Patrick Chames

Natural and designer binding sites made by phage display technology

In the past decade, the drive to develop completely human antibodies for human therapy has led to the development of phage display technology. This technology is able to deliver the ultimate in antibody engineering, that is, high-affinity fully human antibodies to any antigen of choice. Here, this application of phage display technology is reviewed, and the many other antibody-engineering avenues this technology offers are highlighted.

Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC–peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC–peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy. To generate molecules with such a unique fine specificity, we selected a large nonimmune repertoire of phage Fab antibodies on recombinant HLA-A2 complexed with three common antigenic T cell, HLA-A2-restricted epitopes derived from the melanoma differentiation antigen gp100. We were able to isolate a surprisingly large panel of human recombinant Fab antibodies that exhibit a characteristic TCR-like binding specificity to each of the three gp100-derived epitopes, yet unlike TCRs, they did so with an affinity in the nanomolar range. These TCR-like antibodies recognize the native MHC–peptide complex expressed on the surface of antigen-presenting cells. Moreover, they can detect the specific MHC–peptide complexes on the surface of melanoma tumor cells. These results demonstrate the ability to isolate high-affinity human recombinant antibodies with the antigen-specific, MHC-restricted specificity of T cells, and this ability was demonstrated for three different epitopes of the same melanoma-derived antigen.

TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity

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. 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. 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. 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. 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. 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. 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. 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.

Selections on Biotinylated Antigens

Phage antibody library selections on peptides or proteins are usually carried out using antigens directly coated on a plastic surface (e.g. Petri dishes, microtiter plate well, immunotubes, Chapter 9). This straightforward method is easy to perform and has been shown to be very successful for a diverse set of antigens (for review see Winter et al. 1994). However, phage-antibody selections on some proteins and especially on peptides are not always successful, often due to by immobilization-associated features. The main problem observed for selection on peptides is the very poor coating efficiency of some peptides and the altered availability of epitopes on plastic-coated peptides. The direct coating of proteins on plastic is usually more efficient but can also be problematic, because the passive adsorption on plastic at pH 9.6 is a mechanism of protein denaturation. Under these conditions, 95% of adsorbed proteins are non-functional (Butler et al. 1992; Davies et al. 1994). This problem is not very important for a classical ELISA because mostly a small fraction of proteins having a native conformation is still detectable. However, this phenomenon can be very troublesome for phage antibody library selections, because phage antibodies binding to epitopes only present in denatured molecules may be selected.