, in a complementary approach, modulate the immunosuppressive TME by delivering chemokine-encoding mRNA directly to tumor cells. Materials and Methods: LNPs encapsulating in vitro–transcribed mRNA were formulated via a microfluidic system and applied to primary human T cells or solid tumor cell lines. CAR expression and functionality were assessed by flow cytometry and in vitro co-culture assays. Chemokine overexpression and secretion following tumor-cell transfection were measured by multiplex immunoassay. Results: Anti-B7H3 CAR mRNA was optimized for stability and translational efficiency using bioinformatic analysis, leading to enhanced CAR expression and cytotoxic activity. Delivery of chemokine-encoding mRNA to tumor cells is expected to result in efficient chemokine overexpression and secretion, promoting CAR-T cell migration and tumor cell killing. Discussion and Conclusions: Anti-B7H3 CAR mRNA can be efficiently delivered in vitro via LNPs, generating solid tumor–directed CAR-T cells with cytotoxic capacity, which will be further evaluated in vivo. Chemokine-encoding mRNA will be delivered to solid tumor cells in vitro. In vivo validation will provide further insights into the applicability of tumor-targeting LNPs and their potential to sensitize solid tumors for CAR-T cell therapy.

PV018 / #78

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NON-VIRAL CAR/TCR GENE TARGETING

CLINICAL TRANSLATION OF CRISPR-CAS9-MEDIATED TCR-ENGINEERING FOR THE TREATMENT OF VIRAL INFECTIONS

Jonathan Albert¹, Theresa Käuferle^2,3, Andreas Carr¹, Laura Valentiner¹, Umair Anwar¹, Luise Schröter^2,3, Luka Cicin-Sain⁴, Sandra Ammann⁵, Karl Petri⁶, Giandomenico Turchiano⁷, Nathan White⁷, Anja Lindemann⁸, Martin Hildebrandt⁹, Hermann Einsele⁶, Toni Cathomen⁵, Tobias Feuchtinger^2,3, Dirk Busch^1,10, Elvira D’Ippolito^1,10

¹Technical University of Munich (TUM), TUM School of Medicine, Institute For Medical Microbiology, Immunology And Hygiene, Munich, Germany, ²University of Freiburg, Pediatric Hematology, Oncology And Stem Cell Transplantation, Medical Center, Freiburg, Germany, ³University of Freiburg, Center For Cell And Gene Therapy Freiburg, Medical Center, Freiburg, Germany, ⁴Helmholtz Centre for Infection Research, 4 Department Of Viral Immunology, Braunschweig, Germany, ⁵University of Freiburg, Institute For Transfusion Medicine And Gene Therapy, Medical Center, Freiburg, Germany, ⁶University Hospital Würzburg, Würzburg, Medical Department Ii, Würzburg, Germany, ⁷University College London, 7 Infection, Immunity, And Inflammation Teaching And Research Department, London, United Kingdom, ⁸TUM, Tumcells Interdisciplinary Center For Cellular Therapies, School Of Medicine, Munich, Germany, ⁹Klinikum rechts der Isar, Department Of Internal Medicine Iii, Munich, Germany, ¹⁰German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany

AS: Academic Abstract Body: Cytomegalovirus (CMV)-seropositive stem cell transplant (SCT) recipients with CMV-seronegative donors (R⁺/D⁻) are at high risk of CMV reactivation after transplantation. Notably, up to 40% of patients experience late-onset reactivation following discontinuation of letermovir prophylaxis. Adoptive transfer of naturally occurring virus-specific T-cells from seropositive SCT donors has demonstrated strong efficacy against refractory CMV reactivation. However, this option is unavailable for R⁺/D^- SCT patients. Obtaining virus-specific T-cells from HLA-matched third-party donors remains logistically complex and has shown limited efficacy. We therefore aim to generate CMV-specific T-cells from seronegative donors using CRISPR-Cas9–mediated targeted T-cell receptor (TCR) replacement, with predictable, near-physiological function. Lead candidate TCRs recognizing CMV-derived immunodominant epitopes restricted to HLA-A*02:01 and HLA-B*07:02 were selected by evaluating epitope specificity via pMHC multimer staining, peptide sensitivity, and antiviral activity of TCR-T-cells in a physiologically relevant in vitro model of human CMV infection. Characterization of the engineered T-cell product generated under a GMP-aligned platform confirmed a purified CD8⁺ T-cell population without detectable off-target impurities. Residual on-target impurities consisted of CD8⁺CD56⁺ cells that retained stable TCR expression, activation capacity, and antiviral activity upon epitope recognition. To define the genomic safety profile of engineered cells, we applied the CAST-Seq and CLEAR-time platforms to comprehensively map CRISPR-Cas9-mediated off-target sites, translocations, large deletions, and aneuploidy, and to evaluate their oncogenic potential. Taken together, we are preparing a first-in-human phase I trial of prophylactic CMV-specific transgenic-TCR T-cell infusion for CMV-seropositive SCT recipients with seronegative donors, establishing a workflow adaptable to TCR-engineered therapies targeting other viral infections and malignancies.

PV019 / #65

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NON-VIRAL CAR/TCR GENE TARGETING

PRECISE GENOME EDITING TO ENABLE NEXT-GENERATION T CELL THERAPIES

Valentina Carbonaro

AstraZeneca, Oncology, R&d-cell Therapy Biotechnologies, Cambridge, United Kingdom

AS: Industry Abstract Body: The CRISPR-Cas9 system and related Precise Genome Editing (PGE) technologies have been enabling tools in the engineering of primary T cells to support a paradigm shift in T cell-based immunotherapies, and importantly next-generation chimeric antigen receptor (CAR)-T therapies. To this end we have harnessed the power of PGE to generate T cell therapies with improved efficacy and safety. We have developed optimised workflows for both non-viral and viral PGE strategies, allowing for accurate editing of human T cells via endogenous gene Knock-Out (KO) and/or exogenous transgene Knock-In (KI). With an established end-to-end process that spans “clinical-grade” sgRNA design to stringent off-target profiling, phenotypic, functional, and genetic validation, we are well positioned to facilitate the manufacturing of next-generation T cell therapies. This is achieved through the engineering of human T cells to express exogenous CARs, alternative T cell receptors (TCRs), and/or control switches. Additionally, we can enhance these therapies phenotypically by knocking out endogenous genes. PGE presents an exciting future for cell therapy, offering the potential to overcome current obstacles to cancer clearance.

PV020 / #125

Posters Viewing

NON-VIRAL CAR/TCR GENE TARGETING

COMPLETE ENDOGENOUS TCR ABLATION IS REQUIRED TO ELIMINATE CD3 COMPETITION AND MISPAIRING IN TCR GENE THERAPY

Maximilian Koch, Hazar Kahveci, Luciano Ponce, Sabine Heitzeneder

Stanford, Stanford Cancer Institute, Palo Alto, United States of America

AS: Academic Abstract Body: T cell receptor (TCR) specificity is jointly determined by its α- and β-chain heterodimer assembled within the TCR-CD3 complex. Introduction of a therapeutic transgenic TCR into mature T cells inevitably creates competition with the endogenous TCR, enabling mispairing between transgenic and endogenous chains. This generates receptors of unpredictable specificity, reduces surface expression of the therapeutic TCR, and represents a persistent safety and efficacy concern in TCR gene therapy. Notably, individual TCRs differ markedly in their ability to outcompete endogenous receptors. Here, we systematically dissect determinants of TCR mispairing and assembly across >20 clinically relevant cancer- and virus-specific TCRs. In-vitro transcribed mRNA encoding respective α- and β-chains was electroporated into multiple TCR-positive T cell lines as well as engineered α-, β-, and dual-TCR knockout variants, creating a platform for controlled assessment of correct chain pairing. We demonstrate that TCRs differ substantially in re-expression efficiency under competitive conditions. Rather than a simple hierarchy of “dominant” versus “weak” TCRs, our data reveal a spectrum defined by selective versus promiscuous chain pairing properties combined with variable CD3 recruitment capacity. These intrinsic features determine surface expression and mispairing propensity. Consistent with these findings, complete ablation of endogenous TCR chains maximizes engineering efficiency. We benchmarked multiple CRISPR-Cas9 gRNAs targeting TRAC and developed a dual-targeting TRBC1/2 gRNA that achieves superior β-chain knockout compared to previously published strategies. Collectively, our study refines the mechanistic framework of TCR mispairing and provides practical guidance for improving safety and performance of therapeutic TCR engineering.

PV021 / #164

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NON-VIRAL CAR/TCR GENE TARGETING

SCALABLE, HIGH-THROUGHPUT CRISPR SCREENING FOR T CELL ENGINEERING USING THE EXPERT DTX™ ELECTROPORATION PLATFORM

Linda Meggiolaro, Lauren Unsworth, Maxwell Van Buskirk, Isabel Daher, James Brady, Rama Shivakumar

MaxCyte, Inc., Rockville, United States of America

AS: Industry Abstract Body: As demand for advanced therapies accelerates, developers face mounting pressure to innovate despite high costs, long development timelines, and inconsistent gene-editing outcomes. The ExPERT DTx™ Electroporation Platform was designed to address these challenges by delivering rapid, scalable, and customizable electroporation of up to 96 samples in under three minutes, offering high-throughput efficiency, reproducible results, and compatibility with diverse cell types. Importantly, optimized conditions on the DTx^TM can be directly transferred to other MaxCyte systems, enabling seamless scale-up from small-scale discovery to GMP-compliant manufacturing. In this study, we applied the DTx™ to high-throughput CRISPR/Cas workflows. We evaluated guide RNA performance for T-cell knockout across varying RNP concentrations and compared small and medium-sized plasmid donor templates for CD19 CAR knock-in at the TRAC locus. To ensure robustness across biological variability, the optimized workflow was tested in T cells from three healthy donors and successfully scaled from the DTx™ to the GTx^®. Across these applications, the DTx™ demonstrated strong viability, consistent performance, and efficient gene modification. Collectively, these findings establish the DTx™ Electroporation Platform as a powerful and versatile solution for high-throughput gene-editing optimization, providing reliable results and smooth scalability to streamline the transition from early discovery to clinical manufacturing.

PV022 / #168

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NON-VIRAL CAR/TCR GENE TARGETING

SAFE AND SCALABLE SMAR VECTORS: EXPANDING PAYLOAD CAPACITY FOR ADVANCED CELL ENGINEERING

Silvia Rizzato¹, Khwab Sanghvi², Miriam Stenzinger³, Alicia Roig-Merino⁴, Lukas Bunse², Michael Platten², Richard Harbottle¹

¹DKFZ (German Cancer Research Center), Dna Vectors Division, Heidelberg, Germany, ²DKFZ (German Cancer Research Center), Neuroimmunology And Brain Tumor Immunology, Heidelberg, Germany, ³IKTZ (Institute for Transfusion Medicine and Cell Therapy), Heidelberg, Germany, ⁴Maxcyte, Rockville, United States of America

AS: Academic Abstract Body: Next-generation cancer cell therapies increasingly rely on complex, multi-gene constructs to enhance potency, specificity and safety. However, conventional gene delivery methods, particularly viral vectors, are limited by cargo size, integration-associated genotoxic risk and high manufacturing costs. To address these bottlenecks, we developed a minimally immunogenic non-viral DNA nanovector platform based on Scaffold/Matrix Attachment Regions (S/MARs). This system enables stable, integration-free gene expression and was largely validated in human T cells, which show robust cytotoxic activity when engineered with clinically relevant CARs and TCRs. To facilitate clinical translation and demonstrate scalability, we established a GMP-compliant manufacturing protocol tailored to the S/MAR platform, which enables rapid and cost-effective large-scale production of engineered T cells. The workflow integrates the CliniMACS Prodigy automated closed system for cell isolation and culture with the MaxCyte ExPERT GTx electroporator, streamlining T cell engineering into a 6-day process. This is part of the pre-clinical validation supporting our upcoming trial with TCR-engineered T cells, making it the first use of the S/MAR nanovector technology in a clinical application. We now seek to expand the S/MAR platform by developing modular, multi-cassette constructs that support more sophisticated designs and allow fine-tuning of T cell activity through independently regulated expression units. As a proof-of-concept, we generated S/MAR DNA vectors incorporating dual-promoter reporters and dual-CAR/TCR systems. Constructs ranging from 5.9 to 9.1 kb with varied genetic composition were assembled and tested in Jurkat76 cells, where we confirmed efficient parallel expression and no adverse effects upon transfection of larger payloads.

PV023 / #265

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NON-VIRAL CAR/TCR GENE TARGETING

SAFER ΑVΒ6/ΑVΒ8-TARGETING PEPTI-CAR-T CELLS FOR PDAC: SUICIDE SWITCHES AND NON-VIRAL ENGINEERING

Alice Schirru¹, Elisa Sangiovanni¹, Juan Roberto Rodrìguez Madoz², Chiara Bonini³, Felipe Prósper Cardoso⁴, Anna Mondino¹, Arianna Pocaterra¹

¹IRCCS Ospedale San Raffaele, Lymphocyte Activation Unit, Division Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²Cima Universidad de Navarra, Center For Applied Medical Research, Pamplona, Spain, ³Vita-Salute San Raffaele University, Milan, Italy, ⁴Clinica Universidad de Navarra, Pamplona, Spain

LB: Academic Abstract Body: Peptides are versatile molecules for the development of therapeutics and diagnostic agents. We recently developed a novel chimeric antigen receptor (pepti-CAR) incorporating a chromogranin A (CgA)-derived peptide that selectively binds αvβ6 and αvβ8 integrins. Engineered pepti-CAR T cells showed potent cytotoxicity against antigen-expressing tumor cells in vitro and effectively control or eradicated primary and metastatic PDAC lesions in preclinical models. To support the clinical translation of αvβ6/αvβ8-specific pepti-CAR T cells, we incorporated safety switches to limit potential toxicity in humans. We designed two bidirectional lentiviral vectors, each encoding a distinct safety switch systems: Herpes Simplex Virus Thymidine Kinase (HSV-TK) and truncated Epidermal Growth Factor Receptor (EGFRt). Pepti-CAR T cells expressing HSV-TK retained efficient killing of antigen-expressing PDAC cell lines in vitro and anti-tumor activity in an orthotopic patient-derived PDAC organoids model, to a similar extend as the parental construct. Importantly, ganciclovir addition to cocultures and systemic administration in mice enable rapid HSV-TK-meditated elimination of CAR T cells. Similarly, EGFRt expression allowed efficient antibody-mediated depletion of engineered T cells upon cetuximab treatment in vivo. In parallel, to overcome the potential genotoxicity, manufacturing complexity and high costs associated with lentiviral vectors, we investigated a non-viral gene delivery strategy based on the Sleeping Beauty (SB) transposon system. Although preliminary experiments showed lower transduction efficiency, SB-engineered CAR Ts demonstrated comparable anti-tumor activity to lentiviral products. Overall, these findings support the development of a safer, clinically translatable αvβ6/αvβ8 targeting pepti-CAR T cell therapy for PDAC, combining effective suicide switch system with scalable non-viral engineering platforms.

PV024 / #87

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NON-VIRAL CAR/TCR GENE TARGETING

NEXT-GENERATION NON-VIRAL CAR NK ENGINEERING USING EPISOMAL NSMAR NANOVECTORS

Beeke Tappe, Luisa Burger, Sophie Williams, Anastasiia Lokteva, Richard Harbottle

German Cancer Research Centre, Infection, Immunity And Cancer - Dna Vectors, Heidelberg, Germany

AS: Academic Abstract Body: Engineering T cells to express CARs has transformed cancer therapy and accelerated innovation in immune cell engineering. Extending CAR strategies to NK cells is a key objective, as NK cells provide intrinsic tumour reactivity, reduced risk of GvHD and CRS, and complementary effector functions. However, efficient genetic modification of NK cells remains challenging, as their intrinsic antiviral sensing pathways frequently impair viral transduction, and non-viral strategies are limited by electroporation-induced stress, restricted proliferative capacity, and inefficient nuclear DNA delivery. We established a next generation non-viral CAR NK platform based on episomal Scaffold Matrix Attachment Region (SMAR) DNA nanovectors. SMAR elements are cis-acting sequences that tether DNA to the nuclear matrix, enabling stable episomal persistence and mitotic co-segregation without genomic integration. In contrast to mRNA transfer, SMAR DNA vectors support sustained transgene expression while avoiding insertional mutagenesis associated with integrating systems such as transposons. We engineered NK92 cells or primary NK cells from healthy donors via electroporation, or mechanoporation. In NK92 cells, electroporation achieved 86% transgene expression with 18.5% viability and 29% CAR expression with 24% viability. Mechanoporation yielded 20% transgene expression with 70% viability. We furthermore achieved engineering of primary NK cells with electroporation resulted in 38% CAR+ cells at 22% viability and 50% transgene expression at 39% viability. CD19-CAR NK92 cells showed antigen specific IFNγ secretion (112 pg/mL at E:T 1:1 to 477 pg/mL at E:T 1:10) and significant tumour cell killing in live cell assays against K562. Collectively, we established a non-integrating nSMAR-DNA strategy for CAR NK engineering.

PV025 / #121

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OTHER

SHED L1CAM IMPAIRS CAR T CELL EFFICACY AGAINST NEUROBLASTOMA

Lena Andersch^1,2,3, Jasmin Vahid Yousefnia¹, Silke Schwiebert¹, Anika Winkler¹, Kathleen Anders¹, Annette Künkele^1,3

¹Charité-Universitätsmedizin Berlin, Pediatric Oncology, Berlin, Germany, ²German Cancer Research Center (DKFZ), Heidelberg, Germany, ³German Cancer Consortium (DKTK), Partner Site Berlin, Berlin, Germany

AS: Academic Abstract Body: L1CAM-directed CAR-T cell therapy has demonstrated limited efficacy in neuroblastoma. L1CAM is highly expressed in neuroblastoma and tumor-specific glycosylation of the CE7-epitope makes it a suitable target. However, ADAM-metalloproteases constitutively cleave its extracellular domain, releasing CE7-containing L1CAM into circulation. Moreover, soluble L1CAM can bind via its RGD-motif to extracellular matrix or integrins expressed by healthy tissues. We hypothesized that shed L1CAM, either in soluble form or immobilized within tissues, compromises CAR-T cell function by inducing antigen-dependent activation prior to tumor engagement, thereby promoting dysfunction. We demonstrated that soluble L1CAM levels were elevated in serum from neuroblastoma patients and mice harboring neuroblastoma xenografts, confirming in vivo L1CAM shedding. In vitro, immobilized and soluble recombinant L1CAM induced CAR-T cell activation. CAR constructs incorporating a long-spacer domain exhibited enhanced activation compared to short-spacer variants. Plate-bound and soluble L1CAM upregulated inhibitory receptors on L1CAM-CAR-T cells and induced apoptosis. Antigen pre-exposure triggered IFNG secretion, and prior stimulation with immobilized L1CAM reduced subsequent cytotoxicity against L1CAM-positive neuroblastoma cells. Soluble L1CAM also bound to L1CAM-negative tumor cells, presumably via integrins, resulting in CAR-T cell activation. Co-culture with L1CAM-bound tumor cells activated CAR-T cells and induced IFNG-release. Notably, a synNotch-inducible L1CAM-CAR mitigated antigen-induced dysfunction in-vitro. Our findings suggest that shed L1CAM, either soluble, sequestered in extracellular matrix or bound to integrins on healthy tissue impairs L1CAM-CAR-T cell efficacy and may explain poor L1CAM-CAR-T cell persistence and efficacy in the clinic. Ongoing in vivo studies are evaluating whether conditional CAR expression can overcome shedding-mediated impairment and enhance therapeutic efficacy.

PV026 / #257

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OTHER

ENHANCING CAR T MANUFACTURING: INNOVATIVE GIBCO™ SOLUBLE DYNACTIVATOR™ CD3/CD28 PROTOTYPE

Joanna Kern¹, Marta Gabriel², Kyle Zastrow¹, Britt Lindestad², Megan Logan¹, Maxi-Lu Boschen², Hany Meås², Heidi Vebø², Tuva Hereng², Algirdas Grevys²

¹Thermo Fisher Scientific, Grand Island, United States of America, ²Thermo Fisher Scientific, Oslo, Norway

LB: Industry Abstract Body: The Gibco™ Soluble DynActivator™ CD3/CD28 Prototype represents a significant advancement in the field of cell therapy manufacturing. This soluble activation technology is designed to enable the activation and expansion of T cells, a critical component in the development of effective immunotherapies. Traditional methods of T cell activation often face challenges such as inconsistent activation, limited scalability, and high costs. Gibco™ Soluble DynActivator™ CD3/CD28 Prototype addresses these issues by providing a soluble, easy-to-use activator that enable robust and cost-effective T cell activation. In this study, we demonstrate the efficacy of Gibco™ Soluble DynActivator™ CD3/CD28 Prototype in various cell therapy manufacturing processes. Our results show that Gibco™ Soluble DynActivator™ CD3/CD28 Prototype enables gentle activation, robust proliferation rates of T cells and maintains their functionality and viability over extended culture periods. Additionally, the soluble nature of DynActivator™ simplifies the manufacturing workflow, reducing the need for complex and labor-intensive procedures.

