Development and Characterization of a potent Tumor Necrosis Factor-alpha blocking agent
Tumor necrosis factor-α (TNFα), one of the major pro-inflammatory cytokines, plays a pivotal role in an effective inflammation process and immune response. TNFα interacts with two different receptors, TNFR1 and TNFR2, which are differentially expressed on cells to initiate both, distinct and overlapping signal transduction pathways. The diverse signalling pathways lead to different cellular responses, including cell death, survival, differentiation, proliferation and cell migration. Vascular endothelial cells respond to TNFα by undergoing several pro-inflammatory changes which increase leukocyte adhesion, endothelial translocation and vascular leak to promote thrombosis. The central role of TNFα in inflammation has been demonstrated by the ability of agents that block the action of TNFα to treat a range of inflammatory conditions, including Rheumatoid arthritis, Psoriasis, Psoriatic arthritis, Crohn’s disease, Ulcerative colitis, Ankylosing spondylitis etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. Despite having beneficial effects of anti-TNFα agents in inflammation associated diseases, there are some unexpected, aberrant adverse events with all approved TNFα inhibitors, such as opportunistic infections, tuberculosis, demyelinating disease and drug-induced lupus erythematosus. Antibodies have different applications, including, a) medicine (diagnosis, therapy), b) biomedical research, (Western blotting, immunosorbent assays, immunohistochemistry, immunoprecipitation, and flow cytometry). Due to the cardinal role of antibodies in therapies, such as antibody drug conjugates and tumour therapy, development of therapeutic antibodies has become one of the most challenging area of medical biology. Pharmaceutical industries and medical biologist have been trying to use the specificity of antibodies for drug development against a range of diseases. The present study focuses on development of a neutralizing anti-TNFα antibody which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity and cell death. To achieve this goal, we have generated and characterized a library of 13 hybridoma clones secreting monoclonal antibodies (mAbs) against human-TNFα as described in chapter 2. Four of the mAbs rescued L929 fibroblast cells from TNFα triggered cell death as examined by MTT assay, out of 5 which mAb C8 was found to have the highest affinity to human-TNFα and importantly, inhibited TNFα toxicity at very low concentrations. In order to gain insights in to the mechanism by which C8 antibody inhibits human TNFα-mediated toxicity, we attempted to delineate the epitope corresponding to the mAb C8 as described in chapter 3. Thus, towards the objective, truncation and mutational analysis of TNFα revealed that the epitope corresponding to mAb C8 lies in the stretch of amino acids 99-120, and that the amino acids 102-104(QRE) form the core epitope. The Antigen-Antibody complex model also confirmed the result. The data presented in chapter 4 describes studies carried out to compare the binding affinities of mAb C8 with the commercially available Infliximab and also their effectiveness as TNFα inhibitors. Recent progress in the field of antibody engineering opened a new window for generating single-chain variable fragments (scFv). To generate C8 scFv, the variable regions of the heavy (VH) and light chains (VL) were amplified from the hybridoma mRNA and linked together. ELISA results revealed that immobilized scFv binds specifically to hTNFα. However, the scFv did not bind to the antigen when in solution phase, suggesting that further studies are required to stabilize its conformation. The data are presented in chapter 5. In brief, the thesis is presented in five chapters as described above: Chapter 1- ‘Introduction’, provides a brief overview on the discovery of Tumor necrosis factor-α, its’ structure, expression and signalling, TNFα role in physiological condition as well as during inflammation, TNFα associated diseases and available inhibitors of therapeutic activity. Chapter 2- describes the characterization and screening of monoclonal antibodies against human-TNFα and the identification of the highest affinity mAb C8. Chapter 3- presents the identification of the epitope corresponding to the neutralizing mAb C8. Chapter 4- discusses the results obtained from comparative study of mAb C8 and Infliximab (anti-TNFα mAb). And Chapter 5- describes the generation of single-chain variable fragment (scFv) from mAb C8 secreting hybridoma and investigating its binding. In summary, we have obtained a monoclonal antibody to hTNFα that can effectively neutralize TNFα-mediated cytotoxicity and maybe explored for use in anti-inflammation therapy.
- Biochemistry (BC)