Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth.

Neuropilin-1 (NRP1) guides the development of the nervous and vascular systems. Binding to either semaphorins or VEGF, NRP1 acts with plexins to regulate neuronal guidance, or with VEGFR2 to mediate vascular development. We have generated two monoclonal antibodies that bind to the Sema- and VEGF-binding domains of NRP1, respectively. Both antibodies reduce angiogenesis and vascular remodeling, while having little effect on other VEGFR2-mediated events. Importantly, anti-NRP1 antibodies have an additive effect with anti-VEGF therapy in reducing tumor growth. Vessels from tumors treated with anti-VEGF show a close association with pericytes, while tumors treated with both anti-NRP1 and anti-VEGF lack this organization. We propose that blocking NRP1 function inhibits vascular remodeling, rendering vessels more susceptible to anti-VEGF therapy.

Vascular endothelial growth factor a in intraocular vascular disease.

UNLABELLED: The vascular beds supplying the retina may sustain injury as a result of underlying disease such as diabetes, and/or the interaction of genetic predisposition, environmental insults, and age. The vascular pathologic features observed in different intraocular vascular diseases can be categorized broadly as proliferation, exemplified by proliferative diabetic retinopathy, leakage such as macular edema secondary to retinal vein occlusion, or a combination of proliferation and leakage, as seen in neovascular age-related macular degeneration (AMD). The World Health Organization has identified diabetic retinopathy and AMD as priority eye diseases for the prevention of vision loss in developed countries. The pathologic transformations of the retinal vasculature seen in intraocular vascular disease are associated with increased expression of vascular endothelial growth factor A (VEGF), a potent endothelial-specific mitogen. Furthermore, in model systems, VEGF alone is sufficient to trigger intraocular neovascularization, and its inhibition is associated with functional and anatomic improvements in the affected eye. Therapeutic interventions with effect on VEGF include intraocular capture and neutralization by engineered antibodies or chimeric receptors, downregulation of its expression with steroids, or alleviation of retinal ischemia, a major stimulus for VEGF expression, with retinal ablation by laser treatment. Data from prospective randomized clinical trials indicate that VEGF inhibition is a potent therapeutic strategy for intraocular vascular disease. These findings are changing clinical practice and are stimuli for further study of the basic mechanisms controlling intraocular angiogenesis. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Characterization of ML-IAP protein stability and physiological role in vivo.

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics.

PURPOSE: To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. METHODS: Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. RESULTS: After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. CONCLUSIONS: We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.

Blocking neuropilin-2 function inhibits tumor cell metastasis.

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.