FDA's Approval of the First Biosimilar to Bevacizumab.

Passage of the Biologics Price Competition and Innovation Act of 2009 created an abbreviated licensure pathway for biosimilar products. The FDA approved ABP215 (MVASI, bevacizumab-awwb; Amgen) as a biosimilar to U.S.-licensed Avastin (bevacizumab; Genentech) based on an extensive comparative analytic characterization, data obtained in a pharmacokinetic similarity study in healthy subjects, and a comparative clinical study in patients with non-small cell lung cancer. The totality of the evidence for biosimilarity supported extrapolation of the data to support licensure as a biosimilar for other approved indications of U.S.-licensed Avastin, without the need of additional clinical studies. Clin Cancer Res; 24(18); 4365-70. ©2018 AACR.

Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers.

Expression and secretion of human proinsulin-B10 from mouse salivary glands: implications for the treatment of type I diabetes mellitus.

Adenovirus (Ad) mediated expression of therapeutic proteins from salivary glands can result in the delivery of biologically active proteins into the circulation where they impart their physiological function. In recent years, Ad vector delivery to salivary glands (SGs) has emerged as a viable option for gene therapy. Here, we engineered a variant of human proinsulin (hProinsulin-B10) into an Ad vector and demonstrated its ability to transduce cell lines, and express a bioactive protein that induces the phosphorylation of AKT, a key insulin signaling molecule. We also examined its expression in mice following delivery of the vector to the parotid gland (PTG), the submandibular gland (SMG) or to the liver via the tail vein and assessed transgenic protein expression and vector containment for each delivery method. In all cases, hProinsulin-B10 was expressed and secreted into the circulation. Lower levels of circulating hProinsulin-B10 were obtained from the PTG while higher levels were obtained from the tail vein and the SMG; however, vector particle containment was best when delivered to the SMG. Expression of hProinsulin-B10 in the SMG of chemically induced diabetic mice prevented excessive hyperglycemia observed in untreated mice. These results demonstrate that hProinsulin-B10 can be expressed and secreted into the circulation from SGs and can function physiologically in vivo. The ability to remediate a diabetic phenotype in a model of type 1 diabetes mellitus is the first step in an effort that may lead to a possible therapy for diabetes.

Glucagon-like peptide-1 gene therapy.

Glucagon-like peptide 1 (GLP-1) is a small peptide component of the prohormone, proglucagon, that is produced in the gut. Exendin-4, a GLP-1 receptor agonist originally isolated from the saliva of H. suspectum or Gila monster, is a peptide that shares sequence and functional homology with GLP-1. Both peptides have been demonstrated to stimulate insulin secretion, inhibit glucagon secretion, promote satiety and slow gastric emptying. As such, GLP-1 and Exendin-4 have become attractive pharmaceutical targets as an adjunctive therapy for individuals with type II diabetes mellitus, with several products currently available clinically. Herein we summarize the cell biology leading to GLP-1 production and secretion from intestinal L-cells and the endocrine functions of this peptide and Exendin-4 in humans. Additionally, gene therapeutic applications of GLP-1 and Exendin-4 are discussed with a focus on recent work using the salivary gland as a gene therapy target organ for the treatment of diabetes mellitus.

Prevention of radiation-induced salivary hypofunction following hKGF gene delivery to murine submandibular glands.

