Therapeutic monoclonal antibodies (MAb) are produced as secreted complex glycoproteins from mammalian cell systems and represent one of the most important classes of therapeutic medicines for the treatment of a variety of human diseases. Their benefit in health care and high economic impact provide the driving force for the development of improved production levels with the focus of optimizing clinical efficacy. One important issue is the optimization of monoclonal antibody production. A frequent approach used to address this challenge is the engineering of mammalian cell lines to increase antibody production levels through genetic manipulation. Valuable information can then be obtained by monitoring the effects of genetic changes on the biochemistry of the cell associated with MAb production. Global protein expression profiling of mammalian cells used for the production of biopharmaceuticals may reveal key biochemical characteristics associated with MAb-producing cell lines. A better understanding of these characteristics can in turn lead to more rational strategies for cell line and process development. The proposed research relates to a larger NSERC Strategic Network (MAbNet) Grant to develop and establish a novel platform for the large-scale manufacture of specific glycoforms of therapeutic monoclonal antibodies. The efficacy of these recombinant MAbs will be enhanced by the control of their glycosylation profiles. The work presented in this thesis will assist MAbNet in meeting their objectives. Specifically, we use 2D-Differential In-Gel Electrophoresis (2D-DIGE) to quantify protein expression differences between EG2-hFc1-producing Chinese Hamster Ovary cells (CHO-1A7) with its parental cell line (CHO-BRI). Here, we identified 34 unique differentially expressed proteins associated with EG2-hFc1 production that relate to various biological processes including protein processing, carbohydrate metabolism, amino acid metabolism, energy metabolism, apoptosis, and cell proliferation pathways. The majority of identified significant protein expression changes and their associated metabolic processes seem to prioritize energy production in CHO-1A7 cells. Due to the metabolic load of recombinant antibody production, the CHO-1A7 cell line attempts to meet the energy requirements needed for recombinant protein biosynthesis while maintaining cell viability and efficient protein folding mechanisms. A 2-D proteome reference map was also constructed for the CHO-BRI host cell line containing 131 identified protein spots. The map provides information that will further expand our understanding of this particular cell line. It will be a useful tool for studies investigating physiological responses and protein expression patterns of CHO-BRI to genetic and environmental perturbations. The set of identified differentially expressed proteins provides data on the downstream changes in protein expression due to genetic manipulation, and furthermore can provide targets for cell-line specific optimization of antibody production. The work described in this thesis furthers our understanding of antibody production in a specific CHO cell line.

Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.

Transfusion medicine is an important part of modern health care and the provision of reliably phenotyped red blood cells (RBC) is essential for safe and effective blood transfusions. For identification of many RBC antigens, monoclonal antibodies of either murine or human origin are available for use in agglutination assays, in which they perform as well as or better than the human polyclonal antibody preparations which they have replaced. However, the detection of some blood groups is still reliant on the use of human polyclonal antisera, which is a less reliable reagent source with respect to availability, batch to batch variation and bio-safety. The use of recombinant antibody and phage display technology for the discovery of new monoclonal antibodies with specificity for some of these RBC antigens has the potential to deliver an economical, unlimited supply of specific antibody reagents suitable for use in RBC phenotyping. Samples of human B cells from donors producing useful phenotyping antibodies were identified and transformed using Epstein Barr virus into lymphocyte cell lines. Antibody genes were obtained from the cell lines in the form ofRNA which was reverse transcribed, amplified by PCR and cloned into a phagemid vector system to generate several combinatorial antibody libraries. These antibody libraries were displayed on the surface of phage particles and subjected to antigen-driven selection by several rounds of phage display biopanning using soluble and cell based RBC antigens. In addition a large naIve library was biopanned against the same antigens in an attempt to isolate a wide range of antibodies suitable for blood typing. Several high quality combinatorial antibody libraries with respect to size (> 107 clones) and diversity were generated. Biopanning of recombinant libraries resulted in enrichment of phage antibodies specific for RBC antigens, and several clones were isolated which were shown to be specific for Duffy a antigen. The isolated antibodies would be ideal candidates for re-engineering into multivalent antibody molecules capable of direct agglutination of RBC and as such, have the potential to replace human polyclonal sera in the identification of Duffy a RBC antigen phenotyping.

Bibliography: leaves 123-141. / xv, 141 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis developes specific tools to monitor receptor expression in a ligand-independent manner, demonstrates the receptor expression is not static and can be modulated by cytokines, identifies strong evidence in defining the N-terminal domain of IL-3R & chain and B'C' and F'G' loopes of domain 4 of Bc as functional domains involved in ligand binding and function and provides novel potential therapeutics. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1997

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xii, 156 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [83]-98).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 131-149). Also available in print.

Thesis (Ph. D.)--University of Virginia, 2005. / Includes bibliographical references (leaves 196-216). Also available online through Digital Dissertations.

The colony-stimulating factor-1 receptor (CSF-1R) is expressed by the mononuclear phagocytic lineage, and is important for the development of these cells from their progenitors and also for promoting their survival and activation after maturation. The receptor has two ligands, CSF-1 and IL-34, which induce the formation of a stable dimer between two receptor monomers. This leads to intracellular autophosphorylation of tyrosine residues and subsequent signalling cascades, leading to rapid protein expression, cytoskeleton remodelling and cellular motility. Although CSF-1R signalling is crucial for normal embryogenic development and other physiological functions mediated by the phagocytic lineage, it has also been found to promote the pathogenic progression of cancer. Tumour-associated macrophages (TAMs) can comprise a large proportion of the cellular population in several solid tumours. These cells promote several hallmarks of cancer malignancy, such as increased neovascularization, tissue invasion, induction of metastases and immunosuppression. In this work, it was confirmed that CSF-1 had a prominent role in inducing cancer-promoting cellular phenotypes. Both canine cancer cells and macrophages respond to this cytokine, respectively increasing cancer cell proliferation and reducing inflammatory activation. Given the importance of CSF-1R signalling in the tumour microenvironment, antibodies were generated with the objective of blocking receptor function. Mice were immunized with either the extracellular region or the dimerization domain of the CSF-1R. Hybridomas were produced using the primed splenocytes, and monoclonal antibody (mAb) candidates were selected based on their performance in immunostaining and on their capacity to inhibit CSF-1R+ cells. The best antibodies were subjected to speciation. Chimeric antibodies maintained the ability of the parental mAbs to inhibit macrophage proliferation following CSF-1R stimulation. However, the mAbs possessed moderate affinity and specificity for their target, failing to stain monocytes and presenting a degree of cross-reactivity. The binding properties of one of such mAbs were altered by PCR-induced mutations, generating semi-synthetic antibody libraries. These were screened by phage display, yielding novel clones that show reduced cross-reactivity with unrelated proteins and retain the property of inhibiting macrophage survival. These results are a step in the development of therapeutic monoclonal antibodies for cancer treatment in dogs.