Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell s urface. It is defined by a monoclonal antibody, mAb5T4, that recognises a c onformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progressio n and poor clinical outcome. Its restricted expression in non-malignant tis sue makes 5T4 antigen a suitable target for the development of antibody dir ected therapies. The use of murine monoclonal antibodies for targeted thera py allows the tumour specific delivery of therapeutic agents. However, thei r use has several drawbacks, including a strong human anti-mouse immune (HA MA) response and limited tumour penetration due to the size of the molecule s. The use of antibody fragments leads to improved targeting, pharmacokinet ics and a reduced MAMA. A single chain antibody (scFv) comprising the varia ble regions of the mAb5T4 heavy and light chains has been expressed in Esch erichia coli. The addition of a eukaryotic leader sequence allowed producti on in mammalian cells. The two 5T4 single chain antibodies, scFvST4WT19 and LscFvST4, described the same pattern of 5T4 antigen expression as mAb5T4 i n normal human placenta and by FAGS. Construction of a 5T4 extracellular do main-IgGFc fusion protein and its expression in COS-7 cells allowed the rel ative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K-D = 1.8X10(-11) M, as did both single chain antibodies, scFvST4WT19 K-D = 2.3X1 0(-9) M and LscFvST4 K-D = 7.9X10(-10) M. The small size of this 5T4 specif ic scFv should allow construction of fusion proteins with a range of biolog ical response modifiers to be prepared whilst retaining the improved pharma cokinetic properties of scFvs. (C) 2000 Elsevier Science B.V. All rights re served.