Publications by authors named "Hossein Salimi-Moosavi"

27 Publications

  • Page 1 of 1

The serum protein transthyretin as a platform for dimerization and tetramerization of antibodies and Fab fragments to enable target clustering.

J Biol Chem 2020 07 9;295(30):10446-10455. Epub 2020 Jun 9.

Amgen Research, Amgen Inc., South San Francisco, California, USA.

Transthyretin (TTR) is an abundant homotetrameric serum protein and was selected here for engineering higher-valency molecules because of its compact size, simple structure, and natural propensity to tetramerize. To demonstrate this utility, we fused TTR to the C terminus of conatumumab, an antibody that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2, as heavy chains to form antibody dimers and Fab heavy chains to form Fab tetramers. Moreover, we used constant heavy domain 3 heterodimerization substitutions to create TTR-mediated conatumumab tetramers. The conatumumab-TTR fusions displayed substantially enhanced potency in cell-based assays, as well as in murine tumor xenograft models. We conclude that antibody-TTR fusions may provide a powerful platform for multimerizing antibody and Fab fragments to enhance the capabilities of human therapeutics that benefit from target clustering and higher-order antigen-binding valency.
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http://dx.doi.org/10.1074/jbc.RA120.013135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383373PMC
July 2020

Rapid single B cell antibody discovery using nanopens and structured light.

MAbs 2019 Aug/Sep;11(6):1025-1035. Epub 2019 Jun 11.

b Department of Therapeutic Discovery, Amgen Research , Burnaby , Canada.

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.
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http://dx.doi.org/10.1080/19420862.2019.1624126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748590PMC
January 2020

Pharmacokinetic comparison of a diverse panel of non-targeting human antibodies as matched IgG1 and IgG2 isotypes in rodents and non-human primates.

PLoS One 2019 23;14(5):e0217061. Epub 2019 May 23.

Amgen Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, United States of America.

In this study we compared the pharmacokinetic profile of four unrelated antibodies, which do not bind to mammalian antigens, in IgG1 and IgG2 frameworks in both rats and non-human primates (NHP). This allowed for extensive cross comparison of the impact of antibody isotype, complementarity determining regions (CDR) and model species on pharmacokinetics without the confounding influence of antigen binding in the hosts. While antibody isotype had no significant impact on the pharmacokinetics, the CDRs do alter the profile, and there is an inverse correlation between the neonatal Fc receptor (FcRn) affinity and pharmacokinetic performance. Faster clearance rates were also associated with higher isoelectric points; however, although this panel of antibodies all possess basic isoelectric points, ranging from 8.44 to 9.18, they also have exceptional in vivo half-lives, averaging 369 hours, and low clearance rates, averaging 0.18 ml/h/kg in NHPs. This pattern of pharmacokinetic characteristics was conserved between rats and NHPs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217061PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533040PMC
February 2020

Engineering Na1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

ACS Chem Biol 2019 04 27;14(4):806-818. Epub 2019 Mar 27.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel Na1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed Na1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNa1.7 IC = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable Na in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained Na1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a Na1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage Na1.7 in vivo.
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http://dx.doi.org/10.1021/acschembio.9b00183DOI Listing
April 2019

Late-life targeting of the IGF-1 receptor improves healthspan and lifespan in female mice.

Nat Commun 2018 06 19;9(1):2394. Epub 2018 Jun 19.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.

Diminished growth factor signaling improves longevity in laboratory models, while a reduction in the somatotropic axis is favorably linked to human aging and longevity. Given the conserved role of this pathway on lifespan, therapeutic strategies, such as insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibodies (mAb), represent a promising translational tool to target human aging. To this end, we performed a preclinical study in 18-mo-old male and female mice treated with vehicle or an IGF-1R mAb (L2-Cmu, Amgen Inc), and determined effects on aging outcomes. Here we show that L2-Cmu preferentially improves female healthspan and increases median lifespan by 9% (P = 0.03) in females, along with a reduction in neoplasms and inflammation (P ≤ 0.05). Thus, consistent with other models, targeting IGF-1R signaling appears to be most beneficial to females. Importantly, these effects could be achieved at advanced ages, suggesting that IGF-1R mAbs could represent a promising therapeutic candidate to delay aging.
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http://dx.doi.org/10.1038/s41467-018-04805-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008442PMC
June 2018

Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

J Biol Chem 2017 02 19;292(5):1865-1875. Epub 2016 Dec 19.

