Irvith M. Carvajal

A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor.

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 1013 Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 μM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.

Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an AdnectinTM, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PEGylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (KD, 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15-60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.

Dual Inhibition of Interleukin-23 and Interleukin-17 Offers Superior Efficacy in Mouse Models of Autoimmunity

Therapies targeting either interleukin (IL)-23 or IL-17 have shown promise in treating T helper 17 (Th17)–driven autoimmune diseases. Although IL-23 is a critical driver of IL-17, recognition of nonredundant and independent functions of IL-23 and IL-17 has prompted the notion that dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating autoimmune diseases relative to targeting either cytokine alone. To test this hypothesis, we generated selective inhibitors of IL-23 and IL-17 and tested the effect of either treatment alone compared with their combination in vitro and in vivo. In vitro, using a novel culture system of murine Th17 cells and NIH/3T3 fibroblasts, we showed that inhibition of both IL-23 and IL-17 completely suppressed IL-23–dependent IL-22 production from Th17 cells and cooperatively blocked IL-17–dependent IL-6 secretion from the NIH/3T3 cells to levels below either inhibitor alone. In vivo, in the imiquimod induced skin inflammation model, and in the myelin oligodendrocyte glycoprotein peptide–induced experimental autoimmune encephalomyelitis model, we demonstrated that dual inhibition of IL-17 and IL-23 was more efficacious in reducing disease than targeting either cytokine alone. Together, these data support the hypothesis that neutralization of both IL-23 and IL-17 may provide enhanced benefit against Th17 mediated autoimmunity and provide a basis for a therapeutic strategy aimed at dual targeting IL-23 and IL-17.

CT322, a VEGFR-2 antagonist, demonstrates anti-glioma efficacy in orthotopic brain tumor model as a single agent or in combination with temozolomide and radiation therapy.

Glioblastomas are among the most aggressive human cancers, and prognosis remains poor despite presently available therapies. Angiogenesis is a hallmark of glioblastoma, and the resultant vascularity is associated with poor prognosis. The proteins that mediate angiogenesis, including vascular endothelial growth factor (VEGF) signaling proteins, have emerged as attractive targets for therapeutic development. Since VEGF receptor-2 (VEGFR-2) is thought to be the primary receptor mediating angiogenesis, direct inhibition of this receptor may produce an ideal therapeutic effect. In this context, we tested the therapeutic effect of CT322, a selective inhibitor of VEGFR-2. Using an intracranial murine xenograft model (U87-EGFRvIII-luciferase), we demonstrate that CT322 inhibited glioblastoma growth in vivo and prolonged survival. Of note, the anti-neoplastic effect of CT322 is augmented by the incorporation of temozolomide or temozolomide with radiation therapy. Immunohistochemical analysis of CT322 treated tumors revealed decreased CD31 staining, suggesting that the tumoricidal effect is mediated by inhibition of angiogenesis. These pre-clinical results provide the foundation to further understand long term response and tumor escape mechanisms to anti-angiogenic treatments on EGFR over-expressing glioblastomas.

Abstract 2586: Adnectins as a platform for multi-specific targeted biologics: A novel bispecific inhibitor of EGFR and IGF-IR growth factor receptors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor-1 (IGFR) are transmembrane receptor tyrosine kinases that mediate proliferative and invasive cell signaling in cancer. Inhibition of either receptor reduces tumor growth in both mouse models and in human clinical studies. Blocking the EGFR pathway can induce compensatory activation of the IGFR pathway to drive tumor growth and IGFR inhibition can result in activation of EGFR signaling in preclinical models. Therefore, blocking both receptors simultaneously may achieve superior efficacy to blocking either pathway alone. We developed individual optimized Adnectins™ specific for blocking either EGFR or IGFR signaling and engineered them into a single protein that linked both Adnectins together to construct a bi-specific Adnectin targeting the EGFR and IGFR (EI-tandem). The bifunctional molecule blocked activation of EGFR and IGFR, inhibited both EGF and IGF-induced down-stream cell signaling (MAPK and AKT pathways) and was antiproliferative in human cancer cell lines. Potency of the EI-tandem was comparable to anti-EGFR and anti-IGFR antibodies. The EI-tandem demonstrated a synergistic inhibition of IGFR phosphorylation and down-stream cell signaling compared to Adnectins specific for only EGFR or IGFR alone. Although Adnectins bound to the EGFR at a site distinct from the clinically approved anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, they still blocked binding of EGF to the EGFR. PEGylated EI-tandem inhibited the growth of human tumor xenografts driven by both EGFR and IGFR signaling, degraded EGFR and IGFR, and reduced phosphorylation of EGFR in tumors. Treatment of mice with EI-tandem caused increases in levels of the circulating ligands TGFα and IGF1 resulting from blockade of their respective receptors and provided convenient soluble biomarkers of target suppression. These results show that a bifunctional Adnectin can confer improved biological activity compared to monospecific biologics in tumors where growth is driven by multiple growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2586.

Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations

ABSTRACT Protein therapeutics represent a rapidly growing proportion of new medicines being developed by the pharmaceutical industry. As with any new drug, an Occupational Exposure Limit (OEL) should be developed to ensure worker safety. Part of the OEL determination addresses bioavailability (BA) after inhalation, which is poorly understood for protein therapeutics. To explore this, male Sprague‐Dawley rats were exposed intravenously or by nose‐only inhalation to one of five test proteins of varying molecular size (10–150kDa), including a polyethylene glycol‐conjugated protein. Blood, lung tissue and bronchoalveolar lavage (BAL) fluid were collected over various time‐points depending on the expected test protein clearance (8 minutes‐56 days), and analyzed to determine the pharmacokinetic profiles. Since the BAL half‐life of the test proteins was observed to be>4.5h after an inhalation exposure, accumulation and direct lung effects should be considered in the hazard assessment for protein therapeutics with lung‐specific targets. The key finding was the low systemic bioavailability after inhalation exposure for all test proteins (˜≤1%) which did not appear molecular weight‐dependent. Given that this study examined the inhalation of typical protein therapeutics in a manner mimicking worker exposure, a default 1% BA assumption is reasonable to utilize when calculating OELs for protein therapeutics. HIGHLIGHTSFor typical large proteins (10–150kDa), systemic bioavailability in the rat following inhalation is low, at ˜≤1%.Addition of a PEG moiety on a protein can considerably decrease systemic bioavailability.Direct effects on the lung should be considered when assessing the hazard potential for proteins that have lung targets.The application of a default PK factor adjusting for 1% bioavailability is warranted when calculating OELs.

Adnectin–drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors

Abstract Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody–drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens—but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin–drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues. We used an engineered cysteine to conjugate an Adnectin that binds Glypican-3, a membrane protein overexpressed in hepatocellular carcinoma, to a cytotoxic derivative of tubulysin, with the drug-to-Adnectin ratio of 1. We demonstrate specific, nanomolar binding of this Adnectin–drug conjugate to human and murine Glypican-3; its high thermostability; its localization to target-expressing tumor cells in vitro and in vivo, its fast clearance from normal tissues and its efficacy against Glypican-3-positive mouse xenograft models.