Polymeric human Fc-fusion proteins with modified effector functions
1
David N A Mekhaiel
¶,
2,3
Daniel M Czajkowsky
6,7
1
¶
, Jan Terje Andersen4,5¶,, Jianguo Shi ,
6
Marwa El-Faham , Michael Doenhoff , Richard S McIntosh6, Inger Sandlie4, Jianfeng
8
2
3
1*
He , Jun Hu , Zhifeng Shao and Richard J Pleass
1
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.
2
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai Jiao Tong
University, Shanghai 200240, China..
3
Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong
University, Shanghai 200240, China.
4
Centre for Immune Regulation and Department of Molecular Biosciences and Centre for
Immune Regulation, University of Oslo, P.O. Box 1041, N-0371 Oslo, Norway.
5
Department of Immunology, Oslo University Hospital Rikshospitalet and University of Oslo,
PO Box 4956, N-0424 Oslo, Norway.
6
School of Biology, University of Nottingham, Nottingham, NG7 2RD,
7
University of Alexandria, Alexandria, Egypt.
8
Department of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China.
Fig. S1. Purification and characterization of monomeric PfMSP119 fusions to either human IgG1Fc or mouse IgG2a-Fc expressed in CHO-K1 cells. (A) Sandwich ELISA with mAb anti-PfMSP119
(either mAb12.10 for the human reagents or IgG1 C1 for the mouse reagents) to capture and antihuman or anti-mouse (shown in panel a) IgG-Fc-HRP-conjugated Abs detect high secreting CHO-K1
clones. Red bars are negative control supernatants. (B) Size-exclusion chromatography (SEC) analysis
on Superdex-200 10/300GL column showing PfMSP119-mIgG2a-Fc running with an approximate
molecular weight of 90 kDa as expected for a monomeric Fc-fusion protein to PfMSP119. Elution
profiles of molecular weight standards are indicated by the green trace. (C) 5 g of protein from
protein-G eluted fractions were run under non-reducing or reducing conditions on 4-12% bis-Trisacrylamide gradient gels, transferred to nitrocellulose, and detected with either an anti-human IgG-FcHRP (left panel) or an anti-mouse IgG-Fc-HRP (right panel). Separate western blots were also probed
with mAbs specific for PfMSP119 (mAbs 12.10 or IgG1 C1 as above) and are shown on the right hand
side of each panel. Bands of approximately ~90 kDa represent intact fusion proteins run under nonreducing conditions which on reduction run at ~45 kDa as expected.
Fig S2. Purification and characterization of PfMSP119-hIgG1-Fc-TP-LH309/310CL expressed in
CHO-K1 cells. (a) Sandwich ELISA with polyclonal anti-human IgG-Fc or mAb 12.10 to capture and
an HRP-conjugated anti-human IgG-Fc mAb to detect high secreting CHO-K1 clones. Red bars are
negative control supernatants and green bars positive control dilutions of human IgG1. (b) Sizeexclusion chromatography (SEC) analysis on Superdex-200 10/300GL column showing PfMSP119hIgG1-Fc-LH309/310CL-TP (left panel) or the hIgG1-Fc-LH309/310CL-TP empty cassette devoid of
Ag (right panel), running with approximate molecular weights of 540 (H2) and 312 (H1) kDa
respectively (representing hexamers), and with molecular weights of ~160 (D2) and ~100 (D1) kDa
representing dimers (red trace). Elution profiles of molecular weight standards are indicated by the
black trace. (c) 5g of hIgG1 (lane 1), PfMSP119-hIgG1-Fc-LH309/310CL-TP (lane 2) or hIgG1-FcLH309/310CL-TP (lane 3) were run on 6% Tris-glycine gels, transferred to nitrocellulose, and detected
with an anti-human IgG conjugated to HRP. Corresponding bands on the SEC are arrowed.
Fig. S3. Size-exclusion profiles of IgM-Fc fusion proteins (pink trace) run against known molecular
weight markers (blue trace) as in Fig. S2.
Fig. S4. Molecular model of PfMSP119-hIgG1-Fc-LH309/310CL-TP showing key binding
sites on the hexamer. (a) Shown on the left is the top view of the hexamer with all of the
interacting proteins superimposed. The panel on the top right highlights the specific
interaction with FcRn55, and the panel on the lower right is with Protein-G (pink)56. In each
superposition, the Fc region present in the crystal structure of the particular Fc-binding
protein is colored in cyan to show the overlap with the modeled hIgG1 Fc presented here. (b)
Shown on the left is the bottom view of the hexamer with all of the interactions
superimposed. The panel on the right shows the interaction with FcγRIII (green)57 along with
the Fc-region present in this crystal structure (cyan). The coloring of the hexamer is the same
as in Fig. 1B.