From the Biologics Optimization-Therapeutic Discovery, Clinical Immunology, and Process Development, Amgen Inc., Thousand Oaks, California 91320

IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects. Here an attempt was made to engineer an IgG1-based scaffold lacking effector function but with stability equivalent to that of the parent IgG1. Care was taken to ensure that both stability and lack of effector function was achieved with a minimum number of mutations. Among the Asn mutants that result in lack of glycosylation and thus loss of effector function, we demonstrate that the N297G variant has better stability and developability compared with the N297Q or N297A variants. To further improve the stability of N297G, we introduced a novel engineered disulfide bond at a solvent inaccessible location in the CH2 domain. The resulting scaffold has stability greater than or equivalent to that of the parental IgG1 scaffold. Extensive biophysical analyses and pharmacokinetic (PK) studies in mouse, rat, and monkey further confirmed the developability of this unique scaffold, and suggest that it could be used for all Fc containing therapeutics (e.g. antibodies, bispecific antibodies, and Fc fusions) requiring lack of effector function or elimination of binding to Fcγ receptors.
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http://dx.doi.org/10.1074/jbc.M116.748525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290958PMC
February 2017

A bispecific antibody targeting sclerostin and DKK-1 promotes bone mass accrual and fracture repair.

Nat Commun 2016 05 27;7:11505. Epub 2016 May 27.

Cardiometabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA.

Inhibition of the Wnt antagonist sclerostin increases bone mass in patients with osteoporosis and in preclinical animal models. Here we show increased levels of the Wnt antagonist Dickkopf-1 (DKK-1) in animals treated with sclerostin antibody, suggesting a negative feedback mechanism that limits Wnt-driven bone formation. To test our hypothesis that co-inhibition of both factors further increases bone mass, we engineer a first-in-class bispecific antibody with single residue pair mutations in the Fab region to promote efficient and stable cognate light-heavy chain pairing. We demonstrate that dual inhibition of sclerostin and DKK-1 leads to synergistic bone formation in rodents and non-human primates. Furthermore, by targeting distinct facets of fracture healing, the bispecific antibody shows superior bone repair activity compared with monotherapies. This work supports the potential of this agent both for treatment and prevention of fractures and offers a promising therapeutic approach to reduce the burden of low bone mass disorders.
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http://dx.doi.org/10.1038/ncomms11505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894982PMC
May 2016

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics.

Anal Biochem 2015 Feb 27;470:52-60. Epub 2014 Oct 27.

Department of Pharmacokinetics and Drug Metabolism, Amgen, Thousand Oaks, CA 91320, USA.

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.
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http://dx.doi.org/10.1016/j.ab.2014.10.007DOI Listing
February 2015

Inhibition of CRAC with a human anti-ORAI1 monoclonal antibody inhibits T-cell-derived cytokine production but fails to inhibit a T-cell-dependent antibody response in the cynomolgus monkey.

J Immunotoxicol 2015 Apr-Jun;12(2):164-73. Epub 2014 Jul 3.

Department of Inflammation Research .

ORAI1 is the pore-forming component of calcium release-activated calcium (CRAC) channels. CRAC channels are the primary route for calcium ion (Ca(2+)) entry into T-cells following antigen stimulation. This Ca(2+) entry induces proliferation and cytokine production through activation of calcineurin and the nuclear factor of activated T-cells (NFAT) transcription factor along with subsequent cytokine-related genes. It was hypothesized that the in vivo inhibition of T-cell function by blocking ORAI1 or calcineurin would lead to similar functional consequences. To test this hypothesis the activity of 2C1.1, a fully human anti-ORAI1 monoclonal antibody, and cyclosporin A (CsA) were tested in vivo for their suppressive effect on T-cell-derived cytokine production and a T-cell-dependent antibody response (TDAR) using sheep red blood cells (SRBC) in cynomolgus monkeys. Despite showing similar inhibition of ex vivo interleukin (IL)-2 production by stimulated T-cells, both molecules exhibited different pharmacologic effects on the SRBC antibody response. CsA blocked the development of SRBC-specific antibodies, while 2C1.1 failed to inhibit the antigen-specific antibody response. These surprising observations suggest that full inhibition of the CRAC channel is required to inhibit a functional immune response, consistent with findings from human patients with loss of function mutations in ORAI1.
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http://dx.doi.org/10.3109/1547691X.2014.915897DOI Listing
December 2015

Evaluating the impact of matrix effects on biomarker assay sensitivity.

