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. 2011 Dec 20;108(51):20690-4.
doi: 10.1073/pnas.1108360108. Epub 2011 Dec 5.

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Larry Zeitlin  1 , James Pettitt, Corinne Scully, Natasha Bohorova, Do Kim, Michael Pauly, Andrew Hiatt, Long Ngo, Herta Steinkellner, Kevin J Whaley, Gene G Olinger
Affiliations

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

Larry Zeitlin et al. Proc Natl Acad Sci U S A. .

Abstract

No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED(50) = 33 μg). A version with typical heterogenous mammalian glycoforms (ED(50) = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED(50) = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
N-glycosylation profile of different h-13F6 glycoforms and Rituxan as determined by LS-ESI-MS. Numbers represent the presence of the different glyco-species in %. Minor glycoforms (below 5%) are not indicated. N-glycan nomenclature according to www.proglycan.com.
Fig. 2.
Fig. 2.
C1q binding ELISA. Various concentrations of mAb were coated onto ELISA plates. After blocking, 2 μg/mL of human C1q was added. The binding of C1q to the mAb was detected using goat anti-human C1q polyclonal antibody followed with rabbit anti-goat (human adsorbed) HRP-conjugated antibody. Error bars indicate SD (n = 3).
Fig. 3.
Fig. 3.
Surface plasmon resonance sensorgrams showing binding and dissociation of h-13F6 mAbs to murine FcγRs. Approximately 1,000 RUs of HIS-tagged recombinant murine Fcγ receptors were bound to an NTA sensor chip. h-13F6 mAb (5 μg/mL) was subsequently flowed across the surface.
Fig. 4.
Fig. 4.
Summary of dose–response experiment in mice. Mice (n = 10) received mAb i.p. 1 d before i.p. challenge with 1,000 PFU of mouse-adapted Ebola Zaire. *P < 0.05 compared with 3 μg of h-13F6CHO and PBS (Mantel–Cox). **P = 0.08 compared with 30 μg h-13F6CHO; P < 0.001 compared with 30 μg of h-13F6agly and PBS. Murine 13F6 data obtained using identical experimental conditions are plotted from table 1 in ref. 2).
Fig. 5.
Fig. 5.
Survival curves for the low-dose (3 μg) groups of mice. Data are from the groups described in Fig. 4.
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