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Protein Engineering, Vol. 13, No. 5, 353-360, May 2000
© 2000 Oxford University Press

Design and synthesis of germline-based hemi-humanized single-chain Fv against the CD18 surface antigen

Cristina Caldas1, Verônica P.C.V. Coelho2, Daniel J. Rigden3, Goran Neschich3, Ana Maria Moro4 and Marcelo M. Brígido1,5

1 Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70910-900, 2 Laboratório de Imunologia de Transplantes, INCOR/Universidade de São Paulo, São Paulo, SP, 05403-000, 3 EMBRAPA-CENARGEN, Brasília, DF, 70770-900 and 4 Instituto Butantan, São Paulo, SP, 05503-900, Brazil

The 6.7 murine monoclonal antibody (mAb) recognizes the human CD18 antigen and is therefore of interest as an anti-inflammatory agent. The 6.7 heavy variable chain (VH) was humanized using the closest human germline sequence as the template on to which to graft the murine complementary determining regions (CDRs). Two versions were proposed, one in which the residue proline 45 of the murine form was maintained and another in which this framework residue was changed to the leucine found in the human sequence. These VH humanized versions were expressed in the yeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs), with the VL from the murine antibody. The scFv from the murine antibody was also expressed. The binding activities of the murine and both hemi-humanized scFvs were determined by flow cytometry analysis. All the constructions were able to recognize human lymphocytes harboring CD18, indicating successful humanization with transfer of the original binding capability. Some differences between the two hemi-humanized versions were observed. The method used was simple and straightforward, with no need for refined structural analyses and could be used for the humanization of other antibodies.


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