Bioanalysis 2014 Apr;6(8):1081-91

Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

Background: The accuracy of highly sensitive biomarker methods is often confounded by the presence of various circulating endogenous factors in samples causing matrix effects.

Method: This article outlines two different biomarker methods: hepcidin enzyme-linked immunosorbent assay (ELISA) for which an orthogonal assessment of ELISA to liquid chromatography-tandem mass spectrometry was performed to examine the potential matrix effect, and sclerostin ELISA to evaluate the matrix effect.

Results: Although the potential interfering effects of the endogenous hepcidin variants (prohepcidin and clipped) showed that these proteins had >30% immunoreactivity in ELISA, the hepcidin ELISA preferentially measures full-length hepcidin when the molar ratios of full-length to variants remain >1. The correlation of ELISA to liquid chromatography-tandem mass spectrometry results showed full-length hepcidin as the major form in diseased populations.

Conclusion: A fit-for-for-purpose assessment of matrix effect/selectivity was also performed for each method. This article demonstrates the utility of a fit-for-purpose approach to assess the validity of biomarker methods in evaluating the interconnected parameters of matrix effects, sensitivity and selectivity.
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http://dx.doi.org/10.4155/bio.14.55DOI Listing
April 2014

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates.

Blood 2013 Oct 14;122(17):3054-61. Epub 2013 Aug 14.

Department of Oncology.

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.
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http://dx.doi.org/10.1182/blood-2013-06-505792DOI Listing
October 2013

Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

Monoclon Antib Immunodiagn Immunother 2013 Jun;32(3):149-61

Department of Hematology and Oncology, Amgen Inc., Thousand Oaks, California 91320, USA.

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.
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http://dx.doi.org/10.1089/mab.2012.0117DOI Listing
June 2013

Cytokines associated with increased erythropoiesis in Sprague-Dawley rats administered a novel hyperglycosylated analog of recombinant human erythropoietin.

Toxicol Pathol 2014 14;42(3):540-54. Epub 2013 May 14.

1Comparative Biology Safety Sciences, Pathology, Amgen Inc., Thousand Oaks, California, USA.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.
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http://dx.doi.org/10.1177/0192623313486318DOI Listing
December 2014

Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

Toxicol Pathol 2014 14;42(3):524-39. Epub 2013 May 14.

1Comparative Biology Safety Sciences, Pathology, Amgen Inc., Thousand Oaks, California, USA.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.
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http://dx.doi.org/10.1177/0192623313486319DOI Listing
December 2014

Differential enzyme-linked immunosorbent assay and ligand-binding mass spectrometry for analysis of biotransformation of protein therapeutics: application to various FGF21 modalities.

Anal Chem 2013 Mar 19;85(5):2731-8. Epub 2013 Feb 19.

PKDM, Amgen Inc., Thousand Oaks, California 91320, United States.

Novel protein therapeutics have become increasingly important modalities for treating diseases. Such therapeutics include recombinant fusions of pharmacoactive polypeptides to half-life extenders such as monoclonal antibodies, fragments of antibodies, and albumin. Half-life extension can also be achieved via chemical attachment to polymers such as polyethylene glycol. Any of these therapeutics may be susceptible to biotransformation, most notably in vivo proteolytic truncation, and it is vital to understand this phenomenon during early drug development to ensure correct pharmacokinetic profiling and optimize the in vivo stability through re-engineering. In this paper, we describe an integrated approach that combines differential enzyme-linked immunosorbent assay (ELISA) with ligand-binding-mass spectrometry (LB-MS) to provide a thorough understanding of the biotransformation of novel protein therapeutics. Differential ELISA allows for a fast, high-throughput means to reveal gross in vivo proteolytic liabilities. Ensuing LB-MS analysis provides higher resolution details such as specific vulnerable loci to allow design refinement of the molecule. In this work, the power of the approach is elucidated by application to the optimization of a promising drug candidate, FGF21.
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http://dx.doi.org/10.1021/ac303203yDOI Listing
March 2013

Simultaneous analysis of multiple monoclonal antibody biotherapeutics by LC-MS/MS method in rat plasma following cassette-dosing.

