Huynh, Truc

Abstract Introduction Checkpoint blockade revolutionized cancer therapy. However, only a small proportion of cancers respond to checkpoint blockade. B7-H3 is a member of the B7 superfamily, which includes both co-inhibitory and co-stimulatory receptors. B7-H3 is upregulated by many tumors which is generally associated with a worse prognosis. However, the exact mechanism by which B7-H3 suppresses the immune response remains elusive. Methods We transfected human HEK293T cells and assessed the binding to recombinant Siglecs via flow cytometry. We generated monoclonal antibody F7 against human B7-H3 and tested its blocking function using B7-H3 transfected HEK293T cells. We examined the extent of degranulation and NK cell-mediated killing on B7-H3 expressing HEK293T cells in vitro, as well as the CART19 cells mediated killing in relapse model of B7-H3 expressing CD19+ leukemia Nalm6 cells NSG mice. Results We demonstrated that sialylated B7-H3 binds to the inhibitory receptor Siglec-9 on immune cells but not to the related protein Siglec-7. A high affinity monoclonal antibody (mAb) F7 against B7-H3 blocks Siglec-9 binding. Functionally, F7 enhances NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. Conclusion We have now shown for the first time that B7-H3 binds to the inhibitory receptor Siglec-9 and generated a novel monoclonal antibody F7 that target a unique sequence in B7-H3 as compared to other monoclonal antibodies being examined for clinical efficacy. F7 can promote NK cell killing of B7-H3 expressing target cells in vitro and synergize with CART19 to enhance killing of B7-H3 expressing tumor cells in vivo. As B7-H3 is overexpressed in many human cancers, this work is critical toward understanding how B7-H3 mediates immune evasion through Siglec-9 for enhancing anti-tumor immunity. Funding Source R01CA243545-01A1 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Gastrointestinal (GI) cancers are rising in people < 50 and remain major causes of cancer mortality despite therapeutic advances. Chimeric antigen receptor T (CART) cell therapy has transformed treatment of hematological cancers. However, its efficacy in GI cancers has been hindered by antigen heterogeneity and immunosuppressive cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME.) Here we aimed to 1) study CART-CAF interactions and 2) engineer CART cells capable of eliminating CAFs to overcome their inhibitory role and enhance their antitumor activity in GI cancers. We first analyzed primary CAFs from 20 GI cancer patients. CAFs showed high fibroblast activation protein (FAP) expression and significantly suppressed T cells proliferation (26-77%, p≤0.0001). We next generated CART cells capable of safely eliminating both GI cancer cells and CAFs. To target cancer cells, we developed a novel nanobody (VHH)-based CAR against Claudin18.2, a GI cancer specific target. To avoid off-tumor toxicity in healthy FAP+ tissues, we incorporated an inducible FAP-CAR using the synNotch platform, enabling conditional targeting upon engagement with Claudin18.2+ cells, restricting FAP targeting within the TME. VHH Claudin18.2 CART cells showed superior specificity and efficacy vs scFv-based CART cells (hu8E85 or zolbetuximab). In Claudin18.2+ SNU-C2B xenografts, tumors were 92mm3 (VHH) vs 48mm3 (huE85) vs 211mm3 (zolbetuximab) on day 23 post-CART, median survival undefined vs 17 days vs 23 days respectively; p=0005. In Claudin18.2+ LS411N xenografts, tumors were 315mm3 vs 106mm3 vs 382 mm3 respectively at 23 days post-CART, median survival undefined vs 19 days vs undefined respectively; p=0004. VHH Claudin18.2 CART cells showed no off-tumor effects, while hu8E85 CART induced significant toxicity. We next established a co-engraftment model. NSG mice received 1x106 SNU-C2B and 6x106 CAFs from a GI cancer patient. At 500 mm3 tumors, mice received 