PV027 / #127

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OTHER

UNDERSTANDING THE IMMUNOSUPRESSIVE TUMOUR MICROENVIRONMENT DYNAMICS IN NEUROBLASTOMA BY TRANSCRIPTOMIC AND SPATIAL PROTEOMIC ANALYSIS

Catarina Guerra, Noah Bonine, Raquel Gonzalez, Ronald De Krijger, Judith Wienke

Princess Máxima Center, Utrecht, Netherlands

AS: Academic Abstract Body: Introduction: Neuroblastoma is the most prevalent extracranial paediatric solid tumour, and it accounts for about one in six paediatric cancer-related deaths. Adoptive cell therapy (ACT), such as anti-GD2 CAR-T cell therapy, has revealed promising results for the treatment of Neuroblastoma; however, patients with a high tumour burden do not respond to treatment. The immunosuppressive tumour microenvironment (TME) is thought to be one of the main causes of the limited efficacy of ACT. Methods: This project aims to explore the spatial interactions within the TME and cell-cell dynamics to identify novel therapeutic interventions and overcome the inhibitory landscape of the TME. The project comprises three stages: 1) single-cell RNA sequencing (scRNAseq) analysis of paired primary and relapse paraffin-embedded biopsy samples to find inhibitory pathways, 2) spatial proteomic and transcriptomic analysis using the MACSima™ Imaging Cyclic Staining (MICS) and 10x Genomics Xenium technologies to explore receptor-ligand dynamics, and 3) validation of the inhibitory targets in vitro in neuroblastoma organoids and in vivo applying syngeneic and PDX mouse models. The main inhibitory processes that will be explored include limited T cell trafficking and infiltration, unsuccessful T cell activation and proliferation, and T cell exhaustion. Results: The project is still in the early stages and optimisation of the pre-developed MICS panels is currently taking place. Conclusion: The novel insights will be explored to design new immunotherapy treatments in Neuroblastoma by applying engineering approaches to modulate the immunosuppressive interactions and identifying synergistic treatments that can boost CAR-T cell therapy and patient’s prognosis and survival.

PV028 / #153

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OTHER

COMPUTATIONALLY DESIGNED TYROSINE KINASE OFF-SWITCH FOR REVERSIBLE CONTROL OF T CELLS

Morteza Hafezi¹, Aikaterini Semilietof¹, Tiffany X.Y. Que¹, Leila Hadadi², Patrick Reichenbach², Raphael Genolet², Emile Dorchies², Bernhard Gentner², Caroline Arber², George Coukos¹, Vincent Zoete², Olivier Michielin³, Melita Irving¹

¹Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne and Lausanne University Hospital (CHUV); Lausanne, Switzerland, Lausanne, Switzerland, ²Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne and Lausanne University Hospital (CHUV); Lausanne, Switzerland., Lausanne, Switzerland, ³Department of Oncology, Geneva University Hospital, Geneva, Switzerland., Geneva, Switzerland

AS: Academic Abstract Body: Controlling activity levels of engineered T cells is essential for improving therapeutic safety. We computationally reprogrammed the ZAP70 ATP-binding pocket to create a universal off-switch that renders TCR- and CAR-T cells selectively sensitive to subtherapeutic doses of clinically approved kinase inhibitors. We also engineered a ZAP-CAR architecture incorporating the switch, enabling specific, rapid and reversible abrogation of cellular activity. Both strategies are clinically adaptable for tunable regulation of engineered T-cell function.

PV029 / #259

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OTHER

EFFICIENT CD3 DEPLETION USING MAGNETIC BEADS TO ENHANCE ROBUSTNESS AND FLEXIBILITY IN T CELL MANUFACTURING

Ole Landsverk¹, Neda Moharrami¹, Joanna Kern², Jenny Rasmussen¹, Giacomo Pesci¹, Nicolay Nilsen¹, Kyle Zastrow², Megan Logan², Srinivasa Boddeda², Sylvi Oliva Kjaer¹, Hany Meås¹, Evan Zynda², Maxi-Lu Boschen¹, Heidi Vebø¹, Tuva Hereng¹

¹Thermo Fisher Scientific, Oslo, Norway, ²Thermo Fisher Scientific, Grand Island, United States of America

LB: Industry Abstract Body: Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable clinical efficacy; however, their manufacturing remains complex, especially in the context of allogeneic workflows that require the removal of cells expressing endogenous T cell receptors. Here, we present a new magnetic bead–based CD3 depletion technology designed to enable efficient and scalable removal of CD3⁺cells, supporting robust and flexible allogeneic CAR T cell manufacturing workflows. The Gibco™ Dynabeads™ CD3 Depletion Prototype beads leverage high-affinity antibodies conjugated to (para)magnetic Dynabeads™ to support a rapid and reproducible depletion of CD3⁺ cells across a range of input cell compositions. Performance was evaluated using healthy donor leukapheresis material, assessing depletion efficiency, cell recovery and viability. The Gibco™ Dynabeads™ CD3 Depletion Prototype beads enabled consistent and effective CD3 depletion while maintaining high viability and recovery of target cell populations. The technology is compatible with the Gibco™ CTS™ DynaCellect™ Magnetic Separation system for closed and automated manufacturing, aligning with current Good Manufacturing Practice (cGMP) requirements and scalable production needs. In summary, this new CD3 depletion bead technology provides a robust tool for cell processing in allogeneic CAR T cell manufacturing. By enabling efficient removal of CD3⁺ cells the Gibco™ Dynabeads™ CD3 Depletion Prototype supports enhanced process control, flexibility, and scalability, addressing key challenges in the manufacture of next-generation cell therapies. Regulatory statement: Gibco™ Dynabeads™ CD3 Depletion Prototype: For Research Use Only. Not for use in diagnostic procedures. Product is a prototype and performance characteristics of this product have not been established.

PV030 / #50

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OTHER

MULTITARGETED CAR-T CELL THERAPY FOR EPITHELIAL TUMOUR TREATMENT

Ksenia Levchuk¹, Alexey Petukhov²

¹Almazov NMRC, Saint-Petersburg, Russian Federation, ²Nazarbayev University, Department Of Biomedical Sciences, Astana, Kazakhstan

AS: Academic Abstract Body: Being the leading immunotherapy approach for oncohematological malignancies, adoptive CAR-T cell therapy continues to broaden its therapeutic horizons and applications. Although CAR-T cell therapy has revolutionized the treatment of hematologic malignancies, its translation to epithelial tumors remains hindered by the immunosuppressive architecture of the tumor microenvironment. This study explores whether simultaneous targeting of CD87, PD-L1/2, and NKG2D ligands—molecules differentially expressed on tumor cells, myeloid suppressors, and tumor-derived microvesicles—could circumvent this barrier. We engineered three CAR constructs: a classical αCD87 CAR, and receptor-based PD-1 and NKG2D CARs of original design. All three conferred specific cytotoxic activity against their respective targets in vitro, as evidenced by real-time cytotoxicity assays and IFN-γ release. In NSG-SGM3 mice bearing HeLa-CD19 xenografts, NKG2D CAR-T cells exhibited inferior persistence and tumor infiltration compared to conventional αCD19 CAR-T cells, underscoring the need for CAR optimization. A panel of epithelial lines (HeLa, HepG2, Caco-2, H460, A431, T47-D) revealed pronounced heterogeneity in CAR-T susceptibility that could not be attributed solely to IFNγ or TNF receptor density, pointing to more complex resistance mechanisms. To model the multicellular complexity of the tumor niche, we established an original in vitro platform incorporating HepG2 cells (NKG2D ligand-rich), KG-1 myelomonocytic cells (CD87-positive), and PD-L1⁺ microvesicles. This system recapitulates key suppressive elements of the epithelial tumor microenvironment and provides a tractable tool for dissecting the efficacy of multi-pronged CAR-T strategies. Our findings lay the groundwork for combination immunotherapies designed to simultaneously eliminate malignant cells and disable the protective stromal and vesicular networks that enable immune evasion.

PV031 / #169

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ANTIGEN-SPECIFIC ENGAGMENT OF CARS UNCOVERS METABOLIC REPROGRAMMING AND TRANSCRIPTIONAL DYNAMICS OF ‘OFF-THE-SHELF” CD19-CAR-T CELLS

Cristina Maccalli¹, Salim Bougarn², Toufiq Mohammed³, Mohammed Elanbari², Francesco Reggiani⁴, Shana Jacobs⁵, Saroja Kotegar Balayya⁵, Evonne Cin Smith², Alex Issam Tout², Lisa Mathew⁶, Kun Wang⁶, Li Liu⁶, Oleksandr Soloviov⁶, Abdulrahman Salhab⁶, Sara Tomei⁷, Damilola Olagunju⁸, Deepa Subramanian⁸, Suruschi Mohan⁸, Chiara Bonini^9,10, Monica Casucci¹¹, Antonio Uccelli¹²

¹IRCCS AOM Policlinico San Martino, Uo Biotherapies, Genoa, Italy, ²Sidra Medicine, Laboratory Of Immune Biological Therapies, Doha, Qatar, ³The Jackson Lab, Farmington, United States of America, ⁴IRCCS AOM Policlinico San Martino, Gene Expression And Epigenetics, Genoa, Italy, ⁵Sidra Medicine, Metabolic Core, Doha, Qatar, ⁶Sidra Medicine, 5integrated Genomics Laboratories, Doha, Qatar, ⁷Sidra Medicine, Integrated Genomics Laboratories, Doha, Qatar, ⁸Sidra Medicine, Department Of Obstetrics, Doha, Qatar, ⁹IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ¹⁰Vita-Salute San Raffaele University, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ¹¹IRCCS San Raffaele Scientific Institute, Innovative Immunotherapies Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ¹²IRCCS AOM Policlinico San Martino, Scientific Direction, Genova, Italy

AS: Academic Abstract Body: Background: The primary objective remains identifying a substrate that harmonizes potent anti-tumor efficacy with a superior safety profile for 'off-the-shelf' cell therapy applications. Methods: The characterization of the cellular landscape upon antigen-specific vs. antigen unrelated stimulation of Umbilical Cord Blood (UCB)-derived T lymphocytes engineered with either CD19-CD28-z or CD19-4-1BB-z CARs was obtained through scRNAseq (10xGenomics), mass-spectromic metabolomic and methylation profiling (long-read sequencing; Oxford Nanopore Technologies). Results: Single-cell RNA sequencing (scRNA-seq) of CD19-CAR-T cells following co-culture with CD19+ targets identified distinct transcriptional signatures associated with CD4+ and CD8+ lineage-specific reactivity. These data highlighted a high degree of cellular subtype complexity linked to anti-tumor effector functions. Complementary metabolomic profiling revealed significant antigen-dependent shifts (p < 0.001, fold-change > 2.5) in pathways critical for T-cell maturation and the prevention of inflammation including amino acid and amino sugar metabolism, and aminoacyl-tRNA biosynthesis.Furthermore, we identified key metabolites regulating proliferation, survival, and differentiation that were consistently enriched upon antigen-specific stimulation, independent of the initial T-lymphocyte activation method. Notably, differential metabolomic profiles were observed based on the specific co-stimulatory domains within the CAR construct. The integration of these multi-omic datasets provides a unified genomic and functional blueprint of UCB-derived CAR-T cells, identifying specific genetic targets for future editing or reprogramming to further enhance the potency and safety of “off-the-shelf” therapeutics. Conclusions: this multi-omic characterization validates Umbilical Cord Blood (UCB) as a potent, safe, and scalable source for allogeneic CD19-CAR-T cells, identifying specific transcriptomic and metabolic hubs that can be exploited to further enhance therapeutic efficacy and economic sustainability.

PV032 / #263

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CTS DETACHABLE DYNABEADS CD4 AND CD8 ENABLE HIGH-PURITY ISOLATION OF T CELLS FROM WHOLE BLOOD

Diana Navickaitė¹, Lucas Gordon^1,2, Michael Bruno², Kyle Zastrow², Megan Logan², Evan Zynda², Heidi Vebø³, Hany Meås³

¹Thermo Fisher Scientific, Cell Therapy, Vilnius, Lithuania, ²Thermo Fisher Scientific, Cell Therapy, Grand Island, United States of America, ³Thermo Fisher Scientific, Cell Therapy, Oslo, Norway

LB: Industry Abstract Body: T cell manufacturing has become a critical focus in recent years to support the production of immunotherapeutic treatments, particularly chimeric antigen receptor T cell (CAR-T) therapies. As the development of these treatments accelerates, there is increasing demand for a reliable source of T cells. Commonly, T cells are sourced from blood products including buffy coats, leukocyte reduction system chambers, or leukopaks, which contain varying concentrations and purities of target cells. The high concentration of PBMCs in leukopaks offers a stable source of T cells for isolation, manipulation, and expansion in T cell-based therapeutics manufacturing. The production of leukopaks through apheresis requires expensive specialized equipment, which may not be available at many treatment centers, especially those with limited resources. Additionally, the process requires patients to be attached to the machine for several hours, potentially causing discomfort. These challenges highlight the need for alternative T cell sources. To address this, the isolation and expansion of T cells directly from whole blood has been evaluated. Whole blood packs are a low-cost, accessible source of T cells that can be obtained from patients in a typical clinic within ∼15 minutes, without specialized equipment. Using GMP-compliant CTS Detachable Dynabeads CD4 and CD8 with the Gibco CTS DynaCellect Magnetic Separation System, a highly pure population of CD3+ T cells can be isolated directly from whole blood, eliminating additional preprocessing and lysing steps. This method offers a practical alternative starting material for T cell manufacturing, potentially broadening CAR-T therapy availability to patients with limited access to leukapheresis.

PV033 / #88

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DECOUPLING DEAMINASE RECRUITMENT AND TRANSGENE TARGETING RESOLVES TRADEOFFS IN MULTIPLEXED BASE EDITING WORKFLOWS FOR IMMUNE CELLS

Leigh-Anne Thomas¹, Alexis Duringer¹, Jesse Stombaugh², Olga Mielczareck¹, Bronwyn Joubert¹, Amanda Haupt², Immacolata Porreca¹, Robert Blassberg¹, Pablo Perez-Duran¹

¹Revvity, Cambridge, United Kingdom, ²Revvity, Lafayette, United States of America

AS: Industry Abstract Body: The development of advanced immune cell therapies relies heavily on complex, precise genome engineering processes including knockout of multiple targets and site-specific integration of transgenes. Base editors offer great opportunities to address these challenges without requiring the introduction of genotoxic double strand breaks and associated risks to genome stability. The nickase component of base editors can also be re-purposed to target transgene integration in parallel with base editing; however, parallel deployment is limited by unintended base editing at the transgene integration site. To develop a flexible, simplified approach to targeted transgene integration in combination with base editing we developed an RNA encoded, modular base editing platform that assembles via an aptamer encoded in the sgRNA, enabling selective recruitment of editing components to distinct genomic sites. The aptamer-dependent recruitment allows for the multi-purposing of a single Cas nickase component for simultaneous multiplexed base editing and targeted transgene integration; the deaminase component is recruited to the site targeted for base editing by gRNAs including aptamers while efficient integration is achieved in parallel by directing only the Cas component to transgene integration sites with gRNAs without aptamers. By de-targeting the deaminase component of the modular base editor from transgene integration sites it was possible to greatly reduce disruption of the DNA nicking activity required for efficient transgene integration, increasing efficiencies of precise integration from double stranded DNA templates. Base editing efficiencies remained high when combined with targeted transgene integration in both iPSCs and T cells, while avoiding translocations or untargeted donor integration.

PV034 / #114

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DESIGNING NOVEL VERSATILE PROTEASE REGULATABLE CHIMERIC ANTIGEN RECEPTOR (VIPER) T CELL THERAPIES FOR GLIOMA AND OTHER SOLID TUMOURS

Krishneel Prasad^1,2, Melinda Iliopoulos¹, Ryan Cross^1,2, Misty Jenkins^1,2

¹The Walter and Eliza Hall Institute of Medical Research, Personalised Oncology Division, Parkville, Australia, ²The University of Melbourne, Department Of Medical Biology, Parkville, Australia

AS: Academic Abstract Body: High grade gliomas (HGG) are aggressive and often fatal brain cancers that affect both adult and paediatric patients. Early-phase clinical trials of cellular therapies in HGG have demonstrated anti-tumour potency (NCT02208362, NCT04196413, NCT05660369), but also highlight the need for improved safety control and adverse event management. Advances in synthetic receptor design offer solutions for designing safer immunotherapies. Versatile Protease Regulatable CARs (VIPERs) are split CARs that mediate antigen-dependent cytotoxicity and can be pharmacologically switched off using the FDA approved antiviral, Grazoprevir. We first validated VIPER function in vitro using a published CD19 construct and proceeded to develop novel HER2 and Antigen-A targeting VIPERs. These novel VIPERs demonstrate potent cytotoxicity in vitro against HGG cell lines (U251, A172 and DIPG36) and comparable efficacy in additional solid tumour models. Addition of Grazoprevir inhibits novel HER2 and Antigen-A targeting VIPER activity, enabling titratable tumour killing and modulation of inflammatory cytokine secretion (IFNγ and TNFα). Importantly, baseline cytokine production from VIPER T cells is lower than matched second generation Chimeric Antigen Receptor (CAR) T cells, suggesting reduced inflammatory profile. We are currently evaluating in vivo activity of matched scFv CAR and VIPER T cell therapies using mouse models . These studies aim to define whether pharmacologically controllable receptor systems provide a safer framework for translating next-generation, multi-antigen targeted T cell therapies for HGG and other solid tumours.

PV035 / #104

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DESIGNING SYNTHETIC INTRAMEMBRANE PROTEOLYSIS RECEPTORS (SNIPRS) FOR PARALLEL GENE INDUCTION IN T CELLS

Krishneel Prasad^1,2, Ryan Cross^1,2, Misty Jenkins^1,2

¹The Walter and Eliza Hall Institute of Medical Research, Personalised Oncology Division, Parkville, Australia, ²The University of Melbourne, Department Of Medical Biology, Parkville, Australia

AS: Academic Abstract Body: SNIPRs are synthetic receptors that comprise of an antigen binding domain and a cleavable intracellular domain that releases a transcription factor following antigen ligation. We cloned the published CD19 SNIPR Gal4/UAS-tagBFP into our lentiviral vector backbone to facilitate cetuximab enrichment via P2A co-expressed tEGFR protein. Overnight co-culture of CD19⁺ NALM6 target cells and CD19 SNIPR T cells induced TagBFP expression in 80% of cells, with minimal baseline leakiness. To enable parallel induction, SNIPRs require transcription factors that function orthogonally. Therefore, we first validated a previously published panel of 11 synthetic zinc fingers fused to the p65 transcriptional activator (synZF) for cross reactivity. Each synZF was developed to bind a corresponding synthetic DNA binding motif (synDBM) that was computationally selected for absence from the human genome. Using HEK293T cells, we confirmed minimal cross-reactivity across the synZF/synDBM panel. We next replaced Gal4VP64 in the CD19 SNIPR construct with each synZF and cloned the matched synDBM reporter modules into lentiviral vectors. Jurkat cells were co-transduced with paired CD19 SNIPR–synZF and synDBM constructs and stimulated via overnight co-culture with CD19⁺ NALM6 cells. Five of eleven synZF/synDBM pairings demonstrated antigen-dependent activation, producing a 1.5–3-fold increase in the frequency of mCherry⁺ cells. Across most pairings, mCherry mean fluorescence intensity increased 2–6-fold, indicating robust inducibility even where baseline leakiness was detectable. Together, these findings support the feasibility of building multi-channel, orthogonal SNIPR-driven gene circuits in T cells. This platform may enable increasingly sophisticated context-dependent programming of therapeutic lymphocytes to improve both efficacy and safety.

PV036 / #264

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OPTIMIZING T-CELL MANUFACTURING WITH A HIGH-PERFORMANCE MEDIUM FREE OF ANIMAL DERIVED COMPONENTS

Gediminas Rutkauskas¹, Joanna Kern², Lauren Kapus², Kyle Zastrow², Megan Logan², Michael Bruno³, Evan Zynda², Anna Hachmann³

¹Thermo Fisher Scientific, Cell Therapy, Vilnius, Lithuania, ²Thermo Fisher Scientific, Grand Island, United States of America, ³Thermo Fisher Scientific, Cell Therapy, Grand Island, United States of America

LB: Industry Abstract Body: Development of T-cell–based therapies has advanced significantly, with CAR-T products currently used against B-cell malignancies. With increasing demand for T-cell therapies and extensive efforts to target solid tumors, highly controlled and reproducible manufacturing platforms are essential for effective T-cell production. The complex manufacturing process of immune-based therapies includes T-cell isolation, activation, genetic modification, and expansion. Cell culture media are critical across these phases, highlighting the need for enhanced formulations that support the entire process. Precise control over the composition and source of media components reduces the risk of contaminants and variability associated with animal-derived materials (1). In addition, immunotherapies are more effective when memory T-cells remain in a stem-like, less differentiated naïve state, as this subset exhibits enhanced functionality and self-renewal capacity (2,3). Therefore, a cell culture medium that supports expansion of T-cells with an early memory phenotype may improve clinical outcomes. CTS™ OpTmizer™ One was developed to meet these T-cell manufacturing requirements. This formulation is free of animal-derived components and supports robust, consistent T-cell expansion. It mitigates safety concerns associated with animal or human-sourced components and is manufactured according to strict GMP standards. Additionally, CTS™ OpTmizer™ One is easy to use, available in automation-friendly formats with scalable packaging, and can be readily integrated into diverse workflows for both process development and clinical manufacturing.

