Rubins, Leigh Ruth; (2001) Recombinant antibody fragments to vascular associated targets of human tumours. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells expressing the VEGF receptor. VEGF aids tumour growth by promoting tumour angiogenesis and/or stroma generation; neutralisation of VEGF inhibits tumour growth. VEGF reacts with its upregulated receptor on tumour vascular endothelium and this VEGF-receptor complex may provide a tumour selective target. A series of recombinant antibodies to VEGF were generated to investigate the potential of VEGF targeting for antibody directed cancer therapy. Antibodies were produced as scFvs (single chain Fvs) from splenic lymphocytes of Balb/c mice immunised with VEGF165 using filamentous phage technology to produce a combinatorial library of approximately 6.7×10^7 scFv clones. Thirty clones expressing scFvs reactive to VEGF were selected by panning. Four of these were selected for further analysis on the basis of their DNA sequence diversity, and by immunohistochemistry to assess reactivity with blood vessel endothelium. The selected scFvs were subcloned into an expression plasmid incorporating a His6 tag and expressed scFv proteins were purified using metal chelate chromatography. Purified scFvs were biotinylated and used in immunohistochemical studies to examine binding to human placenta, normal human tissues, human colorectal carcinoma and human colorectal xenografts. ScFv clone LL4 showed most selectivity for tumour vasculature in these tests and was more extensively studied. LL4 binds VEGF with an affinity of 3.5×10^7M and, in in vivo studies LL4 showed clear localisation to the endothelial layer of tumour blood vessels. LL4 did not neutralise the proliferative effects of VEGF on human umbilical vein endothelial cells (HUVECs) consistent with the hypothesis that it recognised receptor-bound VEGF rather than interfered with the VEGF/VEGF receptor interaction. LL4 demonstrates that VEGF-reactive scFvs, produced by phage technology, have potential as tumour vascular targeting agents.

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