Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition

Abstract

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric ␣␤ TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virusinfected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax 11-19 epitope bound to HLA A2-HuD 87-95 (K D 120 M by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K D 2.5 nM) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.Peptide major histocompatibility complex (pMHC) 1 antigens displayed on the surface of target cells are recognized by T cells via their specific T cell receptor (TCR) (1). The TCR coreceptors CD8 or CD4 bind to invariant domains of pMHC class I (pMHCI) or pMHC class II (pMHCII) and are known to facilitate the process of antigen recognition by T cells (2). Recent advances have enabled the generation of high quality soluble TCR, pMHCI, pMHCII, CD8, and CD4 proteins; these in turn have allowed the biophysical characterization of the interactions between these molecules. Accordingly, the TCR and CD4/8 co-receptors have been shown to have very low affinities (K D ϳ 10 Ϫ3 -10 Ϫ6 M) for cognate pMHC. Despite these low affinity interactions, however, the process of antigen engagement can initiate T cell recognition of antigen-presenting cells (APCs) bearing fewer than 10 copies of a specific pMHC complex (3, 4). The mechanisms by which these weak recognition events result in such exquisite sensitivity are not fully understood.The production of soluble recombinant ␣␤ T cell receptors has proved challenging. The main technical pitfall is heterodimeric instability in the absence of anchoring transmembrane domains and ␣/␤ pairing through an interchain disulfide bond. One of the commonest protein engineering strategies used in TCR studies to date has been the construction of singlechain TCRs. This technique, which takes advantage of the structural similarities between antibodies and TCRs, is based upon the single-chain Fv technology used to generate antibody fragments (5). In short, for the TCR it involves the cloning and expression of a unique chimerical open reading frame where the variable (V) ␣ and V␤ domains are paired with a protein linker (6, 7). Thes...

Visualization of p53264–272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor

“…Thus, it appears to be possible to improve the avidity/affinity of the 264scTCR molecule for peptideloaded APCs by increasing its valency. The results of this experiment are in accord with recent observations made by Laugel et al (19) that tetrameric TCRs bind to peptide/MHC complexes better than monomeric TCRs. To evaluate the sensitivity of the 264scTCR/multimer as a staining reagent, we performed flow cytometric analysis with T2 cells loaded with various concentrations of cognate peptides.…”

“…Thus, multimerization of the scTCR results in a reagent that binds to peptide/MHC complexes with a half-life of 20 min compared with ϳ7 s for the monomer. Similar observations related to changes in peptide/MHC binding kinetics with this type of molecule by multimerization have been reported by other laboratories (19,34). In addition, we have previously shown that dimerization of 264scTCR using an Ig H chain tail did increase both the K a and K d of the molecule while binding to the p53 264 -272 /HLA-A2.1 complexes.…”

“…However, we found that the 264scTCR/multimers could stain cells pulsed with as little as 12 nM peptide and that the addition of ␤2-microglobulin was not required. Because the A6 TCR has a K D of ϳ1 M for Tax [11][12][13][14][15][16][17][18][19] peptide/HLA-A2.1 complex (19), which is similar to that of the 264scTCR for its peptide/MHC complex, the difference in sensitivity between the 264scTCR and A6 TCR in flow cytometry analyses is not due to large differences in affinities for their cognate peptide/MHC complexes. Whether this reflects a difference in the relative stability of the peptide/MHC complexes per se or structural differences resulting from using a single chain vs a heterodimer format for each TCR multimer is unknown.…”

“…Comparisons between the minimum amounts of exogenous peptide required to achieve TCR staining suggest that the methods described here are significantly more sensitive than those reported by others. For example, Laugel et al (19) reported that staining peptide-loaded APCs with either JM22 (Flu matrixspecific) or A6 TCR (human T cell lymphotropic virus type 1 Tax-specific) multimers required at least 10 M cognate peptide. This detection threshold could be lowered to 1 M peptide following MHC stabilization by exogenous ␤2-microglobulin.…”

A T cell receptor associated with naturally occurring human tumor immunity

“…Immunodominant PCD epitopes have not previously been identified, either from our own previous screens or from other studies (35)(36)(37). We find that previously identified candidates, as well as five new peptides, are naturally processed and presented by Ad-cdr2-immunized HLA-A2.1 transgenic mice.…”