CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth.

Murine mAb 225 was effective against the EGFR tyrosine kinase and inhibited tumor growth in preclinical studies. A phase I trial showed safety, tumor localization, and satisfactory pharmacokinetics. Human:murine chimeric C225 retained biologic activity, which was essential for the conduct of subsequent combination therapy trials and eventual regulatory approval.

The use of phage display peptide libraries for basic and translational research.

Phage display is a molecular technique, whereby genes are displayed in a functional form on the outer surfaces of bacteriophages by fusion to viral coat proteins. The gene product is encoded by a plasmid contained within the virus, which can be recovered and sequenced, linking the genetic information to the function of the protein. Phage display offers a powerful tool for the identification of short peptides or single chain antibodies that can bind and regulate the function of target proteins. One major advantage of phage display lies in its ability to rapidly identify target-specific peptides with pharmacological activity as agonists or antagonists.

Surface-epitope masking (SEM): an immunological subtraction approach for developing monoclonal antibodies targeting surface-expressed molecules.

An immunological subtraction approach, surface-epitope masking (SEM), is described that permits the efficient and selective production of monoclonal antibodies (MAbs) reacting with both known and unknown molecules expressed on the cell surface. The tenet underlying SEM involves blocking (masking) of shared antigens between two target populations, a "driver" and a "tester," and using appropriately modified surface-masked "tester" cells to generate MAbs reacting with surface antigens unique to the "tester population" that differentiate the two antigen sources. SEM has been employed to develop MAbs that react with the multidrug resistance surface-expressed P-glycoprotein (MDR-1) and the human interferon-gamma receptor and two potentially novel tumor-associated antigens (TAAs) expressed on the surface of prostate carcinoma and breast carcinoma cells. In principle, the SEM approach provides an uncomplicated and effective means of developing MAbs, which can also be used to identify genes, associated with important cellular processes involved in normal physiology, such as growth, aging, differentiation, and development. In addition, this strategy is amenable to produce MAbs and identify genes associated with specific disease states, including cancer, neurodegeneration, autoimmunity, and infection with pathogenic agents.

Identification of cancer targets and therapeutics using phage display.

Phage display is a well-established approach for the identification of bioactive peptides and antibody fragments through the use of high diversity libraries. One major advantage of phage display lies in its ability to rapidly identify target-specific reagents with pharmacological activity as agonists or antagonists. Peptides and antibodies have several clinical advantages over traditional small-molecule chemotherapeutics, including specificity, selectivity and potency. Thus, phage display can be used to generate 'targeted therapeutics', which is the current clinical paradigm of choice in the field of oncology.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks.

Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2.

BACKGROUND: Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. RESULTS: Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs) which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75) into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFalpha and TNFbeta and induced an unexpected biological response in a TNFR2-specific manner. CONCLUSIONS: To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

Target validation and drug discovery using genomic and protein-protein interaction technologies.

After the successful completion of the human genome project, mapping of the human proteome has become the next important challenge facing the biotech and pharmaceutical industries. Identification of the 'right' target(s) is now a critical part of the process because of the cost of drug discovery. Compounding this situation is the fact that the pharmaceutical industry faces a further challenge of being able to sustain current and historical growth rates. Hence, the discovery of new drug targets is important for developing new drug leads that can become preclinical drug candidates. Proteomics is the next phase of the effort whereby the human genome can be understood. However, mapping the human proteome presents a daunting challenge. Proteomics involves several essential components with the most significant being the discovery and description of all protein-protein interactions. Once this compendium is available, a secondary and equally important initiative will be to decipher proteins that are differentially expressed in any given disease condition. At this point, the critical focus will be to select the most relevant proteins, understand their partner interactions and then further winnow them to the point where they are relevant pharmaceutical target candidates. This paradigm can be compared to finding the relevant 'needle in the proteome haystack'. This review describes the use of genomic and protein-protein interaction technologies to identify and validate these 'needles' as the first step in the drug discovery process.

Peptides identify the critical hotspots involved in the biological activation of the insulin receptor.

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.