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Title: Isolation of a single chain antibody fragment specific to a molecular target of MLL leukaemias
Author: Chan, N. M.-M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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The isolation of a single chain variable fragment (scFv), a format of intrabody, specific to a downstream molecular target of MLL-AF9 and MLL-ENL transloca­tions is described. Further characterisation allowed assessment of its potential as a therapeutic agent. MLL-AF9t(11;9)(q23,q23) and MLL-ENL t(11;19)(q23;p13.3) are frequently implicated in spontaneous acute leukaemias. In both cases, fusion proteins are produced. MLL-fusion proteins are believed to play a dominant role in leukaemogenesis. AF9 and ENL are related transcriptional activators; their MLL-fusion proteins might possess aberrant transcriptional acti­vating potential and by disrupting normal haematopoietic differentiation programmes leukaemia might result. Two engineered mouse models mimicking MLL-AF9 and NILL-ENL translocations have been described. These mice developed myeloid leukaemia and Hoxa7, Hoxa9 and Hoxa10 were up-regulated in tumour tissues. These are all known targets of human MLL-AF9 and MLL-ENL translocations.  Furthermore, nude mice injected with cell lines derived from these tumour tissues themselves developed tumours. These findings supported the representativeness of these mouse models to the human disease. Hoxa9 was chosen for further study. It is be­lieved to have a dominant role in leukaemogenesis. Using the laboratory’s intracellular antibody capture (IAC) protocol, an scFv targeting the Hoxa9 homeodomain (HD) was isolated. The IAC protocol allows top-down isolation of functional scFvs from a large pool of target-specific binders from scFv libraries. scFvs representing three unique sequences were isolated from in vitro phage display screening, and one was found to bind to the Hoxa9 HD in vivo. Further characterisation of this scFv revealed that both domains are required for antigen-recognition. Hoxa9 belongs to an evolutionarily conserved family. There are 39 HOX proteins in humans and they are highly homologous. Besides the Hoxa9 HD, this scFv also recognised Hoxal0, an­other downstream target of MLL-AF9 and MLL-ENL translocations. Hox HDs consist of three helices. In Hoxa9 these helices were replaced individually by the respective helices of HOX11, which are poorly resemblant to Hoxa9’s. It was determined that all three helices of the Hoxa9 HD are required for scFvIII binding, since scFvIII failed to bind any chimaeric Hoxa9 HD. This also indicates high antigen-binding specificity. scFvIII may touch all three helices. This work provides an example of the isolation of target-specific intrabodies which may neutralise the activity of disease-causing proteins inside cells and hence be used as therapeutic agents in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available