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 5

A CRISPR tiling screen in primary human T cells identifies highly potent sgRNAs for therapeutic use targeting the SH2 domain of SOCS1.

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A CRISPR tiling screen in primary human T cells identifies highly potent...
(A) Experimental schematic of the CRISPR tiling screen for discovering potent SOCS1 sgRNAs. Following in silico removal of sgRNAs predicted to target multiple sites in the genome, a sgRNA library targeting every possible Cas9 cut site of the SOCS1 CDS based on the trinucleotide NGG PAM sequence together with controls was introduced by lentiviral transduction into activated primary human T cells with IL-2. Following introduction of Cas9, sgRNA Lib+ T cells were expanded in the presence of IL-2, with the distribution of sgRNAs following expansion evaluated and compared with input. (B) SOCS1 CRISPR tiling screen results. sgRNAs targeting the olfactory genes are in green, genome multicutters in orange, and sgRNAs targeting the SOCS1 CDS are blue. The SOCS1 protein domain structure is depicted above, with the SH2 and SOCS box domains labeled. NTD, N-terminal domain. sgRNAs targeting the SH2 domain of SOCS1 are depicted in light blue. (C) Editing efficiency of top sgRNAs identified in B was assessed by electroporation of Cas9/sgRNA RNPs into activated primary human T cells in an arrayed format, with editing efficiency of the cut site quantified by Amp-Seq. sgRNAs are labeled along the x axis, with dotted line depicting the threshold of SOCS1 sgRNAs achieving the targeted IL-2–mediated increase in pSTAT5. (D) Activated human primary T cells were edited with Cas9/sgRNA RNPs targeting either SOCS1 or olfactory genes in an arrayed format, with edited T cells stimulated with IL-2 and pSTAT5 signals quantified by FACS and depicted as fold change over sgOlf control. sgRNAs are labeled along the x axis, with dotted line depicting SOCS1 sgRNAs achieving the targeted IL-2–mediated increase in pSTAT5. (E) A comparison between editing efficiency (x axis) and pSTAT induction fold-change (y axis) of evaluated sgRNAs, with the u728, kipc, and qd5u sgRNAs identified as the most potent sgRNAs targeting SOCS1 based on editing efficiency and functional potency.