5x106 VHH Claudin18.2 or inducible Claudin18.2/FAP CART. Dual-targeting CART cells showed greater antitumor response (728mm3 vs 320mm3, p=0.0011 at day 20 post-CART), expansion (61 vs 2,350 CART/µl of blood, p=0.0037), and tumor infiltration (8.71 x 105 vs 1.03 x 106 CART/g of tumor, p<0.0001). This was also observed in gastric, colon, and pancreatic PDX models, with no observable off-tumor cytotoxic effects with inducible dual-targeting Claudin18.2/FAP CART. Our findings demonstrate that our novel VHH-based Claudin18.2 CART cells are highly specific and effective against GI malignancies, and that inducible Claudin18.2/FAP dual-targeting CART cells safely remodel the TME into an immune-permissive state. This approach represents a mechanistically innovative and translationally relevant strategy to overcome CAF-mediated immunosuppression, enhance CART cell efficacy, and improve clinical outcomes in refractory solid tumors. Citation Format: Omar L. Gutierrez Ruiz, Jennifer Feigin, Brooke Kimball, R. Leo Sakemura, Elizabeth L. Siegler, Dominic A. Skeele, Ateka Saleh, Claudia Manriquez Roman, Ismail Can, Olivia Sirpilla, Kun Yun, Carli M. Stewart, Mehrdad Hefazi, Yoh Yamaguchi, Lionel Aurelien Kankeu Fonkoua, Long K. Mai, Truc Huynh, Sophia Y. Goldberg, Grace E. DeFranco, Sara Foote, Ekene Johnkennedy Newton Ogbodo, Hong Xia, Michael Redig, Matthew L. Matthew, Tamiel Nichole Turley, Skyeler Klinge, John Alton Copland, Saad J. Kenderian. Optimized CART cell therapy to remodel the tumor microenvironment and eliminate GI malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1525.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.

Abstract Background: Integrin αE (CD103) has been thought to mediate gut-homing Treg retention during GVHD by facilitating E-cadherin interaction on intestinal epithelial cells. However, only a small percentage of natural Tregs express CD103 in peripheral blood (PB). To leverage this mechanism and potentially enhance Treg therapeutic efficacy, we hypothesized that Tregs overexpressing CD103 (CD103high) would show improved gut-homing and GVHD mitigation. Methods: Murine CD4 Tregs were isolated from C57BL/6 (B6) mice via flow sorting and were retrovirally transduced with murine CD103 or truncated epidermal growth factor receptor as a control. Transduced Tregs were then tested in a murine acute GVHD model (B6 → BALB/c). Mouse organs, including small intestine (SI), colon, spleen, PB, axillary (aLN) and mesentric lymph nodes (mLN) were harvested on day 10 post-transplant and analyzed by flow cytometry, multiplex cytokine assay, histopathology, and single-cell RNA sequencing (scRNA-seq). Ex vivo expanded Tregs were subjected to 13C-glucose tracing assay to evaluate glycolytic flux, TCA cycle, and lipid biosynthesis. Results: Despite comparable FoxP3 expression at the end of ex vivo expansion, CD103high Tregs had significantly higher in vitro suppression compared to control Tregs (54% vs. 33% at Treg:Tcon ratio of 1:8, p=0.004). Upon re-stimulation, CD103high Tregs demonstrated increased proliferation compared to control Tregs (3.8 vs 2.8 fold expansion, p <0.0001). Bulk RNA seq on ex vivo expanded CD103high showed upregulation of immuneregulatory and metabolic programs including GTPase signaling and oxidative stress control. These transcriptomic changes suggest that CD103 intrinsically promotes a regulatory phenotype, stress tolerance and metabolic fitness. In vivo, CD103highTregs significantly decreased the severity and mortality of GVHD, compared to control Tregs, as measured by clinical scores, weight loss, and survival (50% vs 0% on day +60, p<0.0001 for three independent experiments combined; n = 24/group). Day 20 histopathology revealed significantly improved colitis scores in mice treated with CD103high Tregs vs controls (9.5 vs. 14.6, p=0.02). While these findings could be partially