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 disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable 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 hybrid 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 structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition 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. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.

Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands.

We have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display we selected several CTLA-4-based variants capable of binding to human alphavbeta3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities.

A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies.

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library.

To generate antibodies to defined cell-surface antigens, we used a large phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.