Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic.

Laura T Morton, Rogier M Reijmers, Anne K Wouters, Christiaan Kweekel, Dennis F G Remst, Cilia R Pothast, J H Frederik Falkenburg, Mirjam H M Heemskerk

Molecular therapy : the journal of the American Society of Gene Therapy 2020 Jan 08

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Generation of an optimal T cell therapeutic expressing high frequencies of transgenic T cell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRαβ reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRαβ was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating T cell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 T cells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited T cell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRαβ chains can be incorporated in current T cell production protocols and is preferential to ensure an improved and safe clinical therapeutic. Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Citation

Laura T Morton, Rogier M Reijmers, Anne K Wouters, Christiaan Kweekel, Dennis F G Remst, Cilia R Pothast, J H Frederik Falkenburg, Mirjam H M Heemskerk. Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic. Molecular therapy : the journal of the American Society of Gene Therapy. 2020 Jan 08;28(1):64-74