attributed to CD103 gut-homing function, we observed that CD103high Treg absolute number and frequency were higher not only in the the GI organs and mLN, but also in the spleen, PB, and aLN with similar distribution ratios across different organs (~5-fold higher). In vivo CFSE suppression assay, where CFSE labeled donor T cells were cotransferred with Tregs into B6 Rag1ko mice, demonstrated enhanced suppressive function of CD103high Tregs in the spleen (p=0.02), an effect that could not be accounted for by the conventional gut-homing mechanism attributed to CD103. Plasma cytokine analysis showed significantly elevated IL-10 levels in mice treated with CD103high Tregs (p<0.0001), consistent with increased Treg number and frequency. 13C-glucose tracing assay showed lower palmitate levels in CD103high Tregs, indicating lower de novo lipid synthesis, while glycolysis and TCA cycle tracing were similar. Day 10 scRNA-seq on colon samples revealed seven distinct immune clusters. The neutrophil cluster was absent in CD103high Treg group, suggesting reduced inflammation. Donor-derived dendritic cells (DCs) upregulated genes such as Cstb, and Slamf7, while pro-inflammatory genes including Cd52 and Cd74 were downregulated. STAT5 signaling was upregulated, indicating reprograming of myeloid cells toward an immunoregulatory state. Colon epithelial cells from CD103high Treg-treated mice displayed enhanced stress tolerance, with increased oxidative phosphorylation, fatty acid metabolism, and epithelial repair pathways. CellChat analysis revealed that CD103high Tregs reshaped the intestinal communication landscape, with DCs acting as key signaling hubs through interactions such as Ccl6–Ccr2 and Lgals3–Cd44. Enhanced DC-to-DC and DC-to-T cell communication suggest that CD103high Tregs promote a tolerogenic DC network that modulates downstream immune responses and supports epithelial recovery. Conclusion: Tregs overexpressing CD103 via retroviral transduction significantly improve overall survival and GVHD clinical scores in a murine transplant model. CD103High Tregs orchestrate broad remodeling of cell-cell communication across immune and stromal compartments, promoting an immunoregulatory microenvironment that underlies their protective effects in GVHD.

Abstract Description B7-H3 is a member of the B7 superfamily, which includes co-inhibitory receptors such as B7-H1/PD-L1 and co-stimulatory receptors such as B7-1/CD80 and B7-2/CD86. B7-H3 is upregulated by almost all types of tumors and is associated with worse prognosis. However, the exact mechanism by which B7-H3 suppresses anti-tumor immune response remains to be discovered as its ligand has yet to be identified. Sialic acid modifications are also increased in many cancers and are similarly associated with poor outcome. Here, we demonstrate for the first time that sialylated B7-H3 binds to the immune inhibitory receptor Siglec-9. Siglec-9 is expressed on many immune cells, including NK cells and T cells. We have shown that B7-H3 expression on target cells reduces killing by Siglec-9 expressing NK cells in vitro. We then hypothesized that antibodies that block B7-H3 binding to Siglec-9 will be an effective intervention to boost anti-tumor immunity. We have generated a high affinity anti-B7-H3 monoclonal antibody, F7, which blocks B7-H3 binding to Siglec-9. F7 functions to enhance NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. In summary, our work has demonstrated that B7-H3 binds Siglec-9, and blockade of such interaction can enhance anti-tumor immunity. Funding Sources • Mayo Clinic Center for Biomedical Discovery • NIH Glycobiology Common Fund supplement to R01CA243545-01A1 to VSS Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Chimeric antigen receptor T (CART) cell therapy has shown clinical successes in B cell hematological malignancies. Two B cell maturation antigen (BCMA)-directed CART cell therapies treating multiple myeloma (MM) were approved by FDA but most patients relapse within 1-2 years. Ex vivo-expanded antigen specific T cells as cancer treatment rely on TCR-mediated killing of tumors. We hypothesized that arming ex vivo enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions. First, we expanded mucin 1 (MUC1)-activated T cells from patients with MM through ex vivo stimulation by MUC1 peptides-loaded autologous PBMCs for 19 days. Then, we engineered MUC1-activated T cells with 4-1BB-co-stimulated BCMA CAR to generate MUC1 TCR+BCMA CAR+ T cells. Baseline T cells without MUC1 stimulation were engineered with BCMA CAR as control. Baseline T cells, MUC1 T cells, baseline BCMA CART cells, or MUC1 BCMA CART cells were cocultured with luc+ RPMI-8226, a MUC1+BCMA+ MM cell line, for 3 days. MUC1 BCMA CART cells showed stronger tumor killing than BCMA CART or MUC1 T cells in all 3 patients (p<0.0001). MUC1 BCMA CART showed improved proliferation than BCMA CART or MUC1 T cells (p=0.0765, p<0.0001, respectively) after a 5-day stimulation by RPMI-8226. To exclude possibility of metabolic alterations by 19-day PBMC incubation leading to better T cell functions in MUC1 BCMA CART, baseline and MUC1-stimulated T cells from 3 healthy donors were processed for the same assays. Day-19 BCMA CART cells showed inferior tumor killing compared to baseline BCMA CART in all 3 donors (p<0.0001). Lower T cell proliferation was also observed in day-19 BCMA CART (p=0.0838). Our data indicate that improved T cell functions in MUC1 BCMA CART were unlikely due to metabolic changes and required stimulation by tumor antigens prior to CAR engineering likely through MUC1 TCR. Additionally, MUC1 T cells, BCMA CART, or MUC1 BCMA CART cells from tested patients were stimulated by RPMI-8226 for bulk RNA sequencing. Both BCMA CART and MUC1 T cells had distinct transcriptomic features from MUC1 BCMA CART cells. Differential gene expression analysis showed upregulation of T cell function-associated genes (RYR2, ITGA1, GNLY, GZMA, etc.) in MUC1 BCMA CART vs BCMA CART. Enrichment of T cell function-associated pathways was also observed in MUC1 BCMA CART. Analysis between MUC1 T cell and MUC1 BCMA CART showed upregulation of T cell function-associated genes (CSF2, CRTAM, IFNG, GZMB, etc.) and enrichment of immune response pathways in MUC1 BCMA CART. Our findings suggest that equipping ex vivo-enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions in vitro. Importantly, this provides a rationale for a therapeutic regimen where patients receive ex vivo-enriched tumor-induced T cells followed by CAR engineering. Persistence of the engineered T cells is being investigated Citation Format: Kun Yun, R. Leo Sakemura, Ismail Can, Omar Gutierrez Ruiz, Ekene J. Ogbodo, Truc N. Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli M. Stewart, Jennifer M. Feigin, Grace E. DeFranco, Sophia Y. Goldberg, Dominic A. Skeele, Mariam Stein, Dara Missan, Latha Pathangey, Long Mai, Hong Xia, Brooke Kimball, Lionel A. Kankeu Fonkoua, Mehrdad Hefazi, Michael W. Ruff, Elizabeth L. Siegler, Leif Bergsagel, Michael Gustafson, Sandra Gendler, Saad S. Kenderian. BCMA CAR engineering arms T cells with superior functions post ex vivo stimulation by MUC1 peptide-loaded autologous PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6103.