Screening for CD19-specific chimaeric antigen receptors with enhanced signalling via a barcoded library of intracellular domains

Abstract

CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3𝛇, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for antitumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.

Novel Fas-TNFR chimeras that prevents Fas ligand-mediated kill and signals synergistically to enhance CAR T-cell efficacy

“…However, CD40 is also expressed in T cells, similarly functioning in a co-stimulatory manner: activating canonical and noncanonical NF-κB pathways, AP-1 and the AP-1 activator JNK 47 ; generating T-cell memory and ameliorating exhaustion 48,49 . CD40 has been identified in several independent screens for enhancing T-cell function 35,50 ; and has been synthetically incorporated into CAR T cells, either as a separate module or incorporated into the CAR architecture, displaying superior anti-tumour activity compared to conventional CAR T cells, facilitated by enhanced proliferation and maintaining T-cell stemness/memory 29,[51][52][53][54][55][56] . BCMA is expressed in mature B lymphocytes and has been synthetically expressed in CAR T cells, augmenting proliferation 56 ; whereas Fn14, expressed in healthy tissue and particularly in solid tumours such as glioblastoma 57 , has not been previously synthetically expressed in T cells to alter their function.…”

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

“…While domain shuffling constitutes a powerful method for rapidly engineering new diversity in a protein, this can make the choice of screening strategy a challenging ordeal. Previous work for CAR engineering relied on functional screening based on the expression of single reporter genes or proteins (e.g., IL-2, NFAT, CD69) in immortalized cell lines 14,27,[32][33][34] . While these approaches enable highthroughput screening of CAR libraries, they are limited by their unidimensionality: they generally reveal only a single aspect of the effector response and do not capture the full complexity of the deeply interconnected signaling network of T cell activation.…”

“…This lack of diversity is partly due to the low-throughput aspect of rational CAR design as well as the laboriousness of performing in vitro functional assays. Recently, two studies from Gordon et al 14 and Goodman et al 15 developed approaches to generate pooled CAR signaling domain libraries, which were screened by combining fluorescence-activated cell sorting (FACS) using standard T cell markers and amplicon sequencing to identify novel functional variants 14,15 .…”

Cancer immunotherapy with CAR T cells: well-trodden paths and journey along lesser-known routes

“…47 Innovative studies are attempting to address the complexities and unknowns by characterizing multiple intracellular signalling domains in a high throughput manner to identify to the CAR designs that have improved functions compared to clinically used CAR T cells. 55…”