PURPOSE: Salivary glands are significantly affected when head and neck cancer patients are treated by radiation. We evaluated the effect of human keratinocyte growth factor (hKGF) gene transfer to murine salivary glands on the prevention of radiation-induced salivary hypofunction. EXPERIMENTAL DESIGN: A hybrid serotype 5 adenoviral vector encoding hKGF (AdLTR(2)EF1α-hKGF) was constructed. Female C3H mice, 8 weeks old, were irradiated by single (15 Gy) or fractionated (6 Gy for 5 days) doses to induce salivary hypofunction. AdLTR(2)EF1α-hKGF or AdControl was administered (10(8) - 10(10) particles per gland) to both submandibular glands (SG) by retrograde ductal instillation before irradiation (IR). Salivary flow was measured following pilocarpine stimulation. Human KGF levels were measured by ELISA. SG cell proliferation was measured with bromodeoxyuridine labeling. Endothelial and progenitor or stem cells in SGs were measured by flow cytometry. The effect of SG hKGF production on squamous cell carcinoma (SCC VII) tumor growth was assessed. RESULTS: In 3 separate single-dose IR experiments, salivary flow rates of mice administered the AdLTR(2)EF1α-hKGF vector were not significantly different from nonirradiated control mice (P > 0.05). Similarly, in 3 separate fractionated IR experiments, the hKGF-expressing vector prevented salivary hypofunction dramatically. Transgenic hKGF protein was found at high levels in serum and SG extracts. AdLTR(2)EF1α-hKGF-treated mice showed increased cell proliferation and numbers of endothelial cells, compared with mice treated with AdControl. hKGF gene transfer had no effect on SCC VII tumor growth ± radiation. CONCLUSIONS: hKGF gene transfer prevents salivary hypofunction caused by either single or fractionated radiation dosing in mice. The findings suggest a potential clinical application.

Gene therapy of salivary diseases.

For many years, our laboratory has been developing gene transfer approaches for salivary gland disorders that currently lack effective therapy. The purpose of this chapter is to describe key methods used in this developmental process. Specifically, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model.

Systemic delivery of bioactive glucagon-like peptide 1 after adenoviral-mediated gene transfer in the murine salivary gland.

An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7-37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic beta-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1's glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis.

Salivary epithelial cells: an unassuming target site for gene therapeutics.

Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer.

Aberrant localization of ezrin correlates with salivary acini disorganization in Sjogren's Syndrome.

OBJECTIVES: To analyse whether the alterations in the structure and organization of microvilli in salivary acinar cells from SS patients are linked to changes in the expression and/or cellular localization of ezrin. METHODS: Salivary gland (SG) acini from controls and SS patients were used to evaluate ezrin expression by western blot and localization of total and activated (phospho-Thr567) ezrin by IF and EM. RESULTS: In acini from control labial SGs, ezrin was located predominantly at the apical pole and to a lesser extent at the basal region of these cells. Conversely, in acini extracts from SS patients, ezrin showed significantly elevated levels, which were accompanied with localization mostly at the basal region. Moreover, F-actin maintained its distribution in both the apical region and basolateral cortex; however, it was also observed in the acinar cytoplasm. Phospho-ezrin (active form) was located exclusively at the apical pole of acinar cells from control subjects and abundantly located at the basal cytoplasm in SS samples. These results were confirmed by immunogold studies. CONCLUSIONS: The decrease of ezrin and phospho-ezrin at the apical pole and the cytoplasmic redistribution of F-actin suggest an altered interaction between the F-actin-cytoskeleton and plasma membrane in SS patient acini, which may explain the microvilli disorganization. These alterations could eventually contribute to SG hyposecretion in SS patients.

Insulin-mediated acceleration of breast cancer development and progression in a nonobese model of type 2 diabetes.

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

Insulin-like growth factor type 1 receptor and insulin receptor isoform expression and signaling in mammary epithelial cells.

The insulin receptor (IR) isoforms and the IGF type 1 receptor (IGF-1R) share a high degree of structural homology but differ in ligand binding kinetics and functions. We developed a highly specific quantitative PCR assay to quantify and compare IR-A, IR-B, and IGF-1R expression within an RNA population. We determined receptor expression in primary murine mammary epithelial cells (MECs) during postnatal development. Both IR isoform mRNAs were 3- to 16-fold higher than IGF-1R expression at all developmental times. IR protein was also 3- to 10-fold higher than IGF-1R protein; however, significantly less IGF-1R was found in hybrid receptors at early (49%) vs. late (79%) pregnancy, indicating that the amount of hybrid receptor is developmentally regulated. Despite high IR expression, IGF ligands were more effective than insulin in stimulating the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt pathway in acutely isolated MECs from virgin glands. Although approximately 40% of IR transcripts were the IGF-II-sensitive IR-A isoform, IGF-II failed to stimulate IR phosphorylation, and an IGF-1R-specific blocking antibody completely abrogated IGF-II-mediated Akt phosphorylation in the virgin MECs. Taken together, these data suggest that the IGF-1R is more active in signaling than the IR and is the predominant mediator of IGF actions in virgin MECs.

