Multi-phenotype CRISPR-Cas9 screens identify p38 kinase as a target for adoptive immunotherapies

Authors

• D Gurusamy
• S Vodnala
• R Kishton
• A Eidizadeh
• L Jia
• CM Kariya
• TN Yamamoto
• DC Palmer
• Z Yu
• JH Pan
• R Eil
• M Sukumar
• SJ Patel*
• NP Restifo*

Abstract

Adoptive T cell immunotherapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has led to complete durable regression of tumors in approximately 22% of patients with advanced metastatic melanoma. While several factors can contribute to the efficacy of ACT, a major limitation is the induction of a terminally differentiated phenotype and finite proliferative capacity of TIL during current ex vivo expansion protocols. Thus, there is significant interest in identifying T-cell intrinsic and extrinsic negative regulatory pathways that limit the fitness and ex vivo expansion of TIL. Preclinical cancer models of ACT have demonstrated that less differentiated memory T cells can mediate superior anti-tumor responses with improved T cell persistence. Therefore, there is also interest in limiting effector differentiation along with reducing oxidative stress and genomic damage. To identify the T cell intrinsic negative regulatory pathways, we developed a multi-phenotypic genetic screen to systematically target 29 major kinases in preclinical T cell expansion model. We used the CRISPR-Cas9 technology coupled with high throughput flow cytometric analysis to evaluate cell expansion, T cell differentiation, oxidative stress, and DNA damage. Results from our genetic screen identified p38 kinase as a unique multi-phenotypic regulator that limited cellular differentiation, oxidative, and genomic stress while achieving improved cellular expansion. Furthermore, pharmacological inhibition of p38 kinase in murine ex vivo T cell expansion models validated the results from our genetic screen. Pharmacological inhibition of p38 kinase in human melanoma TIL ex vivo cultures improved their memory phenotypes and preserved their TCR clonality during expansion. Cells cultured in the presence of a p38 inhibitor had increased capacity for cytokine production, specifically interferon-γ, and demonstrated improved in vivo persistence. Additionally, cells cultured in the presence of the p38 inhibitor demonstrated enhanced in vivo cell expansion, tumor infiltration, and anti-tumor efficacy in an immunocompetent tumor mouse model. Thus, using CRISPR-Cas9 technology we have identified p38 kinase to be a major regulator of multiple desired characteristics of therapeutic TIL and results from our screen has direct implications for current ACT therapies.

Scientific Focus Area: Immunology

This page was last updated on Friday, March 26, 2021