Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity.

MAbs 2013 Nov-Dec;5(6):872-81. Epub 2013 Aug 29.

Department of Protein Chemistry; Genentech, Inc; San Francisco, CA USA.

Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies. The most advanced uses of this approach, however, have been limited to E. coli as an expression host to produce non-glycosylated antibodies. Here, we applied the technology to mammalian host expression systems to produce glycosylated, effector-function competent heterodimeric antibodies. In our mammalian host system, each arm is secreted as a heavy chain-light chain (H-L) fragment with either the knob or hole mutations to allow for preferential heterodimer formation in vitro with low levels of homodimer contaminants. Like full antibodies, the secreted H-L fragments undergo Fc glycosylation in the endoplasmic reticulum. Using a monospecific anti-CD20 antibody, we show that full antibody-dependent cell-mediated cytotoxicity (ADCC) activity can be retained in the context of a knobs-into-holes heterodimer. Because the knobs-into-holes mutations convert the Fc into an asymmetric heterodimer, this technology was further used to systematically explore asymmetric recognition of the Fc. Our results indicate that afucosylation of half the heterodimer is sufficient to produce ADCC-enhancement similar to that observed for a fully afucosylated antibody with wild-type Fc. However, the most dramatic effect on ADCC activity is observed when two carbohydrate chains are present rather than one, regardless of afucosylation state.

Download full-text PDF

Source
http://www.tandfonline.com/doi/abs/10.4161/mabs.26307
Publisher Site
http://dx.doi.org/10.4161/mabs.26307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896601PMC
January 2015
83 Reads

Publication Analysis

Top Keywords

mammalian host
8
full antibody-dependent
8
adcc activity
8
secreted heavy
4
heavy chain-light
4
knobs-into-holes
4
asymmetric recognition
4
arm secreted
4
host system
4
system arm
4
chain-light chain
4
explore asymmetric
4
knob hole
4
hole mutations
4
technology systematically
4
fragment knob
4
systematically explore
4
recognition indicate
4
h-l fragment
4
chain h-l
4

References

(Supplied by CrossRef)

Spigel DRET et al.
J Clin Oncol 2011

Similar Publications