High level Escherichia coli expression and production of a bivalent humanized antibody fragment.
Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.
Department of Protein Engineering, Genentech Inc., South San Francisco,CA 94080.
P
P
Carter
R F
RF
Kelley
M L
ML
Rodrigues
B
B
Snedecor
M
M
Covarrubias
M D
MD
Velligan
W L
WL
Wong
A M
AM
Rowland
C E
CE
Kotts
M E
ME
Carver
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins