Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1,410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare results of the microarray-based platform with those of a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

Id proteins are a class of dominant-negative antagonists of helix-loop-helix transcription factors and have been shown to control differentiation of a variety of cell types in diverse organisms. Although the importance of Id1 in tumor endothelial cells is well established, the expression and role of the Id1 protein in human cancer cells is controversial. To explore this issue, we developed and characterized a highly specific rabbit monoclonal antibody against Id1 to assess its expression in human breast, prostate, and bladder malignancies. Our results show that in usual types of human mammary carcinomas, the Id1 protein is expressed exclusively in the endothelium. Interestingly, we detected nuclear expression of the Id1 protein in the tumor cells in 10 of 45 cases of poorly differentiated and highly aggressive carcinoma with metaplastic morphology. Similarly, only 1 of 30 prostate cancer samples showed Id1-positive tumor cells, whereas in almost all, endothelial cells showed high Id1 expression. Intriguingly, whereas normal prostate glands do not show any Id1 protein expression, basal layer cells of benign prostate glands in proximity to tumors expressed high levels of the Id1 protein.In contrast to the lack of Id1 expression in the usual types of mammary and prostate cancers, the majority of transitional cell bladder tumors showed Id1 protein expression in both tumor and endothelial cells. These results suggest that further refinement of Id1 expression patterns in a variety of tumor types will be necessary to identify and study the functional roles played by Id1 in human neoplastic processes. (Cancer Res 2006; 66(22): 10870-7)

Rabbit antibodies have been widely used in research and diagnostics due to their high antigen specificity and affinity. Though these properties are also highly desirable for therapeutic applications, rabbit antibodies have remained untapped for human disease therapy. To evaluate the therapeutic potential of rabbit monoclonal antibodies (RabMAbs), we generated a panel of neutralizing RabMAbs against human vascular endothelial growth factor-A (VEGF). These neutralizing RabMAbs are specific to VEGF and do not cross-react to other members of the VEGF protein family. Guided by sequence and lineage analysis of a panel of neutralizing RabMAbs, we humanized the lead candidate by substituting non-critical residues with human residues within both the frameworks and the CDR regions. We showed that the humanized RabMAb retained its parental biological properties and showed potent inhibition of the growth of H460 lung carcinoma and A673 rhabdomyosarcoma xenografts in mice. These studies provide proof of principle for the feasibility of developing humanized RabMAbs as therapeutics.

We present here a label-free microarray-based assay platform that we used to identify inhibitors of vascular endothelial growth factor (VEGF)-kinase-insertion domain receptor (KDR) binding. Supported by a combination of special ellipsometry-based optical detection and small molecule microarrays (SMM), this platform consists of three assays: (1) the first assay detects binding of a target protein with SMM and identifies ligands to the protein as inhibitor candidates; (2) the second assay detects binding of a receptor protein with identical SMM and subsequent binding of the target protein (a sandwich assay) to identify the ligands to the receptor protein that do not interfere with the target-receptor binding; (3) the third assay detects binding of the target protein to the receptor protein in the presence of the ligands of the target protein identified from the first assay, with the receptor protein immobilized to a solid surface through the ligands identified in the second assay, to yield dose-response curves. Using this platform, we screened 7,961 compounds from the National Cancer Institute and found 12 inhibitors to VEGF-KDR (VEGFR2) interactions with IC₅₀ ranging from 0.3 to 60 μM. The inhibitory potency of these inhibitors found in the microarray-based assay was confirmed by their inhibition of VEGF-induced VEGFR2 phosphorylation in a cell-based assay.

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