IGF ligand and receptor regulation of mammary development.

The insulin-like growth factors, IGF-I and IGF-II, have endocrine as well as autocrine-paracrine actions on tissue growth. Both IGF ligands are expressed within developing mammary tissue throughout postnatal stages with specific sites of expression in the epithelial and stromal compartments. The elucidation of circulating versus local actions and of epithelial versus stromal actions of IGFs in stimulating mammary epithelial development has been the focus of several laboratories. The recent studies addressing IGF ligand function provide support for the hypotheses that (1) the diverse sites of IGF expression may mediate different cellular outcomes, and (2) IGF-I and IGF-II are distinctly regulated and have diverse functions in mammary development. The mechanisms for IGF function likely are mediated, in part, through diverse IGF signaling receptors. The local actions of the IGF ligands and receptors as revealed through recent publications are the focus of this review.

Epithelial-specific and stage-specific functions of insulin-like growth factor-I during postnatal mammary development.

Postnatal development of the mammary gland requires interactions between the epithelial and stromal compartments, which regulate actions of hormones and growth factors. IGF-I is expressed in both epithelial and stromal compartments during postnatal development of the mammary gland. However, little is known about how local expression of IGF-I in epithelium or stroma regulates mammary growth and differentiation during puberty and pregnancy-induced alveolar development. The goal of this study was to investigate the mechanisms of IGF-I actions in the postnatal mammary gland and test the hypothesis that IGF-I expressed in stromal and epithelial compartments has distinct functions. We established mouse lines with inactivation of the igf1 gene in mammary epithelium by crossing igf1/loxP mice with mouse lines expressing Cre recombinase under the control of either the mouse mammary tumor virus long-terminal repeat or the whey acidic protein gene promoter. Epithelial-specific loss of IGF-I during pubertal growth resulted in deficits in ductal branching. In contrast, heterozygous reduction of IGF-I throughout the gland decreased expression of cyclins A2 and B1 during pubertal growth and resulted in alterations in proliferation of the alveolar epithelium and milk protein levels during pregnancy-induced differentiation. Reduction in epithelial IGF-I at either of these stages had no effect on these indices. Taken together, our results support distinct roles for IGF-I expressed in epithelial and stromal compartments in mediating growth of the postnatal mammary gland.

Growth factor regulation of cell cycle progression in mammary epithelial cells.

Growth factors are among the critical positive and negative regulators of cell proliferation for normal mammary/breast epithelial cells and for breast cancer cells. The mechanisms by which specific growth factors regulate the cell cycle in mammary/breast epithelial cells is beginning to be understood for several growth factor families, including the epidermal growth factor, insulin-like growth factor, and transforming growth factor-beta families. A critical issue for understanding how growth factors regulate the cell cycle in vivo is how individual factors interact with other growth factors or hormones to enhance or inhibit specific molecular targets in the cell cycle machinery. This review addresses what is currently known about how growth factors regulate the cell cycle in mammary/breast epithelial cells both individually and in coordination with other growth regulators.

Cytokine and chemokine gene expression after primary and secondary respiratory syncytial virus infection in cotton rats.

The induction of pro- and anti-inflammatory cytokines and chemokines was studied in the lungs of cotton rats after primary or secondary infection with respiratory syncytial virus (RSV). Increases in messenger RNA (mRNA) levels of all genes analyzed were observed during the course of primary infection. In general, mRNA expression peaked between postinfection days 1 and 4 and returned to near-normal levels by day 10. During secondary infection, the expression of some genes (i.e., interferon [IFN]-gamma and interleukin [IL]-10) began earlier, some (i.e., IL-1beta and macrophage inflammatory protein-1beta) began later, and some (i.e., IL-1beta, IL-10, growth-regulated protein, and tumor necrosis factor-alpha) showed prolonged expression, whereas 2 genes (i.e., IFN-alpha and IL-6) were not expressed. This study presents evidence of different kinetics of expression of inflammatory mediators during primary and secondary infection that likely coincide with innate and adaptive immune response and complement previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.