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Sequence and structural determinants of efficacious de novo chimeric antigen receptors

ABSTRACT: Advances in generative protein design using artificial intelligence (AI) have enabled the rapid development of binders against heterogeneous targets, including tumor-associated antigens. Despite extensive biochemical characterization, these novel protein binders have had limited evaluation as agents in candidate therapeutics, including chimeric antigen receptor (CAR) T cells. Here, we synthesize variable generative protein design workflows to screen 1,487 novel protein binders targeting BCMA, CD22, and CD19 for efficacy in scalable protein binding and T cell assays. We identify three main challenges that hinder the utility of de novo protein binders as CARs, including tonic signaling, occluded epitope engagement, and off-target activity. We develop computational and experimental heuristics to overcome these limitations, including screens of sequence variants for individual parental structures, that restore on-target CAR activation while mitigating liabilities. Together, our framework accelerates the development of AI-designed proteins for future preclinical therapeutic screening, helping enable a new generation of cellular therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE310399 | GEO | 2026/07/06

REPOSITORIES: GEO

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Project description:Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies.

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Sequence and structural determinants of efficacious de novo chimeric antigen receptors

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