1. ¹Department of Biological Sciences, Imperial College London, Exhibition Road, London SW7 2AZ, UK
2. ²Antisoma Research Laboratories, West Africa House, Hanger Lane, London W5 3QR, UK

Correspondence: Dr MP Deonarain, E-mail: m.deonarain@imperial.ac.uk

³Current address: The Leukaemia Sciences Labs, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.

Revised 24 August 2005; Accepted 28 August 2005; Published online 1 November 2005.

Abstract

The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 nM for the IgG and 10 M for the Fab), with particularly low but similar dissociation rate constants (0.031–0.095 s^-1). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.