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. 2017 Mar 10;292(10):4361-4370.
doi: 10.1074/jbc.M116.770628. Epub 2017 Jan 18.

Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody

Srinath Kasturirangan  1 , G Jonah Rainey  1 , Linda Xu  1 , Xinwei Wang  1 , Alyse Portnoff  1 , Tracy Chen  2 , Christine Fazenbaker  2 , Helen Zhong  2 , Jared Bee  3 , Zhutian Zeng  4 , Craig Jenne  4 , Herren Wu  1 , Changshou Gao  5
Affiliations

Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody

Srinath Kasturirangan et al. J Biol Chem. .

Abstract

Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.

Keywords: Fc receptor; IL-6; antibody; antibody engineering; electron microscopy (EM); macrophage; microscopic imaging; pharmacokinetics; protein complex.

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Conflict of interest statement

S. K., G. J. R., C. G., L. X., X. W., A. P., T. C., C. F., J. B., H. Z., and H. W. own stock in AstraZeneca

Figures

FIGURE 1.
FIGURE 1.
mAb1, mAb2, and BiS3Ab exhibit high-affinity binding for their antigen, hIL-6. A–D, a bispecific antibody was generated in the BiS3Ab format, with mAb1 as IgG and mAb2 as scFv, where the scFv is attached to the carboxyl terminus of the IgG via a 10-amino acid flexible linker. Fab arms are shown in blue (light blue, Hc; dark blue, Lc), with complementarity-determining regions highlighted in salmon (Hc) and red (Lc). The hinge and Fc region of the antibody appear in green. The scFv is represented in orange, with light orange representing Hc and dark orange representing Lc (A). Biacore analysis of mAb1 (B), mAb2 (C), or BiS3Ab (D) was performed with the antibodies immobilized and soluble hIL-6 used as analyte.
FIGURE 2.
FIGURE 2.
BiS3Ab can bind both epitopes of IL-6 simultaneously, and the mAb/BiSAb format determines the order of complex formation. A and B, mAb1 (A) or mAb2 (B) was immobilized on a CM5 BIAcore chip. rhIL-6 was captured by the immobilized mAbs, followed by mAb1, mAb2, or BiSAb3. C–F, mAb2 (C), mAb1 (D), a mixture of the two mAbs (E), or BiS3Ab (F) was mixed with rhIL-6 in a molar ratio of 1:1 IL-6:antibody variable domain to generate immune complexes. The complex generated upon incubation was analyzed by HPLC-SEC.
FIGURE 3.
FIGURE 3.
Negative stain electron microscopy and analytical ultracentrifugation demonstrate the formation of large complexes by BiSAb3 in the presence of IL-6. A and B, negative stain EM analysis was performed on the BiS3Ab alone (A) or Bis3Ab mixed with IL-6 (B). Scale bar = 200 nm. C, mean AED frequency distribution histogram for the samples. More accurate sizing of complexes by analytical ultracentrifugation defines complex size and predicts stoichiometry. D, a broad range of large aggregates formed between rhIL-6 and BISAb3. The approximate mass ranges and percentages of the complexes described in the text are noted as annotations. E and F, rhIL-6 + mAb1 (E) and rhIL-6 + mAb2 (F) exhibit only stoichiometric complexes and no large aggregated species. Excess free rhIL-6 was seen at about 2S in all samples.
FIGURE 4.
FIGURE 4.
Complex formation enhances binding to FcγRs. A–E, binding of Ab-Ag complex generated by the parental mAbs or BiS3Ab with rhIL-6 to immobilized hFcγRs: hFcγRI (A), hFcγRIIA (B), hFcγRIIb (C), hFcγRIIIA-158F (D), and hFcγRIIIA-158V (E) was determined by ELISA.
FIGURE 5.
FIGURE 5.
Induced macrophage-like cells phagocytose BiSAb·Ag complexes. A–D, the Ab-Ag complexes generated with AF488-labeled (green) parental antibody alone (A and B), BiS3Ab (C), or an isotype control R347 (D) were added to PMA-stimulated U937 cells. After 30-min incubation at 37 °C, most of the signal for the parental mAbs incubated with rhIL6 remained on the surface (A and B), whereas the complex generated by incubating rhIL6 with BiS3Ab was shown to be rapidly internalized into the cells (C), confirming phagocytosis of the larger complexes. DAPI staining (blue) shows the nuclei of the cells in each field. Scale bars = 50 μm.
FIGURE 6.
FIGURE 6.
BiS3Ab/IL6 complex is efficiently cleared in vivo. Mice were injected with rhIL-6 alone or rhIL-6 incubated with the individual mAbs or BiS3Ab. The rhIL-6 antigen alone has a short half-life and is cleared rapidly. However, when rhIL-6 was incubated with the parental mAbs, it persisted in the circulation because of the buffering effect of the mAbs. The oligomeric complex generated by incubating rhIL-6 with BiS3Ab was cleared rapidly in vivo. *, p < 0.005 for group-to-group comparison.
FIGURE 7.
FIGURE 7.
Clearance of immune complex takes place in the Kupffer cells in the liver. WT mice were injected intravenously with rhIL-6 + R347 (isotype control), rhIL-6 + mAb2, rhIL-6 + mAb1, or rhIL-6 + BiS3Ab. Intravital imaging of the liver was conducted immediately following injection. A, representative images of rhIL-6 capture 20 min after injection for each group. B, the co-localization efficiencies per field of view between red and blue signals in each group. C, representative image of Fcϵr1−/− mice injected with rhIL-6 + BiS3Ab. D, the co-localization efficiencies of rhIL-6 + BiS3Ab complex within Kupffer cells injected in WT and Fcϵr1−/− mice were compared. For A and C, red indicates IL6 and blue indicates F4/80. Scale bars = 100 μm. Data are expressed as mean ± S.E., n = 3–6 mice/group. For each mouse, three to six random fields of view were analyzed. **, p < 0.01; ****, p < 0.0001.
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