Abstract
Soluble T‐cell receptors (TCRs) have recently gained visibility as target‐recognition units of anticancer immunotherapeutic agents. Here, we improved the thermal stability of the well‐expressed high‐affinity A6 TCR by introducing pairs of cysteines in the invariable parts of the α‐ and β‐chain. A mutant with a novel intradomain disulfide bond in each chain also tested superior to the wild‐type in the accelerated stability assay. Binding of the mutant to the soluble cognate peptide (cp)–MHC and to the peptide‐loaded T2 cell line was equal to the wild‐type A6 TCR. The same stabilization motif worked efficiently in TCRs with different specificities, such as DMF5 and 1G4. Altogether, the biophysical properties of the soluble TCR molecule could be improved, without affecting its expression level and antigen‐binding specificity.
Highlights
Soluble T-cell receptors (TCRs) have recently gained visibility as targetrecognition units of anticancer immunotherapeutic agents
FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies
Sadio et al The TCR elements concerned with antigen binding include primarily the a- and b- variable domains, which has triggered the development of soluble TCR formats, such as a/b heterodimer or only the variable domains connected with a polypeptide linker sequence [15–17], a disulfide bond [4,18] or another dimerization agent, such as the constant domain of antibody kappa-light chain [19]
Summary
Soluble T-cell receptors (TCRs) have recently gained visibility as targetrecognition units of anticancer immunotherapeutic agents. Their use for therapeutic purposes has traditionally relied on their expression as membrane-bound molecules expressed on the surface of T lymphocytes with TCR a- and b-variable domains used for antigen recognition [1,2] Due to their unique target repertoire not accessible to antibodies, soluble TCRs are attractive targeting moieties of agents that have already entered clinical trials [3]. The polypeptide linkage between the variable a-and b- domains allowed their display as single-chain TCRs on the surface of phage particle [5] or yeast cell [6], and directed evolution of the derived libraries enabled the increase of their intrinsic low micromolar binding affinity toward the cognate antigen for several orders of magnitude [7,8] These improved properties promise to expand their potential use in adoptive cell therapies [9,10], and enabled their clinical application as soluble fusion proteins with a single-chain antibody specific for CD3e subunit [11–13]. Folding of the TCR in the endoplasmic reticulum (ER) is critically dependent on the formation of the a/b heterodimer [20], especially the folding of the TCR constant domain Ca, which displays little homology to other Ig domains and strongly deviates from the Ig fold [21]
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