Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Abstract

The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

“…This yielded a detection threshold of *400 pMHC complexes per cell as the minimal number of complexes detected by the D2 Fab antibody. Similar sensitivities were obtained with other TCR-like antibodies targeting tumor or viral complexes isolated and characterized by us using the same antibody phage library used to select the D2 Fab [22,23]. …”

“…These enabled us to screen these large libraries of such vast diversity and size in order to isolate these rare and unique TCR-like recombinant antibodies targeting human tumor and viral epitopes [21,22,[24][25][26].…”

“…Using antibody phage display approaches, we have recently generated such unique molecules, coined TCR-like antibodies, against a variety of tumor and viral epitopes restricted to human HLA-A2, the most frequent MHC allele in humans. These antibodies were utilized to detect, visualize and quantitate the number of specific tumor-and viral-associated HLA-A2/peptide complexes on tumor cells, APC, and virus-infected cells [21][22][23][24][25][26].…”

“…Using antibody phage display approaches, we have recently generated such unique molecules, coined TCR-like antibodies, against a variety of tumor and viral epitopes restricted to human HLA-A2, the most frequent MHC allele in humans. These antibodies were utilized to detect, visualize and quantitate the number of specific tumor-and viral-associated HLA-A2/peptide complexes on tumor cells, APC, and virus-infected cells [21][22][23][24][25][26].Another possible application of TCR-like antibodies is their use as targeting molecules to efficiently eliminate tumor cells due to the fact that they can mimic the unique fine specificity of T cells. This can be achieved by using the antibodies or their fragments as targeting moieties and arming them with effector modalities such as potent toxins, chemotherapeutic drugs, isotopes, or biological entities such as cytokines [27][28][29][30][31][32][33].…”

Novel antibodies as anticancer agents

“…The binding properties and kinetics of antibodies were utilized for studying MHC function, T-cell recognition and antigen processing and presentation (Denkberg et al, 2002b;Cohen et al, 2003c;Yamano et al, 2004). For example, it was assumed that each amino acid in the peptide binds independently of one another to enhance or detract from the overall binding affinity and that residues at the anchor positions (usually positions two and nine) do not affect the conformation of residues elsewhere in the peptide.…”