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Updated: Dec 9, 2025
Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs
Published on: September 10, 2017
A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences
Korbinian N Kropp1, Tim J Schäufele1, Martina Fatho1
1Internal Medicine III, University Cancer Center (UCT), Research Center for Immunotherapy (FZI), University Medical Center (UMC) of the Johannes Gutenberg University and German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany.
Researchers developed a new cloning strategy for generating T-cell receptor (TCR) expression constructs. This method enables efficient preclinical testing of tumor-reactive TCRs, accelerating cancer immunotherapy research.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Preclinical testing of tumor-reactive T-cell receptors (TCRs) requires efficient generation of expression constructs.
- Current cloning strategies can be time-consuming and lack optimization for enhanced TCR functionality.
Purpose of the Study:
- To develop a novel, rapid, and convenient cloning strategy for generating optimized TCR expression constructs.
- To create a versatile vector backbone for seamless cloning of TCR V(D)J regions.
Main Methods:
- A pDONR™221 vector backbone was engineered with Gateway™ compatibility.
- Type IIS restriction enzyme sites (BsmBI and BsaI) were incorporated for V(D)J region cloning.
- TCR constant regions were optimized (murine counterparts, disulfide bond, β-P2A-α arrangement) for enhanced functionality.
Main Results:
- The developed vector backbone successfully generated Gateway™ compatible entry clones for bicistronic αβTCR constructs.
- Seamless cloning of TCRα and TCRβ V(D)J regions was achieved using the engineered restriction sites.
- Functional testing in primary human T cells confirmed the utility of the vector for melanoma-reactive TCRs.
Conclusions:
- The novel vector backbone provides an efficient method for generating optimized TCR expression constructs.
- This strategy facilitates preclinical testing and development of TCR-based immunotherapies.
- The system is adaptable for various TCRs, including those targeting solid tumors like melanoma.