PV037 / #72

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DEVELOPMENT OF A NOVEL CLAUDIN 6-TARGETING CAR-T CELL THERAPY

Ana Shahpazir^1,2, Siret Tahk¹, Paule Hermet-Teesalu¹, Anu Ustav¹, Robin Pau¹, Gaily Kivi¹, Mario Plaas², Andres Männik¹, Joan Teyra¹, Mart Ustav¹, Tanel Mahlakõiv¹

¹Icosagen Cell Factory OÜ, Tartu, Estonia, ²University of Tartu, Tartu, Estonia

AS: Industry Abstract Body: Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of B cell malignancies but remain largely ineffective in solid tumors, primarily due to limited availability of tumor-specific antigens that enable safe and precise targeting. Claudin-6 (CLDN6) is an oncofetal tight junction protein absent in healthy adult tissues and aberrantly overexpressed in multiple solid tumors, making it an attractive immunotherapeutic target. To discover CLDN6-targeting antibodies, chickens were immunized with virus-like particles (VLPs) with a high-density presentation of transmembrane CLDN6. Monoclonal antibodies were screened for CLDN6 specificity on VLPs pseudotyped with Claudin family members. The most promising specific binders able to discriminate between CLDN6 and CLDN9 were humanized via the Qumanize platform and incorporated into a second-generation CAR construct with a 4-1BB costimulatory domain and CD3ζ signaling domain. CLDN6-targeting CAR-T cells were generated via lentiviral transduction and evaluated in vitro for specificity and cytotoxic function. Two CAR constructs selectively recognized and eliminated CLDN6-expressing tumor cells derived from ovarian, lung, and gastric cancer, while sparing CLDN6-negative cells. CLDN6 CAR-T cell activation and interferon-γ secretion occurred only in the presence of CLDN6-positive targets, confirming antigen-specific activity. Importantly, CLDN6 CAR-T cells retained robust cytotoxic capacity after 30 days of continuous exposure to target cells, indicating sustained functional durability. Collectively, these data demonstrate the feasibility of generating highly specific CLDN6-targeting CAR-T cells and present a promising avenue for developing next-generation immunotherapies for CLDN6-expressing solid tumors.

PV038 / #251

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GENERATION OF FUNCTIONAL CAR-T CELLS FROM NON-ENRICHED PBMCS: A SIMPLIFIED MANUFACTURING APPROACH

Emilio Straface¹, Caterina Giordano¹, Debora Gentile¹, Antonio Abatino¹, Costanza Maria Cristiani², Raffaella Gallo¹, Ilenia Aversa¹, Lisa Isdraele Romano¹, Camillo Palmieri¹, Giuseppe Fiume¹

¹University of Catanzaro ‘Magna Graecia’, Department Of Clinical And Experimental Medicine, Catanzaro, Italy, ²University of Catanzaro ‘Magna Graecia’, Department Of Medical And Surgical Sciences, Catanzaro, Italy

LB: Academic Abstract Body: Chimeric antigen receptor T (CAR-T) cell manufacturing typically relies on purified T lymphocytes, requiring additional costs and processing steps. This study aimed to compare two CAR-T production workflows: one based on isolated T cells and another derived from peripheral blood mononuclear cells (PBMCs) without prior T cell enrichment, evaluating feasibility and functional activity. Human PBMCs were obtained from peripheral blood and either subjected to T cell isolation using a commercial selection kit or directly cultured without enrichment. Both cell populations were activated and transduced with a lentiviral vector encoding a CAR construct. Lentiviral particles were produced using standard transient transfection protocols. Transduced cells were expanded in vitro for several days under controlled culture conditions. CAR expression was assessed by flow cytometry. Functional activity was evaluated through co-culture assays with target-expressing cells, measuring cytotoxicity and activation markers. Efficient CAR expression was achieved in both workflows. PBMC-derived cultures retained a heterogeneous composition but were still able to generate functional CAR-T populations. Cytotoxicity assays demonstrated comparable target cell recognition and killing between the two approaches, with some variability in activation kinetics. Expansion capacity was maintained in both conditions. CAR-T cells can be successfully generated from both purified T lymphocytes and non-enriched PBMC populations. While T cell isolation improves culture uniformity, PBMC-based production represents a simplified and potentially cost-effective alternative without compromising functional activity. These findings support the feasibility of streamlined CAR-T manufacturing strategies.

PV039 / #135

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MRNA-MEDIATED LDLR UPREGULATION ENHANCES LENTIVIRAL-BASED CHIMERIC ANTIGEN RECEPTOR T-CELL TRANSDUCTION EFFICIENCY

Koramit Suppipat^1,2, Supannikar Tawinwung^2,3

¹Chulalongkorn University, Excellence Center For Cellular Immunotherapy, Bangkok, Thailand, ²Thailand Hub of Talent in Cancer Immunotherapy, Bangkok, Thailand, ³Chulalongkorn University, Faculty of Pharmaceutical Sciences, Pharmacology And Physiology, Bangkok, Thailand

AS: Academic Abstract Body: Lentiviral transduction using vesicular stomatitis virus G (VSV-G)–pseudotyped vectors remains the dominant platform for chimeric antigen receptor (CAR) T-cell manufacturing. However, gene transfer efficiency in primary T cells is frequently suboptimal, partly due to low expression of the low-density lipoprotein receptor (LDLR), the cellular entry receptor for VSV-G. We hypothesized that transient LDLR upregulation via mRNA electroporation would enhance lentiviral transduction and increase CAR T-cell yield. LDLR mRNA was generated by in vitro transcription and electroporated into activated human T cells. Surface LDLR expression was quantified by flow cytometry. Cells were subsequently transduced with a VSV-G–pseudotyped CD19-CAR lentiviral vector at multiplicities of infection (MOI) 0.3, 1, and 3. CAR expression was assessed by flow cytometry, and cytotoxicity against CD19⁺ Nalm-6 leukemia cells was evaluated in vitro. LDLR mRNA electroporation significantly increased surface LDLR expression compared with non-transfected controls (80.2 ± 9.3% vs. 47.9 ± 15.5%). Enhanced LDLR expression translated into improved CAR transduction efficiency at all MOIs. CAR expression reached 13.6 ± 1.2%, 48.3 ± 19.8%, and 62.5 ± 9.0% at MOI 0.3, 1, and 3, respectively, compared with 6.3 ± 0.09%, 31.4 ± 15.6%, and 47.4 ± 15.9% in controls. Cytotoxic activity against Nalm-6 cells was preserved. Transient LDLR upregulation via mRNA enhances VSV-G lentiviral transduction without compromising CAR T-cell function, providing a scalable, non-integrating strategy to improve manufacturing efficiency and potentially reduce vector usage and production costs.

PV040 / #116

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CD19 MUTATIONAL LANDSCAPE OF TUMOR CELLS RESISTANT TO CD19-4-1BB CAR-T THERAPY

Magdalena Winiarska¹, Marta Krawczyk¹, Klaudyna Fidyt², Xose Puente³, Ana Gutiérrez Fernández³, Monika Pepek⁴, Pablo Bousquets³, Narcis Fernández²

¹Mossakowski Medical Research Institute, Warsaw, Poland, ²Josep Carreras Leukaemia Research Institute, Barcelona, Spain, ³IUOPA - Universidad de Oviedo, Oviedo, Spain, ⁴Faculty of Medicine, University of Rzeszow, Rzeszow, Poland

AS: Academic Abstract Body: CD19-CAR-T-cell therapy revolutionized the treatment of relapsed/refractory (r/r) B-cell malignancies. Although CD19 loss is a major cause of resistance, the effect of the CAR costimulatory domain on CD19 genetic changes is unclear. Here, using in vitro resistance models, we directly compare 4-1BB and CD28 CARs in the context of CD19 mutation–driven resistance. The results show that CD19-4-1BB-CAR-T-cells induced complete resistance of tumor B cells, whereas CD19-CD28-CAR-T-cells only partially reduced tumor sensitivity. Epitope-specific antibody analysis confirmed total CD19/FMC63-specific loss with 4-1BB-CAR-T and moderate reduction with CD28-CAR-T. Transcriptomic analysis revealed that resistance in 4-1BB-exposed cells, but not in CD28-treated cells, was driven by CD19 mutations, loss of heterozygosity (LOH), and alternative splicing (intron 2/6 retention and exon 2 skipping). RNAseq identified a high-VAF frameshift mutation (in exon 3) with LOH in the Raji-4-1BB model, and frameshift (in exon 2) plus missense (R163H) mutations in Nalm-6-4-1BB. Using the limited dilution cloning, we confirmed that both mutations are heterozygous and affect both alleles of CD19 gene. Amplicon-based ultra-deep NGS revealed that both mutations in the Nalm-6-4-1BB model were detectable at ∼2% VAF after 14 cycles and dominated the population after 16 cycles. Mutations disrupted the FMC63 epitope and impaired CD19 detection by FC and immunohistochemistry. These observations were also reported in r/r B-ALL and lymphoma patients after CD19-4-1BB-CAR-T therapy. In clinical settings, CD19-mutation-mediated antigen escape acquired after 4-1BB-CD19-CAR-T treatment cannot be properly predicted, as CD19 mutations that rapidly dominate the tumor population are initially undetectable. Funding: NCN 2020/39/O/NZ6/01434, 2023/49/N/NZ7/03096, 2023/50/A/NZ6/00423; NAWA BPI/PST/2024/1/00127/DEC/1.

PV041 / #46

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BEYOND THE CANONICAL CYTOTOXIC RESPONSE: SINGLE-CELL TCR ANALYSIS UNCOVERS CD⁴⁺ AND VD1+ GDT-CELL ANTI-TUMOR ACTIVITY IN NEUROBLASTOMA

Elisa Zappa¹, Naima Hiddink Verberne¹, Guillem Sánchez-Sánchez², Chin Leng Tan³, Aleksandra Balwierz¹, Wim De Jong¹, Jeff De Martino¹, Philip Lijnzaad¹, Thanasis Margaritis¹, Lideke Van Der Steeg¹, Ronald De Krijger¹, Lieve Tytgat¹, Edward Green³, Monika Wolkers⁴, Wouter Scheper⁵, David Vermijlen², Jan Molenaar¹, Judith Wienke¹

¹Princess Máxima Center, Utrecht, Netherlands, ²Université libre de Bruxelles, Bruxelles, Belgium, ³Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany, ⁴Sanquin, Amsterdam, Netherlands, ⁵Netherlands Cancer Institute (NKI), Amsterdam, Netherlands

AS: Academic Abstract Body: Despite intensive treatment, ∼50% of children with high-risk neuroblastoma relapse, highlighting the need for new therapies. Although T-cell infiltration correlates with improved survival, efforts to enhance natural T-cell responses, e.g. with checkpoint blockade, have failed. Low MHC-I expression likely contributes to this lack of efficacy. Hence, it remains unclear whether (CD8) T-cell responses are even generated and, if so, why they fail to eliminate the tumor. We generated a single-cell atlas of T-cell dynamics in 10 neuroblastoma patients, combining single-cell RNA, αβTCR and γδTCR sequencing of matched blood and tumors pre- and post-chemotherapy. We detected clonally expanded T-cells that persisted throughout chemotherapy. Interestingly, tumor-specific expansion and gene signatures associated with reactivity (TIGIT⁺CTLA4⁺PD-1⁺LAG-3⁺) were confined to CD4⁺ memory T-cells. In contrast, CD8⁺ memory T-cells were expanded in both blood and tumor, but showed reduced activation and cytotoxicity profiles within the tumor. They were characterized by an anergic/resting state, distinct from classical T-cell exhaustion, also reflected by CD28 downregulation. Immunosuppressive interactions, e.g. with Tregs and/or myeloid cells via the 4-1BB/4-1BBL or CXCR4/CXCL12 axes may contribute to this state but require further investigation. Remarkably, among γδT-cells, tumor-infiltrating Vδ1+, but not Vδ2+ cells, showed high engagement of their TCR signaling pathway, suggestive of tumor-reactivity. In conclusion, we demonstrated that CD4⁺ memory and Vδ1⁺ γδT-cells are the key mediators of anti-neuroblastoma responses, which might be strengthened with alternative immune-checkpoint therapy. On the contrary, CD8⁺ memory T-cells showed an anergic/resting dysfunctional profile. Research is underway to develop therapies that may revert this phenotype and enhance their tumor-killing capacity.

PV042 / #48

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COMBINED CAR T CELL THERAPY AND LILRB2 TARGETING FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA

Silvan Brunn, Laura Raas, Morgane Chambovey, Marianna Ponzo, Chiara Magnani

University Hospital Zurich, Medical Oncology And Hematology, Zurich, Switzerland

AS: Academic Abstract Body: CAR T-cell therapies have shown limited efficacy in Acute myeloid leukemia (AML), despite their established clinical benefit in other hematologic malignancies. In this context, the leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) has emerged as an immunosuppressive receptor in AML and other cancers. To date, the impact of LILRB2 on anti-AML CAR T-cell function remains unknown, and its potential as target for CAR T cells is not fully established. As LILRB2 is overexpressed in approximately 45% of patient samples’ blasts, in this study, we combined targeting of LILRB2 receptors with anti-AML CAR T-cell approaches to increase their therapeutic efficacy against AML. We showed that CD33 CAR T-cell activity is impaired in response to LILRB2-overexpressing leukemia in vitro and in vivo, associated with increased differentiation and exhaustion of CAR T cells. Mechanistically, LILRB2 expression by tumor cells leads to downregulation of gene sets associated with immune response in effector and target cells. LILRB2 inhibition by blocking antibodies partially restored CD33 CAR T-cell functionality. CAR T cells directly targeting LILRB2 showed cytotoxic activity and increased cytokine production against AML cell-lines expressing LILRB2 in vitro, and significantly reduced tumor growth in mice engrafted with LILRB2+ AML in vivo. Altogether, our study demonstrates that LILRB2 expression impairs CAR T-cell functional response in AML. Targeting LILRB2, either through pharmacological inhibition or direct targeting by anti-LILRB2 CAR T cells, therefore represents a promising therapeutic option to enhance CAR T-cell therapies in AML.

PV043 / #255

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PEPTIDE-DRIVEN CAR-T CELLS WITH A COMPANION DIAGNOSTIC FOR THE TREATMENT OF SOLID TUMORS

Gaia Fumagalli¹, Filippo Folchini², Flavio Curnis³, Angelo Corti⁴, Anna Mondino², Arianna Pocaterra²

¹Università vita salute San Raffaele, Lymphocyte Activation Unit, Milano, Italy, ²IRCCS Ospedale San Raffaele, Lymphocyte Activation Unit, Division Immunology, Transplantation And Infectious Diseases, Milan, Italy, ³IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁴IRCCS Ospedale San Raffaele, Division Of Experimental Oncology, Milan, Italy

LB: Academic Abstract Body: Peptides are increasingly explored as targeting moieties in tumor therapy, enabling imaging and targeted delivery of therapeutics and theranostics. In addition to antibody-derived fragments, peptides can also be used to redirect chimeric antigen receptor (CAR) specificity. In this study, we aim to develop a novel peptide-guided CAR (pepti-CAR) based on a 27 aminoacid-long peptide previously validated for cancer diagnostics, and pair it with a non-invasive imaging strategy to support precise efficacy and safety assessments. The pepti-CAR construct showed around 70% surface expression in transduced murine T cells, while the target receptor was broadly expressed across multiple solid tumor cell lines. Functionally, pepti-CAR-T cells exhibited potent cytotoxicity against target-expressing tumor cells across various effector-to-target ratios. In vivo evaluation was performed in a subcutaneous glioblastoma model (GL261-Luc). Mice received lymphodepletion with either 5Gy total body irradiation or cyclophosphamide, followed by infusion of pepti-CAR-Ts or mock-transduced T cells. Both preconditioning regimens alone were sufficient to control tumor growth in immunocompetent mice, limiting accurate assessment of CAR-T efficacy. No evidence of off-tumor toxicity was observed, as indicated by the absence of abnormal CD3⁺ cell infiltration in peripheral tissues and physiologic circulating liver enzyme levels. To support the theranostic platform, fluorescently conjugated peptides probes were generated for preclinical validation, with future development of radiolabeled counterparts. Ongoing studies are evaluating the tumor uptake of these probes to enable non-invasive antigen visualization, longitudinal monitoring of therapeutic response, and safety assessment. Additional studies using human CAR-T cells and orthotopic glioblastoma models are being explored to support clinical translation.

PV044 / #141

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IDENTIFICATION AND CHARACTERIZATION OF PROTECTIVE VIRUS-SPECIFIC T-CELL CLONES AFTER ADOPTIVE CELL TRANSFER

Sara Malchow¹, Geoffroy Andrieux², Jakob Arnold³, Vincent Schipperges², Barbara Gerhart¹, Nora Kaltenbach¹, Kerstin Bruder³, Paulina Ferrada Ernst⁴, Tessa Kerre⁵, Giuseppina Lipira⁶, Chiara Agrati⁶, Irene Teichert-Von Lüttichau⁷, Melanie Boerries^2,8, Sagar Sagar³, Semjon Willier¹, Tobias Feuchtinger^1,9, Theresa Käuferle^1,9

¹Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, University of Freiburg, Freiburg, Germany, ²Institute of Medical Bioinformatics and Systems Medicine, University Medical Center Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany, ³Faculty of Medicine, Clinic for Internal Medicine II, Gastroenterology, Hepatology, Endocrinology and Infectious Disease, University Medical Center Freiburg, Freiburg, Germany, ⁴Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital LMU Munich, Munich, Germany, ⁵Department of Hematology, Ghent University Hospital, Department of Internal medicine and Pediatrics, Ghent University, Ghent, Belgium, ⁶Department of Pediatric Oncology, Hematology, Cell and Gene Therapy, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy, ⁷Department of Pediatrics and Children's Cancer Research Center, TUM School of Medicine, Children's Hospital Munich Schwabing, Technical University of Munich, Munich, Germany, ⁸German Cancer Consortium (DKTK), Partner site Freiburg, a partnership between DKFZ and Medical Center - University of Freiburg, Freiburg, Germany, ⁹German Center for Infection Research (DZIF), Freiburg, Germany

AS: Academic Abstract Body: Introduction: Viral infections are a major cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive cell transfer (ACT) of virus-specific T cells (VSTs) can effectively clear infections long-term. However, donor availability limits the use of this therapeutic option and a better understanding of the T-cell response after ACT is required. We aim to characterize protective T-cell clones during viral infections in immunocompromised post-HSCT patients who underwent ACT in the Phase III Clinical Study “TRACE”. Methods: Patient samples were taken at screening, day of VST transfer and during follow-up 1, 2, 4, 8 and 15 weeks post VST transfer. We monitored the frequency of VSTs by virus-specific in vitro re-stimulation of peripheral blood mononuclear cells (PBMCs). Expanding VST populations were isolated using fluorescence activated cell sorting (FACS) followed by 10X single-cell T-cell receptor (TCR) and transcriptome sequencing. Results: We detected VST populations whose expansion correlated with viral clearance and subsequently isolated and sequenced the respective VSTs from selected donor and patient samples. Sequencing results revealed dominant highly expanded T cell clones in each patient sample whereas the VSTs in donor samples were multiclonal. Discussion and Conclusions: The expansion of VSTs in a timely correlation with viral clearance suggests these T cells have a protective function. The highly expanded TCR clones in the patients are thought to be highly functional and will be further characterized in vitro. Additionally, transcriptomic data and the analysis of additional donor and patient samples will improve our understanding of the protective characteristics of VSTs.

