Antibody Name: 1B4 (IgG2a|k)
References: Singer, I.I., Kawka, D.W., DeMartino, J.A., Daugherty, B.L., Elliston, K.O., Alves, K., Bush, B.L., Cameron, P.M., Cuca, G.C., Davies, P. Forrest, M.J., Kazazis, D.M., Law, M., Lenny, A.B., MacIntyre, D.E., Meurer, R., Padlan, E.A., Pandya, S., Schmidt, J.A., Seamans, T.C., Scott, S., Silberklang, M., Williamson, A.R. and Mark, G.E. "Optimal humanization of 1B4, an anti-CD18 murine monoclonal antibody is achieved by correct choice of human V-region framework sequences" J. Immunol. (1993) 150:2844-2857.
Daugherty, B.L., DeMartino, J.A., Law, M-F., Kawka, D.W., Singer, I.I. andMark, G.E. "Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins" Nucleic Acid Res. (1991) 19:2471-2476.
DeMartino, J.A., Daugherty, B.L., Law, M-F., Cuca, G.C., Alves, K., Silverklang, M. and Mark, G.E. "Rapid humanization and expression of murine monoclonal antibodies" Antibod. Immunoconj. Radiopharmaceut. (1991) 4:829-835.
Donor Antibody: mouse
Acceptor Antibody: Human IgG4 | k ("to minimize binding to C1q and FcγReceptor")
Antigen: human B subunit of B-2 integrin (CD18) on leukocytes (different epitope to YFC51.1).
Laboratory: Merck Research Labs, NJ; Molecular Structure, NIH, MD
Design: Search for homologous human sequences. Examine those with highest identity for conservation of packing residues as in Padlan, E.A. "A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties" Mol. Immunol. (1991) 28:489-498. Fidelity of humanisation of each chain tested with "hemichimaeric" mAbs. No modelling. Independent selection of human FRs for each chain.
Frameworks: VH GAL, JON, NEW | VL REI, LEN
CDRs: All 6 VH and VL, Kabat Definition.
Backmutations: Four constructs GAL|REI GAL|LEN JON|REI NEW|REI tested.
GAL|REI with R16G:H (unusual residues in human) W47L:H V109L:H (to increase "packing" identity score) V104L:L
Binding: No difference between CDR-grafted REI or LEN (maybe light chain is unimportant in this antibody?)
GAL/REI with backmutations showed best avidity (IC50) in competition binding with mouse mAb, but GAL/REI (no mutations) selected for further analysis (with 33% binding activity)
Expression: CV1P cells
Comment: "Avidity correlated with overall percent identity between the human and murine VH FRs and, in particular, with conservation of "packing" residues"
REI and LEN were interchangeable. Immunogenicity was anti-idiotypic.
The specificity was not altered by humanization.
"CDR-grafted antibodies directed toward cell-surface molecules may be more immunogenic than those recognizing soluble Ag" due to internalisation and efficient presentation of CDR epitopes to the humoral immune system.
Clinical Indication: Acute inflammatory disease.

explanation of symbols and abbreviations

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