AAPS J 2013 Apr 12;15(2):337-46. Epub 2012 Dec 12.

Departments of PKDM, Amgen Inc, One Amgen Center Drive, Mail Stop 30E-3-B, Thousand Oaks, CA 91320, USA.

We have recently developed a general liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using a stable isotope-labeled (SIL) monoclonal antibody (mAb) as an internal standard (IS) for single-analyte quantification of mAb (Li et al. Anal Chem 84(3):1267-1273, 2012). The method offers an advantage over ligand binding assay in reducing the time and resources needed for bioanalytical support in preclinical stages of drug development. In this paper, we report another marked increase in assay efficiency for multi-analyte bioanalysis using unique surrogate peptides for each analyte and the strategic choice of the SIL-IS peptide. The method was qualified for the simultaneous determinations of four mAbs in rat plasma and applied to samples from discrete- and cassette-dosed rats. The pharmacokinetic parameters of the four mAbs of cassette dosing were comparable to those of discrete dosing and of enzyme-linked immunosorbent assay results. Although there may be limitations and special considerations for cassette-dosing of biologics, these results demonstrate the robust performance of the multi-analyte LC-MS/MS method allowing cassette-dosing that would ultimately reduce animal use and improve efficiency.
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http://dx.doi.org/10.1208/s12248-012-9435-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675729PMC
April 2013

Inhibition of WISE preserves renal allograft function.

J Am Soc Nephrol 2013 Jan 26;24(1):66-76. Epub 2012 Nov 26.

Division of Metabolic Disorders Research, Amgen Inc, Thousand Oaks, California, USA.

Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of β-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.
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http://dx.doi.org/10.1681/ASN.2012010012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537210PMC
January 2013

Bioanalysis of target biomarker and PK/PD relevancy during the development of biotherapeutics.

Bioanalysis 2012 Oct;4(20):2513-23

Amgen Inc., Thousand Oaks, CA 91320, USA.

The majority of biotherapeutic drugs act on specific targets, which may serve as biomarkers to be evaluated for target engagement and validation. Together with subsequent pathway biomarkers, these data can provide proof-of-mechanism and understanding of the biological drug affect. A major task during early development is to predict, for the first first time in human clinical trials, the starting dose and simulate the PK/PD relationship. However, determinations of the biotherapeutic drug and target concentrations are not straightforward due to temporal changes of drug-target binding and challenges in developing reliable methods to measure the free and total drug and target. Herein, the bioanalysis of the target biomarker and the biotherapeutics in the context of PK/PD relevancy during drug development is reviewed. Binding of the target to the biotherapeutic will affect target clearance and drug disposition, resulting in nonlinear PK. Reliable and specific methods are crucial for the correct PK/PD modeling and interpretation.
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http://dx.doi.org/10.4155/bio.12.220DOI Listing
October 2012

Implementation of a universal analytical method in early-stage development of human antibody therapeutics: application to pharmacokinetic assessment for candidate selection.

Bioanalysis 2012 Oct;4(19):2357-65

Department of Pharmacokinetic & Drug Metabolism, Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA.

Background: Effective bioanalytical support for pharmacokinetic assessment of therapeutics in early development remains challenging. Concurrent evaluation of multiple candidates per program is typical, requiring efficient characterization in various preclinical species; an ambitious effort often complicated by assay reagent unavailability and limited sample volume. Accordingly, a universal anti-human Fc assay for human monoclonal antibody and derived therapeutics was developed using a microfluidics platform to address these bioanalytical challenges.

Results: The universal assay with standardized format was qualified for quantitation of human IgG Fc-containing biotherapeutics in matrices from commonly used preclinical species. Results from this assay compared well with those from traditional colorimetric immunoassays. Furthermore, result comparison between the universal and target-specific assays provided additional information on the effect of antidrug antibodies and in vivo drug catabolism.

