IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions

A Corrigendum to this article was published on 19 September 2017

This article has been updated

Abstract

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

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Figure 1: Biochemical and functional properties of antibodies with engineered Fc domains that bind to C1q with exquisite selectivity.
Figure 2: In vitro complement activation by C1q-specific antibodies to CD20.
Figure 3: Killing of CD20+ cells by CDCC.
Figure 4: Quantitative analysis of RA801-mediated CDCC.
Figure 5: CDCP of CD20+ cells.
Figure 6: In vivo activity of RA801.
Figure 7: Structural features of A801-Fc and G801-Fc.

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Change history

  • 27 June 2017

    In the version of this article initially published online, the labels identifying each plot in Figure 1b were missing. The labels are as follows (left to right): CHO, FcγRI, FcγRIIaR131, FcγRIIaH131, FcγRIIb, FcγRIIIaF158 and FcγRIIIaV158. Also, the reference cited in the accompanying legend (ref. 21) is incorrect. The correct reference is ref. 14. The errors have been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

We thank Y. Tanno for assistance with protein expression; A. Bui for assistance with liquid chromatography–tandem mass spectrometry; P. Tucker (University of Texas at Austin) for cancer cell lines; D. Lee (MD Anderson Cancer Center) for patient-derived primary acute lymphocytic leukemia cells; the Macromolecular Crystallography Facility of the University of Texas at Austin; the Berkeley Center for Structural Biology; the Advanced Light Source (supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract DE-AC02-05CH11231); the Proteomics Facility at the University of Texas at Austin (supported by grant RP110782 from the Cancer Prevention Research Training Program); and A. Nicola and the Plate-Forme d'Imagerie Dynamique (Institut Pasteur, Paris) for help with the bioluminescence experiments. Supported by the Clayton Foundation, the Institut Pasteur (P.B. laboratory), the Institut National de la Santé et de la Recherche Médicale (P.B. laboratory), the European Research CouncilSeventh Frame-work Program (ERC-2013-CoG 616050 for the P.B. laboratory), the Pasteur–Paris University International PhD program (B.B.), the Cancer Prevention Research Training Program (RP140108 to M.D.; RP160015 to H.T.; and RP130570 to N.V.), the American Cancer Society (123506-PF-13-354-01-CDD to N.M.), Uehara Memorial Foundation (H.T.), Japan Society for the Promotion of Science (H.T.), Deutsche Forschungsgemeinschaft (CRC1181-A07 to F.N.), the US National Institutes of Health (R01CA174385 to N.V.; and R01 GM104896 to Y.J.Z.) and the Welch Foundation (F-1778 to Y.J.Z.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Cancer Prevention and Research Institute of Texas.

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C.-H.L. and G.G. conceived of and designed the research; C.-H.L., G.R., W.Y., M.W., W.C., B.T., J.L., K.T., M.D., A.L., N.M., M.A.L., O.R.-L.G., B.B., T.H.K., H.T., G.D. and C.A. performed experiments; C.-H.L, G.R., W.Y., O.I.L., R.P.T., F.N., N.V., P.B., Y.J.Z. and G.G. analyzed data; and C.-H.L, G.R., N.V., P.B., Y.J.Z., and G.G. wrote the paper.

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Correspondence to George Georgiou.

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Competing interests

G.G. and C.-H.L. are authors of the approved patent PCT/US2016/017100 9 ('Engineered immunoglobulin Fc polypeptides displaying improved complement activation').

Integrated supplementary information

Supplementary Figure 1 Engineering of C1q-specific IgG antibodies.

