Dataset Information

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

ABSTRACT: 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 TCR?? reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCR?? 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 TCR?? chains can be incorporated in current T cell production protocols and is preferential to ensure an improved and safe clinical therapeutic.

SUBMITTER: Morton LT

PROVIDER: S-EPMC6953894 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Morton Laura T LT Reijmers Rogier M RM Wouters Anne K AK Kweekel Christiaan C Remst Dennis F G DFG Pothast Cilia R CR Falkenburg J H Frederik JHF Heemskerk Mirjam H M MHM

Molecular therapy : the journal of the American Society of Gene Therapy 20191004 1

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 ...[more]

PMID: 31636040

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Project description:Genetically engineered virus-specific T cells (VSTs) are a platform for adoptive cell therapy after allogeneic hematopoietic stem cell transplantation. However, redirection to a tumor-associated antigen by the introduction of a transgenic T-cell receptor (TCR) reduces anti-viral activity, thereby impeding the possibility of preventing or treating two distinct complications-malignant relapse and viral infection-with a single cell therapy product. Availability of CD8αβ co-receptor molecules can significantly impact class I restricted T-cell activation, and thus, we interrogated whether transgenic CD8αβ improves anti-viral activity mediated by native VSTs with or without a co-expressed transgenic TCR (TCR8). Our existing clinical VST manufacturing platform was adapted and validated to engineer TCR+ or TCR8+ VSTs targeting cytomegalovirus and Epstein-Barr virus. Simultaneous anti-viral and anti-tumor function of engineered VSTs was assessed in vitro and in vivo. We used pentamer staining, interferon (IFN)-γ enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), cytotoxicity assays, co-cultures, and cytokine secretion assays for the in vitro characterization. The in vivo anti-tumor function was assessed in a leukemia xenograft mouse model. Both transgenic CD8αβ alone and TCR8 had significant impact on the anti-viral function of engineered VSTs, and TCR8+ VSTs had comparable anti-viral activity as non-engineered VSTs as determined by IFN-γ ELISpot, ICS and cytotoxicity assays. TCR8-engineered VSTs had improved anti-tumor function and greater effector cytokine production in vitro, as well as enhanced anti-tumor function against leukemia xenografts in mice. Incorporation of transgenic CD8αβ into vectors for TCR-targetable antigens preserves anti-viral activity of TCR transgenic VSTs while simultaneously supporting tumor-directed activity mediated by a transgenic TCR. Our approach may provide clinical benefit in preventing and treating viral infections and malignant relapse post-transplant.

Dataset Information

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

Publications

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

Similar Datasets

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