Conclusion: This assay has wide applicability as a default bioanalytical approach in therapeutic candidate selection and preliminary pharmacokinetics evaluation during early-stage therapeutic development.
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http://dx.doi.org/10.4155/bio.12.201DOI Listing
October 2012

Universal immunoassay applied during early development of large molecules to understand impact of immunogenicity on biotherapeutic exposure.

AAPS J 2012 Dec 1;14(4):843-9. Epub 2012 Sep 1.

Clinical Immunology Department, Amgen Inc., One Amgen Center Drive, MS 30E-3-B, Thousand Oaks, California 91320, USA.

Immunogenicity testing during early biotherapeutic development is usually limited by resources needed for assay development, validation, and the necessity for unique product-specific controls and reagents. We describe a unique immunoassay [universal indirect species-specific assay (UNISA)] that can be applied during early phase preclinical studies to support pharmacology, pharmacokinetics (PK), and toxicology evaluation during biotherapeutic antibody candidate assessment. UNISA was evaluated across three animal species: mouse, rat, and cynomolgus monkey. For each species, a unique and specific antibody pair was generated consisting of the secondary antibody and the positive control. The secondary antibody is specific for species anti-IgG antibody while demonstrating no cross-reactivity to human antibody-based biotherapeutics. The positive control is comprised of a species-specific anti-human IgG antibody clone specific for binding to the CH2 domain of all human IgG subtypes. Applications of this platform included: (a) identifying the dose with the least immunogenicity risk; (b) characterizing the impact of immunogenicity on PK exposure profiles across multiple antibody candidates and dose regimens; and (c) characterizing the immune response specificity to the idiotype or non-idiotypic region of the biotherapeutic candidate. Due to its use of universal species-specific reagents, UNISA can overcome resource constraints and avoid extensive validation and development time to support immunogenicity testing during the early research and preclinical phase of programs. Enhanced understanding of the impact of the immunogenicity on biotherapeutic exposure and target-related immunomodulatory effects have been made possible with the use of this assay.
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http://dx.doi.org/10.1208/s12248-012-9403-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3475848PMC
December 2012

Rapid affinity measurement of protein-protein interactions in a microfluidic platform.

Anal Biochem 2012 Jul 26;426(2):134-41. Epub 2012 Apr 26.

Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, CA 91320, USA.

A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to μM range.
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http://dx.doi.org/10.1016/j.ab.2012.04.023DOI Listing
July 2012

General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies.

Anal Chem 2012 Feb 25;84(3):1267-73. Epub 2012 Jan 25.

PKDM, Amgen Inc., Thousand Oaks, California 91320, USA.

Ligand binding assays (LBAs) are widely used for therapeutic monoclonal antibody (mAb) quantification in biological samples. Major limitations are long method development times, reagent procurement, and matrix effects. LC-MS/MS methods using signature peptides are emerging as an alternative approach, which typically use a stable isotope labeled signature peptide as the internal standard (IS). However, a new IS has to be generated for every candidate, and the IS may not correct for variations at all processing steps. We have developed a general LC-MS/MS method approach employing a uniformly heavy-isotope labeled common whole mAb IS and a common immunocapture for sample processing. The method was streamlined with automation for consistency and throughput. Method qualification of four IgG(2) and four IgG(1) mAbs showed sensitivity of 0.1 μg/mL and linearity of 0.1-15 μg/mL. Quality control (QC) data of these eight mAbs were accurate and precise. The QC performance of the whole molecule labeled IS was better than those of synthetic labeled IS peptides tested. The pharmacokinetic results of two mAbs (an IgG(2) and IgG(1) candidate) dosed in rats were comparable to those of LBA. The general LC-MS/MS method approach overcomes the limitations of current methods to reduce time and resources required for preclinical studies.
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http://dx.doi.org/10.1021/ac202792nDOI Listing
February 2012

Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

J Bone Miner Res 2011 Nov;26(11):2610-21

Department of Metabolic Disorders, Amgen Inc., Thousand Oaks, CA, USA.