PV045 / #256

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CAR-T CELL DYNAMICS AND MYELOID-CELL ALTERED PROFILE ARE ASSOCIATED WITH CLINICAL OUTCOME IN ANTI-CD19 CAR-T CELL THERAPY FOR RELAPSED/REFRACTORY B-CELL MALIGNANCIES: A REAL-LIFE COHORT STUDY

Maddalena Noviello¹, Campodonico Edoardo¹, Veronica Valtolina¹, Elena Tassi¹, Laura Rudilosso², Carlo Cipriani², Raffaella Greco¹, Elisabetta Xue¹, Maria Teresa Lupo-Stanghellini¹, Laura Falcone¹, Valeria Beretta¹, Jacopo Peccatori¹, Monica Casucci¹, Andrès Jose Maria Ferreri^1,3, Daniela Cesana², Matteo Carrabba¹, Fabio Ciceri^1,3, Piera Angelillo¹, Chiara Bonini^1,3

¹IRCCS Ospedale San Raffaele, Milan, Italy, ²San Raffaele Telethon Institute for GT (SR-Tiget), IRCCS Ospedale San Raffaele, Milan, Italy, ³Vita-Salute San Raffaele University, Milan, Italy

LB: Academic Abstract Body: Anti-CD19 CAR-T cell therapy is effective in recurrent/refractory B-cell malignancies but is limited by toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), and relapse. We aimed to identify immune biomarkers of efficacy and toxicity in a real-world setting. The infused product (IP) was analyzed by flow cytometry and high-dimensional methods. Peripheral blood samples were collected longitudinally to assess CAR-T pharmacokinetics (PK), clonality, and PBMC phenotype. Among 47 patients enrolled, 20 received axicabtagene ciloleucel (axi-cel), 9 brexucabtagene autoleucel (brexu-cel) and 18 tisagenlecleucel (tisa-cel). The Overall Response Rate was 56% at +90 days. CRS was observed in 44 patients (n=42 grade 1-2, n=2 G3), ICANS in 13 patients (n=9 G1-2, n=4 G3-4). The infusion of early-differentiated CAR-T correlated with complete remission. Interestingly, high frequencies of untransduced T cells expressing Inhibitory Receptors within IP correlated with relapse. PK curves showed the association between higher CAR-T counts at +7 days and complete remission, threatened by higher toxicities. Integration site analyses revealed a significantly lower number of integrations, reduced complexity and persistence of circulating CAR-T in relapsing patients, compared to patients who achieved complete remission. No differences were observed regarding vector integration preferences or enrichment towards specific gene classes. Finally, a myeloid-cell altered profile, characterized by low expression of HLA-DR and CD86 and high levels of CD163, correlated with severe CRS/ICANS and was already detectable before the infusion. These results indicate that IP phenotype, the amount and clonality of circulating CAR-T, and a myeloid-cell altered phenotype can predict clinical outcomes.

PV046 / #248

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DUAL-SPECIFICITY PEPTIDE-GUIDED CAR T CELLS TARGETING ΑVΒ6/ΑVΒ8 INTEGRINS ENABLE THERAPY, IMAGING, AND TUMOR MICROENVIRONMENT REMODELING IN PANCREATIC CANCER

Arianna Pocaterra¹, Elisa Sangiovanni¹, Veronica Basso¹, Martina Mazza¹, Filippo Folchini¹, Chiara Gnasso², Anna Tosi³, Giulia Di Gregorio¹, Beatrice Greco⁴, Martina Spiga⁵, Chiara Balestrieri⁵, Nicoletta Caronni⁶, Marco Genua⁶, Renato Ostuni^6,7, Antonio Rosato^3,8, Maurilio Ponzoni^7,9, Michele Reni^7,10, Massimo Falconi^7,10, Chiara Bonini^5,7, Monica Casucci⁴, Claudio Doglioni⁹, Angelo Corti^7,11, Flavio Curnis¹¹, Anna Mondino¹

¹IRCCS Ospedale San Raffaele, Lymphocyte Activation Unit, Division Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²IRCCS Ospedale San Raffaele, Experimental Imaging Center, Milan, Italy, ³Veneto Institute of Oncology - IOV, Padova, Italy, ⁴IRCCS San Raffaele Scientific Institute, Innovative Immunotherapies Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁵IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁶San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy, ⁷Vita-Salute San Raffaele University, Milan, Italy, ⁸università degli studi di padova, padova, Italy, ⁹IRCCS Ospedale San Raffaele, Pathology Unit, Milan, Italy, ¹⁰IRCCS Ospedale San Raffaele, Pancreas And Transplant Surgical Unit, Pancreas Translational And Clinical Research Center, Milan, Italy, ¹¹IRCCS Ospedale San Raffaele, Division Of Experimental Oncology, Milan, Italy

LB: Academic Abstract Body: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, characterized by limited therapeutic options and poor survival. Here we describe the design and preclinical validation of a peptide-based chimeric antigen receptor (CAR) with dual specificity for integrins αvβ6 and αvβ8, two tumor-associated targets implicated in PDAC progression, metastasis, and immune evasion. Dual targeting was achieved using the RGDL motif derived from human chromogranin A (amino acids 39–63), engineered via an E→L substitution within the canonical RGDE sequence. The RGDL peptide enabled non-invasive imaging of PDAC lesions, positioning it as a potential companion diagnostic for patient selection and treatment monitoring. Incorporation of RGDL into CAR constructs conferred cross-reactivity toward both murine and human αvβ6/αvβ8 integrins. RGDL CAR-T cells exhibited potent antitumor activity across primary, metastatic, and patient-derived orthotopic PDAC models, outperforming αvβ6-restricted RDGE CAR-T cells. Notably, RGDL CAR-T cells also targeted and depleted intratumoral αvβ8-expressing regulatory T cells, suggesting a dual mechanism of action combining direct tumor killing with modulation of the immunosuppressive microenvironment. Importantly, antigen expression is maintained in PDAC samples following chemotherapy, and CAR T cells are efficiently generated from patient-derived PBMCs. Furthermore, functional validation of an integrated safety switch, together with the successful generation of CAR T cells using a non-viral, transposon-based gene delivery system, supports the translational feasibility of this approach. Collectively, these findings establish RGDL-based dual-targeting CAR T cells as a promising therapeutic strategy for PDAC, with strong potential for rapid clinical translation.

PV047 / #155

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PRESERVED PRIMARY AND MEMORY T CELL IMMUNITY DOES NOT EXPLAIN INCREASED INFECTION RISK EARLY AFTER CD19 CAR T-CELL THERAPY

Cilia Pothast¹, Jaap Van Doesum², Els Van Hees¹, Anne Spanjaart³, Sabine Haggenburg³, Paul Den Tex³, Michel Bhoekhan³, Nienke Haverkate³, Michel Kester¹, Sophie Bruins¹, Bram Schoffelmeer³, Rosa De Groot¹, Joost Vermaat¹, Abraham Goorhuis³, Caroline Rutten³, Marie-José Kersten³, Mette Hazenberg³, Mirjam Heemskerk¹, Inger Nijhof³

¹LUMC, Hematology, Leiden, Netherlands, ²University Medical Center Groningen, Groningen, Netherlands, ³Amsterdam UMC, Amsterdam, Netherlands

AS: Academic Abstract Body: CD19-targeting CAR-T cells effectively eliminate malignant B cells and have demonstrated major clinical success. A limitation of CAR T-cell therapy is the risk of patients for severe infections due to the impact of the disease and (pre-)treatment on the immune system. While T-cell reconstitution is variable and may take considerable time, NK cells repopulate rapidly. However, their phenotype and function after therapy remain poorly defined. This study investigated quantitative and qualitative immune reconstitution of T and NK cells in 76 patients. PBMC were collected longitudinally during CAR-T cell therapy to evaluate T- and NK-cell phenotypes, primary responses to SARS-CoV-2 vaccination, and memory T-cell responses to CMV and EBV. Two weeks after infusion, non-CAR T cells exhibited a highly activated phenotype, predominantly comprising effector memory T cells. This activation was partly attributed to CMV-specific T cells, which showed greater activation than EBV-specific T cells. Although CMV/EBV-specific T cells were detectable in many CAR-T cell products, they are unlikely to substantially contribute to CAR-T cell persistence. Among 44 vaccinated patients, primary T-cell responses to SARS-CoV-2 mRNA vaccination were detectable three months post-infusion, also when CD4+ T-cells were <200/ml, with comparable functionality at later vaccination time points. Overall, T cells demonstrated preserved primary and memory function after CAR-T-cell therapy. Despite delayed naïve T-cell recovery and low circulating counts, effective primary responses can be generated. These findings show that although the quantity and composition of T-cells are altered after CAR T-cell therapy, T-cell function is maintained and thus unlikely to explain increased infection risks.

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HLA I DYEMERS FOR RELIABLE IMMUNE MONITORING OF LOW-FREQUENCY VIRUS SPECIFIC T CELLS

Malte Schütz¹, Emely Hauch¹, Rebecca Jonczyk¹, Björn Bütefisch¹, Stephan Immenschuh², Cornelia Blume¹

¹Leibniz Universität Hannover, Institut Für Technische Chemie, Hannover, Germany, ²Medizinische Hochschule Hannover, Institute Of Transfusion Medicine And Transplant Engineering, Hannover, Germany

AS: Academic Abstract Body: CD8⁺ T cells are central mediators of antiviral immunity, recognizing viral peptides presented by human leukocyte antigen (HLA) class I via their T cell receptor (TCR) and eliminating infected cells. In transplant patients, monitoring T cells is critical for guiding immunosuppressive therapy. Moreover, TCR characterization holds promise for personalized T cell therapies. We established a detection platform for low-frequency virus-specific CD8⁺ T cells, based on Dyemers (imusyn GmbH & Co. KG, Germany), modular HLA-I Streptamer-variants consisting of two HLA-I monomers linked via Streptavidin-binding peptide tags to fluorescently labeled Strep-Tactin. Using cytomegalovirus (CMV) as a model system, Dyemer’s staining performance was evaluated in PBMC samples from more than 10 donors by assessing detection frequency and fluorescence intensity. In parallel, a protocol for antigen-specific T cell expansion was established to maximize the yield of detectable cells. Peripheral blood mononuclear cells (PBMCs) were stimulated with CMV-derived peptide pools and cultured with Interleukin-2 to promote selective expansion of virus-specific T cells. Dyemer-based staining demonstrated approximately twofold detection frequency and approximately fivefold fluorescence intensity compared to established Streptamers (Iba Lifesciences, Germany), enabling reliable detection of virus-specific CD8⁺ T cells even in unstimulated PBMCs. Peptide stimulation and subsequent short-term expansion of PBMCs resulted in a >10-fold increase in detectable antigen-specific T cells, reaching up to 70% of total measured lymphocytes compared to unstimulated PBMCs, providing a robust basis for subsequent cell isolation and TCR characterization. Collectively, this approach bridges TCR characterization with clinically applicable immune monitoring, paving the way for personalized T cell–based therapeutic strategies.

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POINT-OF-CARE MANUFACTURE OF BOB1-TCR T CELLS FOR THE TREATMENT OF B-CELL MALIGNANCIES

Johannes Wellershoff¹, Sabrina Veld¹, Lois Hageman¹, Cassandra Verhaeghe¹, Patrick Claus¹, Martijn Willemsen¹, Anne Wouters¹, Ian Johnston², Peter Van Balen¹, Rosa De Groot¹, Mirjam Heemskerk¹

¹Leiden University Medical Center, Hematology, Leiden, Netherlands, ²Miltenyi Biotec B.V & Co. KG, Bergisch Gladbach, Germany

AS: Academic Abstract Body: Chimeric Antigen Receptor (CAR) therapies have shown great promise in the treatment of several B-cell malignancies (BCMs). Irrespective of disease entity, however, relapse rates upon CAR therapy remain high. T-cell receptor (TCR)-based therapy may aid in overcoming challenges associated with CAR therapies. Here, we present the development of a 6-day, Good Manufacturing Practice-compliant, Point-of-Care manufacturing process for autologous TCR T cells targeting a peptide of B Cell Specific Octamer Binding Protein 1 (BOB1) presented in HLA-B*07:02. BOB1-TCR TCR T-cell manufacture makes use of the automated and functionally-closed CliniMACS Prodigy system. Process development entailed a total of 11 engineering manufacturing runs with healthy donor material during which the impact of key process parameters on quality attributes of the obtained infusion products was explored. In process validation, the final manufacturing process yielded drug substances of on average 1.5E+09 CD8 T cells, among which on average 69.9% BOB1-TCR⁺ CD8 T cells. Generated infusion products were of high purity and viability. Vector integration analysis exhibited mean vector copy numbers of 3.8, staying below recommended safety thresholds at all times. BOB1-TCR T cells in infusion products were predominantly of central memory phenotype and exhibited potent anti-tumour activity in vitro against a panel of BCM cell lines. We hence conclude that the manufacturing process for BOB1-TCR T cells generated infusion products of good quality and high in vitro functionality. The manufacturing process is currently employed in a phase I/II clinical trial investigating the safety, feasibility and efficacy of BOB1-TCR T-cell therapy in patients with various BCMs.

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MAPPING BCMA ANTIGEN ESCAPE USING A BASE EDITING SCREENING PLATFORM TO IDENTIFY DRIVERS OF CAR-T RESISTANCE IN MULTIPLE MYELOMA

Daniela Abele¹, Valeria Colon Oliveros¹, Gabriele Pelosi¹, Naile Koleci¹, Rupert Öllinger², Roland Rad², John Doench³, Marcela Maus⁴, Julian Grünewald⁵, Andrea Schmidts¹

¹Technical University Munich, TUM, Department Of Medicine Iii, Hematology And Oncology & Translatum, Munich, Germany, ²Technical University Munich, TUM, Department Of Medicine Ii, Tum University Hospital, Technical University Of Munich, Tum School Of Medicine And Health, Munich, Germany, Germany, ³Broad Institute, Cambridge, United States of America, ⁴Massachusetts General Hospital, Mass General Brigham Cancer Institute, Boston, United States of America, ⁵Technical University Munich, TUM, Department Of Medicine I: Cardiology & Translatum, Munich, Germany

LB: Academic Abstract Body: BCMA-directed CAR-T cell therapy induces high response rates in multiple myeloma; however, relapse remains common. Antigen escape via reduced BCMA expression or mutations impairing CAR-T recognition is a key mechanism of treatment failure. We employed a CRISPR base-editing screening platform to perform saturation mutagenesis of TNFRSF17 to identify regulatory regions controlling BCMA expression and mutations affecting CAR-T recognition after BCMA-directed CAR-T therapy. Adenine and cytosine base editors with a tiled sgRNA library spanning TNFRSF17 generated thousands of single-nucleotide variants across >70% of coding and non-coding regions. MM.1S cells were transduced and sorted by BCMA expression. Next-generation sequencing identified variants linked to altered expression. Candidates were reintroduced for validation and tested for susceptibility to BCMA CAR-T therapies, including flow cytometry-based quantification of BCMA surface density and functional cytotoxicity assays using clinically relevant CAR-T constructs. Screens identified 487 variants with decreased and 196 with increased BCMA expression. Downregulating variants clustered in exon 1, while upregulating variants targeted the promoter, with strongest effects in exon 3. Importantly, a variant with marked downregulation remained susceptible to cilta-cel but resistant to ide-cel cytotoxicity. These results highlight mutation-specific differences in CAR-T susceptibility and suggest differential sensitivity between ide-cel and cilta-cel may be driven by epitope or binding context alterations supporting clinical relevance of antigen escape mechanisms in multiple myeloma therapy resistance evolution processes. These findings uncover functional consequences of TNFRSF17 mutations and genetic drivers of resistance to BCMA CAR-T therapy. Our base editing platform provides a scalable framework for benchmarking and engineering resistance-resilient CAR-T therapies.

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AID-BASED ENGINEERING OF B CELLS THROUGH THE INTEGRATION OF HIV-RECEPTOR EXONS

Ata Ul Wakeel Ahmad^1,2, Kathrin De La Rosa¹, Christoph Ratswohl³

¹Centre for Individualised Infection Medicine (CiiM), a joint venture between the Helmholtz-Centre for Infection Research (HZI) and the Hannover Medical School (MHH), Hannover, Germany, Department Of Personalised Immunotherapy, Hannover, Germany, ²Max Delbrück Center for Molecular Medicine, BERLIN, Germany, ³2Institute of Cell Biology and Immunology Thurgau (BITG), Kreuzlingen, Switzerland

AS: Academic Abstract Body: Antibody diversification plays a key role in fighting infections by producing specific antibodies of high affinity against pathogens. Rarely, B cells can diversify by incorporating pathogen receptors resulting from a genomic insertion in theswitch region. Here, we aim to replicate this natural mechanism using AID and CRISPR-based integration of exon-inserts and their splicing into final antibody mRNAs.

To guide substrate design, we generated recombinant antibodies bearing HIV-specific Llama-VHHJ3, human wtCD4 and CD4v1 domains and confirmed their expression, breadth, and specificity. To enable optimal splicing, a GFP-based fluorescent screening system was developed to identify B cell-specific intronic splice-enhancers (ISE). Utilizing a high-throughput sequencing approach, we identified and validated novel intronic splice enhancer sequences specific to B and T cells.

Building upon this work, we engineered primary human and murine B cells with VHHJ3, wtCD4 and CD4v1 substrates employing AID and CRISPR knock-in strategies. We show successful in vitro engineering of primary human and murine B cells with stable expression of VHHJ3, wtCD4 and CD4v1, respectively. Furthermore, an in vivo study with adoptive transfer of AID and CRISPR-edited B cells exhibited germinal center recruitment and production of CD4-positive antibodies in response to a heterologous immunization with HIV-BG505-gp140 and HIV-Bal-gp140.

Prospectively, AID-based editing of B cells by the addition of receptor domains represents a promising alternative to the CRISPR-based replacement of V(D)J heavy and light chain genes, without affecting the fitness of a cell to take part in the immune response and reducing the chance of off-targets.

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ARMORED EGFRVIII CAR-T CELLS LOCALLY SECRETING B7-H3 T CELL ENGAGERS EXPAND ENDOGENOUS IMMUNITY FOR DURABLE CONTROL OF ANTIGEN-HETEROGENEOUS SOLID TUMORS

Javier Arroyo-Ródenas^1,2,3, Alex Chen⁴, Jason Tonne⁴, Jill Thompson⁴, Thanich Sangsuwannukul⁴, María Rivas-Sánchez⁵, David Sánchez-Alonso⁵, Raquel Tobes⁶, Yin Chen⁷, Eduardo Pareja⁶, Jorge Martínez-Torrecuadrada⁵, Anaïs Jiménez-Reinoso^1,2,8, Michael Shapiro⁷, Nicolas Sarbia⁹, Shane Foo⁹, Virginia Shapiro⁷, Richard Vile^4,7,9, Luis Álvarez-Vallina^1,10,11

¹Centro Nacional de Investigaciones Oncológicas (CNIO), Cnio-hmarbcn Cancer Immunotherapy Clinical Research Unit, Madrid, Spain, ²Hospital Universitario 12 de Octubre, Cancer Immunotherapy Unit (unica), Department Of Immunology, Madrid, Spain, ³Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Immuno-oncology And Immunotherapy Group, Madrid, Spain, ⁴Mayo Clinic, Department Of Molecular Medicine, Rochester, United States of America, ⁵Centro Nacional de Investigaciones Oncológicas (CNIO), Protein Production Unit, Structural Biology Programme, Madrid, Spain, ⁶Alamo Blanco Research Association, Las Gabias, Spain, ⁷Mayo Clinic, Department Of Immunology, Rochester, United States of America, ⁸Instituto de Investigación Biomédica Hospital 12 de Octubre, Immuno-oncology And Immunotherapy Unit, Madrid, Spain, ⁹King's College London, Joan Reece Chair Of Immuno-oncology, Comprehensive Cancer Centre, School Of Cancer & Pharmaceutical Sciences, School Of Immunology And Microbial Sciences, London, United Kingdom, ¹⁰Hospital del Mar Research Institute Barcelona (HMRIB), Barcelona, Spain, ¹¹Banc de Sang i Teixits, Barcelona, Spain

AS: Academic Abstract Body: CAR T cell therapies have shown remarkable efficacy in hematologic malignancies but limited success in solid tumors due to antigen heterogeneity and poor persistence. Dual-targeting CAR-based therapies have improved efficacy compared to single-target CAR-T cells, but still suffer from low persistence and poor responses. In addition to multi-antigen targeting, emerging clinical evidence suggests that endogenous immune activation and subsequent epitope spreading are major contributors to sustained responses in T cell redirection strategies. Here, we present a dual-targeting strategy where T cells locally secrete an anti–B7-H3 T cell engager (STAb-T) in combination with the expression of an EGFRvIII-specific CAR (CAR–STAb-T). In vitro, using heterogeneous mixes of antigen-knockout tumors, CAR–STAb-T cells achieved superior control of antigen-negative tumor escape compared to single-target CAR and STAb-T cells. Furthermore, in immunocompetent mouse models injected with both homogeneous and heterogeneous tumors, CAR–STAb-T cells induced potent cytotoxicity, prevented antigen-loss escape, and, most importantly, expanded the host TCR repertoire against the tumor, promoting the breaking of tolerance to tumor-associated antigens through epitope spreading and long-term tumor-free survival. In fact, local secretion of the TCE from CAR-T cells not only increased in vivo persistence but enhanced the expansion of endogenous immune responses against antigen-negative tumor cells in the lymph nodes. By coupling CAR-driven persistence with TCE-mediated bystander recruitment and immune diversification, CAR–STAb-T cells provide a promising platform to overcome antigenic heterogeneity and relapse in solid tumors, achieving long-term durable responses that support the clinical translation of locally TCE-secreting CAR-T cell therapies.

