Joel H. Martin

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.

ERBB3/HER2 Signaling Promotes Resistance to EGFR Blockade in Head and Neck and Colorectal Cancer Models

EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies. Mol Cancer Ther; 13(5); 1345–55. ©2014 AACR.

Bispecific Antibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681–93. ©2017 AACR.

Characterization of the anti-PD-1 antibody REGN2810 and its antitumor activity in human PD-1 knock-in mice

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti–PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1–dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861–70. ©2017 AACR.

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates

Abstract Background For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

Abstract A131: Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC)

Ado-trastuzumab-emtansine or T-DM1, an antibody drug conjugate (ADC) targeting the well-characterized breast cancer oncogene HER2, has shown benefit for breast cancer patients. However, treatment is not indicated for patients whose tumors express low or intermediate levels of HER2. Thus, additional targets for ADC are needed in breast cancer. The lineage-restricted marker prolactin receptor (PRLR) is also expressed in a subset of breast cancers. Unexpectedly, we found that, unlike HER2, low levels of cell-surface PRLR are sufficient to mediate efficient ADC killing of breast ductal carcinoma cells, including T47D. To understand what properties of PRLR vs HER2 allow for efficient cell killing, we compared intracellular trafficking of these two receptors. We found that approximately 90% of a PRLR antibody was internalized by T47D cells within 1h after treatment, and the internalized PRLR Ab co-localized with the lysosomal marker, Lysotracker Red. In contrast, trastuzumab was restricted to the plasma membrane and did not co-localize with Lysotracker Red. Overnight incubation of T47D cells with PRLR Ab, but not trastuzumab, resulted in accumulation in a late lysosomal compartment, as detected using the pH-sensitive marker, pHrodo. Inhibiting protein synthesis with cycloheximide resulted in almost complete degradation of PRLR after 2h, whereas HER2 was degraded only slightly after 4h. The rapid turnover of PRLR was not significantly affected by adding exogenous ligand (prolactin), or by PRLR antibodies, or by proteasome inhibitors, but was blocked by lysosomal inhibitors including bafilomycin A1, and monensin. The signals for this constitutive PRLR internalization and degradation appear to be contained within its cytoplasmic domain, since substitution of the PRLR extracellular domain by that of HER2 still resulted in degradation rates similar to those of full length PRLR. Moreover, simultaneous substitution of two dileucine lysosomal sorting signals contained in the PRLR cytoplasmic domain (e.g. 283LL and 292LL) to alanine significantly diminished constitutive PRLR turnover. In accordance with these data, PRLR ADC, but not T-DM1, induced cell cycle arrest (proportional to PRLR ADC-induced cell killing) in T47D cells, which was completely abolished by lysosomal inhibitors. Taken together, these data indicate that rapid constitutive ligand-independent turnover of PRLR, but not Her2, can deliver high amounts of ADC to lysosomes, resulting in efficient tumor cell killing. Citation Format: Julian Andreev, NIthya Thambi, Frank Delfino, Joel Martin, Marcus P. Kelly, Jessica R. Kirshner, Douglas MacDonald, Nicholas Popadopoulos, Willian Olson, Gavin Thurston. Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A131.

Abstract 4492: Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models

Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy. Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2014-4492