(a) Schematic illustration of aglycosylated IgG display system for Fc engineering. Soluble C1q-PE competes with non-fluorescent FcγRs-GST for binding to the displayed IgG variants on the surface of bacterial spheroplasts before FACS sorting. (b) Schematic illustration of the construction of the three sub-libraries of mutated Fc domains: S-library: randomization of the focused 15 amino acids; E-library: random mutagenesis on Fc domain by error prone PCR with 1% error rate: SE-library: random mutagenesis of the S-library gene pool by error prone PCR with 1% error rate. The sizes of sub-libraries were 2 × 108 (S-library), 3 × 108 (SE-library), and 1 × 109 (E-library). (c) C1q does not bind to IgG-displaying E.coli spheroplasts in high salt buffer (50 mM phosphate, 330 mM NaCl, pH 7.4). Since E.coli cannot synthesize glycosylated antibodies, E.coli cells were engineered to display an antigen (PA domain 4) on the inner membrane and then the spheroplasted cells were incubated with the very high affinity anti-PA domain 4 antibody, M18. Binding to C1q to the surface-bound C1q was detected by FACS using 10 nM C1q-PE. (d) Flow cytometry analysis of C1q binding onto IgG-displaying E.coli spheroplasts in high salt buffer. (e) Flow cytometry analysis of E.coli library spheroplasts labeled with 10 nM C1q-PE before (Red) and after sorting (Cyan). (f-g) Fluorescent histogram of isolated IgG variants binding to 10 nM of C1q-PE in high salt phosphate buffer (f) or to 10 nM of tetrameric FcγRIIIaV158-PE in PBS (g). (h) MFI and fold increase in MFI following incubation with C1q or tetrameric FcγRIIIaV158 relative to cells unmutated aglycosylated IgG.

Supplementary Figure 2 LC-MS/MS spectra of 801 Fc.

Purified A801 Fc (a-b) or G801 Fc (c-d) was examined without (a & c) or with (b & d) 160 mM of dithiothreitol (DTT). Molecular weight (M.W.) estimated from the LS-MS data are shown; the respective calculated masses were presented at top of each plot. In (c) and (d) multiple predominant species detected are a consequence of glycan heterogeneity.

Supplementary Figure 3 Binding analysis of RA801 or RA802 to human C1q, FcγRs and FcRn.

(a) ELISA analysis of C1q-specific antibody variants binding to human FcγRs. ELISA plates were coated with aglycosylated IgG1 (negative control), glycosylated IgG1 (Rituxan, positive control), RA801 or RA802. 500 nM or 50 nM of his-tagged FcγRI, GST-tagged FcγRIIaH131, GST-tagged FcγRIIaR131, GST-tagged FcγRIIb, GST-tagged FcγRIIIaV158, or GST-tagged FcγRIIIaF158 was added and binding was detected using anti-His IgG conjugated to HRP or anti-GST IgG-HRP, accordingly. Errors bars: standard deviations from triplicate experiments. (b-c) SPR analysis of C1q-specific antibody variants. (b) SPR analysis of antibody binding to purified C1q or to effector Fcγ receptors. Antibody variants were immobilized on CM5 chips. The binding of C1q, or of GST fusion proteins high affinity FcγRI, or low affinity FcγRIIa, FcγRIIb, or FcγRIIIa, all expressed as dimers, to enhance binding, was assayed. (c) pH-dependent binding to human FcRn. In all sensorgrams, x-axis is time (sec) and y-axis is RU (response unit). Data are from one experiment representative of three experiments (a-c).

Supplementary Figure 4 Complement-mediated tumor-cell killing by C1q-specific antibody variants.

(a) CDC assays with Daudi cells. 10 μg/ml of each antibody was incubated with 50 % of PHS and CD20+ Daudi cells for 15 mins and 30 mins. Lysed cells were detected by TO-PRO-3. (b) C1q deposition on mAb-opsonized CD20+ cells. Time course for C1q deposition on CD20+ Raji cells. Raji cells were incubated in 5% NHS and 10 μg/ml mAb for different time periods at 37°C. Cells were washed twice, incubated with FITC-conjugated anti-C1q and assayed by flow cytometry. MFI were converted to molecules of equivalent soluble fluorochrome (MESF) using calibrated beads (Spherotech). (c-d) CD20+ cancer cells killing activities by PBMC or PMNs. Raji (or Ramos) cells incubated with effector cells in RPMI1640 medium without serum (c) or with 25% of C9-depleted serum (d). (e) Effect of α-CR3 or α-CR4 antibodies in CDCC. Rituximab (or RA801)-opsonized Ramos cells were incubated with: 10 μg/ml of α-CR3 Ab (or α-CR4 Ab)-coated effector cells in RPMI1640 medium supplemented with 25% C9-depleted serum. (f) Cell killing of CD20+ cancer cells by effector cells in the presence of serum. Cell lysis of CD20+ cells with effector cells in RPMI1640 medium supplemented with 25% PHS. In c-f, % of cell lysis was determined 4 hours after the addition of cells and antibody. In all assays, the percent of tumor cell lysis was calculated according to the following formula: 100 × (E-S)/(M-S), where E is the fluorescence of the experimental well, S is the fluorescence in the absence of antibody (tumor cells incubated with medium and complement alone), and M is that of tumor cells with lysis buffer (Triton® X-100 at 2% v/v, SDS at 1% w/v, 100 mM NaCl, and 1 mM EDTA). In all assays, PMN cells were stimulated by incubation with 10 ng/mL GM-CSF and trastuzumab-IgG was used as a negative control. Errors bars indicate the standard deviation (s.d.) from triplicate experiments. Data are from one experiment representative of three experiments.

