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 1

Functional CRISPR/Cas9 screens identify SOCS1 as a top target constraining in vitro expansion of TILs and in vivo infiltration of transferred CD8+ T cells in syngeneic solid-tumor models.

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Functional CRISPR/Cas9 screens identify SOCS1 as a top target constraini...
(A) Experimental schematic depicting a CRISPR screen performed on the in vitro expansion of human melanoma TILs under conditions used to manufacture for therapeutic use. Following pre-REP, a sgRNA library was introduced by lentiviral transduction with TILs, then engineered by introduction of Cas9 by electroporation. A REP was then initiated in the presence of allogeneic iPBMC feeders, OKT3, and IL-2. The distribution of sgRNAs was compared prior to and following the REP to identify targets enhancing TIL accumulation. (B) SOCS1 is the top target enhancing the accumulation of TIL under REP conditions. (C) Experimental schematic depicting the workflow of in vivo CRISPR screens using Cas9-Tg × TCR-Tg (OT1 or pmel) CD8+ T cells. CD8+ T cells were activated and transduced to express a sgRNA library, with Cas9-Tg × TCR-Tg T cells transferred into mice bearing 100 mm3 tumors on the flank. Either 14 or 21 days following transfer, tumors were harvested and the sgRNA distribution of T cells within tumors analyzed and compared with the input population of T cells. (D) MAGeCK-MLE identifies SOCS1 as a top target enhancing OT1 T cell infiltration into B16-OVA tumors 14 days following transfer. (E) Enrichment or depletion patterns of sgRNAs targeting known genes by tumor OT1s in comparison with input OT1s. (F) MAGeCK-MLE identifies SOCS1 as a top target enhancing the infiltration of pmel CD8+ T cells 14 days after transfer into the MC38-gp100 solid-tumor model found to be refractory to inhibition of PD-1.