Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains

Ligands specific for B7.1 (CD80) and B7.2. (CD86) have applications in dise ase indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligan d, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody varia ble light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains. We first describe the "rational" design and construction of a VL/CTLA-4 hyb rid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and st ructure-based alignment. This molecule was secreted as a soluble product fr om Escherichia coil, but did not show any binding to B7.1 and B7.2. In a se cond approach we constructed a VL library in which human VL genes derived f rom B-cells were spiked with the CDR3-like loop of CTLA-4 and further diver sified by DNA shuffling. This library was displayed on phage, and after sel ection gave B7.1 binding ligands which competed with CTLA-4. In order to ev aluate the possible general utility of VL domains as binding ligands, we ha ve constructed a non-biased VL library. From this DNA-shuffled human VL lib rary we have selected single VL domains specific for B7.1, B7.2 or human Ig G. Two B7.1-specific YL ligands and one B7.2-specific VL ligand showed comp etition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies. The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermor e, our studies suggest that it is feasible to create specific single VL dom ains to diverse targets as is the case for single VH domains. (C) 2001 Acad emic Press.