Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode.

Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A(∗)0201) complexed with human T cell lymphotropic virus type 111-19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3ß loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3ß loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.

Monoclonal TCR-redirected tumor cell killing.

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Identification and characterization of a membrane receptor for proteolysis-inducing factor on skeletal muscle.

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (K(d), in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (K(d), 1.1 x 10(-7) mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [(35)S]PIF to triton solublized membranes from C(2)C(12) cells provided evidence for a binding protein of apparent M(r) of approximately 40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target.

Stable, soluble T-cell receptor molecules for crystallization and therapeutics.

Antibody and T-cell receptors (TCRs) are the primary recognition molecules of the adaptive immune system. Antibodies have been extensively characterized and are being developed for a large number of therapeutic applications. This has been possible because of the ability to manufacture stable, soluble, monoclonal antibodies which retain the antigen specificity of B cells. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble trans-membrane domain. Characterization and development of TCRs has been hampered by the lack of suitable methods for producing them as soluble and stable proteins. Here we report the engineering of soluble human TCRs suitable for crystallization studies and potentially for in vivo therapeutic use.