Supplementary Figure 5 Time-lapse imaging in nanowell grids (TIMING).

Raji tumor cells and NK cells are stained with PKH26 and PKH67 respectively, deposited en masse on a grid containing thousands of nanowells, immersed in fluorescent annexin V-containing medium and imaged for 6 h by high throughput timelapse microscopy. Imaging data were analyzed as described (Merouane Bioinformatics).

Supplementary Figure 6 Mouse complement activation of C1q-specific antibody variants and FcγR-mediated internalization in CD20+FcγRIIb+ cancer cells.

(a) Binding RA801 and RA802 to mouse FcγRs. Microtiter well plates were coated with aglycosylated IgG1 (negative control), glycosylated IgG1 (Rituxan, positive control), mIgG, RA801, or RA802. His-tagged mFcγRs were added and binding was detected using anti-His IgG conjugated to HRP. Errors bars: standard deviations from triplicate experiments. (b) Lack of binding of RA801 immune complexes to mouse FcγRs expressed on CHO cells. The binding activities of performed ICs made of RA801 or Rituxan with PE-conjugated F(ab’)2 goat anti-human IgG F(ab’)2. (c) CDC assays with pooled mouse serum (PMS). CDC assays of CD20+ cells by RA801 with PMS. Experimental conditions for CDC assays of Ramos cells as in Supplementary Fig. 4. Calcein-loaded EL4-hCD20 cells were incubated with 50 % of PMS and serially diluted antibodies for 4 hrs. EC50 values were presented in the plot (Orange: Rituxan, Blue: RA801). (d) α-CD20 antibody internalization in CD20+FcγRIIb+ cancer cells, HBL-1 or TMD8. CD20+FcγRIIb+ cancer cells were incubated with 100 nM of RA801 or RA802 for 0, 2, 4, or 6 h. Surface bound IgG was detected by flow cytometry using FITC conjugated goat anti-human Fc. (Abcam). In all assays, trastuzumab-IgG was used as a negative control and errors bars indicate the s.d. from triplicate experiments. Data are from one experiment representative of three experiments.

Supplementary Figure 7 Structural analysis of C1q-specific Fc variants.

(a) Crystal packing environment for A801-Fc. The crystallographic packing lattice is shown for A801-Fc (PDB ID: 5V43). The dimer from one asymmetric unit is shown in cartoon representation, with Cγ2A in white; Cγ2B in blue; and Cγ3B in pink. Surrounding asymmetric units are shown in ribbon representation (magenta). No contacts or atomic clashes were observed between Cγ2B and molecules from surrounding asymmetric units. (b) B factors distribution fucosylated-WT Fc (PDB ID: 3AVE), G801-Fc (PDB ID: 5V4E), aglycosylated-WT Fc (PDB ID: 3S7G), A801-Fc (PDB ID: 5V43). The coloring of the figure is based on the B factors gradient with lowest (blue) to highest (red). The dash line represents disordered regions. (c) Distance of FcγR-binding motifs of Fc domain to FcγRI (PDB ID: 4X4M). FcγR is shown as a white transparent surface and the secondary structure of the whole complex is shown in ribbon. Two different regions on the Fc are both identified to be important for FcγR and Fc complex formation: LLPP motif (important residue Leu235 is shown in sticks) and C’E loop. (d-e) Overlaid Cγ2 and 'soft' C'E loop of 801-Fc with wild type Fc. (d) Cγ2 of G801-Fc (PDB 5V4E, purple) was superimposed with fucosylated-WT Fc (orange, PDB: 3AVE). (e) G801-Fc (purple) was superimposed with aglycosylated-WT Fc (green, PDB: 3S7G). The 'soft' C'E loop of G801-Fc was highlighted by arrow. The glycan in PDB 3AVE is shown in white.

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Lee, CH., Romain, G., Yan, W. et al. IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions. Nat Immunol 18, 889–898 (2017). https://doi.org/10.1038/ni.3770

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