An investigation into the folding and assembly of engineered antibodies and novel antibody formats

Monoclonal antibodies and novel antibody formats are currently one of the principal therapeutic in the biopharmaceutical industry worldwide and are widely used in the treatment of autoimmune diseases and cancer. It is for this reason that the productivity and quality of antibody production requires improvement; specifically investigations into the engineering of antibodies and any issues that may arise from the production of these therapeutics. The work presented in this thesis describes an investigation into the folding and assembly of seven antibodies plus the novel antibody format FabFv. IgG is comprised of two identical HCs and two identical LCs. The folding process of immunoglobulin is controlled by the CH1 domain within the HC. The CH1 domain remains in a disordered state and is sequestered by BiP in the endoplasmic reticulum. Upon the addition of a folded CL domain, BiP is displaced, the CH1 domain is able to fold and the complete IgG protein can then be secreted from the cell. The results presented in this thesis however, have outlined an additional mechanism for the folding of the CH1 domain. We have shown that the CH1 domain is able to fold in the absence of LC resulting in the secretion of HC dimers in a VH dependent manner. The proposed mechanism for the secretion of HC dimers suggests that some VH domains can interact with each other in order to bring the CH1 domains in close proximity to enable folding to occur. As HC dimer secretion is a hindrance in antibody production, this result has highlighted an engineering target to improve antibody yield. Examination of the folding of IgG4 with the variable region A33 has revealed the inability to secrete LC dimers, cleavage of the HC during expression and secretion of HC dimers in the Fab, FabFv and full length forms. The attributes described have also been shown to be variable region dependent. This has introduced a new concept that the variable domain is important in determining the expression and secretion of antibodies and their individual chains. Pulse chase and 2D gel electrophoresis analysis of the novel antibody format FabFv has revealed that the folding and expression of the LC and HC causes multimeric species of FabFv to be secreted, as opposed to the monomeric form which is the desired therapeutic. Our hypothesis is that this process occurs via a LC dependent mechanism. The proposed hypothesis suggests that further engineering to the LC could diminish the formation and secretion of FabFv multimers. The results from these investigations can be applied to increase the productivity of therapeutics and increase the biological understanding of the domain interactions of IgG during folding, assembly and secretion.

Every nation under heaven: the importance of the biblical concept of nations for contemporary mission practice

Is there a concept of nationhood in the Bible that can provide us with a framework for cross-cultural Christian mission? This thesis argues that current evangelical missiology has accepted too willingly the categories of the secular Enlightenment understanding of ethnicity and nationhood, and that it needs to rethink its understanding of nations from a biblical standpoint. While the pressures of globalisation are seen by some as rapidly eclipsing the nation-state, this thesis will argue that we need to move beyond the narrower secular categories of citizenship, political power and the boundaries of the state to recover a more biblical understanding of nationhood. By reference to Genesis 10-11, Acts 2:1-11 and those passages in the Book of Revelation that discuss the destiny of the nations, it will show that the biblical understanding of nations includes deeper ideas of shared history, culture and language as the essential components of nationhood. It will explain how nations are part of the created order, and explore the impact of the Babel narrative on our understanding of nations in relation to God. It will demonstrate that Pentecost did not reverse the curse of Babel, but served rather to honour the dignity and value of nations and their languages. It will also argue that nations have a destiny in the New Creation according to the Book of Revelation. This biblical concept of nationhood has significant implications in several areas: the development of a public theology; a Christian response to nationalism; the question of how urban mission fits within mission to the nations; and the importance of indigenous languages in cross-cultural mission, especially in the multicultural cities of Europe.