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Zhao S.,Johnson and Johnson Pharmaceutical Research and Development LLC | 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. Source


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. Source


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. Source


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. Source


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. Source

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