Wachter, F. | Search bioRxiv

bioRxiv2026

Unbiased, systematic surfaceome profiling of Acute Leukemia to identify novel immunotherapy targets

Cell surface proteomics provides a direct topological assessment of the outer membrane of cells and enables the capture of low abundance proteins that may be missed by whole cell proteomics. Here we present an unbiased atlas of the whole cell and surface proteomes of 25 commonly used leukemic cell lines, encompassing both lymphoid and myeloid lineages, and a variety of driver mutations. Paired-wise analysis highlights recurrent surface proteins that are not detected by whole cell proteomics. Coupling this dataset to RNA-sequencing, we also discovered genes where protein and RNA abundances are discordant. In KMT2A-rearranged AML, CD70 expression was increased across cell lines and validated in primary patient samples, supporting CD70 as a candidate therapeutic target in this disease. Several proteins are enriched in the surface proteomes but lack surface annotation, adding to the growing list of potential non-canonical cell surface proteins. These findings reveal a substantial pool of proteins absent from conventional surface annotations, including RNA-binding proteins, an emerging class of candidate immunotherapeutic targets.

molecular biology↗

bioRxiv2025

Precision Editing of Cyclophilin A Generates Cyclosporine and Voclosporin Resistant Cellular Therapies

Recipients of allogeneic transplants or patients with autoimmune disease require immune suppression, often with calcineurin inhibitors. There is an expanding repertoire of immune effector cell therapies, including CD19 CAR-T cells and viral-specific T cells, deployed in these patients; however, ongoing calcineurin inhibition may be detrimental to cell therapy function. We developed a CRISPR/Cas9-based approach to engineer dual cyclosporine/voclosporin resistant cell therapies by targeting PPIA (encoding cyclophilin A), a critical binding partner for both drugs. Because Cyclophilin A has homeostatic functions in T cells, a complete knock-out is detrimental to cell viability. We thus targeted its C-terminus, disrupting drug binding while leaving the majority of the protein intact. C-terminal editing was stable throughout expansion and preserved Cyclophilin A expression. Edited CD19 CAR-T cells retained effector function in the presence of cyclosporine and voclosporin, including proliferation, cytokine production, and target cell killing, resulting in improved survival in murine models of CD19+ leukemia. Edited CMV-specific T cells also demonstrated preserved antigen-specific proliferation and cytokine production in the presence of these drugs. C-terminal editing of Cyclophilin A offers a promising avenue for developing next-generation cell therapies for patients receiving calcineurin inhibitors.

immunology↗