425

Antibody Name: 425 (VH SGIIB | VL kIV/VI)
References: Kettleborough, C.A., Saldanha, J., Heath, V.J., Morrison, C.J. and Bendig, M.M. "Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation." Prot. Engng. (1991) 4: 773-783.
Donor Antibody: mouse
Acceptor Antibody: Human IgG1 | k
Antigen: Polypeptide epitope on external domain of human Epidermal Growth Factor Receptor (EGFR)
Laboratory: E. Merck, Germany; MRC Collaborative Centre, UK
Design: Select homologous consensus sequence. "Where no single amino acid was listed, the most commonly occurring amino acid at that position was selected. If there was no preferred amino acid at a particular position in the human consensus sequence, the amino acid that was found at that position in the (mouse) sequence was selected."
Nine versions of VH and two of VL were made.
Frameworks: VH SGI consensus (Kabat Ed IV 1987) | REI (SGI consensus) (modified as in Campath-1.)
CDRs: All 6 VH and VL, Kabat Definition.
Backmutations: VH L71V R66K V67A S30T V48I
VL F71Y
Binding: In competition-binding assay with mouse mAb to purified antigen, humanised mAb (best version VHg | VLb) had ~60% avidity.
Expression: transient expression in cos cells
Comment: Some confusion over Kabat numbering of residues 66,67:H. The molecular model was used to post-rationalise the changes rather than aid in the initial design. Position 71 in both VH and VL again showed the importance of retaining canonical residues. S30T was presumed to be due to direct binding with antigen.
Clinical Indication: Enhanced EGFR expression is found on malignant tissue from a variety of sources, thus making 425 a possible agent for the diagnosis and treatment of human tumours, particularly colorectal carcinomas and gliomas.

explanation of symbols and abbreviations

José Saldanha © 1997-8. Birkbeck College, London WC1E 7HX.