Selection of nanobodies that target human neonatal Fc receptor

Abstract

FcRn is a key player in several immunological and non-immunological processes, as it mediates maternal-fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation. Thus, there is an intense interest in studies of how FcRn binds and transports its cargo within and across several types of cells, and FcRn detection reagents are in high demand. Here we report on phage display-selected Nanobodies that target human FcRn. The Nanobodies were obtained from a variable-domain repertoire library isolated from a llama immunized with recombinant human FcRn. One candidate, Nb218-H4, was shown to bind FcRn with high affinity at both acidic and neutral pH, without competing ligand binding and interfering with FcRn functions, such as transcytosis of IgG. Thus, Nb218-H4 can be used as a detection probe and as a tracker for visualization of FcRn-mediated cellular transport.

Figure 1. Immunization of a llama and selection of anti-hFcRn phage particles.

(A) Serum samples collected before and after immunization of a llama with recombinant hFcRn. Serial dilutions of sera were incubated in hFcRn coated ELISA wells and bound antibodies were detected using a anti-llama IgG from mouse followed by an anti-mouse IgG secondary antibody from rabbit. (B) Representative ELISA results showing binding of selected phage clones to hFcRn at pH 6.0 and pH 7.4. The numbers given represent the mean of duplicates.

Figure 2. Bacterial production of Nbs and binding to hFcRn.

(A) Nb candidates were produced in E. coli and subsequently purified and analyzed by 12% SDS-PAGE. Lane 1 shows a molecular weight (MW) standard, lane 2 shows Nb218-H4, lane 3 shows Nb203-G8, lane 4 shows (Nb218-H4)₂, and lane 5 (Nb203-G8)₂. (B) Binding of GST-tagged hFcRn to the Nb variants captured on an anti-c-Myc antibody at pH 6.0 and pH 7.4. Bound receptor was visualized using an HRP-conjugated anti-GST antibody. (C) Binding of the Nb variants to human β2-microglobulin coated in wells and detected with an anti-c-Myc antibody. An HRP-conjugated anti-human β2-microglobulin antibody was included as a positive control. The numbers given represent the mean of triplicates. (D–E) Anti-hFcRn Nbs bind hFcRn independently of the presence of the ligands. Representative SPR responses showing binding of hFcRn alone or in complex with anti-hFcRn Nbs or a control Nb to (D) human IgG and (E) human albumin immobilized on a CM5 sensor chip. Injections were done at pH 6.0 at 25°C with a flow rate of 50 μl/min. n = 3–4. All data are presented as mean±s.d.

Figure 3. Binding properties of monomeric and dimeric forms of anti-hFcRn Nb218-H4.

Serial dilutions of Nb218-H4 injected over immobilized hFcRn at either pH 7.4 or pH 6.0. The equilibrium binding responses obtained at (A) pH 7.4 and (B) pH 6.0 were fitted to a steady state affinity binding model supplied with the BIAevaluation software. (C) A representative sensorgram showing the overlays of serial dilutions of Nb218-H4 injected over immobilized hFcRn at pH 7.4. The derived binding kinetic values are presented in Table 1. (D) A representative sensorgram showing binding of equal amounts of monomeric Nb218-H4 and bivalent (Nb218-H4)₂ to immobilized hFcRn at pH 6.0. Injections were performed at 25°C with a flow rate of 50 μl/min. n = 3–4. All data are presented as mean±s.d.

Figure 4. Conjugation to Nb218-H4 and detection of hFcRn.

Binding of hFcRn to titrated amounts of (A) human IgG1 and (B) albumin coated in wells. Binding of hFcRn to the ligands at pH 7.4 and pH 6.0 was visualized using HRP-conjugated Nb218-H4. The numbers given represent the mean of triplicates. HeLa cells transfected to over-express hFcRn (hFcRn-RFP and human β2-microclobulin) were stained with (C) Alexa-conjugated (Nb218-H4)₂ or (D) a control bivalent Nb. (C–D) Cells were also allowed to take up human albumin conjugated to Alexa647. (E) HeLa cells transfected with a plasmid encoding only the RFP-fused hFcRn HC and then incubated with Alexa-conjugated (Nb218-H4)₂ or a control Nb. All cells were inspected by confocal microscopy as described in Material and Methods. n = 3. All data are presented as mean±s.d.

Figure 5. The impact of Nb218-H4 on FcRn-mediated IgG transcellular transport.(D) Polarized human T84 cells were seeded in a Transwell system, and grown to high transcellular resistance (1000–1500 Ω/cm²).

Samples of human IgG1-WT or IgG1-IHH were added to the apical reservoir. At time 0 h and 4 h, medium samples were collected from the basolateral reservoirs. The amounts of IgG transported across the cellular layer were quantified by ELISA. The same assay was performed with WT IgG1 in the presence of a >15-fold excess amount of Nb218-H4 or a control Nb. The numbers given represent the mean of triplicates. n = 3-. All data are presented as mean±s.d.