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PV054 / #75

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GLYCOSYLATION-DEPENDENT TIM-3 RECOGNITION ENABLES SELECTIVE DUAL CD33/TIM-3 CAR-CIK THERAPY IN ACUTE MYELOID LEUKEMIA

Marta Biondi¹, Corinne Arsuffi², Erica Grassenis², Camilla Firpo², Antonio Alviano³, Beatrice Cerina¹, Bruna My⁴, Andrea Lia⁴, Andrea Biondi¹, Antonio Galeone⁴, Sarah Tettamanti¹, Marta Serafini²

¹Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy, ²School of Medicine and Surgery, University of Milano-Bicocca, Milan, Italy, ³Pathology, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy, ⁴Institute of Nanotechnology, National Research Council (CNR-NANOTEC). Tecnomed Puglia - Tecnopolo per la medicina di precisione (Biotech Lecce Hub), Lecce, Italy

AS: Academic Abstract Body: CAR cytokine-induced killer (CIK) cell therapy for acute myeloid leukemia (AML) is challenged by disease heterogeneity, on-target/off-tumor toxicity and the persistence of leukemic stem cells (LSCs). To overcome these issues, we have focused on developing next-generation CARs directed against antigens more restricted to LSCs. T-cell immunoglobulin mucin-3 (TIM-3) is an attractive target, enriched on AML-LSCs while absent on healthy hematopoietic stem cells, although expressed on certain normal immune cell subsets. Using our non-viral Sleeping Beauty transposon system, we designed a novel TIM-3.CAR derived from an antagonistic, ligand-blocking monoclonal antibody. We observed that our TIM-3.CAR targets a protein-proximal TIM-3 epitope whose binding is selectively enhanced by AML-specific N-glycan modifications. This selectivity was confirmed both functionally, as inhibiting fucosylation reduced CAR binding and cytotoxicity, and structurally, through analyses showing that AML blasts display hyper-fucosylated and hyper-sialylated TIM-3 glycoforms, which are absent on normal TIM-3⁺ immune cells. To address AML heterogeneity and further improve safety, we employed an IF-BETTER combinatorial strategy to simultaneously target TIM-3 and CD33. We paired a second-generation CAR with a cytokine-costimulatory receptor (CCR) developing two Dual CAR designs: CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR. Both Dual CARs demonstrated potent and selective cytotoxicity against AML cell lines and primary patient samples, effectively controlled leukemia in vivo, and spared healthy hematopoietic and immune cell subsets. Together, our findings suggest that AML-specific TIM-3 glycosylation enhances CAR-antigen avidity and promotes selective leukemia targeting. Moreover, integrating glycosylation-modulated antigen recognition into an IF-BETTER dual CAR platform might offer a promising and potentially safer immunotherapeutic strategy for AML.

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BIOCHEMICAL DRIVERS OF CAR TONIC SIGNALING

Philipp Brunmayr¹, Fiona Spreitzer¹, Michael Traxlmayr^1,2

¹BOKU University, Department Of Natural Sciences & Sustainable Resources, Institute Of Biochemistry, Vienna, Austria, ²CD Laboratory for Next Generation CAR T Cells, Vienna, Austria

AS: Academic Abstract Body: Background The extracellular domain of chimeric antigen receptors (CARs) plays a critical role in determining receptor surface expression, tonic signaling, and downstream T-cell fitness. While these domains are typically engineered for antigen sensitivity and specificity, their influence on ligand-independent (tonic) signaling and receptor expression is not yet fully understood. In practice, tonic signaling has been linked to poor CAR-T cell persistence and early exhaustion. Moreover, previous analyses have relied on a small set of individual single-chain variable fragments (scFvs), restricting broader generalization. We therefore generated a site-specifically randomized CAR library to systematically investigate sequence determinants of CAR expression and tonic signaling. Methods The CAR library was constructed using a conserved extracellular scaffold incorporating randomized amino acids at predefined positions within the binding domain. These sites were selected based on their contribution to scaffold stability and antigen interaction. The library was stitched onto a CD28ζ CAR backbone and packaged into lentiviral vectors. Jurkat Nur77 reporter cells were transduced and sorted based on CAR expression and tonic signaling levels. Enriched variants were analyzed by next-generation sequencing. Results Initial analyses reveal broad variation in CAR surface expression and tonic signaling across library variants, despite a shared extracellular framework and signaling backbone. Sequence analysis suggests that intrinsic biochemical features within the extracellular library influence tonic signaling and receptor surface expression. Ongoing deep sequencing analyses aim to uncover broader structural and biochemical principles associated with tonic signaling propensity and receptor expression.

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OPTIMIZED MELOE-1 TCR EXPRESSION IN PRIMARY T-CELLS LEADS TO ENHANCED CLEARANCE OF METASTATIC MELANOMA

Gwenann Cadiou¹, Thibault Leray^1,2, Ali Telet¹, Tiffany Beauvais^3,4, Sébastien Wälchi¹, Nathalie Labarrière^3,4, Else Marit Inderberg¹

¹Translational Research Unit, Section for Cellular Therapy, Department of Oncology, Oslo University Hospital, Oslo, Norway, ²Medical Faculty, University Of Oslo, Oslo, Norway, ³Nantes Université, Université d'Angers, INSERM, CNRS, Immunology and New Concepts in ImmunoTherapy, INCIT, UMR 1302/EMR6001, Nantes, France, ⁴LabEx IGO, Université De Nantes, Nantes, France

AS: Academic Abstract Body: Among cellular immunotherapies in the treatment of metastatic melanoma, T-cell Receptor (TCR) therapy is one of the most promising. However, two longstanding challenges remain; the proper expression of the TCR by the T cells and selection of a tumor-specific antigen, both crucial for the success of such a therapy. In this study, we focused on a TCR specific of MELOE-1 -an immunogenic non-conventional melanoma antigen- derived from a peripheral blood CD8⁺ T cell clone. Primary T cells were retrovirally transduced with natural or murinized versions of this TCR. Results showed that murinization of both ɑ and β-constant domains improved TCR expression (fold change>4). Functional assays demonstrated that increased TCR expression was consistent with an increased response against endogenous MELOE-1 antigen in melanoma cell lines showing superior cytotoxicity capacity. More interestingly, although this TCR is CD8-dependent, the combined CD4⁺ and CD8⁺ TCR transduced T cells were as effective as the CD8⁺ alone, reinforcing the potential of this therapeutic strategy. This contribution of CD4⁺ T-cells in the co-culture appears to rely on both cellular and molecular mechanisms. Finally, TCR efficacy was assessed in vivo in NXG mice injected with 2 melanoma cell lines, and engineered combined CD8⁺ and CD4⁺ TCR-T cells were able to control tumor growth. Additionally, no toxicity against melanocytes was detected, suggesting that our optimized TCR remains melanoma-specific and safe. In summary, these data support the further development of an encouraging TCR-based T-cell therapy for metastatic melanoma treatment and can be combined with other therapeutic approaches.

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CELL AVIDITY: THE NEXT-GEN BINDING ASSAY TO ADVANCE IMMUNE-BASED THERAPEUTIC DEVELOPMENT

Justin Moser, Andrea Candelli

LUMICKS, Amsterdam, Netherlands

AS: Industry Abstract Body: While conventional assays such as affinity, cytokine secretion, and cytotoxicity provide valuable data at a molecular level, this information is insufficient to fully characterize and select the best cellular therapies. There is still a lack of understanding about the biophysical cell-cell interactions that drive functional processes. Cell avidity, the integrated strength of multivalent interactions between an effector cell and its target, can help elucidate the mechanism of action for therapeutic candidates. Our platform challenges these interacting pairs using contactless force and quantifies the strength of binding between effector and target cells to distinguish productive from unproductive cell binding in a physiological context. This biophysical metric provides a unique view into cell binding characteristics to interrogate binding potency, selectivity, sensitivity, and kinetics. Here, we review recent publications highlighting how researchers have used Cell Avidity to: Fine-tune the affinity/cell avidity of CAR-T cells to mitigate on-target off-tumor toxicity in renal cell carcinoma. Format-tune bispecific T cell engagers to enhance efficacy against renal cell carcinoma Validate binding mechanism of tandem CAR-T cells to overcome tumor heterogeneity Engineer CAR-T cells to overcome a challenging tumor microenvironment in pancreatic adenocarcinoma. Elucidate mechanism of action of tandem CAR-T targeting heterogenous solid tumors Phenotype the tumor-primed NK cells for cell binding and function We developed a Cell Avidity platform that enables the characterization and screening of various binder modalities, including antibodies, small molecules, and cell therapies. Cell Avidity provides essential information revealing potency, selectivity, sensitivity, and kinetics, offering key biophysical insights into the MOA for cell therapies.

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SIMULTANEOUS ORTHOGONAL CELL ENGINEERING BY A SINGLE CRISPR-CAS9 POLYFUNCTIONAL EDITOR

Deborah Cipria¹, Tania Baccega^1,2, Miriana Rizzo¹, Piergiuseppe Quarato¹, Alice Reschigna^1,3, Rita El Khoury^4,5, Martino Alfredo Cappelluti¹, Sandra Ammann^6,7, Valeria Mollica Poeta¹, Matteo Conti¹, Sara Valsoni¹, Pietro Spinelli⁸, Ivan Merelli^1,9, Toni Cathomen^6,7,10, Monica Casucci⁴, Angelo Lombardo^1,5

¹San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan, Italy, ²Veneto Institute of Oncology IOV – IRCCS, Padua, Italy, ³LMU University Hospital, Department Of Ophthalmology, Munich, Germany, ⁴IRCCS San Raffaele Scientific Institute, Innovative Immunotherapies Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁵Vita-Salute San Raffaele University, Milan, Italy, ⁶University of Freiburg, Institute For Transfusion Medicine And Gene Therapy, Medical Center, Freiburg, Germany, ⁷University of Freiburg, Center For Cell And Gene Therapy Freiburg (cgf), Medical Center, Freiburg, Germany, ⁸nChroma Bio, Boston, United States of America, ⁹Institute for Biomedical Technologies, National Research Council, Segrate, Italy, ¹⁰University of Freiburg, Faculty Of Medicine, Freiburg, Germany

AS: Academic Abstract Body: The simultaneous disruption of multiple genes coupled with targeted transgene insertion offers a powerful strategy for precise and efficient T cell engineering. However, such orthogonal editing typically involves multiple DNA breaks, raising safety concerns due to the potential risk of chromosomal rearrangements and translocations. Here, we present a polyfunctional CRISPR-Cas9–based platform that enables concurrent transgene integration and targeted epigenetic silencing at distinct genomic sites in a single treatment, without inducing chromosomal translocations. This is achieved through an optimized all-in-one editor design, combined with length-tuned guide RNAs (gRNAs) that direct transgene knock-in to one genomic site while suppressing Cas9 nuclease activity at loci designated for epigenetic silencing. Using this approach, we obtained off-the-shelf T cells at high efficiency, engineered to carry multiplexed epigenetic silencing of clinically relevant genes along with targeted insertion of an NY-ESO1-specific TCR into the TRAC locus or an anti-CD19 CAR and an immunomodulatory receptor into the B2M locus. According to the mechanism of action of the technology, epigenetic silencing was accompanied by CpG hypermethylation at gene promoters and proved to be remarkably stable, withstanding the multiple rounds of cell division associated with antigen recognition. Importantly, no reciprocal chromosomal translocations were observed among edited loci, and poly-edited T cells retained their cytotoxic activity against cancer cells both in vitro and in vivo. This polyfunctional editing approach offers a flexible and safer strategy for multiplexed genome and epigenome engineering, with transformative potential for cancer immunotherapy and beyond.

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CD39 DISRUPTION INDUCES METABOLIC REPROGRAMMING IN TCR-EDITED T CELLS FOR ADOPTIVE T CELL THERAPY

Alessia Potenza¹, Laura Conte¹, Lorenzo Da Dalt², Fabrizia Bonacina², Martina Spiga¹, Barbara Camisa¹, Chiara Balestrieri¹, Elena Vezzoli³, Paolo Monti⁴, Danilo Norata², Eliana Ruggiero¹, Chiara Bonini¹

¹IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²Università degli Studi di Milano, Department Of Pharmacological And Biomolecular Sciences, Milan, Italy, ³IRCCS San Raffaele Scientific Institute, Advanced Light And Electron Microscopy Bioimaging Centre (alembic), Milan, Italy, ⁴IRCCS San Raffaele Scientific Institute, Transplantation Immunology Unit, Milan, Italy

AS: Academic Abstract Body: The adenosine (Ado) pathway critically regulates the functionality of T cells infiltrating primary and metastatic colorectal cancer. Upon T cell receptor (TCR) engagement, T cells release adenosine triphosphate (ATP) into the tumor microenvironment (TME), where it is converted by CD39 and CD73 into immunosuppressive Ado. We previously demonstrated that the genetic disruption of CD39 enhances the efficacy of TCR-edited (TCR_ED) T cells in the context of adoptive T cell therapy (ACT), as HER2-redirected TCR_EDCD39_KO T cells showed enhanced perforin and granzyme production when exposed to target cells, resulting in superior cytotoxic potential. However, the mechanisms underlying this functional improvement remains unclear. CD39 activity depends on extracellular ATP availability: thus, we hypothesized that CD39 knockout reshapes T cell energetic metabolism, thereby influencing efficacy. Seahorse analysis showed that, after antigen-specific stimulation, TCR_EDCD39_KOHER2 T cells displayed lower oxygen consumption (OCR) and extracellular acidification (ECAR) rates, and produced ATP at levels comparable to unstimulated cells. This trend was more pronounced under glucose- and oxygen-deprived conditions mimicking the TME, in which reactive oxygen species production by TCR_EDCD39_KOHER2 T cells was reduced. These metabolic changes were accompanied by mitochondrial adaptations. Transmission electron microscopy showed remodeling of mitochondrial cristae upon antigen stimulation, and gene expression analysis suggested increased mitochondrial fission along with reduced use of long-chain fatty acids as an energy source. In parallel, TCR_EDCD39_KOHER2 T cells exhibited increased lysosomal content, consistent with their enhanced cytotoxic profile. Together, these data support a direct link between CD39 disruption, reshaped T cell metabolic profile, and improved cytotoxic function.

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INTERLEUKIN-3 AND GM-CSF KNOCK-OUT IN CAR-T CELLS AGAINST AML TO PREVENT CYTOKINE-MEDIATED RESISTANCE

Maruška Demšar¹, Semjon Willier¹, Julian Färber¹, Jannika Seiferling¹, Theresa Käuferle², Ulrike Burk¹, Tobias Feuchtinger¹

¹Children's Hospital, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Department Of Pediatric Hematology, Oncology And Stem Cell Transplantation, Freiburg, Germany, ²University of Freiburg, Center For Cell And Gene Therapy Freiburg, Medical Center, Freiburg, Germany

AS: Academic Abstract Body: Background: Acute Myeloid Leukemia (AML) remains a significant clinical challenge in both pediatric and adult patients, characterized by high relapse rates and dismal outcomes. Novel therapeutic options are urgently required. While immunotherapy demonstrates potential in B-cell lineage malignancies, its translation into effective AML therapies is limited. Recent findings by Bhagwat et al. published in Nature Medicine (2024) indicate, that myeloid-supporting cytokines secreted during CAR-T therapy promote AML resistance and drive CAR-T cell exhaustion. Methods: Hypothesizing that CRISPR/Cas9-mediated disruption of GM-CSF and IL-3 secretion in CD33-targeting CAR-T cells would increase their anti-AML functionality, we first identified a subset of GM-CSF and IL-3 sensitive AML cell lines. Next, we identified crRNA sequences leading to efficient genetic disruption of GM-CSF and IL-3 gene loci in activated primary human T cells. These edited T cells were then transduced with a previously validated CD33-specific CAR construct. In vitro co-culture experiments using GM-CSFKO and IL-3KO antiCD33-CAR T cells from multiple donors and AML cell lines asses cytotoxicity, activation and exhaustion of CAR-T cells and cytokine secretion. Functional analyses are performed using the IncuCyte live-cell imaging system, flow cytometry, and multiplex ELISA. Results: Among six AML cell lines reported to exhibit GM-CSF- and IL-3-dependent proliferation, three demonstrated clear dose-dependent growth responses to cytokine stimulation. CRISPR guide screening identified efficient crRNAs for both targets, with selected guides achieving ≥68% knockout efficiency in CAR-T cells. Sources: Bhagwat, Anand S et al. “Cytokine-mediated CAR T therapy resistance in AML.” Nature medicine vol. 30,12 (2024): 3697-3708. doi:10.1038/s41591-024-03271-5

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INVESTIGATING THE DISTINCTIVE ROLES OF LAT IN CAR AND NATIVE T-CELL SIGNALING

Laura Dénes, Andrea Nunez, Zhengmin Yang, Jesse Goyette

UNSW, Department Of Molecular Medicine, Sydney, Australia

AS: Academic Abstract Body: Chimeric antigen receptor (CAR) T-cell technology has transformed cancer therapy; however, it is also associated with multiple side effects that can be life threatening and limit efficacy. Furthermore, CAR receptors are less effective in initiating T-cell activation than the T-cell receptor (TCR), requiring increased antigen number and higher binding affinity. Understanding the molecular mechanisms of CAR T-cell signaling and comparing it with TCR signaling can help us design better CARs with improved outcomes. According to recent evidence the key differences between CAR and TCR signaling is the level of recruitment of Linker for Activation of T-cells (LAT) protein. LAT recruits multiple signaling molecules and is necessary for TCR signaling in T-cells, but how CAR T-cells can bypass the recruitment of LAT remains unclear. In this work we investigated the significance of LAT in CAR T-cell signaling and we utilized synthetic protein structures to influence the signaling processes of CAR T-cells. We implemented a single-vector CRISPR/Cas9 lentiviral strategy to knock-out LAT in CAR T-cells. Our findings suggest that the absence of LAT leads to a decreased, but still significant, activation of CAR T-cells. Additionally, expression of protein adaptors bridging between CAR and LAT lead to a significantly enhanced CAR T-cell sensitivity. Our findings reveal major differences between signaling initiated by CARs and the TCR and emphasize synthetic signaling protein engineering as a strategy to improve CAR designs.

PV062 / #133

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A DUAL-TARGETING STAB-T PLATFORM ENABLING MULTI-EFFECTOR IMMUNOTHERAPY THROUGH LOCAL SECRETION OF NK- AND T-CELL ENGAGERS

Jaime Franco-Mansilla¹, Rodrigo Lázaro-Gorines¹, Ivana Zagorac¹, Carlos Carrasco-Padilla², Pedro Roda-Navarro², Luis Álvarez-Vallina¹

¹Spanish National Cancer Research Centre (CNIO), Hospital del Mar Research Institute Barcelona (HMRIB), Cnio-hmarbcn Cancer Immunotherapy Clinical Research Unit, Madrid, Spain, ²Complutense University of Madrid, Madrid, Spain

AS: Academic Abstract Body: Brief Introduction: Bispecific antibodies (BsAbs), including T cell engagers (TCEs), can redirect cytotoxic lymphocytes against cancer, but systemic dosing often yields tumor exposure and pharmacokinetics that are inadequate in solid tumors. The STAb-T (Secretion of T cell Engaging Antibodies) platform addresses this by engineering T cells to secrete BsAbs for localized delivery. To broaden immune engagement and limit immune escape, we developed NKCE-HA, a novel NK-cell engager that recruits NKR+ immune subsets against HA-expressing tumors. Material and Methods: NKCE-HA was produced as a purified recombinant protein and characterized for HA binding specificity, developability/stability (including monomeric state), and antigen-dependent cytotoxicity. NKCE-HA was then evaluated in STAb-T by engineering T cells to secrete NKCE-HA alone or together with an HA-targeting TCE (TCE-HA). Results: Recombinant NKCE-HA mediated HA-dependent killing and showed specific HA recognition without detectable off-target binding under the tested conditions. NKCE-HA remained monomeric, did not trigger HA internalization, and retained activity in the presence of serum IgG. Beyond NK cells, NKCE-HA engaged multiple NKR+ effector subsets, expanding immune recruitment. STAb-T–secreted NKCE-HA preserved functional activity, promoted NK recruitment, and achieved tumor killing comparable to TCE-HA. Dual STAb-T cells co-secreting NKCE-HA and TCE-HA produced a greater-than-additive antitumor effect versus either engager alone. Main Conclusions: NKCE-HA is a stable, highly specific NK-cell engager that preserves HA surface accessibility, activates NK and other NKR+ effector populations, and remains functional as a recombinant protein and via STAb-T secretion. Activity in serum IgG and synergy with TCE-HA support NKCE-HA for dual-engager, multi-effector immunotherapy strategies in solid tumors.

