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1.
Cancer Discov ; 7(9): 1030-1045, 2017 09.
Article in English | MEDLINE | ID: mdl-28526733

ABSTRACT

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.


Subject(s)
Antineoplastic Agents/therapeutic use , Cadherins/antagonists & inhibitors , Kidney Neoplasms/drug therapy , Ovarian Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Cadherins/genetics , Cadherins/metabolism , Female , Humans , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Macaca fascicularis , Mice, Nude , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Rats , Xenograft Model Antitumor Assays
2.
Int Immunopharmacol ; 37: 16-22, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26627559

ABSTRACT

Endotoxin removal using detergent washes and extractions are well-established, efficient, and cost-effective methods; however, removing residual detergent post treatment has been shown to be a challenge. In this communication, we show a simple and fast method for determining the detergent concentration in a protein solution post treatment and highlight strategies for detergent removal to achieve levels below the critical micelle concentration (CMC), the minimum concentration at which detergent micelles form.


Subject(s)
Detergents/analysis , Endotoxins/chemistry , Endotoxins/isolation & purification , Octoxynol/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Animals , CHO Cells , Cricetulus , Detergents/isolation & purification , Methods , Micelles , Octoxynol/isolation & purification , Solutions
3.
Cell Rep ; 10(2): 239-52, 2015 Jan 13.
Article in English | MEDLINE | ID: mdl-25558064

ABSTRACT

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.


Subject(s)
Cytokines/pharmacology , Goblet Cells/drug effects , Lung/pathology , Receptor, Notch2/metabolism , Animals , Cell Culture Techniques , Cell Differentiation/drug effects , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Goblet Cells/cytology , Goblet Cells/metabolism , Hepatocyte Nuclear Factor 3-gamma/genetics , Hepatocyte Nuclear Factor 3-gamma/metabolism , Humans , Interleukin-13/genetics , Interleukin-13/metabolism , Interleukin-13/pharmacology , Interleukin-17/genetics , Interleukin-17/metabolism , Interleukin-17/pharmacology , Lung/metabolism , Metaplasia , Mice , Mice, Inbred BALB C , Mucin 5AC/genetics , Mucin 5AC/metabolism , Mucin-5B/genetics , Mucin-5B/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology
4.
Protein Expr Purif ; 108: 80-84, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25514202

ABSTRACT

Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis.


Subject(s)
Chromatography, Affinity/methods , Chromatography, Gel/methods , Recombinant Fusion Proteins/isolation & purification , Animals , CHO Cells , Cricetulus , Recombinant Fusion Proteins/chemistry
5.
Biotechnol Prog ; 30(6): 1497-501, 2014.
Article in English | MEDLINE | ID: mdl-25079968

ABSTRACT

The removal of bacterial endotoxins from biological samples is critical to avoid the potentially fatal pyrogenic response possible when introduced to mammalian systems. Endotoxins have a variety of specific characteristics that can be exploited to target their isolation and subsequent removal, but one that has not been extensively characterized is their difference in size from that of monoclonal antibodies. Here, we present a study which utilizes gel filtration chromatography as a method for endotoxin removal from both aggregated and nonaggregated antibody preparations, outlining a mechanistically simple method for removal of this impurity.


Subject(s)
Antibodies, Monoclonal/chemistry , Chromatography, Gel/methods , Endotoxins/isolation & purification , Animals , Antibodies, Monoclonal/isolation & purification , CHO Cells , Cricetinae , Cricetulus , Micelles
6.
J Immunol Methods ; 393(1-2): 81-5, 2013 Jul 31.
Article in English | MEDLINE | ID: mdl-23612045

ABSTRACT

The expanding use of monoclonal antibodies in the biopharmaceuticals industry has brought the need for new analytical tools. We have developed a coupled affinity and gel-filtration high-performance liquid chromatography method to simultaneously analyze titer and quality of monoclonal antibodies. Before this assay, available analytical methods for protein aggregation required highly purified proteins. This assay can qualitatively describe a protein from a clarified cell culture solution by trending protein aggregation over time while measuring protein titer. It can be used to assess proteins in both early- and late-stage culture due to its dynamic range and sensitivity. This assay is a sensitive technique that overcomes the time limitations of previous approaches. It provides an essential tool to accomplish process optimization.


Subject(s)
Antibodies, Monoclonal/chemistry , Chromatography, Affinity/methods , Chromatography, Gel/methods , Chromatography, High Pressure Liquid/methods , Antibodies, Monoclonal/analysis , Calibration
7.
Biotechnol J ; 7(12): 1509-16, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23081824

ABSTRACT

The removal of endotoxin from protein solutions and its prevention are key to the success of recombinant protein production due to the possible pyogenic response in mammals caused by contaminated samples. In the pre-clinical situation, protein production is often carried out in a non-good manufacturing practice (GMP) setting, utilizing bacterial DNA for transient transfection and non-validated cleaning techniques. Here, we present our findings evaluating various options for endotoxin removal, and propose strategies for endotoxin prevention with emphasis on chromatographic separations, endotoxin-removing membranes and on-column wash strategies.


Subject(s)
Biological Products/standards , Biotechnology/methods , Endotoxins/isolation & purification , Recombinant Proteins/standards , Biological Products/chemistry , Biological Products/isolation & purification , Biotechnology/standards , Chromatography, Liquid/methods , Drug Industry , Endotoxins/chemistry , Escherichia coli/metabolism , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
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