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, CAR T-cell therapy still has low durable response rates and common associated toxicities such as cytokine release syndrome (CRS). We recently reported that sequential therapy with the CD19 monoclonal antibody tafasitamab and CD19-directed CAR T (CART19, 41BBζ costimulated) cells significantly ameliorated CAR T-cell apoptosis and tumor cell pyroptosis, improving CAR T safety and efficacy (PMID: 37879074). Building on these findings, we hypothesized that controlling massive initial CAR T-cell stimulation by diminishing extensive tumor cell pyroptosis is critical to improve CAR T safety and durability. To test this hypothesis, we evaluated several caspase (Casp) inhibitors, including Casp1, Casp3, Casp7, and Casp11, in combination with CAR T cells. We found that the Casp1 inhibitor VX765 did not inhibit CAR T antigen-specific cytotoxicity and thus focused on VX765 in subsequent assays. We first assessed CAR T proliferation upon stimulation with the CD19+ JeKo-1 mantle cell lymphoma cell line in the presence of VX765. CAR T with vehicle control showed greater proliferation at day 3, but CAR T with VX765 showed higher proliferation at day 5 (p=0.03, absolute CD3+ counts by flow cytometry). We then co-cultured CAR T and JeKo-1 for 24 hours and observed a significant reduction in apoptotic CAR T (Annexin V+ and 7-AAD- via flow cytometry) in VX765-treated conditions (p=0.05). Additionally, there was significantly reduced pyroptosis (Annexin V+ 7-AAD+) (p=0.02) but higher apoptosis (Annexin V+ 7-AAD-) in JeKo-1 in VX765-treated conditions (p=0.04). VX765-treated CAR T showed higher Bcl2 expression via flow cytometry (p=0.03). We also studied tumor cell pyroptosis by measuring the level of HMGB-1. CART19, JeKo-1, and VX765 or vehicle control were co-cultured for 24 hours, and the level of HMGB-1 was found to be significantly reduced in VX765-treated conditions, indicating reduced tumor pyroptosis (p=0.006). We then assessed the impact of Casp1 inhibition in our in vitro model of CAR T exhaustion. We stimulated CAR T with CD19+ JeKo-1 every other day for 2 weeks and then assessed CCR7 and CD45RA expression on CAR T. VX765-treated CAR T showed higher central memory (p=0.08) and naïve (p=0.02) and lower terminally differentiated effector memory phenotypes (p=0.03). We next studied the effect of Casp1 inhibition with VX765 in vivo. To recapitulate CAR T-cell toxicities, including CRS, we utilized an immunocompetent mouse model. First, we generated murine CART19 (mCART19, CD28ζ costimulated) cells. Recipient mice were then engrafted with luciferase+ murine CD19+ TBL12 cells, a murine lymphoma cell line. Mice were treated 12 days later with cyclophosphamide (300 mg/kg, ip) and 24 hours after mice were imaged and randomized to receive 1 x 107, 1) untransduced T cell control (UTD), 2) mCART19 + vehicle control, or 3) mCART19 + VX765 (25 mg/kg). VX765 was given immediately before the CART19 injection and on day 2 post-CAR T injection. Mice were serially monitored and weighed to assess CAR T-cell toxicity. Tumor burden was assessed via weekly bioluminescent imaging. Mice treated with vehicle control showed a significant reduction in weight compared to the UTD control (p=0.02), while mice treated with VX765 maintained their weight and remained active without signs of CRS, including motor weakness and hunched body. UTD mice reached humane endpoints within 3 weeks after T-cell injection. Three satellite mice from each group were euthanized and spleens were harvested. Flow cytometric analysis showed a significant reduction in apoptotic CAR T (p=) and pyroptotic tumor cells in mice treated with VX765 (p=0.03). Western blot showed lower cleaved GSDME and GSDMD in mice treated with VX765. VX765-treated mice demonstrated prolonged CAR T persistence with durable anti-tumor activity. In summary, Casp1 inhibition with VX765 significantly reduced CAR T apoptosis, improved memory phenotype, and increased delayed CAR T proliferation. VX765 also reduced tumor cell pyroptosis and CRS incidence without impacting CAR T anti-tumor activity in vivo. Given the favorable preclinical outcomes and existing clinical data on VX765, this approach holds substantial translational potential for enhancing the safety and efficacy of CAR T-cell therapy in patients with large B-cell lymphoma.