PV063 / #93

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DEVELOPMENT OF DUAL-TARGETED STAB-T CELLS FOR CANCER IMMUNOTHERAPY

Eva García-Veros^1,2,3,4, Anaïs Jiménez-Reinoso^1,2,3,4, Luis Álvarez-Vallina^1,2,3,4

¹Immunology Department, Hospital Universitario 12 de Octubre, Cancer Immunotherapy Unit (unica), Madrid, Spain, ²Spanish National Cancer Research Centre (CNIO), Hospital del Mar Research Institute Barcelona (HMRIB), Cnio-hmarbcn Cancer Immunotherapy Clinical Research Unit, Madrid, Spain, ³Banc de Sang i Teixits, Barcelona, Spain, ⁴Instituto de Investigación Biomédica Hospital 12 de Octubre, Immuno-oncology And Immunotherapy Unit, Madrid, Spain

AS: Academic Abstract Body: T cell-redirecting strategies, such as adoptive therapy with engineered T cells expressing chimeric antigen receptors (CAR-T), have revolutionized the treatment of hematological malignancies, but the prognosis remains poor, since 30-60% of patients relapse after treatment. A promising approach based on the production of bispecific T cell-engagers (TCE) by modified T cells (STAb-T therapy) is improving the efficacy of CAR-T cells in hematological tumors. The development of engineered T cells secreting CD19-TCEs, named STAb-T19 cells, has shown excellent results in vitro and in vivo. However, optimal T cell activation requires not only antigen-specific stimulation, but also ligand-specific co-stimulatory signals and appropriate cytokine production. To enhance STAb-T effector functions, proliferative capacity and persistence, we have developed a novel dual-targeting strategy based on “double-bladed” STAb-T cells (STAb-T^db) engineered to simultaneously secrete CD19-TCEs and cell surface-anchored antigen-specific chimeric co-stimulatory receptors (CCRs) against a second tumor associated antigen (HA1), bearing CD28 and/or 4-1BB. Thus, CD19-TCE binding to both of its targets would provide the specific T cell activation signal while CCR interaction with its specific antigen (HA1) would effectively trigger the costimulatory signal to promote optimal activation, effector functions and in vivo persistence. Our results demonstrate that STAb-T^db cells show a superior performance versus STAb-T cells in terms of activation capacity, cytotoxic responses, viability, cytokine secretion, and resistance to functional exhaustion, ultimately preventing the escape of low antigen-expressing tumor cells and achieving superior and durable tumor control in vivo.

PV064 / #94

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PROGRAMMABLE HUMAN B CELLS ENGINEERED VIA BCR-EDITING OR CAR REDIRECTION MEDIATE ROBUST ANTI-TUMOR ACTIVITY IN SOLID TUMORS

Lucia Sereni^1,2,3, Marta Freschi^1,2,4, Silvia Rizzato², Andrea Bianchi^1,2,4, Giada Zambonini^1,2, Ciro Improta¹, Sofiya Vinogradova¹, Denise Klatt^1,2,3, Christian Brendel^1,2,3, Pietro Genovese^1,2,3

¹Boston Children's Hospital, Hematology/oncology, Boston, United States of America, ²Dana-Farber Cancer Institute, Pediatric Oncology, Boston, United States of America, ³Harvard Medical School, Boston, United States of America, ⁴Milano-Bicocca University, Milan, United States of America

AS: Academic Abstract Body: B-cells are abundant tumor-infiltrating lymphocytes in solid tumors and exert both pro- and anti-tumor functions. While regulatory B-cells suppress immunity within the tumor, antigen-experienced B-cells produce tumor-specific antibodies, present antigen, secrete cytokines, and organize tertiary lymphoid structures that sustain T-cell activation. In HER2⁺ breast cancer (BC), HER2-autoreactive B-cells correlate with improved clinical outcomes; however, these rare cells are difficult to isolate/expand, limiting therapeutic exploitation. We developed genetic engineering strategies to reprogram B-cell specificity ex vivo. By optimizing cytokine stimulation, timing, and AAV serotype, we established a CRISPR/Cas9-based BCR editing platform, achieving ∼60% integration of an anti-HER2 antigen-binding fragment from a clinical antibody into the endogenous IgH locus. BCR-edited cells stably expressed a functional HER2-specific BCR, responded to antigen-mediated signaling, underwent class switching, secreted engineered antibody, and effectively stimulated T-cells. In parallel, we generated anti-HER2 CAR-B cells using Baboon envelope–pseudotyped lentiviral vectors that efficiently transduce primary B-cells. CD40L-based activation enhanced editing efficiency, expansion, and acquisition of an antigen-presenting phenotype. In PBMC-humanized NSG mice bearing orthotopic HER2⁺-BC, both BCR-edited and CAR-B cells trafficked to lymphoid tissues and tumors, persisted after infusion, and significantly reduced tumor burden. Tumor control correlates with increased intratumoral T-cell infiltration, rather than antibody production, and T-cell IFNγ responses upon ex-vivo re-challenge were enhanced. In contrast to CD8 T-cells, CD4 T-cells alone were essential for robust engraftment of engineered B-cells, secretion of the BCR-edited anti-HER2 IgM/IgG, and for tumor control. These findings position engineered B-cells as programmable cellular adjuvants that combine antibody production, antigen presentation, and immune orchestration.

PV065 / #59

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WT1 SPECIFIC T-CELL RECEPTOR THERAPIES FOR THE TREATMENT OF ACUTE MYELOID LEUKAEMIA (AML)

Sayali Gore¹, Joshua Halpin¹, Brian Gloss², Pooja Laxman¹, Koon Lee^2,3,4, Parul Saini², Geraldine O’Neill¹, Emily Blyth⁴, Kenneth Micklethwaite², Kavitha Gowrishankar^1,5

¹Kids Research, Sydney Children's Hospital Network, Children's Cancer Research Unit, Westmead, NSW, Australia, ²Westmead Institute of Medical Research, Sydney, Australia, ³Westmead Hospital, Centre For Blood Transplant And Cell Therapy, Sydney, Australia, ⁴University of Sydney, Sydney Medical School, Sydney, Australia, ⁵University of Sydney, Soms, Sydney, Australia

AS: Academic Abstract Body: T-cell receptor (TCR) therapies are an emerging treatment modality showing great promise for heterogenous malignancies with huge clonal diversity, such as acute myeloid leukaemia (AML) and solid tumours expressing a wide range of intracellular antigens. Wilms tumour 1 (WT1), a high priority intracellular antigen, is overexpressed in AML and linked to poorer prognosis and survival. This study aimed to generate TCR T-cells targeting HLA-A*02:01/WT1_37-45 or WT1_235-243. Single reactive T-cells from cytotoxic T-lymphocyte cultures stimulated with WT1 peptides were captured either using tetramers or IFNγ beads and TCRab sequences identified using the 10x Chromium platform. Full length sequences determined from IMGT/V-Quest generated 6 unique TCRs which were cloned into piggyBac transposon vectors. Additional C-terminal pairing moieties of modified leucine zippers were added to the murine constant regions with di-sulphide bridges. The generated transgenic TCR specificity was confirmed through tetramer binding, with additional pairing features resulting in better binding. K562 and Raji cells were engineered to express HLA-A*02:01 with or without additional WT-1 full length antigen and used as targets to confirm functional responses with intracellular cytokine release flow cytometry and Calcein AM cytotoxicity assays. The leading WT1 specific TCR candidate showed strong specific response to A*02:01^+ve peptide loaded cells and no cross-reactivity to other HLAs tested. It also demonstrated similar levels of specific cytotoxicity against cell lines endogenously expressing WT1 to a previously published TCR. Novel strategies for dual TCR expression are currently being tested, to extend therapeutic efficacy through multi-antigen targeting for AML.

PV066 / #41

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DEVELOPMENT OF IL-3 AND WILMS’ TUMOUR-1 TARGETING COMBINATORIAL ARTIFICIAL RECEPTOR THERAPIES (CO-ART) FOR AML

Joshua Halpin¹, Sayali Gore¹, Koon Lee², Crystal Fischer¹, Pooja Laxman¹, Emily Blyth³, Kenneth Micklethwaite², Kavitha Gowrishankar¹

¹Sydney Children's Hospital, Children's Cancer Research Unit, Sydney, Australia, ²University of Sydney, Sydney Medical School, Sydney, Australia, ³Westmead Institute of Medical Research, Sydney, Australia

AS: Academic Abstract Body: Acute myeloid leukemia (AML) has a dismal 5-year survival rate of ∼30% patients responding poorly to current standard treatment and single-antigen CAR T-cell therapy. Major barriers include tumour antigen heterogeneity, shared antigens with normal haematopoietic cells and the predominance of intracellular targets. A suite of therapeutic cellular therapies with multiple receptor combinations is an attractive solution to address the immunophenotypic and mutational diversity of AML. We hypothesise that Combinatorial Artificial Receptor (Co-ART) expressing therapeutic cells will be superior to single antigen targeting therapies for AML. To this end, we have developed CAR T-cells targeting various surface antigens and TCRs targeting intracellular antigen Wilms’ Tumour -1 (WT-1). Our anti-CD123 CAR utilises the ligand-cytokine IL-3, containing two engineered-mutations, K116W and E22R, to enhance binding of the α-subunit of CD123 receptor and to prevent proliferative signalling. Results show specific T-cell activation, Th1 cytokine release, cytotoxicity against target cells and survival advantage in AML murine models. Using a bi-directional promoter system 30-50% of the cells were able to express a secondary anti-B7-H3 CAR that showed comparable in vitro cytotoxicity against dual antigen (CD123+B7-H3+) expressing AML targets to the single antigen CAR T-cells. In parallel, TCR T-cells with in-house discovered TCR sequences recognising HLA-A*02:01/WT-1_37-45 and WT-1_235-243 with enhanced α/β pairing moieties were expanded with an unique universal expansion tool and irradiated feeder cells. Endogenous TCRα/β CRISPR knockout with simultaneous knock-in of the CAR constructs and WT-1 TCR is being optimised to achieve precise and stable genomic integration, to generate a novel Co-ART for AML.

PV067 / #122

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ENGINEERING T CELLS TO OVERCOME MIGRATION BARRIERS AND IMPROVE ANTI-TUMOR POTENCY IN SOLID TUMORS

Jeffrey Chung, Jessica Olivas-Corral, Ashley Wood, Ashton Sigler, Edward Ning, Michelle Allen, Kayla Fairweather, Jordan Jacobelli

University of Colorado School of Medicine, Immunology & Microbiology, Aurora, United States of America

AS: Academic Abstract Body: Physical barriers within solid tumors, such as abnormal vasculature and a collagen-dense extracellular matrix, impede the infiltration of T cells into the tumor mass. This has limited the clinical impact of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cell therapies against solid tumors. Here, we address this bottleneck by engineering T cells to overexpress Formin-like-1 (FMNL1), a cytoskeletal regulator that drives T cell extravasation and motility through restrictive environments. In vitro, FMNL1-overexpressing T cells had increased chemotactic migration through confined spaces. In vivo, FMNL1-engineered tumor-specific TILs and CAR-T cells consistently showed greater intratumoral accumulation in multiple restrictive solid tumor mouse models. Furthermore, FMNL1 overexpression preserved T cell antigen-driven reactivation and target cell killing, indicating that enhanced trafficking was achieved without compromising effector functions. Importantly, in both immunocompetent and immunodeficient melanoma models, compared to control CAR-T cells, transferred FMNL1-overexpressing CAR-T cells limited tumor growth and extended survival of tumor-bearing mice. These findings establish cytoskeletal augmentation via FMNL1 overexpression as a novel generalizable strategy to overcome vascular and stromal tumor barriers to increase intratumoral T cell bioavailability and improve adoptive cell therapy outcomes in patients with solid tumors. Because FMNL1 acts independently of antigen recognition, FMNL1 overexpression can function as a complementary cell trafficking enhancer that can be combined with diverse TIL or CAR-T cell platforms to boost efficacy in various solid tumor types.

PV068 / #149

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EXPANDING THE LIBRARY OF NPM1 NEOANTIGEN-SPECIFIC TCRS FOR IMMUNOTHERAPY OF ACUTE MYELOID LEUKEMIA

Georgia Koutsoumpli¹, Nadine Struckman¹, Dyantha Van Der Lee¹, Eva Argiro¹, Nina Groenland¹, Maria Honders¹, Rob De Jong¹, Renate Hagedoorn¹, Dennis Remst¹, Hendrik Veelken¹, Peter Van Veelen², Mirjam Heemskerk¹, Frederik Falkenburg¹, Ian Johnston³, Marieke Griffioen¹

¹Leiden University Medical Center, Department Of Hematology, Leiden, Netherlands, ²Leiden University Medical Centrer, Center For Proteomics And Metabolomics, Leiden, Netherlands, ³Miltenyi Biotec B.V & Co. KG, Bergisch Gladbach, Germany

AS: Academic Abstract Body: Approximately 30-35% of acute myeloid leukemia (AML) patients harbor 4bp frameshift mutations in the nucleophosmin-1 (NPM1) gene, leading to an aberrant protein with its C-terminal end translated in an alternative reading frame. We previously identified HLA-binding NPM1 neopeptides on AML and isolated two T-cell receptors (TCRs) targeting CLAVEEVSL in HLA-A*02:01 or AVEEVSLRK in HLA-A*11:01. Both TCRs efficiently redirected CD8 T-cells to target NPM1-mutated AML in vitro, but only the HLA-A*02:01-restricted TCR-T cells induced an anti-tumor response in AML-engrafted immunodeficient mice, suggesting that the reactivity of the HLA-A*11:01-restricted TCR-T cells may not be strong enough to induce anti-tumor responses in patients. Therefore, we continued searching for new NPM1 neoantigen-specific TCRs. Here, we used peptide-HLA tetramers containing AVEEVSLRK in HLA-A*03:01 or HLA-A*11:01 and isolated for each neoantigen one T-cell clone with strong reactivity against NPM1-mutated AML. The TCRs of the clones were sequenced and analyzed for their potential to target NPM1-mutated AML after transfer to CD8 T cells. Similar to the parental clones, T-cells engineered with the newly-identified TCRs recognized NPM1-mutated AML, and induced specific lysis of patient-derived AML cells with different progenitor phenotypes. Importantly, in contrast to the previously-isolated HLA-A*11:01-restricted TCR, T-cells engineered with the new HLA-A*11:01-restricted TCR induced a strong anti-tumor response in AML-engrafted immunodeficient mice similar to the HLA-A*02:01-restricted TCR, which is currently under clinical evaluation in a phase I/II study. In conclusion, we expanded the library of NPM1-specific TCRs with two new TCRs which may be relevant for immunotherapy to treat HLA-A*03:01- or HLA-A*11:01-positive patients with NPM1-mutated AML.

PV069 / #148

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T-CELL RECEPTORS FOR FOUR NEW HEMATOPOIETIC-RESTRICTED MINOR HISTOCOMPATIBILITY ANTIGENS TO TARGET ACUTE MYELOID LEUKEMIA AFTER ALLOGENEIC STEM CELL TRANSPLANTATION

Georgia Koutsoumpli, Marian Van De Meent, Annemieke Boeringa, Benjaminas Kaper, Kyra Fuchs, Maria Honders, Michel Kester, Frederik Falkenburg, Marieke Griffioen

Leiden University Medical Center, Department Of Hematology, Leiden, Netherlands

AS: Academic Abstract Body: Acute myeloid leukemia is a major indication for allogeneic stem cell transplantation (alloSCT). After HLA-matched alloSCT, donor-derived T cells recognize polymorphic HLA-binding peptides on patient cells that are absent from donor cells. These minor histocompatibility antigens (MiHAs) are created by genetic differences between patient and donor. While donor T cells directed against MiHAs on the patient’s malignant cells trigger anti-tumor responses, side effects occur when MiHAs are attacked on healthy non-hematopoietic tissues. MiHAs with selective expression on hematopoietic cells constitute attractive targets, since donor-derived T cells for these antigens target patient’s malignant cells, while non-hematopoietic tissues and healthy hematopoietic cells, which are of donor origin after alloSCT, are spared. In previous research, we isolated T-cell clones from transplanted patients and identified eleven new hematopoietic-restricted MiHAs. Here, we sequenced and cloned T-cell receptors (TCRs) for four of these MiHAs, including LB-MYO1G-2M (HLA-C*03:03/C*03:04), LB-LILRB4-1G (HLA-B*07:02), LB-IL10RA-1R (HLA-B*07:02), and LB-ITGB2-1 (HLA-B*15:01). T cells engineered with these TCRs bound cognate peptide-HLA tetramers and recognized patient EBV-B cells, while no reactivity was observed against donor EBV-B cells and skin fibroblasts. Using a large panel of antibodies against early stem cell and myeloid progenitor markers, we demonstrated that TCR-T cells against LB-MYO1G-2M, LB-IL10RA-1R, and LB-ITGB2-1 induced specific lysis of AML cells arrested at different stages in myeloid differentiation, while TCR-T cells for LB-LILRB4-1G specifically killed AML cells with more mature phenotypes. These results highlight the therapeutic potential of TCR-T cells targeting hematopoietic-restricted MiHAs, supporting their development as novel immunotherapies to treat AML patients after HLA-matched alloSCT.

PV070 / #147

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DEVELOPMENT OF A DRUG-INDUCIBLE MODULAR STAB-T IMMUNOTHERAPY FOR CONTROLLED T CELL REDIRECTION

Susana Luengo-Arias^1,2,3, Carmen Domínguez-Alonso^1,3, Anaïs Jiménez-Reinoso^1,2,3, Luis Álvarez-Vallina^2,4

¹Immunology Department, Hospital Universitario 12 de Octubre, Cancer Immunotherapy Unit (unica), Madrid, Spain, ²Spanish National Cancer Research Centre (CNIO), Hospital del Mar Research Institute Barcelona (HMRIB), Cnio-hmarbcn Cancer Immunotherapy Clinical Research Unit, Madrid, Spain, ³Instituto de Investigación Biomédica Hospital 12 de Octubre, Immuno-oncology And Immunotherapy Unit, Madrid, Spain, ⁴Banc de Sang i Teixits, Barcelona, Spain

AS: Academic Abstract Body: T cell-based redirecting immunotherapies have transformed cancer treatment through two main approaches: the adoptive transfer of genetically modified T cells expressing chimeric antigen receptors (CAR-T) and the systemic infusion of bispecific T cell-engaging antibodies (TCE). A novel strategy based on the endogenous in situ secretion of TCE by genetically modified T cells, termed STAb-T, aims to combine the active trafficking of adoptive transferred engineered T cells with the TCE-mediated polyclonal T cell recruitment, addressing the short half-life of small antibody fragment-based TCEs. However, these therapies are limited by adverse effects such as cytokine release syndrome, neurotoxicity in hematological malignancies, and on-target/off-tumor toxicity in solid tumors. To regulate engineered T cell activity, we developed a drug-controlled STAb-T system using a split TCE (TCE^ON) targeting a tumor associated antigen (TAA). The TCE^ON design physically separates its modules (scFv anti-TAA and scFv anti-CD3), which dimerize upon exposure to a inductor drug. Results showed that TCE^ON modules were efficiently secreted by human cells, recognized their cognate antigens as conventional TCE (TCE^C), and formed heterodimers only in the presence the inductor drug in a dose-dependent manner. They also induced T cell activation against TAA-positive tumor cells and formed immunological synapses morphologically similar to TCE^C in the presence of the inductor drug. In primary transduced T cells (STAb^ON), cytotoxicity was comparable to TCE^C-transduced cells (STAb^C) while releasing lower IFN-γ levels. Overall, these findings suggest that STAb^ON therapy could provide a safer approach to overcome the limitations of conventional T cell-redirecting immunotherapies.

PV071 / #144

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ENGINEERING CAR T CELLS TO TARGET LIGAND-ACTIVATED EGFR

Markus Dobersberger¹, Lina Maltrovsky¹, Delia Sumesgutner¹, Charlotte Zajc², Benjamin Salzer^3,4, Elisabeth Laurent⁵, Dominik Emminger^3,4, Elise Sylvander^1,4, Elisabeth Lehner^1,4, Magdalena Teufl^4,6, Jacqueline Seigner^6,7, Madhusudhan Bobbili^8,9, Renate Kunert⁷, Manfred Lehner^3,4,10, Michael Traxlmayr^1,4

¹BOKU University, Department Of Natural Sciences & Sustainable Resources, Institute Of Biochemistry, Vienna, Austria, ²Medical University of Vienna, Vienna, Austria, ³St Anna CCRI, Vienna, Austria, ⁴CD Laboratory for Next Generation CAR T Cells, Vienna, Austria, ⁵BOKU University, Boku Core Facility Biomolecular & Cellular Analysis, Vienna, Austria, ⁶BOKU University, Department Of Chemistry, Institute Of Biochemistry, Vienna, Austria, ⁷BOKU University, Department Of Biotechnology, Institute Of Animal Cell Technology And Systems Biology, Vienna, Austria, ⁸BOKU University, Department Of Biotechnology, Institute Of Molecular Biotechnology, Vienna, Austria, ⁹Research Ceneter in Cooperation with AUVA, Ludwig Boltzmann Institute For Traumatology, Vienna, Austria, ¹⁰St. Anna Children' Hospital, Medical University of Vienna, Department Of Pediatrics, Vienna, Austria

AS: Academic Abstract Body: Despite the remarkable response rates of chimeric antigen receptor (CAR) T cells in treating hematological malignancies, their efficacy in solid tumors remains limited.

A central challenge is the lack of truly tumor-specific antigens, as many tumor-associated targets are also expressed in healthy tissues, causing on-target/off-tumor toxicities. The epidermal growth factor receptor (EGFR) is a tumor-associated antigen representing this problem. It is frequently overexpressed in solid tumors, but also expressed at low levels in healthy tissues, complicating current EGFR-targeted therapy approaches. Addressing this issue, we investigated a novel strategy to selectively target ligand-bound EGFR. As many solid tumors promote EGFR activation through increased secretion of its ligands, we hypothesized that targeting ligand-EGFR complexes may increase tumor-specificity. To test this concept, we employed yeast surface display to engineer non-antibody binding domains specific for the ligand-induced conformation of EGFR. Selected binders were characterized by SEC-HPLC, DSC, and titration assays on EGFR-positive human cell lines. The engineered domains were then incorporated into CAR T cells and evaluated for ligand-dependent cytotoxicity and IFN-γ release. Biochemical analyses demonstrated that the engineered binders were monomeric, thermally stable, and displayed no detectable aggregation. Importantly, they recognized EGFR-positive cells in a ligand-dependent manner. When integrated into CARs, these domains enabled T cells to discriminate between inactive EGFR and activated ligand-EGFR complexes, resulting in selective cytotoxicity and cytokine release upon ligand engagement. Overall, we successfully engineered CAR T cells to selectively recognize activated ligand-EGFR complexes. This approach may reduce on-target/off-tumor toxicities, thereby addressing a key challenge of conventional EGFR-targeted therapies.

PV072 / #262

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SUV39H1 DISRUPTION IN 1XX CAR T CELLS IMPROVES PERSISTENCE AND DURABLE ANTITUMOR RESPONSES IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Silvia Menegatti¹, Anne Laure Privat¹, Kyle Raymond¹, Silva Lisseth¹, Denis Malaise², Zelia Gouveia¹, Sebastian Amigorena¹, Marion Alcantara¹, Jaime Fuentealba¹

¹Institut Curie, Cellaction, Saint Cloud, France, ²Institut Curie, Ocular Oncology, Paris, France

LB: Academic Abstract Body: Patients with primary central nervous system lymphoma (PCNSL), an aggressive B-cell malignancy confined to the brain, cerebrospinal fluid, spinal cord, and/or eye, have poor outcomes, especially at relapse. While CD19-directed CAR-T cell therapies are feasible and safe, over half of patients relapse, highlighting the need for improved CAR-T cell persistence and function within the immunosuppressive CNS environment. To address this challenge, we engineered second-generation CD19 CAR-T cells combining ITAM-tuned “1XX” CAR signaling with CRISPR/Cas9-mediated knockout of SUV39H1, a histone methyltransferase that restricts memory T cell differentiation. These dual-engineered CAR-T cells displayed an enhanced memory phenotype and sustained tumor control upon repeated stimulation in a 3D PCNSL spheroid model. In an orthotopic PCNSL xenograft model, these cells showed superior tumor control and prolonged survival compared to conventional CAR-T cells. Spectral flow cytometry confirmed increased memory T cell populations and reduced exhaustion. To further assess long-term functional persistence, we established a primary vitreoretinal lymphoma rechallenge model: SUV39H1-deficient CAR-T cells were capable of eliciting effective recall responses, achieving complete tumor control following tumor rechallenge. To identify SUV39H1 genomic targets, we are performing ChIP-seq to map its binding across the genome and analyze loci associated with T cell memory and exhaustion, aiming to define its epigenetic regulatory network. Overall, these results support the development of next-generation CAR-T cells for PCNSL through epigenetic reprogramming and optimized signaling. A GMP-compatible manufacturing process incorporating the RQR8 safety switch is being finalized in preparation for a phase I/II clinical trial.

PV073 / #244

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DRIVING ENGINEERED CARGOS AGAINST MULTIPLE MYELOMA

Benjamin Motais¹, Thomas Kimman¹, Marta Cuenca¹, Ralph Tieland¹, Corine Pleijte¹, Maiken Verstappen¹, Monique Minnema², Victor Peperzak¹

¹University Medical Center Utrecht, Center For Translational Immunology, Utrecht, Netherlands, ²University Medical Center Utrecht, Department Of Hematology, Utrecht, Netherlands

LB: Academic Abstract Body: CAR-T therapy has revolutionized the treatment of hematological malignancies, yet patients still relapse due to tumor resistance mechanisms. In multiple myeloma (MM), we found that overexpression of the pro-survival protein MCL-1 impairs anti-BCMA (B-cell maturation antigen) CAR-T cell-mediated killing. While MCL-1 inhibitors can restore sensitivity to therapy, their clinical use is limited due to cardiac toxicity. To address this, we developed the CARgo technology to locally deliver the endogenous MCL-1 antagonist, NOXA, directly to tumor cells. We engineered anti-BCMA CAR-T cells to co-express granzyme B bound to NOXA through a cathepsin D-cleavable linker. This way, NOXA is trafficked to lytic granules and enters perforin pores upon synapse formation with a target cell. Proper cargo localization and transfer were confirmed using fluorescent reporters and live-imaging. NOXA-CARgo-T cells demonstrated enhanced cytotoxicity and apoptosis induction in MM cell lines and primary samples compared to an inactive control. In vivo, they achieved improved and durable tumor control in MM xenograft models. Importantly, NOXA-CARgo-T cells did not affect the viability of non-target bone marrow stromal cells or iPSC-derived cardiomyocytes. These results demonstrate that targeted delivery of NOXA overcomes apoptosis resistance and enhances CAR-T efficacy without systemic toxicity. This strategy paves the way to testing additional CARgo payloads that could further enhance tumor cell killing and trigger alternative death pathways, including immunogenic cell death. To this end, we are currently implementing biosensor-based approaches to characterize the underlying mechanisms of CARgo-T cell induced tumor cell death.

PV074 / #67

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ENABLING AUTOMATED MANUFACTURING OF GENE-MODIFIED T CELLS WITH LIPID NANOPARTICLES

Mara-Kristin Nuding, Caroline Brandes, Nihal Kus, Lydia Kopplin, Boris Engels, Dominik Lock

Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany

AS: Industry Abstract Body: Lipid nanoparticles (LNPs) provide a novel and scalable non-viral delivery modality for adoptive T cell therapies. Unlike electroporation, LNP-mediated transfection preserves T cell viability, minimizes cellular stress, and enables efficient nucleic acid transfer. Their favorable tolerability enables genetic modification at multiple stages of cell therapy manufacturing processes, offering high flexibility beyond conventional transfection techniques. In this study, we established and optimized an automated process for the scaled manufacture of LNP-engineered T cells. Initially, primary human T cells were transfected using LNPs encapsulating CRISPR/Cas9 mRNA and TRAC-targeting guide RNAs for endogenous TCR disruption. Small-scale optimization yielded TCR-knockout efficiencies of up to 98%, while maintaining viability above 90%. Next, we evaluated the feasibility of combining lentiviral CD33-CAR transduction with LNP-mediated TCR knockout. Co-modification achieved 52.0 ± 3.6% knockout efficiency and 67.3 ± 9.6% CAR transduction. An automated, closed manufacturing workflow was subsequently established on the CliniMACS Prodigy^® TCT platform using healthy donor frozen leukapheresis material. CD4+ and CD8+ T cells were isolated, activated, transduced and edited with LNPs. Within eight days, the process yielded >1.6 × 10⁹ ± 2.6 × 10⁷ TCR-negative, CAR-positive T cells, demonstrating clinically relevant scalability. Final products exhibited high viability (97.4 ± 1.8%) and a favorable early-memory phenotype. CAR-engineered T cells gained strong functional potency, selectively lysing CD33-positive target cells. LNP-mediated transfection was effective across multiple manufacturing time points (day 2: 87.6%, day 3: 92.1%, day 7: 70.8%), supporting process flexibility. Overall, we present an innovative, robust, reproducible, and highly flexible LNP-based process for the (co-)modification of T cells, enabling efficient integration into clinical manufacturing workflows.

PV075 / #247

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MODULAR DELIVERY OF CO-STIMULATORY SIGNALS THROUGH A PD-1-BASED IMMUNOSWITCH RECEPTOR IMPROVES THE FUNCTIONALITY OF HEPATITIS B VIRUS-SPECIFIC ENGINEERED T CELLS

Luis Olguin Contreras¹, Johanna Heep¹, Lisa Schiller¹, Eva Loffredo-Verde¹, Marvin Festag¹, Kai Metzger¹, Stephanie Färber¹, Susanne Wilde², Karin Wisskirchen², Elfriede Nößner³, Ulrike Protzer¹

¹Technical University of Munich, Institute Of Virology, Munich, Germany, ²SCG Cell Therapy GmbH, Planegg, Germany, ³Helmholtz Center Munich, Munich, Germany

LB: Academic Abstract Body: Chronic hepatitis B virus infection (cHBV) creates a highly suppressive liver microenvironment that undermines antiviral immunity and contributes directly to the development of hepatocellular carcinoma (HCC). Engineered T-cell therapies offer a promising route to restore immune control, yet their effectiveness is limited by dominant inhibitory pathways—most notably the PD-1/PD-L1 axis—that accelerate T-cell dysfunction and exhaustion. This work introduces PD-1–based immunoswitch receptors, a modular platform designed to convert PD-L1 engagement from an inhibitory cue into productive co-stimulation, delivered independently of antigen recognition. By decoupling co-stimulatory input from CAR or TCR signaling and exploiting ligand distributions unique to chronic infection and cancer, PD-1-based immunoswitch receptors re-establish a more physiological and durable activation architecture. Our findings demonstrate that these switches enhance cytokine production, cytotoxicity, and antigen sensitivity, while reducing exhaustion and improving persistence in vitro and in a chronic HBV mouse model in vivo. Among the variants tested, PD-1_4-1BB induced signaling and functional profiles associated with sustained antiviral responses. The modular co-stimulation concept provides a flexible and adaptable strategy to strengthen T-cell therapies for cHBV and HCC and offers a broadly applicable framework for overcoming inhibitory microenvironments in chronic infections and solid tumors.

PV076 / #143

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MECHANISTIC DISSECTION OF ANTIGEN-DEPENDENT SYNNOTCH ACTIVATION AGAINST NEUROBLASTOMA ANTIGENS

Jennie Padlo¹, Jasmin Yousefnia¹, Emily Ohlendorf¹, Lena Andersch², Kathleen Anders¹, Dimitrios Wagner³, Uta Höpken⁴, Annette Künkele¹

¹Charite-Universitätsmedizin, Department Pediatric Oncology And Hematology, Berlin, Germany, ²Charité-Universitätsmedizin Berlin, Pediatric Oncology, Berlin, Germany, ³Berlin Center for Regenerative Therapy (BCRT), Berlin, Germany, ⁴Max Delbruck Center for Molecular Medicine, Department Pediatric Oncology And Hematology, Berlin, Germany

AS: Academic Abstract Body: Synthetic Notch (SynNotch) receptors enable programmable, antigen-dependent transcriptional control and represent a powerful strategy for combinatorial CAR T cell therapies in solid tumors such as neuroblastoma. However, SynNotch receptors targeting different antigens exhibit variable activation efficiencies and thresholds, limiting rational receptor design. The mechanistic basis for these differences remains unclear. We investigate two hypotheses to explain variability in SynNotch activation across neuroblastoma-associated antigens (CD19, GD2, and B7H3). First, we propose that ligand-binding kinetics, specifically the dissociation rate (K_off), determine the efficiency of ADAM10-mediated ectodomain shedding and subsequent γ-secretase cleavage. We hypothesize that lower K_off values, reflecting more stable receptor–ligand interactions, enhance force transmission across the Notch core and promote productive proteolytic cleavage. By comparing SynNotch constructs with defined binding affinities, we aim to correlate K_off values with reporter induction and activation thresholds. Second, we hypothesize that distinct SynNotch receptors differ in their dominant activation mechanism. While CD19-targeting SynNotch receptors may be efficiently activated through ligand-induced pulling forces at the plasma membrane, receptors targeting GD2 or B7H3 may rely more heavily on receptor endocytosis and cleavage within endosomal compartments. To dissect these mechanisms, we employ pharmacologic inhibitors of endosomal trafficking, ADAM10, and γ-secretase and assess transcriptional output alongside receptor surface expression. Overall, we seek to define the biophysical and cellular determinants governing SynNotch activation in the context of neuroblastoma antigens. Elucidating how binding kinetics and cleavage location shape receptor performance will enable rational tuning of SynNotch design and inform the development of next-generation logic-gated T cell therapies for pediatric solid tumors.

PV077 / #97

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CAR-MEDIATED TARGET RECOGNITION LIMITS TCR-MEDIATED TARGET RECOGNITION OF TCR- AND CAR-DUAL-RECEPTOR-EDITED T CELLS

Teuntje Poortvliet, Tassilo Wachsmann, Miranda Meeuwsen, Dennis Remst, Anne Wouters, Renate Hagedoorn, Frederik Falkenburg, Mirjam Heemskerk

Leiden University Medical Center, Hematology, Leiden, Netherlands

AS: Academic Abstract Body: Unfortunately, relapse has been shown to be rule rather than exception in patients with multiple myeloma (MM) treated with BCMA-CAR-T therapy. Antigen escape can drive relapse after T-cell-based therapies and is commonly observed in BCMA-CAR-T treated MM patients. Targeting of multiple antigens can help limit antigen escape. By combining CAR- and TCR-mediated antigen targeting, the spectrum of targetable antigens in MM can be broadened significantly. Here, we explored whether dual antigen targeting in MM can be achieved by installing dual antigen specificity on T cells using combined TCR- and CAR-engineering. We report the generation of CD8 T cells that express both an HLA-B*07:02-restricted TCR specific for a peptide derived from transcriptional coactivator BOB1 and a BCMA-targeting CAR. These T cells, named TRaCR T cells, efficiently recognized target cells expressing either only TCR- or only CAR-cognate antigen. Thus, demonstrating general dual-specificity of the TRaCR cells. In the presence of both TCR- and CAR-relevant antigen, however, target cell recognition was preferentially conferred via the CAR resulting in a survival advantage for BCMA-negative tumour cells observed both in vitro and in vivo. TRaCR T cells displayed higher avidity for CAR-antigen expressing targets than TCR-antigen expressing targets, which could be reversed by provision of increased levels of TCR-antigen. Thus, indicating that differences in affinity and abundance of CAR- or TCR-antigen interactions likely dictate preferential target recognition by TRaCR T cells. In conclusion, we demonstrate general dual-specificity of TRaCR T cells, but advise caution when using TRaCR T cells as strategy to target heterogenous tumours.

PV078 / #89

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ALLOGENEIC TGFΒ-RESISTANT CAR-T PRECURSORS: TOWARD A TRUE OFF-THE-SHELF T CELL IMMUNOTHERAPY

Fabio Raineri¹, Chloé Ventujol², Abrial Pauline¹, Tobias Abel¹, Inna Menkova¹, Julien Marie²

¹Allogenica, LYON, France, ²Cancer Research Center of Lyon (CRCL) UMR INSERM1052, Cnrs 5286, Lyon, France

AS: Industry Abstract Body: Adoptive T cell therapies have reshaped the treatment landscape for hematologic malignancies, with CD19 CAR-T cells achieving outstanding responses in B-cell cancers. However, autologous CAR-T products remain constrained by T-cell exhaustion, the profound immunosuppression mediated by TGFβ in the tumor microenvironment, and the inherent limitations of patient-specific manufacturing designed to prevent graft-versus-host disease (GVHD). We developed a next-generation strategy based on genetically engineered T cell precursors (pre-T cells) expressing a CD19 CAR combined with a dominant-negative TGFβ receptor II (TGFβRIIDN), designed to overcome both the immunosuppressive influence of TGFβ and key drawbacks of autologous approaches. In vitro, CD19 CAR-TGFβRIIDN T cells demonstrated robust cytotoxic activity and improved exhaustion control. In NSG mice, CAR-pre-T cells exhibited efficient thymic homing and subsequently matured in the thymus, where negative selection promoted immune tolerance and continuous replenishment of a functional T cell pool - processes normally impaired by TGFβ signaling. Within weeks, animals displayed rapid peripheral T cell reconstitution without GVHD signs, alongside effective clearance of B-cell lymphoma. These results support the development of a fully allogeneic CAR-T platform empowered by TGFβ-resistant precursor T cells, paving the way for scalable and clinically impactful off-the-shelf immunotherapies.

PV079 / #120

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LOCALLY SECRETED T CELL ENGAGERS INDUCE POLYCLONAL IMMUNE ACTIVATION TO OVERCOME RESISTANCE IN SMALL CELL LUNG CANCER

Lucía Rivas Gómez^1,2, Ivana Zagorac², Belén Blanco^1,3, Carolina Pola¹, Luis Paz-Ares^4,5, Luis Álvarez-Vallina^2,6

¹STAb Therapeutics, Madrid, Spain, ²CNIO-HMarBCN Cancer Immunotherapy Clinical Research Unit, Clinical Research Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain, ³Department of Advanced Therapy Drug Development, Instituto de Salud Carlos III, Madrid, Spain, ⁴Medical Oncology Department, Hospital Universitario 12 de Octubre (H12O), Madrid, Spain, ⁵H12O-CNIO Lung Cancer Clinical Research Unit, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain, ⁶Banc de Sang i Teixits, Madrid, Spain

AS: Academic Abstract Body: Small cell lung cancer (SCLC) is an aggressive malignancy with poor prognosis and limited therapeutic options, underscoring the need for new immunotherapeutic strategies. T cell redirecting strategies, including chimeric antigen receptor (CAR) T cells and systemically administered bispecific T cell engagers (TCEs), hold promise but remain challenged by solid-tumor barriers and the need for sustained activity at the tumor site. Engineered T cells that secrete TCE antibodies in vivo (STAb-T cells) represent a complementary strategy that integrates adoptive cell therapy with continuous, localized engager delivery and broad recruitment of non-transduced bystander T cells, thereby amplifying antitumor immunity within the tumor microenvironment. Here, we present “Solid-STAb-T,” an all-in-one adoptive cell immunotherapy for SCLC based on endogenous secretion of a tumor antigen-directed TCE. Solid-STAb-T cells released soluble, functional TCEs and mediated potent tumor cell killing. In both 2D and 3D cytotoxicity assays, Solid-STAb-T consistently outperformed matched CAR-T cells, achieving superior tumor control with lower IFN-γ secretion and efficient bystander T cell recruitment, supporting a favorable efficacy–safety balance. In comparison with the corresponding soluble TCE delivered exogenously, Solid-STAb-T achieved comparable activity only under continuous antibody availability, highlighting the advantage of sustained, on-site TCE production. Consistently, persistent CD3 decoration translated into improved cytotoxic performance relative to intermittent exposure to soluble TCE. Collectively, these data support Solid-STAb-T as a promising strategy for SCLC and warrant further preclinical development toward clinical translation.

