Production, characterization, and in vivo half-life extension of polymeric IgA molecules in mice

Abstract

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.

Keywords: Asialoglycoprotein receptor (ASGPR); N-linked glycan; polymeric IgG receptor (pIgR); serum half-life; transcytosis.

Figure 1.

Protein sequences of human IgA heavy chain constant domains and J chain. (A) Alignment of protein sequences for the human heavy chain constant domains CH1, CH2, CH3, hinge,¹² and tailpiece of IgA1, IgA2m1 and IgA2m2.¹³ Mismatches relative to the IgA1 sequence are highlighted gray, N-linked glycosylation motifs are boxed, and asterisks indicate amino acid differences in IgA2m2 from IgA1 and IgA2m1 in the tailpiece. (B) Protein sequence of the human J chain with the N-linked glycosylation motif boxed. (C) Schematic of IgA oligomeric states with light chain (LC, yellow), heavy chain (HC, blue), and joining chain (JC, green). IgA polymers represent trimer, tetramer, and pentamer species.

Figure 2.

The oligomeric state of recombinantly produced IgA is affected by the amount of J chain DNA used in transfection and the heavy chain tailpiece sequence. (A-C) Overlay of normalized analytical size-exclusion chromatograms of affinity-purified IgA from small-scale transient transfections performed with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA for the following isotypes/allotypes: (A) IgA1 (B) IgA2m1 or (C) IgA2m2. Monomer (M), dimer (D), and polymer (P) peaks are indicated. Values were normalized based on the highest signal of each chromatogram. (D-F) Relative amounts of monomer, dimer, and polymer (trimer/tetramer/pentamer) species produced for IgA variants, quantified by analytical SEC. (D) The effect of mutations in the IgA tailpiece of IgA1, IgA2m1, and IgA2m2 at positions 458 and 467 on polymer formation. The effect of mutations which remove N-linked glycosylation sites in (E) IgA1 or (F) IgA2m2 on polymer formation.

Figure 3.

Biophysical and structural characterization of recombinant IgA oligomers. (A) Overlay of analytical size-exclusion chromatograms of purified IgA1, IgA2m1, IgA2m1 P221R, and IgA2m2 monomers, dimers, and tetramer. (B) SDS-PAGE analysis of non-reduced (-DTT) and reduced (+DTT) IgA1, IgA2m1, IgA2m1 P221R, and IgA2m2 monomers (M), dimers (D) and tetramer (T). Heavy chain (HC), light chain (LC), and joining chain (*) are indicated in reduced samples, and the LC-LC dimer of IgA2m1 is indicated with an arrowhead. (C-D, upper panels) Reference-free 2D classes from negative stain electron microscopy for (C) IgA2m2 dimer or (D) IgA2m2 tetramer. (C-D, lower panels) A raw image particle compared to its assigned 2D class is presented next to a model of IgA superimposed on the 2D class with the Fc domains (orange) and Fab fragments (green) highlighted.

Figure 4.

Recombinantly produced IgA oligomers are stable and functional in vitro. (A) In vitro transcytosis of anti-mIL-13 hIgA monomers, dimers and tetramer in MDCK cells transfected with human pIgR. IgA oligomers transcytose, while monomers do not. (B) Thermostability of anti-mIL-13 IgAs, IgG1, and IgG1 Fab are measured by differential scanning fluorimetry (DSF). Only one melting transition was observed for all samples.

Figure 5.

Recombinant IgA oligomers demonstrate rapid serum clearance in vivo. (A) Serum-time concentration profiles of IgA or IgG in mice. The overall serum exposures of Balb/c mice administered with a single 5 mg/kg intravenous (IV) dose of IgA or IgG molecules at 5 min, 15 min, 30 min, 1 h, 1 day, 3 day, 7, and 14 days post dose. All mice were bled retro-orbitally under isoflurane to evaluate serum concentration profile. Human serum IgA monomer was administered at 10 mg/kg and is shown as a dashed line. (B-C) Tissue distribution of IgA or IgG in mice at 1 h post injection. All graphs are means ± SEM for each group with n = 4. %ID/g: Injected Dose/gram. (B) Concentrations of intact antibodies were subtractive blood normalized per tissue, except blood, as¹²⁵I  (%ID/g tissue). (C) Concentrations of catabolized antibody values were determined by subtracting the¹²⁵I (%ID/g tissue) from the¹¹¹¹In (%ID/g tissue).

Figure 6.

Incomplete glycosylation of recombinant IgA molecules. (A) Schematic of N-linked glycan processing. (B) Global N-linked glycan analysis of recombinant IgA and IgA purified from human serum. Glycan analysis was done by mass spectrometric analysis after antibody deglycosylation and subsequent glycan enrichment. While human serum IgA shows greater than 90% sialylation, all recombinantly expressed IgA molecules have less than 60% sialylation. (C) Site-specific N-linked glycan analysis of the IgA2m1 dimer reveals heterogenous glycan composition between the different N-linked glycosylation sites on the IgA2m1 heavy chain (HC) and joining chain (JC).

Figure 7.

IgG1-IgA2m1 Fc fusions and aglycosylated IgA2m2 show improved serum exposures compared to wild-type IgA in vivo and demonstrate the ability to transcytose in vitro. (A) Schematic of IgA2m2 tetramer with light chain (LC, yellow), heavy chain (HC, blue) and joining chain (JC, green) with 41 N-linked glycosylation sites (gray) (left) or aglycosylated (right). (B) Schematic of IgG1, IgA2m1 dimer, or IgG1-IgA2m1 Fc dimer formats with LC (yellow), IgG1 HC (orange), IgA2m1 HC (blue), JC (green), and N-linked glycosylation (gray). (C) Analytical SEC of iodinated IgG1-IgA2m1 Fc dimers or tetramer after 0 h (black), 24 h (orange) or 96 h (blue) incubation in mouse plasma. The initial IgG1-L-P221R IgA2m1 Fc tetramer and dimer show degradation similar to the peak of anti-HER2 IgG1 (Trastuzumab) control, whereas the reengineered IgG1$\mathrm{\Delta }$K-P221 IgA2m1 Fc or IgG1$\mathrm{\Delta }$K-C242 IgA2m1 Fc dimers are stable. (D) Serum-time concentration profiles of IgA or IgG in mice. The overall serum exposures of Balb/c mice administered with a single 30 mg/kg IV dose of IgA molecules. The in-house concentration data of a typical human IgG1 (anti-gD) previously dosed as a single intravenous (IV) injection at 30 mg/kg are shown as a dashed line. All mice were bled retro-orbitally or via cardiac puncture under isoflurane to evaluate serum concentration profile. (E) In vitro transcytosis of hIgA in MDCK cells transfected with human pIgR.