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Carlson K.R.,Health Center | Pomerantz S.C.,Janssen Research and Development LLC | Li J.,Biologics Research | Vafa O.,Janssen Research and Development LLC | And 4 more authors.
BMC Biotechnology | Year: 2015

Background: The therapeutic use of α-amidated peptides (e.g. calcitonin, glucagon-like peptide) has increased dramatically, but there are major impediments to wider use of such peptides. Larger peptides are expensive to synthesize, and short plasma half-lives frequently limit the clinical circumstances in which the peptides would be useful. Both problems are potentially solved by producing peptides as fusions with the Fc region of human immunoglobulin. Methods: Glucagon-like peptide 1 (GLP1), peptide YY (PYY) and neuromedin U (NMU) were expressed and purified from stable CHO lines; since the α-amide group is essential for full biological potency of many peptides, Fc-fusion peptides were expressed in CHO lines stably expressing the α-amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3). Purified fusion proteins were analyzed intact and after HRV3C rhinovirus protease cleavage, at a site in the linker separating the Fc region from the peptide, by mass spectrometry and amide-specific immunoassays. Results: The Fc fusions were expressed at 1-2.5 μg/mg cell protein and secreted at 5-20 % of cell content per hour, in a peptide-specific manner. CHO cells express measurable endogenous PAM activity, amidating 25 % of Fc-PYY and almost 90 % of Fc-GLP1. Expression of exogenous PAM increased the level of peptide amidation to 50 % of Fc-PYY and 95 % of Fc-NMU. The Fc-GLP1 fusions were 10,000-fold less active than synthetic GLP1 in a cell-receptor cyclic AMP-based assay, as expected since the amino terminal of GLP1 is essential for full biological activity. The Fc-PYY fusions were 100-fold less active than PYY-NH2 but 10-fold more active than non-amidated PYY-Gly. Conclusions: This type of approach can be used for the production of stabilized α-amidated peptides aimed at clinical trials. © 2015 Carlson et al. Source


Carlson K.R.,Health Center | Pomerantz S.C.,Janssen Research and Development LLC | Li J.,Biologics Research | Vafa O.,Janssen Research and Development LLC | And 4 more authors.
BMC Biotechnology | Year: 2015

Background: The therapeutic use of aα-amidated peptides (e.g. calcitonin, glucagon-like peptide) has increased dramatically, but there are major impediments to wider use of such peptides. Larger peptides are expensive to synthesize, and short plasma half-lives frequently limit the clinical circumstances in which the peptides would be useful. Both problems are potentially solved by producing peptides as fusions with the Fc region of human immunoglobulin. Methods: Glucagon-like peptide 1 (GLP1), peptide YY (PYY) and neuromedin U (NMU) were expressed and purified from stable CHO lines; since the aα-amide group is essential for full biological potency of many peptides, Fc-fusion peptides were expressed in CHO lines stably expressing the aα-amidating enzyme, peptidylglycine aα-amidating monooxygenase (PAM: EC 1.14.17.3). Purified fusion proteins were analyzed intact and after HRV3C rhinovirus protease cleavage, at a site in the linker separating the Fc region from the peptide, by mass spectrometry and amide-specific immunoassays. Results: The Fc fusions were expressed at 1-2.5μg/mg cell protein and secreted at 5-20% of cell content per hour, in a peptide-specific manner. CHO cells express measurable endogenous PAM activity, amidating 25% of Fc-PYY and almost 90% of Fc-GLP1. Expression of exogenous PAM increased the level of peptide amidation to 50% of Fc-PYY and 95% of Fc-NMU. The Fc-GLP1 fusions were 10,000-fold less active than synthetic GLP1 in a cell-receptor cyclic AMP-based assay, as expected since the amino terminal of GLP1 is essential for full biological activity. The Fc-PYY fusions were 100-fold less active than PYY-NH2 but 10-fold more active than non-amidated PYY-Gly. Conclusions: This type of approach can be used for the production of stabilized aα-amidated peptides aimed at clinical trials. © Carlson et al. 2015. Source


Chiu M.L.,Biologics Research
Current Protocols in Protein Science | Year: 2012

Membrane proteins (MPs)mediate important physiological processes for the cell via extracellular and intracellular interactions. To better understand the biochemical and structural bases of these interactions, well-characterized preparations of purified MPs are required. This introduction reviews common problems encountered in MP preparation. © 2012 John Wiley & Sons, Inc. Source


Zheng S.,Biologics Clinical Pharmacology | Moores S.,U.S. Biology Oncology | Jarantow S.,Biologics Research | Pardinas J.,Biologics Research | And 3 more authors.
mAbs | Year: 2016

abstract: Multispecific proteins, such as bispecific antibodies (BsAbs), that bind to two different ligands are becoming increasingly important therapeutic agents. Such BsAbs can exhibit markedly increased target binding and target residence time when both pharmacophores bind simultaneously to their targets. The cross-arm binding efficiency (χ) describes an increase in apparent affinity when a BsAb binds to the second target or receptor (R2) following its binding to the first target or receptor (R1) on the same cell. χ is an intrinsic characteristic of a BsAb mostly related to the binding epitopes on R1 and R2. χ can have significant impacts on the binding to R2 for BsAbs targeting two receptors on the same cell. JNJ-61186372, a BsAb that targets epidermal growth factor receptor (EGFR) and c-Met, was used as the model compound for establishing a method to characterize χ. The χ for JNJ-61186372 was successfully determined via fitting of in vitro cell binding data to a ligand binding model that incorporated χ. The model-derived χ value was used to predict the binding of JNJ-61186372 to individual EGFR and c-Met receptors on tumor cell lines, and the results agreed well with the observed IC50 for EGFR and c-Met phosphorylation inhibition by JNJ-61186372. Consistent with the model, JNJ-61186372 was shown to be more effective than the combination therapy of anti-EGFR and anti-c-Met monovalent antibodies at the same dose level in a mouse xenograft model. Our results showed that χ is an important characteristic of BsAbs, and should be considered for rationale design of BsAbs targeting two membrane bound targets on the same cell. © 2016 Jansenn R&D. Source


Schaffner F.,Scripps Research Institute | Schaffner F.,University of Strasbourg | Yokota N.,Scripps Research Institute | Carneiro-Lobo T.,Scripps Research Institute | And 6 more authors.
PLoS ONE | Year: 2013

Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR+ cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR+ cells or the heterogenous mixture of EPCR+ and EPCR- cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment. © 2013 Schaffner et al. Source

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