Identifying the structures that contribute to monoclonal antibody (mAb) binding sites (epitopes) within native G protein-coupled receptors (GPCRs) can be useful for developing topological models of the accessible receptor surface, for selecting the most relevant mAbs for therapeutic, diagnostic, and research applications and for distinguishing the intellectual property positions of otherwise similar mAbs. While conventional site-directed mutagenesis studies can identify individual amino acid residues that are critical to mAb binding, defining comprehensive epitopes is difficult and time-consuming for these structurally complex proteins. For example, in studies over the past decade, 13 residues (cumulatively) in the GPCR CCR5 have been reported to contribute to the interactions of five well-studied mAbs. 1-5 However, crystallographically defined epitopes contain an average of 20 contact residues each, 6 so these 13 residues likely represent only a portion of all the amino acids that constitute these five epitopes. Because of the importance of CCR5 in HIV infection and inflammation,7 more mAbs have been raised against the native form of this receptor than most other GPCRs, providing a useful set of tools to map its immunodominant structural regions. Here, we have used a high-throughput structure-function analysis strategy, which we refer to as "shotgun mutagenesis", to comprehensively map the critical residues, and in some cases the critical atoms, for these five epitopes of CCR5.To map mAb epitopes, we used an arrayed library of mutations covering nearly all the amino acids in the protein to identify amino acid changes that resulted in loss of mAb reactivity. This approach enabled each epitope to be rapidly mapped within a period of weeks. To create the mutant library, a parental CCR5 plasmid was first created, containing the full length (1059 bp) cDNA for wild type CCR5, flanked by a N-terminal HA epitope tag and a C-terminal V5 epitope tag. Cellular expression of the wild type tagged construct was confirmed by Western blot, immunofluorescence, and flow cytometry. Random mutations were next introduced into the parental CCR5 cDNA using a PCR-based method (Diversify PCR Random Mutagenesis kit, Clontech). Sequenced clones, most exhibiting one to two substitutions, were then selected from these random mutants to create a library with substitutions spanning the entire protein.The final library comprised 734 mutant CCR5 plasmids with substitutions in 346 of the 352 residues of CCR5 (>98% coverage). The average mutation rate per clone was 1.86 amino acids, and each amino acid position was substituted multiple times (an average of 3.95) across the entire library.We used this selective library of CCR5 mutants to map the epitopes of the anti-CCR5 mAbs CTC8, 45523, 45529, 45533 (R&D Systems), and 2D7 (Becton Dickinson). All five mAbs were originally derived, in three independent immunizations, by injecting mice with cells transiently overexpressing human CCR5. 4, 8 These mAbs are therefore representative of the murine immu...

Rapid proliferation of squamous cell carcinomas of the head and neck (SCCHN) during therapy may contribute to treatment failure. We have investigated the presence of p53 abnormalities in patients with SCCHN as a correlate of proliferation rate and other pathologic and clinical variables. p53 Mutation, as determined by polymerase chain reaction and single-strand conformation polymorphism analysis of microdissected frozen sections of tumor biopsies, was significantly associated with a high labeling index, as determined by in vivo infusion of IUdR and BrdU (P = 0.017). p53 Protein expression was detected by immunohistochemistry with two different antibodies, followed by quantitative image analysis. Many cases exhibited strong p53 protein expression in the absence of mutations within the conserved region of the gene, and expression was not related to proliferation. The presence of p53 mutations was related to tumor differentiation in this group of patients.

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