Mohamed-Ali, Nada; (2025) Investigation of novel camel-derived antibodies and VHH fragments in targeting fibrotic disease pathways. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Systemic sclerosis (SSc) is a chronic, autoimmune disorder characterised by fibrosis of the skin and internal organs. Highest mortality rates are observed in patients with pulmonary comorbidities such as SSc-associated pulmonary fibrosis (SSc-PF), and SSc-associated pulmonary arterial hypertension (SSc PAH). Pro-inflammatory cytokines interleukin-6 (IL-6) and connective tissue growth factor (CTGF) have been implicated in the pathogenesis of SSc. Current therapeutics include monoclonal antibodies (mAbs), such as Tocilizumab, targeting these cytokines and their signalling molecules. Often lack of specificity of mAbs render them more prone to adverse side effects. Studies have suggested that heavy chain only (HcAb) camelid antibodies and fragments of their hypervariable binding regions (VHH fragments) have advantageous properties. Owing to their smaller size and differences in their tertiary structures, HcAbs and VHH fragments and have been suggested to have greater stability and higher tissue penetrative ability in comparison to their conventional counterparts. Additionally, it has been suggested that these antibodies may target novel epitopes not recognized by conventional antibodies. This study identified cytokine targets specific for SSc-PF, SSc-PAH and SSc-NLD (no lung disease) using proteomics. Elevated levels of MCP-1, MCP-3 and MCP-4 were found in SSc-PF, compared to all other groups. No robust discriminatory cytokines were found for SSc-PAH or SSc-NLD. Measurement of IL-6 and MCP-1 serum concentrations by ELISA confirmed proteomics results. Investigation of IL-6 trans signalling molecules found increased pro-inflammatory sIL-6R in the pulmonary groups. Secondly, the project aimed to set up in vitro tissue culture models to further clarify the fibrotic signalling pathways. In primary skin fibroblasts, IL-6 with the sIL-6R at pharmacological levels was found to induce Type I Collagen expression and phosphorylation of STAT3. Treatment with CTGF was found to induce IL-6 release through phosphorylation of ERK1/2. Increased IL-6 was found to induce production of MCP-1. The most inflammatory treatment in terms of Type I Collagen production was CTGF with sIL-6R. Finally, this project produced and characterised available, novel, camelid conventional, HcAb and VHH fragment antibodies targeting sIL-6R and CTGF, to attenuate these fibrotic pathways. Camel-derived anti-sIL-6R conventional and HcAbs were found to significantly reduce the expression of pSTAT3 and pERK1/2 in a similar manner as Tocilizumab. However, anti-sIL-6R VHH fragment indicated high specificity towards the ERK pathway, significantly dampening pERK1/2 expression but with little effect on pSTAT3. In skin fibroblasts, a combination of anti-CTGF and anti-sIL-6R conventional and HcAbs reduced secretion of IL-6 to a greater extent than Tocilizumab, and depleted MCP-1 expression.

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