CHOK1SV GS-KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

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

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.

Cloning, expression, and selective inhibition of canine cyclooxygenase-1 and cyclooxygenase-2.

Cyclooxygenase (COX) performs the critical initial reaction in the arachidonic metabolic cascade, leading to formation of proinflammatory prostaglandins, thromboxanes, and prostacyclins. The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2. Cyclooxygenase-2 inhibitors are also being developed for canine applications. To assess the compound potency on canine enzymes, canine COX-1 and COX-2 were cloned, expressed, and purified. Cyclooxygenase-1 was cloned from a canine kidney complementary DNA (cDNA) library, with 96 % sequence homology to human COX-1. Cyclooxygenase-2 was cloned from canine kidney and lipopolysaccharide-stimulated macrophage cDNA libraries, with a 93 % sequence homology to human COX-2. The arachidonic acid Michaelis constants for canine COX-1 and COX-2 were 4.8 and 6.6 micrometer, respectively, compared with 9.6 and 10.2 micrometer for ovine. Inhibition results indicated that, for all compounds tested, there was no significant difference between potencies determined for canine enzymes and those for human enzymes.

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.

Dual functional monoclonal antibody PF-04605412 targets integrin alpha5beta1 and elicits potent antibody-dependent cellular cytotoxicity.

Integrin α5ß1 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 α5ß1-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.

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'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane ß-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]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.

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

INTRODUCTION: The 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. METHODS: Using a phage display library, we identified several high-affinity fully human monoclonal antibodies with inhibitory activity against both human and rodent IGF.1Rs. RESULTS: These 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. CONCLUSION: These fully human antibodies therefore have the potential to provide an effective anti-tumor biological therapy in the human clinical setting.