An IgA mimicry of IgG that binds Polymeric Immunoglobulin Receptor for mucosa transcytosis

Abstract

Most pathogens establish infection through mucosa, where secretary IgA (sIgA) plays an "immune exclusion" role in humoral defense. Extravasation of intravenously administrated therapeutic IgG mainly relies on convection and/or FcRn-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA's full binding activity towards Polymeric Immunoglobulin Receptor (pIgR) that mediates mucosa transcytosis. In a Diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-DT IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Keywords: IgA; IgG; mucosa; pIgR; transcytosis.

Figure 1

Differential binding of various IgG-IgA hybrids to pIgR. (A) Illustration of Constructs A–F. The N-terminal anti-DT single-chain variable fragment (ScFv) is all the same. Constructs A and B encode and assemble into the same products except that J chain is expressed either by pDirect7.0 vector or in a pre-inserted site via ‘Toggle-In’, as is in Construct E. The staining intensities for Constructs C and D are >60-fold weaker than those for A, B and F. The protein sequences of these constructs are disclosed in Supplementary data. (B) FACS staining of CHO-pIgR cells with Protein A purified Constructs A–F (1.0 μg/mL), followed by anti-human IgG secondary antibody. Empty area: control staining; shaded area: staining with each IgG-IgA hybrid, respectively. Data represent at least three different experiments.

Figure 2

In vivo protection of DT-mediated depletion of PD-L2 + DTR+ cells by IgG-IgA hybrids in a defined window of a peritoneal transcytosis model. (A) Peritoneal IgM + CD5^int B1a B cell population contains PD-L2+ cells that are genetically tagged with TdTomato and DTR in CD19-Cre^+/−PZTD^+/+ mice [9]. DT treatment (i.p., 25 ng/g of body weight) depleted PD-L2+TdTomato+ population from 45.5 to 3.2% of the gated B1a population. (B) i.v. injection of 25 μg (upper panel) or 1 μg (lower panel) of anti-DT antibodies in 0.1 mL PBS 1 h before DT injection protected PD-L2+DTR+ cells from depletion. Due to the confounding omental vascular leakage, the differential effect of different antibody forms can only be shown with the least amount of antibody administrated (1 μg, low panel). (C) Protection efficiency by anti-DT antibodies (1 μg) on DT-mediated depletion of PD-L2+TdTomato+DTR+ cells is calculated based on depletable PD-L2+TdTomato+ population size as: [antibody treated−DT only (3.2%)]/[PBS only (45.5%)−DT only (3.2%)] × 100%.

Figure 3

Model of cross-epithelium transcytosis of IgG(tp)J by pIgR. As normal IgG, extravasation of intravusly administrated IgG(tp)J also relies on convection (more at discontinuous and fenestrated capillaries) and/or FcRn-mediated transcytosis from circulation into interstitial space. At subepithelial stroma of tissues with rich blood flow, serum IgG could transcytose epithelium via FcRn, although in a bidirectional manner. Locally produced secretory dimeric IgA in complex with J chain can be specifically bound by pIgR on the basolateral side of epithelial cells, and unidirectionally transcytose the epithelium onto mucosa surface. Likewise, the novel form of IgG(tp)J can bind to pIgR equally well and efficiently transcytose the epithelium; hence, the biodistribution of IgG(tp)J includes not only circulation, but also mucosa.