Development of humanized rabbit monoclonal antibodies against vascular endothelial growth factor receptor 2 with potential antitumor effects.

Vascular endothelial growth factor-A (VEGF-A) plays a critical role in physiologic and pathologic angiogenesis through its receptors especially through VEGFR2. The lack of cross-reactivity of monoclonal antibodies with human VEGFR2/mouse Flk-1 is a major obstacle in preclinical developments. In this study, using a unique hybridoma technique, we generated a panel of 30 neutralization anti-VEGFR2 rabbit monoclonal antibodies (RabMAbs) either blocking VEGF/VEGFR2 interaction or inhibiting VEGF-stimulated VEGFR2 tyrosine kinase phosphorylation. Among 18 RabMAbs with human/mouse VEGFR2 cross-reactivity, we humanized one lead candidate RabMAb by Mutational Lineage Guided (MLG) method and further demonstrated its potent inhibition of tumor growth in xenograft mouse model. Our study suggests that RabMAbs are highly relevant for therapeutic applications.

Reassessment of id1 protein expression in human mammary, prostate, and bladder cancers using a monospecific rabbit monoclonal anti-id1 antibody.

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.

Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform.

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 1410 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 the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

Discovering small molecule ligands of vascular endothelial growth factor that block VEGF-KDR binding using label-free microarray-based assays.

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.

A humanized anti-VEGF rabbit monoclonal antibody inhibits angiogenesis and blocks tumor growth in xenograft models.

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.

Overexpression of the transcription factor Foxo4 is associated with rapid glucose clearance.

Leptin treatment ameliorates lipoatrophic diabetes in animal models and humans. Transgenic mice overexpressing leptin (LepTg) are lipoatrophic but not diabetic and thus represent a model for elucidating mechanisms of leptin-mediated glucose homeostasis. In this communication, we show that LepTg mice overexpress the forkhead transcription factor foxo4 in their remnant adipose tissue. To further characterize the role of foxo4 in adipose tissue, we generated transgenic mice overexpressing a constitutive active form of foxo4 (A3foxo4) under the control of the aP2 promoter/enhancer. aP2-A3foxo4 mice are not lipoatrophic but are able to clear glucose rapidly similar to LepTg mice. In addition, both LepTg and A3foxo4 mice show in their adipocytes increased AMP-activated protein kinase (AMPK) phosphorylation, suggesting a link between AMPK, glucose clearance, foxo4 and the leptin axis. These studies shed new light on mechanisms by which leptin treatment improves glucose disposal.

An mRNA loop/bulge in the ferritin iron-responsive element forms in vivo and Was detected by radical probing with Cu-1,10-phenantholine and iron regulatory protein footprinting.

Messenger RNA (mRNA) regulatory elements often form helices specifically distorted by loops or bulges, which control protein synthesis rates in vitro. Do such three-dimensional RNA structures form in vivo? We now observe formation of the internal loop/bulge (IL/B structure) in the IRE (iron-responsive element) of ferritin mRNA expressed in HeLa cells, using radical cleavage with Cu-phen (Cu-1,10-phenantholine), and protection of the loop/bulge by the regulatory protein (IRP), expressed by cotransfection. Cu-phen, a metal coordination complex (MC) selected because of binding and cleavage at the IL/B in solution, recognized the same site in mRNA in HeLa cells. Endogenous reductants apparently substituted for the sulfhydryl activation of Cu-phen cleavage in solution. Selective RNA IL/B recognition by Cu-phen in vivo is emphasized by resistance to cleavage of a mutated, IL/B IRE in ferritin mRNA. Development of small MCs even more selective than Cu-phen can exploit three-dimensional mRNA or viral RNA structures in vivo to manipulate RNA function. Formation in vivo of the IL/B in the ferritin IRE, which is associated in vitro with greater repression than single IRE structures in other mRNAs, likely contributes to larger derepression of ferritin synthesis in vivo triggered by signals for the IRE/IRP system.

Overexpression of leptin in transgenic mice leads to decreased basal lipolysis, PKA activity, and perilipin levels.

Transgenic mice overexpressing leptin (LepTg) exhibit substantial reductions in adipose mass. Since the binding of leptin to its receptor activates the sympathetic nervous system, we reasoned that the lean state of the LepTg mice could be caused by chronic lipolysis. Instead, the LepTg mice exhibited a low basal lipolysis state and their lean phenotype was not dependent on the presence of beta3-adrenergic receptors. In their white adipose tissue, protein levels of protein kinase A, hormone-sensitive lipase, and ADRP were not impaired. However, compared to normal mice, perilipin, perilipin mRNA, and cAMP-stimulated PKA activity were significantly attenuated. Overall, we demonstrate that the lean phenotype of the LepTg mice does not result in a chronically elevated lipolytic state, but instead in a low basal lipolysis state characterized by a decrease in perilipin and PKA activity in white fat.