Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells.
We recently constructed a humanized bispecific antibody (BsF(ab')2v1) by separate E. coli expression of each Fab' arm followed by directed chemical coupling in vitro. BsF(ab')2 v1 (anti-CD3/anti-p185HER2) was demonstrated to retarget the cytotoxic activity of human CD3+ CTL in vitro against the human breast-tumor cell line, SK-BR-3, which over-expresses the p185HER2 product of the proto-oncogene HER2. Our minimalistic humanization strategy is to install as few murine residues as possible into a human antibody in order to recruit antigen-binding affinity and biological properties comparable to that of the murine parent antibody. This strategy proved very successful for the anti-p185HER2 arm of BsF(ab')2 v1. In contrast BsF(ab')2 v1 binds to T cells via its anti-CD3 arm much less efficiently than does the chimeric BsF(ab')2 which contains the variable domains of the murine parent anti-CD3 antibody. Here we have constructed additional BsF(ab')2 fragments containing variant anti-CD3 arms with selected amino acid replacements in an attempt to improve antibody binding to T cells. One such variant, BsF(ab')2 v9, was created by replacing 6 residues in the second hypervariable loop of the anti-CD3 heavy chain variable domain of BsF(ab')2 v1 with their counterparts from the murine parent anti-CD3 antibody. BsF(ab')2 v9 binds to T cells (Jurkat) much more efficiently than does BsF(ab')2 v1 and almost as efficiently as the chimeric BsF(ab')2. This improvement in the efficiency of T-cell binding of the humanized BsF(ab')2 is an important step in its development as a potential therapeutic agent for the treatment of p185HER2 over-expressing cancers.
Rodrigues
M L
ML
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080.
Shalaby
M R
MR
Werther
W
W
Presta
L
L
Carter
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001666:Binding Sites, Antibody; D017252:CD3 Complex / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D000090063:Proto-Oncogene Mas; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D017252:CD3 Complex; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C000715470:MAS1 protein, human; D009838:Oligodeoxyribonucleotides; D000090063:Proto-Oncogene Mas