Abstract 1153: IL-4 depletion leads to the improvement of CART cell therapy
Authors: Stewart Carli , Cox Michelle J. , Sakemura Reona , Ogbodo Ekene J. , Can Ismail , Roman Claudia Manriquez , Yun Kun , Sirpilla Olivia , Girsch James H. , Huynh Truc , Siegler Elizabeth L. , Kim Jenny J. , Mattie Mike , Scholler Nathalie , Filosto Simone , Kenderian Saad S. 
Published: April 2023

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 &lt; 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.