Brexucabtagene autoleucel (brexu-cel), a CD28-costimulated CD19-targeted chimeric antigen receptor T cell (CART19) product, was FDA-approved in 2020 after demonstrating remarkable efficacy in the pivotal ZUMA-2 trial of patients with mantle cell lymphoma (MCL). However, despite the impressive initial activity of CART19 cell therapy in MCL, most patients relapse within the first 3 years following treatment. Myeloid cells, such as monocytes and macrophages, have been demonstrated to be major inhibitors of other immune cells, including T cells. Growing evidence shows that monocytes and macrophages contribute to CART cell failure, although the exact mechanisms are not fully understood. To better understand the mechanisms contributing to CART treatment resistance in MCL, we first utilized brexu-cel products from patients with MCL on the ZUMA-2 trial to uncover their transcriptomic features by single cell RNA sequencing (scRNAseq) analysis. To understand if myeloid cells had negative impacts on clinical outcomes, we then analyzed autologous baseline peripheral blood mononuclear cell (PBMC) samples by scRNAseq. Our analyses of the brexu-cel samples revealed reduced response to IL-1β and its downstream signaling pathway as well as impaired T cell functions in non-responders. Transcriptomic analyses of the autologous baseline monocyte populations showed downregulation of IL-1β production and its associated pathways as well as upregulation of M2 phenotype-associated genes and IL-1 receptor antagonist (IL-1ra) in non-responder monocytes, implying a potential role of myeloid cells in CART resistance through impairment of IL-1 signaling in brexu-cel products. To validate our findings from the ZUMA-2 samples, we further studied the interactions between macrophages, MCL cells, and CART19 cells in preclinical models. First, CD28-costimulated CART19 cells generated from healthy donors were cocultured with JeKo-1, a CD19+ MCL cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression by M2-like macrophages was contact-independent. Cytokine profile analysis of coculture supernatants showed significant elevation of IL-1ra in M2-like macrophage cocultures (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292). This led us to hypothesize that M2-like macrophages inhibit CART19 by secreting IL-1ra to block IL-1 signaling in CART19. To test this hypothesis, we interrogated the role of IL-1ra in CART19. CART19 + JeKo-1 cocultures were supplemented with IL-1β + IL-1ra or IL-1β + IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 proliferation was improved by IL-1β (vehicle control vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra, p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Next, we assessed the impact of IL-1ra on CART functions in mouse MCL xenografts in the presence of M2-like macrophages. NOD-SCID-γ-/- (NSG) mice were subcutaneously injected with 0.5 million of human macrophages and 1 million of luciferase+ JeKo-1. Mice were then randomized once tumor engraftment was confirmed by bioluminescence imaging (BLI), followed by treatment with 2 million CART19 with 10 mg/kg IL-1ra neuAb or control IgG for 3 weeks. Combination of CART19 cells and IL-1ra neuAb led to improved antitumor activity based on BLI measurements compared to control IgG (IL-1ra neuAb vs control IgG, p=0.0058). Additionally, expression of IL-1RI, receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which might potentially desensitize CART response to IL-1β, resulting in further suppression in CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Overall, our study elucidates a potential mechanism of IL-1 pathway modulation mediated by myeloid cells in contributing to CART19 failure in MCL.

