Selection of functional T cell receptor mutants from a yeast surface-display library

Abstract

The heterodimeric ␣␤ T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the ␣-and ␤-chains and in the ␤-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeastdisplayed mutant TCRs bind specifically to the peptide͞MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.T cell receptors (TCRs) and antibodies have evolved to recognize different classes of ligands. Antibodies function as membrane-bound and soluble proteins that bind to soluble antigens, whereas TCRs function only as membrane-bound molecules that bind to cell-associated peptide͞MHC antigens. All of the energy of the antibody:antigen interaction focuses on the foreign antigen, whereas a substantial fraction of the energy of the TCR:peptide͞MHC interaction seems to be directed at the self-MHC molecule (1). In addition, antibodies can have ligand-binding affinities that are orders of magnitude higher than those of TCRs, largely because of the processes of somatic mutation and affinity maturation. In their normal cellular context, TCRs do not undergo somatic mutation and the processes of thymic selection seem to operate by maintaining a narrow window of affinities (2). The association of TCRs at the cell surface with the accessory molecules CD4 or CD8 also may influence the functional affinity of TCRs (3). Despite these differences, the three-dimensional structures of the two proteins are remarkably similar, with the hypervariable regions forming loops on a single face of the molecule that contacts the antigen (4-7).Based on their structural similarities, it is somewhat surprising that there have been significant differences in the success of producing soluble and surface-displayed forms of the extracellular domains of TCRs and antibodies in heterologous expression systems. Many antibodies have now been expressed at high yield and solubility as either intact or Fab-fragment forms or as single-chain (sc) fragment-variable (Fv) proteins. In addition, there are numerous antigen-binding Fv fragments that have been isolated de novo and͞or improved through...

Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR

“…To this end, large libraries can be composed of cDNAs encoding for any antigen/MHC specificity for which human CTL clones are or become available, they can be generated from in vivo isolated T lymphocytes, generated in vitro by specific stimulation, or created from TCR ␣␤ or ␥␦ display libraries. 33,34 In conclusion, genetic engineering of T lymphocyte specificity with chimeric TCR ␣␤ provides an efficient and reliable tool to produce MHC-restricted, antigenspecific human T lymphocytes that may prove of significant use for disease-specific immune gene therapy of, for example, cancer and viral infections.…”

Exploring Biochemistry and Cellular Biology with Protein Libraries

“…Among the many examples of biologically derived library methods, one of the most common is phage display whereby members of a peptide library are fused to ("displayed" on) bacteriophage coat proteins. Other biologically based methods also rely on display of fusion-peptides, such as yeast surface display (Kieke et al, 1999;Yeung and Wittrup, 2002), E. coli surface display (Daugherty et al, 1999), ribosome display (Hanes and Pluckthun, 1997) and mRNA display (Wilson et al, 2001) (Table 1). The key feature of these biological libraries is a direct link between the phenotype (the displayed peptide) and the genotype (DNA encoding the peptide) that allows for selection and amplification of desired peptides.…”

A phage display selected Fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes

“…Thus far, several approaches have been explored to obtain molecules that bind MHC-restricted antigens on tumor cells. First, TCR libraries specific for peptide/MHC complexes were generated, [27][28][29] but engineering of TCR proved difficult and the TCR were of low binding affinity. 30,31 Second, monoclonal antibodies (mAbs), with MHC-restricted specificity were generated by immunizing mice with soluble peptide-MHC complexes or antigen-presenting cells.…”