Single-chain VαVβ T-cell receptors function without mispairing with endogenous TCR chains

Area of Science:

• Immunology
• Cell Therapy
• Molecular Engineering

Background:

• Adoptive T-cell therapy using exogenous T-cell receptor (TCR) genes shows promise for cancer and viral diseases.
• A significant challenge is the mispairing of exogenous and endogenous TCR chains, leading to graft-versus-host reactions.
• Current engineering methods to reduce mispairing, like cysteine modification, are insufficient.

Purpose of the Study:

• To develop a T-cell receptor (TCR) engineering strategy that completely prevents mispairing with endogenous TCRs.
• To create a functional T-cell therapy that avoids graft-versus-host reactions caused by TCR mispairing.
• To investigate the potential of a single-chain TCR (scTv) approach for safer adoptive T-cell therapies.

Main Methods:

• Engineering exogenous TCR constant regions with cysteine to promote proper pairing.
• Developing a stabilized Vα/Vβ single-chain TCR (scTv) by deleting constant regions.
• Linking high-affinity scTv to intracellular signaling domains (Lck, CD28) for T-cell activation.
• Assessing T-cell responses in the absence of CD3 subunits and co-receptors.

Main Results:

• Cysteine engineering reduced but did not eliminate mixed heterodimer formation.
• Deletion of TCR constant regions to create scTv completely avoided mispairing.
• scTv, linked to signaling domains, activated T cells independently of CD3 subunits.
• Transduced T cells responded to target antigens without acquiring new specificities.

Conclusions:

• Single-chain TCR (scTv) technology offers a complete solution to TCR mispairing in adoptive T-cell therapy.
• This approach enhances the safety profile of T-cell therapies by preventing graft-versus-host reactions.
• scTv-based T-cell therapies can be engineered for targeted antigen recognition and activation, independent of endogenous TCR signaling pathways.