PV080 / #253

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PEPTIDE-DRIVEN RECOMBINANT RECEPTORS FOR ENHANCED CELLULAR THERAPIES AGAINST SOLID TUMORS

Elisa Sangiovanni¹, Alice Schirru¹, Veronica Basso¹, Alessia Potenza², Martina Spiga², Chiara Bonini³, Antonio Rosato⁴, Monica Casucci⁵, Flavio Curnis⁶, Angelo Corti⁶, Arianna Pocaterra¹, Anna Mondino¹

¹IRCCS Ospedale San Raffaele, Lymphocyte Activation Unit, Division Immunology, Transplantation And Infectious Diseases, Milan, Italy, ²IRCCS San Raffaele Scientific Institute, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ³Vita-Salute San Raffaele University, Experimental Hematology Unit | Immunology, Transplantation And Infectious Diseases, Milan, Italy, ⁴Istituto Oncologico Veneto, Padova, Italy, ⁵Innovative Immunotherapies Unit, Division Of Immunology, Transplantation And Infectious Disease, Ircss San Raffaele Scientific Institute, Milan, Italy, ⁶IRCCS Ospedale San Raffaele, Division Of Experimental Oncology, Milan, Italy

LB: Academic Abstract Body: Peptides have recently emerged as effective targeting moieties for redirecting chimeric antigen receptors (CARs) specificity. In this context, our group recently developed two second-generation peptide-based CARs (pepti-CARs): a monospecific CAR (MonoCAR) targeting αvβ6 integrin, and a bispecific CAR (Bi-CAR) targeting both αVβ6 and αVβ8 integrins, tumor-associated antigens overexpressed by multiple solid tumors and upregulated within the tumor microenvironment. This study aimed to define the specificity, avidity and relative contribution of integrins-targeting to pepti-CAR function, and to develop a novel anti-αvβ6/αvβ8 peptide-based chimeric costimulatory receptor (pepti-CCR). Functional responses were assessed using Incucyte live-cell imaging, Z-movi avidity analyses and multiplex cytokine profiling. Antitumor activity was evaluated in vivo using subcutaneous PDAC and prostate models. Bispecific pepti-CAR Ts demonstrated enhanced functional responses and higher avidity than monospecific CAR-Ts on PDAC cells. The relative contribution of each integrin to CAR-Ts activity was investigated through CRISPR-Cas9 mediated knockout of αVβ6 and/or αVβ8 in PDAC cells. Data indicate that αVβ6 is the main driver of antitumor activity; however, only dual integrin targeting achieved complete tumor eradication. Furthermore, co-expression of the bispecific anti-αvβ6/αvβ8 pepti-CCR with an anti-PSMA CAR enhanced T-cell antitumor activity and pro-inflammatory cytokine release in vitro and in vivo in a subcutaneous prostate model. Ongoing studies are extending these findings to PDAC models in combination with benchmark anti-CEA CAR T cells. In summary, we provided mechanistic insight into peptide-based CAR function and introduced a novel peptide-based CCR platform. This strategy holds broad therapeutic potential to enhance CAR-Ts efficacy against solid tumors.

PV081 / #107

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USE OF TIM-3-BASED IMMUNOSWITCH RECEPTORS TO IMPROVE T CELL THERAPY IN CHRONIC HBV INFECTIONS AND HBV-DRIVEN HEPATOCELLULAR CARCINOMA

Sophie Schmitt¹, Luis Olguin-Contreras¹, Jana Schmitt¹, Stephanie Färber¹, Susanne Wilde², Karin Wisskirchen², Elfriede Nößner³, Ulrike Protzer^1,4

¹TUM, Institute Of Virology, Munich, Germany, ²SCG Cell Therapy GmbH, Planegg, Germany, ³Helmholtz Center Munich, Tissue Control Of Immunocytes, Neuherberg, Germany, ⁴Helmholtz Center Munich, Institute Of Virology, Neuherberg, Germany

AS: Academic Abstract Body: Around 254 million people worldwide live with chronic Hepatitis B virus (cHBV) infection. Because infection outcomes are largely determined by T cell responses, TCR-engineered T cell therapy represents a promising strategy for treatment and cure. However, in cHBV- and HBV-driven hepatocellular carcinoma (HCC), the liver's immunosuppressive microenvironment impairs T cell function via inhibitory receptors such as TIM-3, PD-1, and TIGIT. Using a synthetic biology approach, we developed TIM-3–based immunoswitch receptors that convert T-cell inhibitory signals into co-stimulation. These chimeric receptors consist of the TIM-3 extracellular domain fused to intracellular domains of CD28, 4-1BB, or OX40. Immunoswitches were then co-expressed with a low-avidity HBV-specific TCR (WL12) in primary human PBMCs. Functional activity was assessed in co-cultures using hepatocellular carcinoma cell lines expressing low (HepG2.2.15) or high (HepG2-SML) HBV antigen levels and CEACAM1, a membrane-bound TIM-3 ligand. T cell function was evaluated by IFN-γ secretion (ELISA) and cytotoxicity (xCelligence real-time analysis). Initial structural comparisons of TIM-3_CD28 constructs differing in their transmembrane domains (TMDs) demonstrated superior performance when the CD28 TMD was incorporated, highlighting a critical functional role for this region. Subsequent TIM-3_4-1BB and TIM-3_OX40 designs, which incorporated each corresponding co-stimulatory TMDs, consistently enhanced IFN-γ secretion and cytotoxicity. Furthermore, signaling assays using Jurkat 76 triple-parameter reporter (TPR) cells revealed stronger NF-κB activation induced by TIM-3_4-1BB and TIM-3_OX40 than by TIM-3_CD28, suggesting signaling advantages that promote T-cell persistence and memory development. Overall, structurally optimized TIM-3 immunoswitches enhance HBV-specific T cell functionality and represent a promising strategy to overcome checkpoint-mediated dysfunction in cHBV and HBV-associated HCC.

PV082 / #113

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ENGINEERING TIGIT-BASED SWITCH RECEPTORS TO ENHANCE T CELL FUNCTIONALITY IN CHRONIC HEPATITIS B VIRUS INFECTION AND HBV-DRIVEN HEPATOCELLULAR CARCINOMA

Jana Schmitt¹, Luis Olguin-Contreras¹, Sophie Schmitt¹, Stephanie Färber¹, Susanne Wilde², Karin Wisskirchen², Elfriede Nößner³, Ulrike Protzer^1,4

¹Technical University of Munich, Institute Of Virology, Munich, Germany, ²SCG Cell Therapy GmbH, Planegg, Germany, ³Helmholtz Center Munich, Tissue Control Of Immunocytes, Neuherberg, Germany, ⁴Helmholtz Center Munich, Institute Of Virology, Neuherberg, Germany

AS: Academic Abstract Body: Chronic hepatitis B virus infection (cHBV) accounts for nearly half of all hepatocellular carcinoma (HCC) cases worldwide. Persistent infection is characterized by dysfunctional CD8⁺ T cells with reduced antiviral efficacy and upregulation of inhibitory receptors, including PD-1, CTLA-4, Tim-3, and TIGIT. TIGIT binds to CD155 and CD112, which are expressed in the liver during cHBV and HCC (including Kupffer cells and liver sinusoidal endothelial cells), delivering inhibitory signals to CD8⁺ T cells and contributing to immune dysfunction in the tolerogenic hepatic environment. While TCR- and CAR-engineered T cell therapies are under development for cHBV and HCC, their efficacy remains compromised by the immunosuppressive liver microenvironment. To address this, we engineered synthetic immunoswitch receptors that convert inhibitory TIGIT signals into co-stimulation by fusing TIGIT's extracellular domain to the intracellular domains of CD28, 4-1BB, or OX40. Primary human CD8⁺ T cells were transduced with TIGIT-CD28, TIGIT-OX40, or TIGIT-4-1BB immunoswitches, alone or combined with low- or high-avidity HBV-specific TCRs (4G and WL12) or the second-generation HBsAg-targeting S-CAR. Functional assays against HepG2 hepatoma cells expressing low or high HBsAg levels showed significantly enhanced IFN-γ secretion, cytotoxicity, and proliferation compared to controls. TIGIT-CD28 provided the strongest enhancement, followed by TIGIT-OX40 and TIGIT-4-1BB. Importantly, immunoswitch co-expression lowered the activation threshold of the low-avidity WL12 TCR, improving sensitivity in suboptimal antigenic contexts. These results support the use of TIGIT-based immunoswitch receptors as a promising modular strategy to overcome liver-specific immunosuppression and enhance T cell-based immunotherapy for cHBV and HBV-driven HCC.

PV083 / #136

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B7H3–TARGETED CAR T CELLS INCORPORATING INTRACELLULAR INTERLEUKIN-7 RECEPTOR ALPHA SIGNALING FOR CHOLANGIOCARCINOMA

Supannikar Tawinwung¹, Atittaya Wakwatanyoo¹, Koramit Suppipat²

¹Chulalongkorn University, Faculty of Pharmaceutical Sciences, Pharmacology And Physiology, Bangkok, Thailand, ²Chulalongkorn University, Center Of Excellence For Cellular Immunotherapy, Bangkok, Thailand

AS: Academic Abstract Body: Cholangiocarcinoma (CCA) is an aggressive malignancy with limited therapeutic response to current treatments. Given the challenges of CAR T-cell persistence and functional durability in solid tumors, we extended our previously developed B7-H3–IL7 receptor alpha CAR T platform to evaluate its potential in CCA and to define the optimal co-stimulatory options to combined with IL7Rα for anti-tumor function in CCA. B7-H3–specific CAR constructs incorporating intracellular IL-7Rα signaling and CD3ζ were generated with distinct co-stimulatory domains (CD28, CD27, or 4-1BB). CAR T cells were produced by gammaretroviral transduction and characterized for transduction efficiency, expansion kinetics, and memory phenotype. Anti-tumor activity was evaluated using in vitro co-culture assays against HuCCT1 and KKU055 CCA cell lines. All constructs demonstrated comparable expansion and preservation of memory-associated phenotypes, although CD28-IL7Rα CAR T cells showed lower transduction efficiency. CD27-IL7Rα CAR T cells exhibited enhanced degranulation, reflected by increased CD107a expression compared with 4-1BB–based constructs in HuCCT1 co-cultures. Under repeated tumor rechallenge assay, CD28-IL7Rα CAR T cells demonstrated superior proliferative capacity and sustained expansion despite lower initial cytotoxicity. These findings indicate that incorporate IL-7Rα signaling in the B7H3-CAR provide anti-tumor eficacy against CCA with CD28-IL7Rα CAR T cells exhibit higher proliferative capacity.

PV084 / #261

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A HIGH-THROUGHPUT FUNCTIONAL SCREENING PLATFORM FOR DE NOVO BINDERS AGAINST BCMA SURFACE VARIANTS

Silvia Toscani¹, Leonardo Dozal Blanco², Daniela Abele¹, Noah Holzleitner³, Gabriele Pelosi¹, Julian Grünewald³, Andrea Schmidts¹

¹Technical University Munich, TUM, Department Of Medicine Iii, Hematology And Oncology & Translatum, Munich, Germany, ²Ludwig Maximilian University, Faculty Of Biology, Munich, Germany, ³Technical University Munich, TUM, Department Of Medicine I: Cardiology & Translatum, Klinikum Rechts Der Isar, Munich, Germany

LB: Academic Abstract Body: De novo protein design is an emerging approach for CAR T therapy, enabling rapid development of small, stable binding domains via tools like RFdiffusion and BindCraft. While the dissociation constant (Kd) remains a common metric for affinity measurements, it requires specialized equipment and fails to capture real-time functional activation or the impact of tumor escape mutations. To address this, we are developing a versatile high-throughput functional screening platform using engineered Jurkat reporter cells. This integrated system utilizes a barcoded library and a triple-reporter readout (NFAT, NF-κB, AP-1) alongside CD69 upregulation to provide a multi-parametric, cell-based measure of binder-receptor interaction. We are currently validating this platform by screening 32 de novo binders against a panel of K562 cells expressing 20 distinct BCMA surface mutations, including the clinically relevant R27P variant. Using enrichment analysis of sorted activated cells, we aim to map the functional landscape of these binders across this mutational library. Preliminary results demonstrate the potency of this platform, with lead candidates achieving activation levels comparable to benchmark scFv-based CAR T cells in both MM1.s and ectopic BCMA-expressing K562 co-cultures. Notably, our lead candidate, BC44, exhibited highly specific activation, characterized by an 8-fold increase in CD69 surface expression and a dramatic shift in GFP reporter positivity from <1% to >75% upon WT target encounter. This scalable, cell-based platform bypasses the constraints of cell-free affinity measurements to identify escape-resistant candidates. By directly linking antigen escape profiling to binder redesign, this platform establishes an iterative pipeline toward increasingly resilient next-generation CAR T therapies.

PV085 / #145

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BURNOUT PREVENTION FOR CAR T CELLS: HOW DEGRADATION-CONTROLLED CAR RESTING SUSTAINS T-CELL FUNCTION

Ronja Van Berkum¹, Rajeev Rai², Jan Molenaar³, Max Jan⁴, John Anderson², Judith Wienke³

¹Prinses Maxima Centrum, Utrecht, Netherlands, ²Great Ormond Street Hospital and Institute of Child Health, London, United Kingdom, ³Princess Máxima Center, Utrecht, Netherlands, ⁴Harvard Medical School, Boston, United States of America

AS: Academic Abstract Body: Achieving durable responses with chimeric antigen receptor (CAR) T-cell therapies in paediatric solid tumours like neuroblastoma remains challenging, an occurrence frequently attributed to CAR T-cell exhaustion. Immunomodulatory imide drugs (IMiDs) can function as molecular glues inducing protein degradation. We engineered anti-B7H3 CAR T-cells containing a degron enabling reversible, IMiD-mediated CAR depletion, thereby transiently switching CAR signalling off. We tested whether periodic, controlled functional rest mitigates exhaustion and enhances long-term CAR T-cell efficacy, to define optimal rest intervals to support clinical translation. CAR T-cells were challenged in 4-week co-cultures with neuroblastoma cells with bi-daily tumour rechallenges to drive exhaustion. Multiple IMiD-induced ON/OFF intervals were tested. IMiD treatment induced rapid, reversible CAR protein degradation, resulting in markedly reduced cytotoxicity during OFF phases. Extended rest intervals significantly improved exhaustion profiles, with reduced PD-1, TIM-3, LAG-3 and CD39 expression compared the those longer ON. Notably, the 7 × 7 interval, but not shorter rest schedules, resulted in enhanced cytotoxicity compared to constitutively active CAR T-cells, across 4 weeks of tumour rechallenge. These benefits were not observed in IMiD-treated CAR T-cells lacking the degron tag, indicating a rest-dependent mechanism of action. 7 × 7-rested CAR T-cells exhibited significantly increased intracellular IL-2, TNF-α and IFN-γ expression to continuously active CAR T-cells. These data identify a defined, reversible CAR resting schedule (7 days ON, 7 days OFF) that preserves CAR T-cell functionality, limits exhaustion and enhances sustained cytotoxicity under chronic antigen pressure. This inducible resting strategy provides a tuneable approach to improving CAR T-cell durability and functional persistence in solid tumours.

PV086 / #47

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GENERATION OF “OFF-THE-SHELF “CAR-T CELL THERAPY TO DEPLETE PATHOGENEIC T CELL SUBFRACTIONS IN TYPE 1 DIABETES (T1D)

Anna Nimke¹, Abdelsalam Amer¹, Karl Moukarzel^1,2, Nadja Nehls^1,2,3, Matthias Warkotsch^1,2, Mateusz Poltorak¹, Dirk Busch^1,2,4

¹Technical University of Munich (TUM), TUM School of Medicine and Health, Munich, Germany, Institute Of Microbiology, Immunology And Hygiene, Munich, Germany, ²DZKJ, German Center for Child and Adolescent Health, Munich, Germany, ³TUM-Klinikum rechts der Isar of the Technical University of Munich, Germany, Center For Pediatric And Adolescent Medicine, Munich, Germany, ⁴German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany

AS: Academic Abstract Body: CAR-T cell therapies have been successful in malignant diseases and recently demonstrated efficacy in B cell–mediated autoimmune diseases such as SLE. However, extending depleting CAR-T cell therapies to T cell–mediated autoimmune-diseases like T1D remains challenging, as pan–T cell depletion would cause unacceptable toxicity. Therefore, a more feasible strategy is the selective depletion of disease-driving T cell subfractions. To this end, we are exploring the targeting of subfraction-defining TCR germline components such as TRBC1/2 or TCR-V-segments. Additionally, all currently approved CAR-T products are autologous, a process that is slow, expensive and carries the risk of reinfusing pathogenic T cells. An allogeneic “off-the-shelf” approach could overcome these limitations. As a first step, we generated an anti-human TRBC2-CAR. For specificity validation, TCR-knockout T cells were transduced and co-cultured with TRBC1/TRBC2 T cells. Anti-TRBC2 CARs demonstrated selective killing of target cells, while sparing TRBC1-T cells, providing proof-of-concept for targeted subset depletion. We are currently expanding this repertoire to additional human and mouse T cell subfraction-depleting CARs. For allogeneic donor cells, we follow a recently by our group developed innovative HLA-reduction approach retaining HLA-A and non-classical MHC-I expression to preserve adaptive immune surveillance and regulate NK cell recognition, respectively. Using base editing, we generated multiplex-edited T cells lacking HLA-B, HLA-C, MHC-II, and TCR. High editing efficiencies were confirmed by next generation sequencing. Unlike β2m-knockout controls, this quadruple-edited product resisted NK-cell–mediated killing. By pre-screening healthy donors for the most frequent HLA-A haplotypes, this strategy could support a safe and broadly applicable allogeneic “off-the-shelf” treatment option.

PV087 / #105

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IDENTIFICATION AND CHARACTERIZATION OF TYPE 1 DIABETES DRIVING T CELL RECEPTORS AS POTENTIAL TARGETS FOR CAR-T CELL THERAPY

Tom Nolte¹, Karl Moukarzel^1,2, Martina Ciccimarra^1,3, Abdelsalam Amer¹, Nadja Nehls^1,2,4, Anna Nimke¹, Matthias Warkotsch^1,2, Mateusz Poltorak¹, Dirk Busch^1,2

¹Technical University of Munich (TUM), TUM School of Medicine, Institute For Medical Microbiology, Immunology And Hygiene, Munich, Germany, ²DZKJ, German Center for Child and Adolescent Health, Munich, Germany, ³DKFZ, German Cancer Research Center, Heidelberg, Germany, ⁴TUM-Klinikum rechts der Isar of the Technical University of Munich, Germany, Center For Pediatric And Adolescent Medicine, Munich, Germany

AS: Academic Abstract Body: Type 1 diabetes (T1D) is a common autoimmune disease in which autoreactive T cells infiltrate and destroy insulin-producing β cells. Despite insulin-replacement therapy, patients still develop long-term complications. Newer immunomodulatory therapies, such as monoclonal antibodies, have failed to prevent or significantly delay the onset of T1D in high-risk individuals, partly due to insufficient targeting of disease driving T cells. Developing a CAR-T cell product to deplete T cell subfractions containing pathogenic clones, while preserving the rest of the T cell compartment, presents a novel therapeutic approach. To this aim, we sought to characterize the TCR repertoire of non-obese diabetic (NOD) mice against two peptides involved in the autoimmune response, insulin and IGRP. We obtained TCR sequences from diabetic NOD mice by sc-TCR sequencing and single cell cloning. Both IGRP- and insulin-specific T cell responses were highly oligoclonal with the insulin responses consisting largely of a single clone in the repertoire. Interestingly, IGRP-specific TCRs showed a highly conserved usage of the alpha chain featuring the Va16N and Ja42 segments and were largely TRBC2+, offering a promising target. In vitro, IGRP-specific TCRs showed high functional reactivity, as opposed to the weaker response of insulin-specific TCRs, which show a clear negative selection signature. Additionally, we show for IGRP-TCRs a gradient of affinity from the pancreatic lymph nodes to the pancreas, with only the high affinity TCRs able to infiltrate the pancreas. Overall autoreactive TCRs in T1D appear to exhibit restricted clonal diversity, suggesting that selective, deletion-based targeting may be feasible.

PV088 / #40

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ANTIGEN-SPECIFIC MODULATION OF AUTOIMMUNE DISEASES BY TCR-LIKE ANTIBODIES TARGETING AUTOREACTIVE T-CELL EPITOPES

Yoram Reiter, Alona Goor, Maya Cohen

Technion-Israel Institute of Technology, Haifa, Israel

AS: Academic Abstract Body: The development and application of human TCR-like (TCRL) antibodies recognizing disease-specific MHC-peptide complexes may prove as an important tool for basic research and therapeutic applications. Multiple sclerosis is characterized by aberrant CD4 T-cell response to self-antigens presented by MHC class II molecules. This led us to select a panel of TCRL Abs targeting the immunodominant autoantigenic epitope MOG_35-55 derived from myelin oligodendrocyte glycoprotein (MOG) presented on HLA-DR2, which is associated with multiple sclerosis (MS). We demonstrate that these TCRL Abs bind with high specificity to human HLA-DR2/MOG_35-55-derived MHC class II molecules and can detect APCs that naturally present the MS-associated autoantigen in the humanized EAE transgenic mouse model. The TCRL Abs can block ex vivo and in vivo CD4 T-cell proliferation in response to MOG_35-55 stimulation in an antigen-specific manner. Most significantly, administration of TCRL Abs to MOG_35-55-induced EAE model in HLA-DR2 transgenic mice both prevents and regresses established EAE. TCRL function was associated with a reduction in autoreactive pathogenic T-cell infiltration into the CNS, along with modulation of activated CD11b+ macrophages/microglial APCs. Collectively, these findings demonstrate the combined action of TCRL Abs in blocking TCR-MHC interactions and modulating APC presentation and activation, leading to a profound antigen-specific inhibitory effect on the neuroinflammatory process, resulting in regression of EAE. Our study constitutes an in vivo proof of concept for the utility of TCR-like antibodies as antigen-specific immunomodulators for CD4-mediated autoimmune diseases such as MS, validating the importance of the TCR-MHC axis as a therapeutic target for various autoimmune and inflammatory diseases.