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Title: Development of a versatile antibody cloning and expression system
Author: Dodev, Tihomir
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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Over the last three decades, recombinant monoclonal antibodies (mAbs) have become the key tool for basic research, diagnosis and treatment of human diseases. This has required the selection of recombinant antibodies with high affinity for appropriate epitopes on the target antigen and other desirable characteristics, such as their isotype and effector functions. In Biopharmaceutical communities specialised in antibody production, expression levels of grams per litre have been reached. However, lack of a suitable manufacturing platform, which ensures consistent antibody production, has always been one of the major impediments to the development of recombinant antibody material in academia. To overcome this barrier, we have developed a unique cloning method for one-step assembly of antibody heavy- and light-chain DNAs in a single mammalian expression vector. The DNA fragments assembled in this system do not rely on restriction enzyme- and ligase-dependant methods, thus minimising the steps involved in the cloning procedure. This allows the reproducible generation of fully functional recombinant antibodies of any species and isotype with any desired specificity. In less than four weeks, tens of milligrams per litre can be achieved, from cloning through to harvesting of transfected cell supernatants, providing an unbiased manufacturing platform compared to the currently available antibody expression methods in academia. The system proved to be very efficient and readily adaptable for the high-throughput screening of melanoma patient-derived antibody candidates with clinical potential. It enabled the parallel comparative functional studies between IgE and IgGl isotypes in an in vivo xenograft model of melanoma. The IgE isotype showed superior efficacy in restricting tumour growth, which encouraged us to continue developing antibody discovery methods, and pursuing melanoma antigen-specific antibodies as future effective therapies of this disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available