The activity of T cells seems to be dependent on the affinity of the TCR/MHC interaction and/or the dissociation rate of the TCR from the peptide/MHC complex ( 15, 16). Interestingly, TCRs, the molecules that have been selected by evolution to bind pMHC complexes, always display very low affinities for their targets (10 to 0.1 μM) ( 14). Nevertheless, most therapeutic applications, including targeting of toxins or cytokines and adoptive immunotherapy, are likely to demand a higher affinity. Despite a moderate affinity of 250 nM, the selected Ab fragment Fab-G8 is highly specific for the HLA-A1/MAGE-A1 complex and does not bind to HLA-A1/MAGE-A3, a complex with a peptide that differs in only three residues from MAGE-A1. We have recently taken advantage of this technique to select a human Fab capable of binding an Ag of strong clinical value ( 1), the tumor-related pMHC complex HLA-A1/melanoma-associated Ag (MAGE)-A1 ( 13). However, it has recently been suggested that in vitro selection of phage libraries displaying Ab fragments could be one of the most efficient ways to select specific pMHC binders ( 3). Specific TCR, engineered from cloned T cells of known pMHC specificity, have been used to visualize cell surface pMHC complexes ( 4, 5), and MHC-restricted, peptide-specific mAb, identified by various immunization and screening schemes, have been isolated and used similarly ( 5, 6, 7, 8, 9, 10, 11, 12). Two potent classes of reagents have been developed to bind to such complexes. Molecules that bind specifically to these peptide MHC (pMHC) 4 complexes, which are involved in the molecular and cellular processes of Ag presentation, have a wide variety of applications, including direct visualization of the pMHC complexes (both intracellular and at the cell surface), specific masking of complexes involved in autoimmune disease, targeted delivery of toxins and drugs, and adoptive transfer of CTLs expressing pMHC-specific molecules involved in the immune response against cancer or viral infections ( 3). The recent increase in understanding of the immune responses associated with cancer and autoimmune disorders can further be advanced by recognizing the importance of the role played by peptide/MHC complexes in these diseases, and such knowledge provides various strategies for immunotherapy ( 1, 2). This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells.
Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells.
Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies.
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