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Title: Investigating dual-targeted domain antibody fusion proteins in a cancer model
Author: Papple, Alex G.
ISNI:       0000 0004 7962 3613
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2015
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Developing efficacious, highly specific therapeutics is an ongoing challenge. The domain antibody (dAb) fragment platform has shown promise targeting a dAbfused therapeutic moiety to a specific tissue. This investigation sought to conclude on the optimum binding affinity combination to maximise tissue-targeting specificity and localised therapeutic potency by using a panel of dAb fusion proteins with varying affinity combinations of asialoglycoprotein receptor (ASGPR)-targeting dAb mutants and human interferon (hIFN) mutants - hIFN-ASGPRdAbs. The hIFN-ASGPRdAbs were engineered, expressed, 1,4,7- triazacyclononanetriacetic acid (NOTA)-conjugated and purified with binding affinities and potencies characterised. Gallium-68 radiolabelling of the NOTA-hIFNASGPRdAbs was optimised using a high activity fraction in a 10 minute reaction at ambient temperature in pH4.4 sodium acetate. HepG2 xenograft mouse models were injected with 68Ga-NOTA-hIFN-dAbs for biodistribution analysis and PET/CT imaging, followed by mRNA expression analysis of the xenograft tissue. ASGPR binding affinities of the NOTA-hIFN-ASGPRdAbs ranged from 0.73pM to ~528nM, and their hIFN potency ranged from 6.79pM to ~3.87nM. Targeting of HepG2 cells was driven by the dAb-mediated ASGPR targeting. Ga-68 radiolabelling efficiencies up to 98.4% and specific activities up to 2.47MBq/μg were achieved. Xenograft uptake was significantly increased through dAb-mediated ASGPR targeting compared to the non-ASGPR targeted control 68Ga-NOTA-hIFNCTRLdAb, achieving 2.42%ID/g vs. 0.68%ID/g, respectively. A trend of increasing xenograft uptake correlated with increasing ASGPR binding affinity for the panel of mutants, in spite of very high murine liver uptake. Increased xenograft uptake also correlated with a more potent hIFN-mediated anti-proliferative mRNA response in the xenograft tissue. A dAb with a mid-range affinity for ASGPR and maximum affinity hIFN was concluded to be the optimum combination. These results showed that the efficacy of a dAb fusion protein can be influenced by the two intrinsic binding affinities. Hence, dAb-mediated tissue targeting of a fused therapeutic and engineered affinity synergism strategies may hold the key to novel, selectively cytotoxic biopharmaceutical drugs.
Supervisor: Not available Sponsor: Queen Mary University of London ; GlaxoSmithKline (GSK) ; EPRSC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medicine ; therapeutics ; cytotoxic biopharmaceutical drugs.