Raymond K. Tong

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.

Neuropilin-1 Binds to VEGF121 and Regulates Endothelial Cell Migration and Sprouting

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF165. Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF165 binding to NRP1, surprisingly reduces VEGF121-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF121 binds directly to NRP1; however, unlike VEGF165, VEGF121 is not sufficient to bridge the NRP1·VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF165, but not VEGF121 binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.

Robo4 Maintains Vessel Integrity and Inhibits Angiogenesis by Interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.

CCBE1 Is Essential for Mammalian Lymphatic Vascular Development and Enhances the Lymphangiogenic Effect of Vascular Endothelial Growth Factor-C In Vivo

Rationale: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. Objective: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. Methods and Results: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1+ lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C–mediated lymphangiogenesis in a corneal micropocket assay. Conclusions: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1+ lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.

Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates

Bispecific antibodies engineered to both bind to the primate transferrin receptor and inhibit β-secretase are taken up by the nonhuman primate brain and reduce brain β-amyloid. A Two-Pronged Approach for Central Nervous System Therapeutics The brain has been considered off-limits to antibody therapies because of the blood-brain barrier (BBB), which protects the brain from circulating toxins while selectively transporting essential molecules into the brain. Efforts to use natural transport mechanisms to deliver antibody therapies into the brain have been successful in rodents. Whether a similar approach can be used in primates, including humans, remains unknown. Using bispecific antibodies with one arm binding to the transferrin receptor and the other to an Alzheimer’s disease drug target, we show that therapeutic antibodies can effectively and safely cross the BBB and enter the primate brain, thus paving the way for antibody therapeutics to treat central nervous system diseases in humans. Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target β-secretase (BACE1) can cross the BBB and reduce brain amyloid-β (Aβ) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aβ in a TfR affinity–dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aβ both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

The safety of therapeutic bispecific antibodies that use TfR for delivery to the brain can be improved by reducing affinity for TfR and eliminating antibody effector function. Averting Roadblocks En Route to the Brain The blood-brain barrier represents a formidable blockade preventing therapeutic antibody delivery into the brain. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting therapeutic antibody uptake into the brain. Although TfR can act as a molecular lift to promote brain uptake, little is known about the safety ramifications of this approach. Building on a pair of studies published in Science Translational Medicine, Couch and colleagues now report that when mice were dosed with therapeutic TfR antibodies, the animals showed acute clinical reactions and a reduction in immature red blood cells, known as reticulocytes. TfR bispecific antibodies engineered to lack Fc interactions with immune cells eliminated adverse acute clinical reactions and reduced reticulocyte loss; the extent of reticulocyte loss was also influenced by binding to TfR and interaction with the complement cascade. Because reticulocytes express high levels of TfR, other cell types that express high levels of TfR were also investigated. The authors observed, for example, that the blood-brain barrier remained completely intact after TfR antibodies were administered to mice, despite the high expression of TfR in brain endothelial cells. Finally, multiple doses of TfR/BACE1 bispecific antibodies reduced amyloid-β, a toxic protein implicated in Alzheimer’s disease, with minimal sustained toxicity. Investigation of monkey and human TfR levels in circulating reticulocytes suggested that loss of these cells may be less likely to occur in primates than in mice. The translational implications of these discoveries suggest that the blood-brain barrier is not the only obstacle to surmount on the way to the brain, at least when using TfR as a molecular lift. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2

No going back The mammalian spinal cord coordinates neuronal systems across the body. Axons that cross the spinal cord midline during development first need permission to cross and then instruction not to keep crossing back and forth. Jaworski et al. studied the axonal guidance receptor ROBO3 and found a ligand NELL2 in mice that appears to help in this process. Science, this issue p. 961 Multiple fail-safe signaling pathways ensure that developing axons cross the spinal cord midline no matter what. Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2–mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)–mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

Pharmacokinetic and Pharmacodynamic Analysis of Circulating Biomarkers of Anti-NRP1, a Novel Antiangiogenesis Agent, in Two Phase I Trials in Patients with Advanced Solid Tumors

Purpose: MNRP1685A is a monoclonal antibody to neuropilin-1 (NRP1). We evaluated blood-based pharmacodynamic biomarkers of MNRP1685A in two phase I studies to assess exposure/response relationships to inform target dose and regimen selection. Experimental Design: The phase I studies evaluated escalating doses of MNRP1685A as a single agent or in combination with bevacizumab. Plasma placental growth factor (PlGF), VEGF, and circulating NRP1 (cNRP1) were evaluated at multiple time points using meso-scale discovery (MSD) assays and ELISA, respectively. Plasma PlGF was also measured in a phase I/II trial of bevacizumab in metastatic breast cancer (AVF0776). The association between PlGF and MNRP1685A dose was described by a sigmoid Emax model. cNRP1 and MNRP1685A PK profiles were described using a two-target quasi-steady state (QSS) model. Results: A dose- and time-dependent increase in plasma PlGF and cNRP1 was observed in all patients treated with MNRP1685A. PK/PD analysis showed that bevacizumab and MNRP1685A had an additive effect in elevating PlGF. Predictions based on the two-target QSS model showed that the free drug concentration to maintain greater than 90% saturation of membrane NRP1 (mNRP1) and cNRP1 is about 8 μg/mL. Conclusion: These data show that MNRP1685A inhibits the VEGF pathway in humans as assessed by an increase in plasma PlGF. MNRP1685A seems to enhance bevacizumab-mediated VEGF pathway blockade, as showed by an increase in the magnitude of PlGF elevation when combined with bevacizumab. PK/PD analysis of biomarkers in the phase I population allowed identification of doses at which apparent maximal pathway modulation was observed. Clin Cancer Res; 18(21); 6040–8. ©2012 AACR.

MR angiogenesis imaging with Robo4- vs. αVβ3-targeted nanoparticles in a B16/F10 mouse melanoma model

The primary objective of this study was to utilize MR molecular imaging to compare the 3‐dimensional spatial distribution of Robo4 and αVβ3‐integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16‐F10 melanoma‐bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4‐ or αVβ3‐targeted paramagnetic nanoparticles. The percentage of MR signal‐enhanced voxels throughout the tumor volume was low and increased in animals receiving αVβ3‐ and Robo4‐targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4‐ and αVβ3‐targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with αVβ3‐integrin. Robo4‐ and αVβ3‐targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that αVβ3‐integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but αVβ3‐integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with αVβ3‐integrin expression, may help define tumor character prior to and following cancer therapy.—Boles, K. S., Schmieder, A. H., Koch, A. W., Carano, R. A. D., Wu, Y., Caruthers, S. D., Tong, R. K., Stawicki, S., Hu, G., Scott, M. J., Zhang, H., Reynolds, B. A., Wickline, S. A., and Lanza, G. M. MR angiogenesis imaging with Robo4‐ vs. αVβ3‐targeted nanoparticles in a B16/F10 mouse melanoma model. FASEB J. 24, 4262–4270 (2010). www.fasebj.org