Enhancing the expression and function of an EBV-TCR on engineered T cells by combining Sc-TCR design with CRISPR editing to prevent mispairing

Xue, Shao-An; Chen, Yuan; Voss, Ralf-Holger; Kisan, Virad; Wang, Bo; Chen, Keke; He, Fengqin; Cheng, Xiao-Xia; Scolamiero, Laura; Holler, Angelika; Gao, Liquan; Morris, Emma; Stauss, Hans J.

doi:10.1038/s41423-020-0396-9

Cited by 6 publications

(5 citation statements)

References 9 publications

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“…A common modification is the use of alternative constant regions, typically to reduce the likelihood of mispairing with endogenous TCRs when introducing an exogenous TCR. This can take the form of swapping either whole (63,64) or partial (65,66) constant regions with their orthologous equivalents from different species (e.g. swapping human for mouse sequences), swapping whole or partial sequences between loci within a species (e.g.…”

Section: Discussionmentioning

confidence: 99%

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

Heather

Spindler

Alonso

et al. 2021

Preprint

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The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systemizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

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Section: Discussionmentioning

confidence: 99%

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

Heather

Spindler

Alonso

et al. 2021

Preprint

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“…To achieve the best results of maximally reducing mis-pairing and enhancing expression and function of the tumor-specific TCR, we have recently combined multiple strategies by knocking out endogenous TCR using CRISPR/Cas9 together with transduction of a single-chain EBV-specific TCR (EBV-Sc-TCR) ( 91 ). This almost eliminated mis-pairing between the introduced EBV-Sc-TCR and endogenous TCR chains, and we further enhanced tumor-specific TCR expression, functional avidity, and IL-2 production by introducing an extra intra-chain disulfide bond between the Vα and the poly-linker ( 91 ).…”

Section: Overcoming the Challenges Of Tcr-t Cancer Immunotherapymentioning

confidence: 99%

Emerging Strategies in TCR-Engineered T Cells

et al. 2022

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Immunotherapy of cancer has made tremendous progress in recent years, as demonstrated by the remarkable clinical responses obtained from adoptive cell transfer (ACT) of patient-derived tumor infiltrating lymphocytes, chimeric antigen receptor (CAR)-modified T cells (CAR-T) and T cell receptor (TCR)-engineered T cells (TCR-T). TCR-T uses specific TCRS optimized for tumor engagement and can recognize epitopes derived from both cell-surface and intracellular targets, including tumor-associated antigens, cancer germline antigens, viral oncoproteins, and tumor-specific neoantigens (neoAgs) that are largely sequestered in the cytoplasm and nucleus of tumor cells. Moreover, as TCRS are naturally developed for sensitive antigen detection, they are able to recognize epitopes at far lower concentrations than required for CAR-T activation. Therefore, TCR-T holds great promise for the treatment of human cancers. In this focused review, we summarize basic, translational, and clinical insights into the challenges and opportunities of TCR-T. We review emerging strategies used in current ACT, point out limitations, and propose possible solutions. We highlight the importance of targeting tumor-specific neoAgs and outline a strategy of combining neoAg vaccines, checkpoint blockade therapy, and adoptive transfer of neoAg-specific TCR-T to produce a truly tumor-specific therapy, which is able to penetrate into solid tumors and resist the immunosuppressive tumor microenvironment. We believe such a combination approach should lead to a significant improvement in cancer immunotherapies, especially for solid tumors, and may provide a general strategy for the eradication of multiple cancers.

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“…To reduce the risk of mispairing, another strategy is to transduce a stabilized Vα/Vβ single-chain TCRs (Sc-TCRs) ( Aggen et al, 2012 ). The combination of Sc-TCRs transduction and CRISPR disruption was reported to almost completely eliminate TCR mispairing ( Xue et al, 2020 ). The first clinical trial testing the safety and feasibility of CRISPR/Cas9 editing TCR-T cells in patients with refractory cancer was reported in 2020 ( Stadtmauer et al, 2020 ).…”

Section: Application Of Crispr/cas9 System In T Cell Receptor (Tcr)-based Adoptive T Cell Therapymentioning

confidence: 99%

CRISPR/Cas9 Gene-Editing in Cancer Immunotherapy: Promoting the Present Revolution in Cancer Therapy and Exploring More

Yin

et al. 2021

Front. Cell Dev. Biol.

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In recent years, immunotherapy has showed fantastic promise in pioneering and accelerating the field of cancer therapy and embraces unprecedented breakthroughs in clinical practice. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system, as a versatile gene-editing technology, lays a robust foundation to efficiently innovate cancer research and cancer therapy. Here, we summarize recent approaches based on CRISPR/Cas9 system for construction of chimeric antigen receptor T (CAR-T) cells and T cell receptor T (TCR-T) cells. Besides, we review the applications of CRISPR/Cas9 in inhibiting immune checkpoint signaling pathways and highlight the feasibility of CRISPR/Cas9 based engineering strategies to screen novel cancer immunotherapy targets. Conclusively, we discuss the perspectives, potential challenges and possible solutions in this vivid growing field.

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Enhancing the expression and function of an EBV-TCR on engineered T cells by combining Sc-TCR design with CRISPR editing to prevent mispairing

Cited by 6 publications

References 9 publications

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

Emerging Strategies in TCR-Engineered T Cells

CRISPR/Cas9 Gene-Editing in Cancer Immunotherapy: Promoting the Present Revolution in Cancer Therapy and Exploring More

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