Gerald Fries Casperson

Targeting Activin Receptor-Like Kinase 1 Inhibits Angiogenesis and Tumorigenesis through a Mechanism of Action Complementary to Anti-VEGF Therapies

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

PF-03732010: a fully human monoclonal antibody against P-cadherin with antitumor and antimetastatic activity.

Purpose: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. Experimental Design: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. Results: PF-03732010 selectively inhibits P-cadherin–mediated cell adhesion and aggregation in vitro. In the P-cadherin–overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3′-[18F]fluoro-3′-deoxythymidine–positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane β-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3′-[18F]fluoro-3′-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. Conclusions: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials. Clin Cancer Res; 16(21); 5177–88. ©2010 AACR.

Dual Functional Monoclonal Antibody PF-04605412 Targets Integrin α5β1 and Elicits Potent Antibody-Dependent Cellular Cytotoxicity

Integrin α5β1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5β1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies.

Human monoclonal antibodies to the insulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies

IntroductionThe insulin-like growth factor type 1 (IGF-1) receptor contributes importantly to transformation and survival of tumor cells both in vitro and in vivo, and selective antagonists of the IGF-1 receptor (IGF-1R) activity represent an attractive experimental approach for human cancer therapy.MethodsUsing a phage display library, we identified several high-affinity fully human monoclonal antibodies with inhibitory activity against both human and rodent IGF.1Rs.ResultsThese candidate therapeutic antibodies recognized several distinct epitopes and effectively blocked ligand-mediated receptor signal transduction and cellular proliferation in vitro. They also induced IGF-1R downregulation and catabolism following antibody-mediated endocytosis. These antibodies exhibited activity against human, primate, and rodent IGF-1Rs, and dose-dependently inhibited the growth of established human tumors in nude mice.ConclusionThese fully human antibodies therefore have the potential to provide an effective anti-tumor biological therapy in the human clinical setting.

Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics

ABSTRACT Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.

Abstract 3811: Robust anti-tumor activity through targeting integrin α5β1 and eliciting ADCC with a dual functional monoclonal antibody PF-04605412

Integrin α5β1 and its ligand fibronectin are significantly and coordinately over-expressed in tumor associated blood vessels and cancer cells. These molecules have been reported to mediate angiogenesis, tumor survival and metastasis. α5β1 has also been implicated as a poor prognostic marker in a number of human tumor types including NSCLC and ovarian carcinoma. Thus, α5β1 is a potential target for cancer therapy. Accumulating evidence suggests an important role of innate immune effector cells in tumor killing via antibody dependent cellular cytotoxicity (ADCC). Taking advantage of both of these mechanisms, we generated an α5 integrin-specific neutralizing monoclonal antibody (mAb, PF-04605412) with high affinity for Fcγ receptors. In vitro, PF-04605412 potently inhibited α5β1-mediated intracellular signaling, cell adhesion, migration, endothelial cell tubulogenesis, and induced apoptosis of endothelial and tumor cells. Compared to a wild type IgG1 of identical antigen specificity (wt IgG1), PF-04605412 induced a significantly greater tumor and endothelial cell cytotoxicity. ADCC activity correlated with the abundance of NK cells within PBMCs of human donors, but was independent of donor FcγRIIIa polymorphism. In in vivo studies using human xenograft, immuno-xenograft and syngeneic tumor models, PF-04605412 (and an anti-murine α5 surrogate mAb) displayed robust and dose-dependent anti-angiogenesis and anti-tumor efficacy including tumor growth delay, regression and metastasis inhibition, all of which were superior to effects observed with an IgG2 or wt IgG1 of identical antigen specificity. Western blotting and immunohistochemistry analysis of tumor samples showed that anti-tumor efficacy was associated with reduction of total and phosphorylated focal adhesion kinase and positively correlated with α5 expression, infiltrating macrophages, markers of effector cell activity, and apoptosis. Furthermore, depletion of host macrophages abrogated anti-tumor efficacy, indicating a critical contribution of ADCC/phagocytosis to the anti-tumor activity of PF-04605412. The PK-PD relationship, efficacy of PF-04605412 in combination with anti-angiogenic and tumor targeting agents will also be reported. The distinct dual mechanism of action and robust anti-tumor efficacy of PF-04605412 supports the clinical development of this agent for the treatment of human cancers. Short Title: Dual functional mAb PF-04605412 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 3811.