Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

Abstract

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157–165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3α and CDR2β amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1+/HLA-A*02+ tumor cell lines by TCR gene-modified CD4+ T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3α chain was identified that enhanced Ag-specific reactivity in gene-modified CD4+ and CD8+ T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27–35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4+ T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

Ca2+ Release from the Endoplasmic Reticulum of NY-ESO-1–Specific T Cells Is Modulated by the Affinity of TCR and by the Use of the CD8 Coreceptor

“…Because natural tumor Ags elicit relatively weak T cell responses, two approaches currently being investigated are the optimization of the MHC class I anchor residues in tumor epitopes, to enhance binding of the peptide to the MHC class I molecule (1)(2)(3), and site-directed mutagenesis of TCRs to enhance T cell effector function of adoptively transferred T cells (4). Although in vitro and in vivo data with peptide analogs and mutagenized TCR showed larger Ag-specific CTL expansions than T cell proliferation obtained with wild-type peptides (5,6) or wild-type TCRs (7), the use of peptide agonists and mutated TCRs in clinical applications needs to be carefully monitored, because such strategies may result in the loss of Ag specificity due to the enhanced T cell reactivity, as reviewed by Iero et al (8). Recent results have extended this notion by demonstrating Ag cross-reactivity when CD8 + T cells were transfected with a higher-affinity variant of the NY-ESO 157-165 A2-restricted 1G4 TCR (7), whereas the same soluble higher-affinity 1G4 TCR was capable of specifically recognizing NY-ESO 157-165 -pulsed target cells (9).…”

“…Although in vitro and in vivo data with peptide analogs and mutagenized TCR showed larger Ag-specific CTL expansions than T cell proliferation obtained with wild-type peptides (5,6) or wild-type TCRs (7), the use of peptide agonists and mutated TCRs in clinical applications needs to be carefully monitored, because such strategies may result in the loss of Ag specificity due to the enhanced T cell reactivity, as reviewed by Iero et al (8). Recent results have extended this notion by demonstrating Ag cross-reactivity when CD8 + T cells were transfected with a higher-affinity variant of the NY-ESO 157-165 A2-restricted 1G4 TCR (7), whereas the same soluble higher-affinity 1G4 TCR was capable of specifically recognizing NY-ESO 157-165 -pulsed target cells (9).…”

T-cell receptor affinity and avidity defines antitumor response and autoimmunity in T-cell immunotherapy

“…Substantial evidence indicates a correlation between T-cell functional activity and TCR affinity (12)(13)(14)(15)(16)(17)(18). Subsequently, efforts have been taken to generate modified high-affinity TCRs for clinical studies (19)(20)(21). However, this correlation remains controversial: high-affinity TCRs have been shown to lead to stronger (22), plateaued (12,17), or even attenuated (23)(24)(25) T-cell responses.…”

Engineered Drug Resistant Cell-Mediated Immunotherapy

“…Furthermore rearrangements between the chimeric and the naïve TCR chains can induce new and unwanted reactivities. To combat such undesired consequences TCR negative lymphocytes have been modified to harbor αβ transgenic TCR while the sequences of such chimeric αβ T C R s h a v e b e e n redesigned to reduce cross competition within T lymphocytes harboring naïve TCR (Kuball et al, 2007;Robbins et al, 2008).…”