Walsh, Z. H. | Search bioRxiv

bioRxiv2025

Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

Chimeric antigen receptor (CAR) T cells targeted to CD19 are an effective therapy for B-lineage malignancies. However, about half of patients relapse and this therapeutic, often with antigen-negative disease, warranting the targeting of other antigens. CD22 represents another promising target, with highly restricted but ubiquitous expression across the B-lineage. However, despite promising preclinical work by several groups with CD22-targeted CAR T cells targeting of this antigen in the clinic has proven difficult, with many patients relapsing with CD22Lo leukemia, contrasting to complete loss of CD19 expression post CD19-CAR. While prior work has demonstrated that a CAR with so-called "tonic" antigen-independent signaling properties has proven to be highly efficacious, tonic signaling has been shown be detrimental to long-term T cell function. Here, we demonstrate a balance between binding affinity and antigen-independent tonic signaling (as determined by length of flexible linker) in determining CAR function. We show that maximal CAR function in the settings CD22Lo and WT leukemia is maintained by boosting binding affinity without shortening flexible linker to induce tonic signaling, establishing rational modification of antigen binding domain as an important approach for modulating the function of cellular therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/643183v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1d543e3org.highwire.dtl.DTLVardef@5bf226org.highwire.dtl.DTLVardef@76cd2forg.highwire.dtl.DTLVardef@198fd37_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells

Base editing enables generation of single nucleotide variants, but large-scale screening in primary human T cells is limited due to low editing efficiency, among other challenges1. Here, we developed a high-throughput approach for high-efficiency and massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale screens editing 102 genes with central functions in T cells and full-length tiling mutagenesis of selected genes, and read out variant effects on hallmarks of T cell anti-tumor immunity, including activation, proliferation, and cytokine production. We discovered a broad landscape of gain- and loss-of-function mutations, including in PIK3CD and its regulatory subunit encoded by PIK3R1, LCK, AKT1, CTLA-4 and JAK1. We identified variants that affected several (e.g., PIK3CD C416R) or only selected (e.g. LCK Y505C) hallmarks of T cell activity, and functionally validated several hits by probing downstream signaling nodes and testing their impact on T cell polyfunctionality and proliferation. Using primary human T cells in which we engineered a T cell receptor (TCR) specific to a commonly presented tumor testis antigen as a model for cellular immunotherapy, we demonstrate that base edits identified in our screens can tune specific or broad T cell functions and ultimately improve tumor elimination while exerting minimal off-target activity. In summary, we present the first large-scale base editing screen in primary human T cells and provide a framework for scalable and targeted base editing at high efficiency. Coupled with multi-modal phenotypic mapping, we accurately nominate variants that produce a desirable T cell state and leverage these synthetic proteins to improve models of cellular cancer immunotherapies.

immunology↗