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

The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated <i>SOCS-3</i> gene induction through inactivation of the transcription factor c-Jun

Induction of the suppressor of cytokine signalling 3 (SOCS-3) gene is vital to the normal control of inflammatory signalling. In order to understand these processes we investigated the role of the proto-oncogene component of the AP-1 transcription factor complex, c-Jun, in the regulation of SOCS-3 gene induction. We found that cyclic AMP stimulation of HUVECs promoted phosphorylation and activation of JNK MAP kinase and its substrate c-Jun. The JNK responsive element of the human SOCS-3 promoter mapped to a putative AP-1 site within 1000 bp of the transcription start site. The PKC inhibitors, GF-109203X, Gö-6983 and Ro-317549, were all found to inhibit AP-1 transcriptional activity, transcriptional activation of this minimal SOCS-3 promoter and SOCS-3 gene induction in HUVECs. Interestingly, Ro-317549 treatment was also found to promote PKC-dependent activation of ERK and JNK MAP kinases and promote JNK-dependent hyper-phosphorylation of c-Jun, whereas GF-109203X and Gö-6983 had little effect. Despite this, all three PKC inhibitors were found to be effective inhibitors of c-Jun DNA-binding activity. The JNK-dependent hyper-phosphorylation of c-Jun in response to Ro-317549 treatment of HUVECs does therefore not interfere with its ability to inhibit c-Jun activity and acts as an effective inhibitor of c-Jun-dependent SOCS-3 gene induction

IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design

Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation. This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence. Here we show that some humanised heavy chains can fold, form dimers and be secreted even in the absence of light chain. Quality control of recombinant antibody assembly in vivo is thought to rely upon folding of the heavy chain CH1 domain. This domain acts as a switch for secretion, only folding upon interaction with the light chain CL domain. We show that the secreted heavy-chain dimers contain folded CH1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This subversion of the normal quality control process is dependent upon the heavy chain variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. This discovery will impact on the efficient production of both humanised antibodies as well as the design of novel antibody formats