The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.
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http://dx.doi.org/10.1002/jbmr.472DOI Listing
November 2011

Bioanalytical approaches to quantify "total" and "free" therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development.

AAPS J 2011 Mar 15;13(1):99-110. Epub 2011 Jan 15.

Amgen Inc, One Amgen Center Drive, Thousand Oaks, California 91320, USA.

The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono- and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.
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http://dx.doi.org/10.1208/s12248-011-9251-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032085PMC
March 2011

Novel approaches using alkaline or acid/guanidine treatment to eliminate therapeutic antibody interference in the measurement of total target ligand.

J Pharm Biomed Anal 2010 Apr 3;51(5):1128-33. Epub 2009 Dec 3.

Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320, United States.

Measurement of the total target ligand can help to provide pharmacokinetic (PK) and pharmacodynamic (PD) informations. However, the presence of monocloncal antibody therapeutics (ThAs) interferes with ELISA determinations of the total target proteins. The interferences can cause over- or under-estimation of the target protein analysis. The nature of interferences was dependent upon the ThA, target protein, antibody reagents and assay conditions of the ELISA. We have developed novel alkaline and acid/guanidine treatment approaches to dissociate the protein binding and preferentially denature the ThA. The neutralized target proteins can be determined by ELISA. These methods provide reproducible measurements of total target protein without ThA interference. Serum samples, standards and QCs containing target protein and ThA were treated with alkaline buffer (pH>13) containing casein or acid/guanidine buffer (pH<1). Total target proteins for two different ThA systems were successfully measured and interferences were completely eliminated by the treatments. These methods were successfully applied to analysis in pre-clinical serum samples.
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http://dx.doi.org/10.1016/j.jpba.2009.11.021DOI Listing
April 2010

An in vivo platform for translational drug development in pancreatic cancer.

Clin Cancer Res 2006 Aug;12(15):4652-61

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center and the Sol Goldman Pancreatic Cancer Research Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-0113DOI Listing
August 2006

Protein separation and surfactant control of electroosmotic flow in poly(dimethylsiloxane)-coated capillaries and microchips.

J Chromatogr A 2002 Feb;947(2):277-86

Department of Chemistry, University of Alberta, Edmonton, Canada.

A thermally pyrolyzed poly(dimethylsiloxane) (PDMS) coating intended to prevent surface adsorption during capillary electrophoretic (CE) [Science 222 (1983) 266] separation of proteins, and to provide a substrate for surfactant adsorption for electroosmotic mobility control was prepared and evaluated. Coating fused-silica capillaries or glass microchip CE devices with a 1% solution of 100 cSt silicone oil in CH2Cl2, followed by forced N2 drying and thermal curing at 400 degrees C for 30 min produced a cross-linked PDMS layer. Addition of 0.01 to 0.02% Brij 35 to a 0.020 M phosphate buffer gave separations of lysozyme, cytochrome c, RNase, and fluorescein-labeled goat anti-human IgG Fab fragment. Respective plates/m typically obtained at 20 kV (740 V cm(-1)) were 2, 1.5, 1.25, and 9.4-10(5). In 50 mM ionic strength phosphate, 0.01% Brij 35 running buffer, the electroosmotic flow observed was about 25% of that in a bare capillary, and showed no pH dependence between pH 6.3-8.2. Addition of sodium dodecylsulfate (SDS) or cetyltrimethylammonium bromide (CTAB) to this running buffer allowed ready control of electroosmotic mobility, mu(eo). Concentrations of SDS between 0.005 to 0.1% resulted in mu(eo) ranging from 3 to 5 x 10(-4) cm2 V(-1) s(-1). Addition of 1 to 2.3 x 10(-4)% (2.7-6.3 microM) CTAB caused flow reversal. CTAB concentrations between 3.5 x 10(-4) and 0.05% (0.0014-1.37 mM) allowed control of mu(eo) between -1 x 10(-4) and -5.0 x 10(-4) cm2 V(-1) s(-1). For both surfactants the added presence of 0.01% Brij 35 provided slowly varying changes in mu(eo) with charged surfactant concentration.
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http://dx.doi.org/10.1016/s0021-9673(01)01601-6DOI Listing
February 2002