Zhao S.,Centocor |
Lu J.,Centocor R and D Inc.
Molecular Immunology | Year: 2010
Determination of framework regions (FRs) and complementarity determining regions (CDRs) in an antibody is essential for understanding the underlying biology as well as antibody engineering and optimization. However, there are no computational algorithms available to delimit an antibody sequence or a library of sequences into FRs and CDRs in a coherent and automatic fashion. Based upon the mapping relationships among mature antibody sequences and their corresponding germline gene segments, a novel computational algorithm has been developed for automatic determination of CDRs. Even though a human can make more than 10 12 different antibody molecules in its preimmune repertoire to fight off invading pathogens, these antibodies are generated from rearrangements of a very limited number of germline variable (V) gene, diversity (D) gene and joining (J) gene segments followed by somatic hypermutation. The framework regions FR1, FR2 and FR3 in mature antibodies are encoded by germline V gene segments, while FR4 is encoded by J gene segments. Since there are only a limited number of germline gene segments, these genes can be pre-delimited to generate a knowledge base of FRs and CDRs. Then for a given antibody sequence, the algorithm scans each pre-delimited gene in knowledge base, finds the best matching V and J segments, and accordingly, identifies the FRs and CDRs. The described algorithm is stringently tested using nearly 25,000 human antibody sequences from NCBI, and it is proven to be very robust. Over 99.7% of antibody sequences can be delimited computationally. Of those delimited sequences, only 0.28% of them have somatic insertions and deletions in FRs, and their corresponding delimited results need manual checking. Another feature of the algorithm is that it is CDR definition independent, and can be easily extended to other CDR definitions besides the most widely used Kabat, Chothia and IMGT definitions. In addition to delimitation of antibody sequences into FRs and CDRs, the described algorithm is good for sequence annotation and sequence quality control by detecting unusual sequence patterns and features. Furthermore, it has been suggested that the algorithm may easily be embedded into other applications, such as to create a gene family specific PSSM (Position Specific Scoring Matrix) for antibody engineering, and to automatically number an antibody sequence. © 2010 Elsevier Ltd. All rights reserved.
Richards T.J.,University of Pittsburgh |
Kaminski N.,University of Pittsburgh |
Baribaud F.,Centocor R and D Inc. |
Flavin S.,Centocor R and D Inc. |
And 9 more authors.
American Journal of Respiratory and Critical Care Medicine | Year: 2012
Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology with a variable and unpredictable course. Objectives: The aim of this study was to identify and validate plasma proteins that are predictive of outcome in IPF. Methods: Plasma samples were available for 241 patients with IPF (140 derivation and 101 validation). In the derivation cohort, concentrations of 92 proteins were analyzed using a multiplex beadbased immunoassay and concentrations of matrix metalloproteinase (MMP)-7, MMP-1, and surfactant protein D were assessed by ELISA. In the validation cohort concentrations of intercellular adhesion molecule (ICAM)-1, IL-8, and vascular cell adhesion molecule (VCAM)-1 were assessed by bead-based multiplex assay, and S100A12 and MMP-7 by ELISA. Associations of biomarkers with mortality, transplant-free survival, and disease progression were tested in the derivation and validation cohorts using nonparametric methods of survival analysis and the Cox proportional hazards model, and an integrated risk prediction score was derived and tested. Measurements and Main Results: High concentrations of MMP-7, ICAM-1, IL-8, VCAM-1, and S100A12 predicted poor overall survival, poor transplant-free survival, and poor progression-free survival in the derivation cohort. In the independent validation cohort high concentrations of all five were predictive of poor transplant-free survival; MMP-7, ICAM-1, and IL-8 of overall survival; and ICAM-1 of poor progression-free survival. The personal clinical and molecular mortality prediction index derived in the derivation cohort was highly predictive of mortality in the validation cohort. Conclusions: Our results suggest that plasma proteins should be evaluated as a tool for prognosis determination in prioritization of patients for lung transplantation and stratification in drug studies. Copyright © 2012 by the American Thoracic Society.
Brezski R.J.,Centocor R and D Inc. |
Jordan R.E.,Centocor R and D Inc.
mAbs | Year: 2010
The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the "nick" of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts. This suggested a pathway by which tumors might avoid host immune surveillance under a cloak of proteolytically- generated, dysfunctional antibodies that block competent IgG binding. The host immune system cannot be blind to this pathway since there exists a widespread, low-titer incidence of anti-hinge (cleavage-site) antibodies in the healthy population. The prevalence of anti-hinge reactivity may reflect an ongoing immune recognition of normal IgG catabolism. Tumor growth and bacterial infections potentially generate hostile proteolytic environments that may pose harsh challenges to host immunity. Recent findings involving physiologically-relevant proteases suggest that the potential loss of key effector functions of host IgGs may result from subtle and limited proteolytic cleavage of IgGs, and that such events may facilitate the incursion of invasive cells in local proteolytic settings. © 2010 Landes Bioscience.
Vagin A.,University of York |
Teplyakov A.,University of Maryland Biotechnology Institute |
Teplyakov A.,Centocor R and D Inc.
Acta Crystallographica Section D: Biological Crystallography | Year: 2010
MOLREP is an automated program for molecular replacement that utilizes a number of original approaches to rotational and translational search and data preparation. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron density, structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calculated from the input data. © 2010 International Union of Crystallography Printed in Singapore - all rights reserved.
Bastiaansen-Jenniskens Y.M.,Rotterdam University |
Clockaerts S.,Rotterdam University |
Clockaerts S.,University of Antwerp |
Feijt C.,Rotterdam University |
And 8 more authors.
Annals of the Rheumatic Diseases | Year: 2012
Objective: Adipose tissue is known to release inflammatory cytokines and growth factors. In this exploratory study, the authors examined whether the infrapatellar fat pad (IPFP) closely located to cartilage in the knee joint can affect cartilage metabolism. In addition, the authors analysed whether the macrophage types present in IPFP could explain the effect on cartilage. Methods: IPFP explants obtained during total knee replacement of 29 patients with osteoarthritis (OA) were used to make fat-conditioned medium (FCM). Explants of bovine cartilage were cultured with or without FCM. Nitric oxide (NO) and glycosaminoglycan release and gene expression of matrix-degrading enzymes in cartilage were analysed. To stimulate catabolic processes in the cartilage, the authors added interleukin 1β, and the effect of six FCMs was evaluated. The presence of different types of macrophages (CD68+, CD86+ and CD206+) in OA IPFPs was compared with subcutaneous adipose tissue samples and IPFP samples from patients with an anterior cruciate ligament rupture. Results: FCM alone reduced NO and glycosaminoglycan release and matrix metalloproteinase (MMP)1 gene expression by the cartilage. Moreover, when catabolic conditions were enhanced with interleukin 1β, FCM inhibited NO production as well as MMP1 and MMP3 gene expression and increased collagen type II gene expression. Significantly more CD206+ cells were present in OA IPFP samples than in subcutaneous fat or anterior cruciate ligament IPFP samples. Conclusion: In contrast to the authors' expectations, medium conditioned by end-stage OA IPFP inhibited catabolic processes in cartilage. CD206+ cells present in the IPFPs used for making the FCM might have contributed to the inhibition of catabolic processes in the cartilage.
Curran M.E.,Centocor R and D Inc. |
Platero S.,Centocor R and D Inc.
Pharmacogenomics | Year: 2011
On 1-2 December 2010 worldwide leaders in the field of co-diagnostics gathered in Boston, MA, USA, to discuss the state of predictive medicine and the complexities of biomarker implementation and partnering. Flanking the primary conference were three workshops where participants could increase their understanding of companion diagnostic commercialization, best practices from well-known case studies and regulatory guidance and practice. The primary conference reviewed here consisted of 18 lectures and one round table discussion. Topics for the summit included biomarker discovery and validation, partnering between pharmaceutical development and diagnostics companies, commercialization models and evolving regulatory perspectives and guidance. While it is clear that there are many difficulties remaining in making personalized medicine a broad-based reality for all therapeutic areas, it is equally clear that much progress has already been made and that the conference participants are optimistic and dedicated to fulfilling the personalized medicine promise of bringing the right drug to the right patient at the right time. © 2011 Future Medicine Ltd.
Zhao S.,Johnson and Johnson Pharmaceutical Research and Development L.L.C |
Lu J.,Centocor R and D Inc.
Molecular Immunology | Year: 2011
A challenge to antibody engineering is the large number of positions and nature of variation and opposing concerns of introducing unfavorable biochemical properties. While large libraries are quite successful in identifying antibodies with improved binding or activity, still only a fraction of possibilities can be explored and that would require considerable effort. The vast array of natural antibody sequences provides a potential wealth of information on (1) selecting hotspots for variation, and (2) designing mutants to mimic natural variations seen in hotspots. The human immune system can generate an enormous diversity of immunoglobulins against an almost unlimited range of antigens by gene rearrangement of a limited number of germline variable, diversity and joining genes followed by somatic hypermutation and antigen selection. All the antibody sequences in NCBI database can be assigned to different germline genes. As a result, a position specific scoring matrix for each germline gene can be constructed by aligning all its member sequences and calculating the amino acid frequencies for each position. The position specific scoring matrix for each germline gene characterizes " hotspots" and the nature of variations, and thus reduces the sequence space of exploration in antibody engineering. We have developed a bioinformatics pipeline to conduct analysis of human antibody sequences, and generated a comprehensive knowledge database for in silico antibody engineering. The pipeline is fully automatic and the knowledge database can be refreshed anytime by re-running the pipeline. The refresh process is fast, typically taking 1. min on a Lenovo ThinkPad T60 laptop with 3G memory. Our knowledge database consists of (1) the individual germline gene usage in generation of natural antibodies; (2) the CDR length distributions; and (3) the position specific scoring matrix for each germline gene. The knowledge database provides comprehensive support for antibody engineering, including de novo library design in selection of favorable germline V gene scaffolds and CDR lengths. In addition, we have also developed a web application framework to present our knowledge database, and the web interface can help people to easily retrieve a variety of information from the knowledge database. © 2011 Elsevier Ltd.
Wuchner K.,Centocor R and D Inc. |
Buchler J.,Centocor R and D Inc. |
Spycher R.,Centocor R and D Inc. |
Dalmonte P.,Centocor R and D Inc. |
And 2 more authors.
Journal of Pharmaceutical Sciences | Year: 2010
The development of a Microflow digital imaging (MDI) method to detect and monitor protein particulates in a high concentration IgG1 monoclonal antibody formulation is presented. The MDI assay was optimized and qualified as a characterization assay in terms of accuracy and precision of particle size and number using polystyrene standards (5-300 μm) and protein particles (2 to >100 μm). The stability profile of a 90 mg/mL IgG1 formulation stored at 2-88C and -70°C for up to 18 months was then investigated. The MDI assay results showed improved sensitivity to detect subvisible particulates (≥5 μm) compared to conventional light obscuration detection, presumably due to the translucent nature of the protein particles. For evaluation of visible protein particles (>70 μm), a good overall correlation was observed for MDI, inverted microscopy and visual assessments. Long-term stability data for a high concentration IgG1 monoclonal antibody formulation demonstrated an accumulation of protein particles in certain size categories with a concomitant increase in the overall particle size distribution over time. The weight amount of protein particulates in the IgG1 formulation was measured experimentally as ∼0.022% (∼20 μg/mL) after storage at 2-8°C for 16 months. Similar results were obtained by calculation from the MDI particle data indicating a low level of protein particulates by weight. The nature and composition of the protein particulates formed during storage were further characterized by a combination of inverted microscopy, FTIR microscopy, and SEM-EDX. Particulates were identified as protein with silicone, although some particles also contained other elements such as aluminum. The combination of MDI results and protein characterization studies have provided an enhanced understanding of protein particulate formation during long-term storage of a high concentration IgG1 monoclonal antibody formulation. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association.
Puzzo D.,Columbia University |
Puzzo D.,University of Catania |
Privitera L.,Columbia University |
Privitera L.,University of Catania |
And 12 more authors.
Annals of Neurology | Year: 2011
Objective: The goal of this study was to investigate the role of endogenous amyloid-β peptide (Aβ) in healthy brain. Methods: Long-term potentiation (LTP), a type of synaptic plasticity that is thought to be associated with learning and memory, was examined through extracellular field recordings from the CA1 region of hippocampal slices, whereas behavioral techniques were used to assess contextual fear memory and reference memory. Amyloid precursor protein (APP) expression was reduced through small interfering RNA (siRNA) technique. Results: We found that both antirodent Aβ antibody and siRNA against murine APP reduced LTP as well as contextual fear memory and reference memory. These effects were rescued by the addition of human Aβ42, suggesting that endogenously produced Aβ is needed for normal LTP and memory. Furthermore, the effect of endogenous Aβ on plasticity and memory was likely due to regulation of transmitter release, activation of α7-containing nicotinic acetylcholine receptors, and Aβ42 production. Interpretation: Endogenous Aβ42 is a critical player in synaptic plasticity and memory within the normal central nervous system. This needs to be taken into consideration when designing therapies aiming at reducing Aβ levels to treat Alzheimer disease. © 2011 American Neurological Association.
Szalma S.,Centocor R and D Inc. |
Koka V.,Centocor R and D Inc. |
Khasanova T.,Genego, Inc. |
Perakslis E.D.,Centocor R and D Inc.
Journal of Translational Medicine | Year: 2010
Background: The growing consensus that most valuable data source for biomedical discoveries is derived from human samples is clearly reflected in the growing number of translational medicine and translational sciences departments across pharma as well as academic and government supported initiatives such as Clinical and Translational Science Awards (CTSA) in the US and the Seventh Framework Programme (FP7) of EU with emphasis on translating research for human health.Methods: The pharmaceutical companies of Johnson and Johnson have established translational and biomarker departments and implemented an effective knowledge management framework including building a data warehouse and the associated data mining applications. The implemented resource is built from open source systems such as i2b2 and GenePattern.Results: The system has been deployed across multiple therapeutic areas within the pharmaceutical companies of Johnson and Johnsons and being used actively to integrate and mine internal and public data to support drug discovery and development decisions such as indication selection and trial design in a translational medicine setting. Our results show that the established system allows scientist to quickly re-validate hypotheses or generate new ones with the use of an intuitive graphical interface.Conclusions: The implemented resource can serve as the basis of precompetitive sharing and mining of studies involving samples from human subjects thus enhancing our understanding of human biology and pathophysiology and ultimately leading to more effective treatment of diseases which represent unmet medical needs. © 2010 Szalma et al; licensee BioMed Central Ltd.