Humanization of an anti-p185HER2 antibody for human cancer therapy.
The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080.
P
P
Carter
L
L
Presta
C M
CM
Gorman
J B
JB
Ridgway
D
D
Henner
W L
WL
Wong
A M
AM
Rowland
C
C
Kotts
M E
ME
Carver
H M
HM
Shepard
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000627:therapeutic use; D001483:Base Sequence; D001943:Breast Neoplasms; D002455:Cell Division; D002461:Cell Line, Transformed; D002678:Chimera; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000276:immunology*; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007167:Immunotherapy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000276:immunology*; D011518:Proto-Oncogene Proteins / Q000276:immunology*; D011519:Proto-Oncogenes; D018719:Erb-b2 Receptor Tyrosine Kinases; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D066246:ErbB Receptors; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases