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The site-specific modification of therapeutic proteins produced in Escherichia coli : implementation of a combinatorial bispecific antibody fragment library against human breast carcinomas

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Call No.	Ph.D 2010
Author	Isett, Kevin D.
Title	The site-specific modification of therapeutic proteins produced in Escherichia coli : implementation of a combinatorial bispecific antibody fragment library against human breast carcinomas
Date	2010
Abstract	The ability to site-specifically modify therapeutic proteins for the attachment of pharmacokinetic-enhancing molecules is the principal goal of many research programs. Site-specific modalities are often introduced to provide an additional functionality, increase serum half-life, decrease proteolysis, and reduce immunogenicity. Many of the current conjugation methods are non-specific and, therefore, typically result in heterogeneous molecular compositions. Product heterogeneity can further complicate biomolecular characterization of activity, and preclinical safety and efficacy. An ideal production system would have the capability to site-specifically modify protein targets in such a way as to aid in the efficiency of downstream conjugation, purification, and preclinical process streams. Accordingly, we developed methods for the site-specific modification of therapeutic proteins, specifically antibody fragments, produced in E. coli. In one method, an E. coli production strain was endowed with the genetic elements necessary to encode for gene-products pertaining to protein glycosylation. The glycan was utilized as a unique chemical handle for oxidative activation and subsequent conjugation reactions, such as, but not limited to site-specific PEGylation and chemically-defined protein-protein conjugation. Another method targeted engineered cysteine sulphydryls for PEGylation and for chemically-defined protein-protein conjugation. Herein, we developed a rapid method to modify antibody fragments for the production of a combinatorial bispecific/bivalent antibody fragment library. We sought to implement this therapeutic library towards human breast carcinoma receptor targets. We initially hypothesized that combinatorial antibody fragment libraries possess the power to elucidate new and potentially novel therapeutic compositions having completely unique pharmacokinetic behavior against a vast array of human diseases, which would otherwise, not be rationally predicted. From our combinatorial antibody fragment library evaluation, we have discovered several single-molecule, bispecific antibody fragments that possess synergist anti-proliferative effects towards several human breast carcinoma cell lines. Considering the complexity of disease-mediated signaling networks, our approach represents considerable promise for a rapid pre-clinical method towards discovering novel antibody combinations having homogenous molecular compositions against a multitude of human diseases.
Description	xix, 197 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	Gerngross, Tillman U. Griswold, Karl, Hoopes, P. Jack, Sethuraman, Natarajan
Subject	Immunoglobulins -- Synthesis. -- Escherichia coli -- Biotechnology -- Protein engineering -- Breast -- Cancer -- Immunotherapy.
College	Thayer School of Engineering. .
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.394

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	The Site-specific Modification of Therapeutic Proteins Produced in Escherichia coli: Implementation of a Combinatorial Bispecific Antibody Fragment Library Against Human Breast Carcinomas A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy By KEVIN D. ISETT Thayer School of Engineering Dartmouth College Hanover, NH 17th August, 2010 Examination Committee Chairman:________________________________ Professor Tillman U. Gerngross Member:_________________________________ Professor Karl Griswold Member:_________________________________ Professor P. Jack Hoopes Member:_________________________________ Dr. Natarajan Sethuraman ________________________________ Professor Brian W. Pogue Dean of Graduate Studies © 2010 Trustees of Dartmouth College

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The site-specific modification of therapeutic proteins produced in Escherichia coli : implementation of a combinatorial bispecific antibody fragment library against human breast carcinomas

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