Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens
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
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
View: Text | PDF
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

×

Figure 4

SOCS1 is a key checkpoint in the accumulation of Texint and Texeff cells from Texprog subsets within tumors with mechanisms distinct from PD-1.

Options: View larger image (or click on image) Download as PowerPoint
SOCS1 is a key checkpoint in the accumulation of Texint and Texeff cells...
C57BL/6 mice bearing 100 mm3 B16-OVA tumor cells with a median size of 100 mm3 were treated with 3 × 106 SOCS1 (sgSocs1), PD1 (sgPD-1), or OLF1 (sgOlf) engineered OT1s, with editing efficiencies for target genes 82% for sgPD-1, 95% for sgSocs1, and 80% for sgOlf. scRNA-Seq was performed on CD45+ cells isolated from the TME from each treatment group. (A) UMAP visualization of T cell clusters. (B) Projection of OT1s onto T cell clusters based on TCR sequencing. (C) Bar plot of treatment DEGs between treatment-group OT1s, adjusted P < 0.1, abs(avg_log2FC ≥ 0.25). (D) Pseudobulk analysis on OT1s, with DEGs between treatment groups depicted. (E) GSEA by projecting pseudobulk DEGs from between indicated treatment groups in D onto Miller et al. Tex subset gene signatures (41). (F) UMAP visualization depicting the expression of indicated transcripts by T cell clusters (G) Correlogram between indicated transcripts (*P < 0.001). (H) STARTRAC TCR clonal expansion by T cell cluster. (I) Tex subset annotation by cluster. CD8, terminally differentiated Tex subsets. (J) Tex subset frequency by treatment group. Clusters 2, 7, 3 and 5 reflect OT1 frequencies, with cluster 1 containing non-OT1 cells and included for reference. (K) Number of DEGs within each Tex subset as indicated and between depicted treatment groups. (L) Heatmap of Tex subset–defining transcripts by subset and by treatment group.