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Title: Development of human recombinant MT1-MMP antibodies for the diagnosis and treatment of cancer
Author: Basu, B. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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MT1-MMP domains were expressed and purified as Calmodulin (CaM)-tagged recombinant proteins to serve as targets for identification of specific antibodies. V(ariable) domain phage display techniques were employed to isolate single chain V domain antibody fragments (scFvs) against MT1-MMP hemopexin domain from a naïve human V gene library. Following 3 rounds of phage selection using solid phase panning, unique antibody clones were identified that are specific for MT1-MMP and do not react with soluble MMPs. Sequencing of the V genes and epitope mapping studies by surface plasmon resonance identified two lead scFvs, CHA and CHL that recognised different epitopes on MT1-MMP. The V gene segments from these scFvs were sub-cloned for expression and purification using a Drosophila S2 expression system. Affinity measurements by ELISA and surface plasmon resonance technology (BIAcore) revealed an equilibrium dissociation constant (KD) of 10 nM for CHA and 170 nM for CHL. There is 99.5% sequence homology at the amino acid level between human and murine MT1-MMP hemopexin domain, and equal binding of the scFvs to the recombinant hemopexin domains of both species is observed. Immunofluorescence studies showed binding of both CHA and CHL antibodies to membrane-expressed MT1-MMP on cancer cell-lines. In addition, the antibodies modified binding of recombinant MT1-MMP hemopexin domain to Type I collagen by competition ELISA. Antibody CHA also showed functional effects in cell-based assays that evaluate MT1-MMP function such as Type I collagen degradation and shedding of the adhesion molecule CD44 by HT1080 fibrosarcoma cells over-expressing MT1-MMP. Moreover CHA inhibited invasion of HT1080 cells through Type I collagen in two-dimensional assays.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available