CD19-targeted chimeric antigen receptor T (CART19) cell therapy has shown outstanding clinical success. However, patients can develop CART-induced toxicities, including cytokine release syndrome (CRS) and neurotoxicity. Studies have found that activation of myeloid cells by granulocyte monocyte-colony stimulating factor (GM-CSF) can contribute to toxicity development. GM-CSF depletion leads to reduced myeloid activation, prevention of CART-induced toxicities, and improved CART efficacy. CART cells upregulate cytokine receptors upon activation, and our results indicate that CART19 cells significantly upregulate GMCSFR upon antigen-specific stimulation (<10% vs >30%, p<0.01). This led us to hypothesize that GMCSFR signaling contributes to CART cell activity. To understand the role of GM-CSFR upregulation on CART cell functions, we compared the impacts of neutralizing GM-CSF or blocking GM-CSF receptor alpha (GM-CSFRα) on CART19. First, 4-1BB-costimulated CART19 cells generated from healthy donors were stimulated with CD3/CD28 beads at a 1:3 ratio for 6 days. CART cells were maintained at 1 x 106/mL throughout the culture and supplemented with IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade at 10 μg/mL, 20 μg/mL, or 100 μg/mL. On day 6, cells were debeaded and rested for 2 days. Then, CART cells were cocultured with NALM6, a CD19+ acute lymphoblastic leukemia cell line, at a ratio of 1:1 for 3 days. The cocultures were supplemented with the same doses of antibodies listed above. Antigen-specific proliferation of CART cells was measured by flow cytometry. Our data suggested that CART19 proliferated is not impacted by GM-CSF neutralization but is inhibited by GM-CSFRα blockade (IgG isotype vs 10 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 20 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 100 μg/mL GM-CSFRα blockade, p=0.009). CART cells were assessed in a cytotoxicity assay with NALM6 luciferase+ cells as well, which did not show significant impacts from GM-CSF neutralization or GM-CSFRα blockade. To evaluate the impacts of GM-CSF neutralization and GM-CSFRα blockade in a mouse model, we engrafted NSG mice with 1 million NALM6 luciferase+ cells intravenously. Mice were then treated with 1.4 million CART19 plus 10 mg/kg IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade intraperitoneally. GM-CSFRα blockade drastically suppressed antitumor activities of CART (CART19+GM-CSFRα blockade vs CART19+IgG isotype control, p<0.0001; CART19+GM-CSFRα blockade vs CART19+GM-CSF neutralizing antibody, p<0.0001; CART19+ GM-CSFRα blockade vs untransduced T cell, n.s.). Serum cytokines were analyzed 2 days after CART treatment, showing that mice with GM-CSFRα blockade had significantly lower levels of human IFNγ (p=0.0189) and IL-2 (p=0.0121) compared to IgG isotype control group, while GM-CSF neutralization did not show significant impacts. Mice treated with CART plus IgG isotype control or GM-CSF neutralizing antibody showed prolonged survival compared to mice treated with GM-CSFRα blockade or untransduced T cells (p=0.0285). To unravel the mechanisms behind functional differences between GM-CSFRα blockade and GM-CSF neutralization, CART cells (n = 3 biological replicates) were cocultured with NALM6 at a 1:1 ratio supplemented with 10 μg/mL GM-CSF neutralizing antibody, GM-CSFRα blockade, or IgG isotype control overnight, followed by T cell isolation, bulk RNA isolation, and RNA sequencing (RNAseq) analysis. Our RNAseq data indicated significant upregulation (Log2FC>0.5, padj. <0.05) of T cell exhaustion- and dysfunction-associated genes (RGS16, PDCD1, CTLA4, NR4A3, CD101, LAG3) as well as negative regulators of T cell functions (G0S2, PIK3IP1, LGALS3) in the GM-CSFRα blockade group compared to GM-CSF neutralizing antibody or IgG isotype control groups. T cell activation-associated pathways were enriched in the GM-CSFRα blockade group. Overall, our data show that GM-CSFRα blockade impacts CART functions differently from GM-CSF neutralization. Specifically, GM-CSFRα blockade inhibits CART proliferation in vitro and antitumor activity in vivo. Interestingly, GM-CSFRα blockade leads to enrichment of T cell exhaustion- and dysfunction-associated genes in CART19, which is worth further investigation. These findings may be applicable to other cytokine receptors upregulated on activated CART cells.