A cell-based phenotypic library selection and screening approach for the de novo discovery of novel functional chimeric antigen receptors

Julie K Fierle,Vasileios Atsaves,Patrick Reichenbach,Melita Irving,Steven M Dunn,Mariastella De Tiani,Johan Abram-Saliba,George Coukos,Matteo Brioschi

doi:10.1038/s41598-022-05058-5

Abstract

Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings. Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. However, it is becoming increasingly apparent that not all antibodies are equal in this regard, with a growing appreciation that ‘optimal’ CAR performance requires a consideration of multiple structural and contextual parameters. Thus, antibodies raised by classical approaches and intended for other applications often perform poorly or not at all when repurposed as CARs. With this in mind, we have explored the potential of an in vitro phenotypic CAR library discovery approach that tightly associates antibody-driven bridging of tumor and effector T cells with an informative and functionally relevant CAR activation reporter signal. Critically, we demonstrate the utility of this enrichment methodology for ‘real world’ de novo discovery by isolating several novel anti-mesothelin CAR-active scFv candidates.

Highlights

Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings
We explored whether a generic 2nd generation modular chimeric antigen receptors (CARs) scaffold could tolerate a direct translational fusion to a fluorescent protein (FP) reporter as a contiguous multi-domain polypeptide
In order to circumvent the known propensity of many FP proteins to dimerize weakly or form higher order oligomers, potentially leading to undesirable aggregative clustering and signalling of CAR fusion molecules in the absence of antigen target, we used a mutated monomeric variant of GFP and fused this downstream of a modular scFv-28ζ 2nd generation CAR cassette separated by a 13-amino acid synthetic cloning spacer

Summary

Introduction

Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. The authors were able to confirm a clone enrichment discordance between CAR cell sorting on the basis of soluble antigen binding and that due to phenotypic activation/signalling, and could identify individual VH CDR3 mutations that dramatically reduced apparent scFv affinity while conferring an improved CAR discrimination for a H ER2high cell line Despite such encouraging studies using model antibodies, it has yet to be demonstrated that phenotypic reporter approaches have practical utility for the de novo discovery of novel, functionally active scFvs CAR leads, and that they can replace or complement classical affinity-driven screening paradigms. From large starting naïve library repertoires, we show that phenotypically enriched clones are functionally active by design, recognising and killing cell lines bearing the target antigen at endogenous levels and in native cell surface contexts

Methods

Results

Conclusion