Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy

Abstract

In the absence of cell-surface cancer-specific antigens, immunotherapies such as chimeric antigen receptor (CAR) T cells, monoclonal antibodies, or bispecific T cell engagers typically target lineage antigens. Currently, such immunotherapies are individually designed and tested for each disease. This approach is inefficient and limited to a few lineage antigens for which the on-target/off-tumor toxicities are clinically tolerated. Here, we sought to develop a universal CAR T cell therapy for blood cancers directed against the pan-leukocyte marker CD45. To protect healthy hematopoietic cells, including CAR T cells, from CD45-directed on-target/off-tumor toxicity while preserving the essential functions of CD45, we mapped the epitope on CD45 that is targeted by the CAR and used CRISPR adenine base-editing to install a function-preserving mutation sufficient to evade CAR T cell recognition. Epitope edited CD45 CAR T cell were fratricide-resistant and effective against patient-derived acute myeloid leukemia, B cell lymphoma, and acute T cell leukemia. Epitope edited hematopoietic stem cells (HSCs) were protected from CAR T cells and, unlike CD45 knockout cells, could engraft, persist, and differentiate in vivo. Ex vivo epitope editing in HSCs and T cells enables the safe and effective use of CD45-directed CAR-T cells and bispecific T cell engagers for the universal treatment of hematologic malignancies and might be exploited for other diseases requiring intensive hematopoietic ablation.

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection

“…2a-c). Structural analysis and high-throughput screening techniques have been used to design epitope-edited receptors that evade a specific antibody and remain functional [32][33][34] . We hypothesized that epitope editing of SEED payloads would allow for the enrichment of cells with a transgene that would otherwise be depleted through SEED-Selection.…”

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

“…To identify mimotopes for FMC63, we employed a yeast surface display library 32 presenting ∼5x10 8 randomized linear peptides of 10 amino acids (AA, 10mer) for screening against recombinant FMC63 expressed as a full-length IgG (hereafter, FMC63 IgG , Fig. 2B ).…”

“…Further, the CD19 extracellular domain (ECD) is difficult to express and prone to misfolding 29 , making it challenging to use recombinant CD19 for manufacturing an amphiphile vaccine to stimulate CD19 CAR-T cells. As it is common for CAR binding domains to recognized complex conformational epitopes [30][31][32] , we sought to establish a general strategy to generate simple surrogate ligands for any CAR. We hypothesized that generation of mimotopes, linear synthetic peptides that are also recognized by the CAR binding domain in addition to its native target antigen, could be an ideal option (Fig.…”

Targeting CD45 by gene-edited CAR-T cells for leukemia eradication and hematopoietic stem cell transplantation preconditioning

“…Recently an elegant alternative approach to target CD45 was reported, based on base-editing of HSC to eliminate the epitope targeted by BC8 CAR 62 .…”