Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1

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 n for the IgG and 10 μ 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.

Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate

“…Using confocal microscopy, internalization of HuHMFG1 into an EA cell line (OE19) was shown (Figure 6 ). The punctate intracellular pattern is similar to the pattern seen in previous work where endosomal co-localization of HuHMFG1 was demonstrated in a breast cancer cell line [ 21 ].…”

“…Administration led to endogenous production of anti-MUC1 IgG in some patients, but there was no survival benefit in those in whom this occurred [ 20 ]. HuHMFG1 undergoes cell internalization [ 21 ] and was considered as a vehicle for an antibody-drug conjugate (ADC) approach using the potent cytotoxin, calicheamicin. Reasonable efficacy was seen but in this example the overall therapeutic window was low as calicheamicin was not well tolerated at the higher doses [ 22 ].…”

Molecular imaging with nanoparticles: giant roles for dwarf actors

“…Unlike antibodies, their development must generally take place on a case-by-case system, inhibiting the establishment of a generally applicable corpus of techniques. Their major advantage over all other targeting systems, that they target oligosaccharide conWgurations, is not absolute: clinically relevant diVerential glycosylations can be recognised by antibodies (Pericleous et al 2005). Many intercellular recognition functions are lectin-mediated (for example, the selectin recognition system, which initiates inXammation).…”

Population pharmacokinetics of the humanised monoclonal antibody, HuHMFG1 (AS1402), derived from a phase I study on breast cancer

“…Metastatic breast cancer remains incurable and there is a need for new active treatments in this setting. HuHMFG1 is a recombinant DNA-derived humanised monoclonal human milk fat globule-1 antibody that targets the immunodominant epitope of the MUC1 gene product (Pericleous et al, 2005). It was engineered by grafting the complementary determining regions of the parental murine antibody (HMFG1) into the consensus framework of a human IgG1.…”