Modifications to rhesus macaque TCR constant regions improve TCR cell surface expression

Area of Science:

• Immunology
• Cellular Therapy
• Biotechnology

Background:

• T cell immunotherapy efficacy relies on sufficient surface antigen receptor expression on engineered cells.
• Challenges include optimizing engineering, coding sequences, and preventing mispairing of exogenous and endogenous T cell receptor (TCR) chains.
• Preclinical models are vital for refining engineering designs and assessing safety.

Purpose of the Study:

• To enhance rhesus macaque T cell receptor (TCR) surface expression in primary cells for improved TCR-based cellular immunotherapy models.
• To evaluate five alternative TCRαβ constant region constructs for their impact on surface expression and function.

Main Methods:

• Generated five distinct TCRαβ constant region constructs for a SIV Gag-specific TCR in rhesus macaque primary cells.
• Constructs included human codon-optimized rhesus macaque TCR, disulfide linkage, minimal murine substitutions, full murinization, and murinization with FG loop modification.
• Assessed surface expression levels and cytokine production upon antigen-specific stimulation.

Main Results:

• Murinization or minimal amino acid substitutions to the constant region significantly increased rhesus macaque TCR surface expression compared to wild-type.
• All engineered TCR constructs maintained their ability to induce cytokine production in response to cognate peptide antigen stimulation.
• Specific modifications, particularly murinization, proved most effective in enhancing surface expression.

Conclusions:

• Modifications to the TCR constant region, especially murinization, are effective strategies for increasing surface TCR expression in rhesus macaque models.
• These optimized TCR constructs retain antigen-specific functionality, supporting their use in preclinical immunotherapy studies.
• Findings inform the design of TCRs for advanced rhesus macaque models of TCR-based cellular immunotherapy.