Juan Estrada

AMG 706, an Oral, Multikinase Inhibitor that Selectively Targets Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors, Potently Inhibits Angiogenesis and Induces Regression in Tumor Xenografts

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.

Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. Mol Cancer Ther; 9(10); 2641–51. ©2010 AACR.

Naphthamides as novel and potent vascular endothelial growth factor receptor tyrosine kinase inhibitors: design, synthesis, and evaluation.

A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure-activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure-activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.

Evaluation of a Series of Naphthamides as Potent, Orally Active Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitors¶

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.

Novel 2,3-dihydro-1,4-benzoxazines as potent and orally bioavailable inhibitors of tumor-driven angiogenesis.

Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration. Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors of intrinsic KDR activity (IC 50 < 0.1 microM) and human umbilical vein endothelial cell (HUVEC) proliferation with IC 50 < 0.1 microM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC: 4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety as a promising platform for generating kinase-based antiangiogenic therapeutic agents.

Anti-tumor activity of motesanib in a medullary thyroid cancer model

Background: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. Aim: The aim of this study was to investigate the effects of motesanib on wild-type and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. Methods/Results: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC50=66 nM), while it had limited activity against mutant Ret C634W (IC50=1100 nM) or Ret M918T (IC50>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. Conclusions: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

Innate and adaptive immunity contribute to the anti-tumor mechanisms of action of OncoVEXmGM-CSF

Meeting abstracts Talimogene laherparepvec, an investigational oncolytic immunotherapy, is a modified herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells. Intralesional administration of talimogene

Abstract 2351: Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models

Background: Talimogene laherparepvec is an oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1), designed to kill neoplastic cells through two mechanisms: a) direct lysis and b) stimulation of a tumor antigen-specific adaptive immune response by way of GM-CSF expression. In order to support mechanism of action studies in a mouse melanoma model relevant to talimogene laherparepvec9s approved U.S. indication, we expressed the HSV-1 entry receptor Nectin1 on B16F10 melanoma cells. B16F10 cells have previously been shown to be resistant to HSV-1 due to lack of entry receptor expression (Miller et al., Mol Ther, 2001). The goal of our work was to determine if mNectin1 expression in B16F10 melanoma cells renders them sensitive to talimogene laherparepvec treatment. For preclinical studies we used OncoVEXmGM-CSF, an HSV-1 modified similarly to talimogene laherparepvec except that murine GM-CSF is expressed instead of human GM-CSF. Herein we describe studies to assess the efficacy of OncoVEXmGM-CSF in a novel B16F10-mNectin1 syngeneic melanoma model. Methods: Parental B16F10 cells were transduced with lentivirus expressing either mNectin1, or eGFP as a control. In vitro sensitivity was determined by plating both cell types in 96-well plates, infecting with serially diluted OncoVEXmGM-CSF and then measuring ATP 72 hours later. For efficacy studies, B16F10 or B16F10-mNectin1 cells were injected subcutaneously on the right flank of female C57BL/6 mice. On day 10, mice were randomized into treatment groups based on tumor volume (n = 10/group). Talimogene laherparepvec was delivered intratumorally (5×106 PFU/dose,) every 3 days during the first week. Tumor growth and body weight were measured twice per week throughout the experiment. Survival analysis was performed using a Kaplan-Meier estimator. Results: B16F10 cells were insensitive to OncoVEXmGM-CSF up to 100 MOI (multiplicity of infection). In contrast, two other syngeneic cell lines, A20 and CT-26, showed MOI IC50 of 1 and 20. B16F10 cells expressing mNectin1 showed high sensitivity to OncoVEXmGM-CSF with an MOI IC50 of 0.01. In contrast, B16F10 cells expressing eGFP were insensitive up to an MOI of 100. In vivo, B16F10 mNectin1 cells showed a similar growth pattern as the parental B16F10 when injected subcutaneously. However, OncoVEXmGM-CSF treatment of B16F10-mNectin1 tumors caused a highly significant (p Conclusion: Expression of mNectin 1 in the B16F10 melanoma cell line confers sensitivity to OncoVEXmGM-CSF in vitro and extends median overall survival of mice bearing B16F10-muNectin1 tumors. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Petia Mitchell, Yannick Bulliard, Pedro J. Beltran. Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2351.

Abstract 2346: Intratumoral administration of OncoVEXmGM-CSF results in local innate immune alterations and induces systemic anti-tumor effects in syngeneic mouse tumor models

Talimogene laherparepvec, is a modified oncolytic herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors, with lytic cell destruction promoting the local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally and is designed to recruit and stimulate antigen presenting cells to further enhance systemic antitumor immune response. While available evidence suggests that talimogene laherparepvec can induce effects in distant tumors via an immune-mediated effect, additional mechanistic evidence is being sought to help better understand the putative systemic effect. For preclinical mechanism of action studies we employed OncoVEX mGM-CSF , an HSV-1 virus modified in the same manner as talimogene laherparepvec except that murine GM-CSF is expressed. We utilized this virus to further dissect the underlying innate and adaptive immunity following viral administration in syngeneic tumor models. Intratumoral administration of OncoVEX mGM-CSF into established murine tumors induced regressions in the injected lesion but also resulted in significant anti-tumor effects in contralateral (uninjected) lesions. We have previously demonstrated by immunohistochemistry that the systemic effects observed in the contralateral lesions are not driven by systemic spread of the virus, but instead correlate with the infiltration of CD3+ T cell populations. Here we report the full phenotypic characterization of immune infiltrates by flow cytometry in both the injected and contralateral lesions. In a time-course fashion, this characterization revealed the initial infiltration of the injected tumor by innate effector cells and a concomitant induction of a potent type I interferon response. This in turn drives the local release of proinflammatory cytokines and chemokines, resulting in the recruitment of adaptive immune subsets to the injected tumor and enhancing systemic anti-tumor reactivity. Additionally, both the primary oncolytic replication of the virus and the activation of type I interferon pathway are directly correlated with STING activity. In conclusion, in vitro and in vivo observations suggest that intratumoral injection of OncoVEX mGM-CSF activates multiple immune-mediated mechanisms of action leading to T-cell activation and induction of anti-tumor immunity in mice. Citation Format: Keegan Cooke, Karen Fitzgerald, Becky Yang, Juan Estrada, Brian Belmontes, Pedro Beltran, Achim K. Moesta. Intratumoral administration of OncoVEX mGM-CSF results in local innate immune alterations and induces systemic anti-tumor effects in syngeneic mouse tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2346.

Abstract 1022: Development and preclinical testing of AMG 780, a fully human antibody targeting angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2)

Targeting the angiopoietins with trebananib (a peptibody that inhibits the interaction between the endothelial cell-selective Tie2 receptor and its ligands Ang1 and Ang2) in combination with paclitaxel was associated with clinical benefit in a phase 3 ovarian cancer clinical trial (TRINOVA 1). Herein, we describe the generation of AMG 780, a fully human IgG2 antibody targeting Ang1 and Ang2. We compared AMG 780 to trebananib in affinity/neutralization ELISA assays and in vivo studies assessing inhibition of tumor growth and endothelial cell proliferation. AMG 780 is a derivative of an Ang2-binding antibody (Ab X). Ab X was generated by panning a human phage display Fab library (Dyax Corporation) against recombinant Ang2. The Ab X Fab was affinity matured by individually substituting every possible amino acid at every position in the light and heavy chain complementarity determining regions (CDRs). Resulting phage clones were interrogated for improved Ang1- and Ang2-binding activity. Clones with improved potency were converted to full antibodies. The most improved heavy chain clones were paired with the most improved light chain clones. The resulting IgGs were tested for neutralization of the interaction between the angiopoietins and their receptor, Tie2, as measured by homogenous time-resolved fluorescence (HTRF). Three resulting fully human IgG2 antibodies (AMG 780, Ab Y, and Ab Z) and the parental clone (Ab X) with potent Ang2 inhibitory activities (IC50 [nM], 0.05-0.10) and a range of Ang1 inhibitory activities (IC50 [nM], 0.31-547.00) were selected for xenograft studies. Mice were implanted with 5x106 Colo205 (human colon carcinoma) cells. When tumors were approximately 200 mm3, 300 µg of each of the four antibodies intraperitoneally or 14 µg of trebananib subcutaneously were administered twice weekly. For viable tumor fraction analyses, treated tumors at the end of the xenograft study were harvested and processed for paraffin histology. To examine endothelial cell proliferation, treated tumors were enzyme digested and stained with anti-CD31, anti-CD45, and anti-BrdU antibodies. The percentage of BrdU-positive tumor-associated endothelial cells (CD31high/CD45neg) was determined by flow cytometry. Relative to an isotope control antibody, all four IgG2 antibodies and trebananib inhibited tumor growth (P Citation Format: James V. Bready, Kyung Lee, Rick Jacobsen, Kevin Graham, Juan Estrada, Stephen A. Kaufman, Dongyin Yu, Angela Coxon, Jon Oliner. Development and preclinical testing of AMG 780, a fully human antibody targeting angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). [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 1022. doi:10.1158/1538-7445.AM2014-1022