Filosto, Simone

Abstract While chimeric antigen receptor T (CART) cell therapy has shown remarkable success, the development of exhaustion limits durable response. We identified a role for interleukin (IL)-4 in the development of CART cell exhaustion through three independent approaches including: 1) a genome-wide CRISPR knockout screen using healthy donor CART cells in an in vitro model for exhaustion, 2) RNA and ATAC sequencing on freshly produced and chronically stimulated healthy donor CART cells, and 3) RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the Zuma-1 clinical trial that led to the FDA approval of axi-cel. Further, in vitro validation studies revealed that CD19 directed CART (CART19) cells chronically stimulated in the presence of human recombinant IL-4 (hrIL-4) displayed signs of exhaustion such as 1) decreased proliferation (p = 0.01), 2) increased coexpression of inhibitory receptors (p = 0.01), and 3) decreased production of IL-2 and interferon (IFN)-γ (p= 0.02, p = 0.002). Encouragingly, CART19 cells combined with an IL-4 monoclonal antibody improved antitumor activity (p = 0.045) and expansion (p = 0.01) while also decreasing the co-expression of inhibitory receptors (p = 0.02) in a mantle cell lymphoma xenograft mouse model. Building on these results, we asked if IL-4 driven exhaustion results from a direct impact of IL-4 on CART cells. To test this, we used a tumor-free assay where CART19 cells were chronically stimulated with CD19 beads in the presence of hrIL-4 or diluent. CART cells treated with hrIL-4 displayed an exhausted phenotype characterized by increased co-expression of inhibitory receptors (p = 0.04) and decreased production of IL-2 (p = 0.01). Next, we asked if IL-4 driven CART cell exhaustion is dependent on the costimulatory domain. We tested the impact of IL-4 on both CD28ζ and 41BBζ costimulated CART19 cells. Similar to our previous studies with CART19-28ζ cells, chronic stimulation of CART19-BBζ cells in the presence of hrIL-4 enhanced the exhausted phenotype as seen by increased co-expression of inhibitory receptors (p = 0.04) and decreased production of IL-2 and IFN-γ (p = 0.08 and p = 0.007). Finally, we asked if IL-4 induces exhaustion independently of its classic role in Th2 polarization of CD4 CART cells. Following CART production, we isolated CD8 cells and chronically stimulated them in the presence of hrIL-4 or diluent. CD8 CART cells treated with hrIL-4 displayed an enhanced exhausted profile as seen by 1) decreased proliferative ability (p < 0.0001), 2) increased co-expression of inhibitory receptors (p = 0.01), and 3) decreased production of IL-2 and IFN-γ (p < 0.0001, p = 0.004). Together, our data indicates a novel role for IL-4 in the development of CART cell exhaustion that is independent of tumor cells, costimulatory domain, and CD4 cells. As such, we believe IL-4 neutralization may be a widely applicable and actionable approach to improve the durable response to CART cell therapy. Citation Format: Carli M. Stewart, Elizabeth L. Siegler, Truc N. Huynh, R. Leo Sakemura, Brooke Kimball, Long Mai, Kun Yun, James H. Girsch, Jennifer Feigin, Omar Gutierrez Ruiz, Makena Rodriguez, Ekene Ogbodo, Ismail Can, Claudia Manriquez Roman, Olivia Sirpilla, Hong Xia, Jenny Kim, Justin Budka, Mike Mattie, Nathalie Scholler, Simone Filosto, Saad S. Kenderian. IL-4 drives CART cell exhaustion in a CD4 independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 37.

Abstract While chimeric antigen receptor T-cell therapy targeting CD19 (CART19) has shown remarkable success in the treatment of hematological malignancies, the durable response rates remain approximately 40% and there are limited solutions for CART cell therapy in the treatment of solid tumors. To further understand mechanisms of resistance, including CART cell exhaustion, we employed three independent approaches: 1) RNA and ATAC sequencing on unstimulated vs. exhausted healthy donor CART19 cells by utilizing an in vitro model for exhaustion, 2) RNA and ATAC sequencing on pre-infusion CART19 cell products from responders and non-responders in the Zuma-1 clinical trial that led to the FDA approval of axi-cel CART19 therapy, and 3) a genome-wide CRISPR knockout screen in healthy donor CART19 cells using our in vitro model for exhaustion. In each of these approaches, IL-4 was identified as a regulator of CART cell dysfunction. In approach 1, ingenuity pathway analysis of genes that were both differentially accessible and expressed in exhausted compared with unstimulated CART19 cells revealed IL-4 as a top upstream regulator (p = 5E-6). In approach 2, IL-4 was one of two genes that were both upregulated and more accessible in CART19 cell products from non-responders (p < 5E-2). Finally, in approach 3, gene ontology enrichment analysis of genes that were positively selected during the genome-wide CRISPR knockout screen, revealed regulation of the IL-4 pathway as one of the top affected pathways (p = 1E-4). Together, our data indicates a role for IL-4 in CART cell dysfunction caused by exhaustion. Investigating this mechanism further, we saw an increase in the production of IL-4 as CART cells became exhausted (p = 4E-3). Treatment of CART19 cells with human recombinant IL-4 (hrIL-4) resulted in dysfunction as evident by a decrease in antigen specific cytotoxicity (p = 4E-3) and proliferative ability (p= 6.5E-2), as well as exhaustion-specific signs of dysfunction such as an increase in the expression of the inhibitory receptor, TIM-3 (p = 3E-3) and an increase in the transcription of the exhaustion-related transcription factor EOMES (p = 1E-2). Finally, we tested whether IL-4 neutralization enhances CART19 cell functions. Using a CD19+ JeKo-1 xenograft mouse model, we compared the combination treatment of CART19 cells and an IL-4 neutralizing monoclonal antibody (10 mg/Kg, clone # MP4-25D2) to CART19 cells and an IgG control. IL-4 neutralization in combination with CART19 cells resulted in reduced tumor burden (p = 4.6E-2), increased CART cell proliferation (p = 8E-3), and prolonged overall survival (p= 5E-2). In summary, our data indicates that 1) IL-4 induces CART cell dysfunction through a state of exhaustion and 2) IL-4 neutralization with a monoclonal antibody enhances CART cell therapy. As such, this novel combination therapy holds the potential to be translated to the clinic to improve durable responses from CART cell therapy. Citation Format: Carli Stewart, Michelle J. Cox, Reona Sakemura, Ekene J. Ogbodo, Ismail Can, Claudia Manriquez Roman, Kun Yun, Olivia Sirpilla, James H. Girsch, Truc Huynh, Elizabeth L. Siegler, Jenny J. Kim, Mike Mattie, Nathalie Scholler, Simone Filosto, Saad S. Kenderian. IL-4 depletion leads to the improvement of CART cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1153.