Fig. S5. Binding of hexamer (upper panel) or dimer (lower panel) hIgG1-Fc-LH309/310CL-TP to
human FcRI by surface plasmon resonance analysis.
Fig. S6. In vivo half-life of PfMSP119-hIgG1-Fc-LH309/310CL-TP in mice. Injected antibodies
were detected by ELISA at the time points indicated as described in methods. The in vivo half-life of
PfMSP119-hIgG1-Fc-LH309/310CL-TP was approximately 18 hours.
Fig. S7. C1q (left panel) and C5-9 deposition (right panel) to Fc-fusions determined by
ELISA. Each point represents the mean optical density (+/-SD) of duplicate wells for each
mouse within a given group. Data from one of three replicate experiments are shown.
Fig. S8. PfMSP119-specific Ab responses. Mice were immunized with (A) PfMSP119-mIgG2a-Fc-TP
(open symbols) or mIgG2a-Fc-TP (closed symbols) (B) PfMSP119-hIgG1-Fc-TP (group 1 CD64 Tg &
group 2 WT Balb/c) or hIgG1-Fc-TP (groups 3 CD64 Tg and group 4 WT Balb/c). Each point
represents mean optical densities (+/-SD) from duplicate wells of sera from individual animals.
Fig. S9. Dose response curves and effects of Alum co-administration on Ag-specific Ab-titres
generated by immunization with PfMSP119-mIgG2a-Fc in the presence (open symbols) or
absence (closed symbols) of Alum. (A) Pre- and (B) Post-challenge total IgG responses are shown.
Each point represents mean optical densities (+/- SD) of pooled serum from 4 animals per group.
Fig. S10. Immunization with PfMSP119-mIgG2a-Fc bolsters antibody titres post challenge.
Groups of four BALB/C mice were immunized three times with either PBS or 10 g PfMSP119mIgG2a-Fc in a final volume of 200 l at fortnightly intervals. 2 weeks after the last immunization
mice were either challenged with 10,000 infected erythrocytes or terminally bled for antibodies.
Fig. S11. Cercarial elastase (CE)-specific Ab titres. Individual Balb/c mice were immunized i.p. with
a 50 g dose followed by two further doses of 25 g on three separate occasions of CE-mIgG2a-Fc or
recombinant histidine tagged CE (CE-His) in the presence of an equal volume (200l) of Alum. Each
point shows mean optical densities obtained from duplicate wells at each dilution of sera. Each curve
represents the mean of 5 animals per group.
Fig. S12. Enhanced Green Fluorescent Protein (EGFP)-specific Ab titres. Groups of 4 Balb/c mice
per group were immunized with EGFP-mIgG2a-Fc-TP (open symbols) or mIgG2a-Fc-TP (closed
symbols). Each point represents the mean optical densities (+/-SD) from duplicate wells derived from
each animal.
Fig. S13. Electrostatic potential calculation of a PfMSP119-hIgG1-Fc-LH309/310CL-TP
monomer extracted from the hexameric complex. The upper panel shows a view from the
top of the molecule (the TP region closest to the viewer), and the lower panel shows the view
from the side. The depicted contours reflect 1.8 kT/e, with positive potential coloured blue
and negative potential coloured red.
Fig. S14. Fc-fusion proteins expressed by CHO-K1 cells are sialylated. 5g of reduced proteins
were run on 4-12% Bis-Tris gels and transferred to PVDF membranes and blocked with 1% Roche
blocking reagent as described previously31. Membranes were then incubated with a 1/200 dilution of
biotinylated Sambucus nigra bark lectin (Vector laboratories) and developed with streptavidin-HRP
(Serotec) as previously31. Lanes 1) Plasmodium yoelii MSP119-GST negative control, 2) recombinant
human IgG1 (C1), 3) human serum IgG, 4) PfMSP119-IgM-Fc, 5) PfMSP119-mIgG2a-Fc, 6) hexameric
PfMSP119-hIgG1-Fc-TP-LH309/310CL,
monomeric PfMSP119-hIgG1-Fc-TP.
7)
dimeric
PfMSP119-hIgG1-Fc-TP-LH309/310CL,
8)
Fig. S15.
Size-exclusion chromatography (SEC) profiles of affinity purified IgG from Gambian
(green trace) and healthy UK donors (blue trace). IgG was purified on protein-G sepharose affinity
columns from pooled plasma from 14 Gambians or 5 healthy UK controls. IgG was eluted from the
protein-G sepharose beads with 0.1M glycine pH2, and neutralized with 1M Trizma-HCl pH9 for 15
min prior to separation by SEC on a high-performance Superdex-200 10/300GL column using an
AKTA FPLC (GE Healthcare). Eluted fractions were compared against known high MW gel filtration
standards (Biorad). Area under the curve analysis revealed that high MW circulating immunecomplexes (fractions A6-A9) accounted for 10-30% of plasma Ab in both plasma preparations.