Toward high-throughput engineering techniques for improving CAR intracellular signaling domains

Abstract

Chimeric antigen receptors (CAR) are generated by linking extracellular antigen recognition domains with one or more intracellular signaling domains derived from the T-cell receptor complex or various co-stimulatory receptors. The choice and relative positioning of signaling domains help to determine chimeric antigen receptors T-cell activity and fate in vivo. While prior studies have focused on optimizing signaling power through combinatorial investigation of native intracellular signaling domains in modular fashion, few have investigated the prospect of sequence engineering within domains. Here, we sought to develop a novel in situ screening method that could permit deployment of directed evolution approaches to identify intracellular domain variants that drive selective induction of transcription factors. To accomplish this goal, we evaluated a screening approach based on the activation of a human NF-κB and NFAT reporter T-cell line for the isolation of mutations that directly impact T cell activation in vitro. As a proof-of-concept, a model library of chimeric antigen receptors signaling domain variants was constructed and used to demonstrate the ability to discern amongst chimeric antigen receptors containing different co-stimulatory domains. A rare, higher-signaling variant with frequency as low as 1 in 1000 could be identified in a high throughput setting. Collectively, this work highlights both prospects and limitations of novel mammalian display methods for chimeric antigen receptors signaling domain discovery and points to potential strategies for future chimeric antigen receptors development.

Keywords: 41BB; OX40; chimeric antigen receptor; directed evolution; mammalian display.

FIGURE 1

CAR-induced NF-κB and NFAT reporter construct activation in Jurkat cells. (A). Schematic representation of reporter cells with NF-κB- and NFAT-inducible CFP and eGFP expression developed by Rosskopf et al., combined with retroviral CAR introduction to produce CAR J76-TPR cells. CAR constructs contain the positive selection marker hCD19. (B). Representative flow biplots depicting fluorescence intensity of CFP and eGFP with (right) and without (left) stimulation of reporter cells with PMA and ionomycin (P/I). Panel A created in Biorender.

FIGURE 2

Positions, identities, and expression of 4-1BB, DAP10, and CD3ζ sequence variants. (A). 4-1BB alanine substitutions of TRAF-binding residues. (B). Dap10 alanine substitutions of YXXM residues, preventing phosphorylation and binding to PI3K and Grb2. (C). CD3ζ alanine substitution of the ITAM sites, preventing phosphorylation and binding to Zap-70. (D–G). Expression levels. (D). Median Fluorescent Intensity (MFI) of the positive transduction marker, CD19, of unstained, untransduced and CD3ζ CAR-transduced cells serving as negative and positive controls, respectively. (E). Representative CD19 fluorescent histograms for these positive and negative controls. (F). Median Fluorescent Intensity (MFI) of the positive selection marker, CD19, of each CAR construct population following transduction. Error bars represent standard deviation of three technical replicates from one of three biological replicates. (G). Representative CD19 fluorescence histograms of reporter cells transduced with these CAR constructs.

FIGURE 3

CAR-mediated activation of NF-κB and NFAT in signaling domain variants. (A). Representative flow biplots of B7H6-stimulated (50 μg/mL) wildtype and K/O CARs showing activation of NF-κB and NFAT (left panel). (B). MFI signal of NF-κB (top) and NFAT (bottom) in each CAR construct population across a titration of plated B7H6. Error bars represent standard deviation of three technical replicates from one of three biological replicates.

FIGURE 4

Enrichment of 4-1BB CAR reporter cells in a spike-in proof-of-concept mock sort. (A). Schematic of needle in a haystack experiment to demonstrate the ability to retrieve rare variants that exhibit improved signaling profiles from among a larger population with reduced activity. (B). Quantification of control fluorescent 4-1BB K/O cells spiked into an excess of non-fluorescent 4-1BB K/O cells before and after a mock selection based on NFAT and NFkB activation. (C). Quantification of fluorescent 4-1BB cells spiked into an excess of non-fluorescent 4-1BB K/O cells before and after a mock selection for NFAT and NFkB activation for the 4-1BB spiked into 4-1BB K/O condition (right). Prevalence was calculated as the fraction of total CAR T cells as determined by CD19 expression. Pre-and-post sort labels refer to the mock sort on ungated and gated cells. (D). Enrichment of cells was calculated by fold change in non-gated vs. gated populations. (E). Theoretical enrichment was calculated using observed population distributions based on NFAT and NFkB activation of unmixed cell populations. Error bars represent standard deviation of three technical replicates from one of three biological replicates. Panel A created in Biorender.