Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens
Michael R. Schlabach, … , Louise Cadzow, Micah J. Benson
Michael R. Schlabach, … , Louise Cadzow, Micah J. Benson
Published December 15, 2023
Citation Information: J Clin Invest. 2023;133(24):e163096. https://doi.org/10.1172/JCI163096.
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Research Article Immunology

Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens

Abstract

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell–enhancing target. In murine CD8+ T cell–therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.

Authors

Michael R. Schlabach, Sharon Lin, Zachary R. Collester, Christopher Wrocklage, Sol Shenker, Conor Calnan, Tianlei Xu, Hugh S. Gannon, Leila J. Williams, Frank Thompson, Paul R. Dunbar, Robert A. LaMothe, Tracy E. Garrett, Nicholas Colletti, Anja F. Hohmann, Noah J. Tubo, Caroline P. Bullock, Isabelle Le Mercier, Katri Sofjan, Jason J. Merkin, Sean Keegan, Gregory V. Kryukov, Caroline Dugopolski, Frank Stegmeier, Karrie Wong, Fiona A. Sharp, Louise Cadzow, Micah J. Benson

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Figure 2

Inactivation of SOCS1 by CRISPR/Cas9 in transferred CD8+ T cells drives enhanced efficacy in syngeneic mouse models with durable persistence as Tcm cells.

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Inactivation of SOCS1 by CRISPR/Cas9 in transferred CD8+ T cells drives ...
C57BL/6 mice bearing 100 mm3 B16-OVA tumors on the flank were treated with 3 × 106 OT1 CD8+ T cells engineered to inactivate either OLF1 (sgOlf), PD-1 (sgPD-1), or SOCS1 (sgSocs1). Results of statistical analysis depicted between sgOlf versus sgPD-1 and sgOlf versus sgSocs1. (A) Tumor growth curves of each group over time are depicted. (B) Mice treated with sgSocs1 OT1s undergoing complete tumor rejection were rechallenged with B16-OVA tumor cells 61 days following initial transfer, with 10 naive mice included as controls. Tumor growth of the indicated treatment groups is depicted. (C) The frequency of sgSocs1 OT1s as peripheral blood CD8+ T cells prior to (day –10) and following (days 8 through 84) B16-OVA rechallenge. FACS plots are depicted, with OT1s defined as CD8+Va2+Vβ5.1+cells. (D) CD44+CD62L+ and CD44+CD62L– phenotypes of sgSocs1 OT1s in C prior to (day –10) and following B16-OVA rechallenge (days 8 and 84) were quantified by FACS and depicted. (E) C57BL/6 mice bearing MC38-gp100 tumors with a median size of 100 mm3 were treated with 7 × 106 pmel CD8+ T cells inactivated with either SOCS1 (sgSocs1), PD1 (sgPD-1), or OLF1 (sgOlf). Tumor growth curves depicted by treatment group on graph on the left, and final tumor size in individual mice depicted on graph on the right. Results of 2-way ANOVA were used to determine statistical significance between treatment groups in A and E. ****P < 0.0001. Data in A–D are representative of 2 independent experiments.