Construction, expression and characterization of humanized antibodies directed against the human alpha/beta T cell receptor.
Completely humanized antibodies with specificity for the human alpha/beta TCR have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 CD regions and human EU framework regions were synthesized and replaced into previously isolated genomic fragments. These fragments were inserted into mammalian expression vectors containing the human kappa and gamma 1 C region exons. Two variants were constructed each containing selected BMA 031 amino acids within the human frameworks. The humanized genes were transfected into Sp2/0 hybridoma cells by electroporation and transfectomas secreting humanized antibody were isolated. Levels of antibody expression up to 7 pg/cell/24 h were obtained. The humanized antibody, BMA 031-EUCIV2, competed poorly with murine BMA 031 for binding to T cells. BMA 031-EUCIV3, however, bound specifically to T cells and competed effectively with both the murine BMA 031 antibody and a previously constructed chimeric BMA 031 antibody for binding to these cells. The relative affinity of BMA 031-EUCIV3 was about 2.5 times lower than BMA 031. The ability to promote antibody dependent cell-mediated cytolysis was significantly enhanced with the engineered antibodies as compared to murine BMA 031. Humanized BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft vs host disease, and autoimmunity.
Shearman
C W
CW
Genzyme Corporation, Framingham, MA 01701.
Pollock
D
D
White
G
G
Hehir
K
K
Moore
G P
GP
Kanzy
E J
EJ
Kurrle
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005091:Exons; D006801:Humans; D007135:Immunoglobulin Variable Region; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA