Abstract
As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell’s HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.
Highlights
The emergence of chimeric antigen receptor (CAR)-T cell therapy has changed the paradigm of cancer immunotherapy by virtue of its durable remission with manageable toxicity profile
In vitro mixed lymphocyte reactions revealed that alloresponses in responder cells were dampened against HLA-I/II-negative T cells compared to HLA-I-negative cells, solidifying a conceptual framework that narrates the role of HLA-II plays in infused, therapeutic allogeneic cells during host-mediated rejection
Off-the-shelf CAR-T cells produced from healthy allogeneic donor T cells with the “fittest” phenotypes can increase the likelihood of therapeutic response and overcome the barriers of the autologous CAR-T cell therapy
Summary
The emergence of chimeric antigen receptor (CAR)-T cell therapy has changed the paradigm of cancer immunotherapy by virtue of its durable remission with manageable toxicity profile. A recent report in a trial of autologous CAR-T cell therapy documented the unintentional transduction of a single leukemic B cell with anti-CD19 CAR during manufacturing and its product masking the target antigen, thereby escaping CAR-T recognition and resisting the therapy[8]. These limitations could be overcome by employing allogeneic T cells obtained from “fitted” healthy donors as a source of universal CAR-T cell production. In vitro mixed lymphocyte reactions revealed that alloresponses in responder cells were dampened against HLA-I/II-negative T cells compared to HLA-I-negative cells, solidifying a conceptual framework that narrates the role of HLA-II plays in infused, therapeutic allogeneic cells during host-mediated rejection
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