Enhancing IgG distribution to lung mucosal tissue improves protective effect of anti-Pseudomonas aeruginosa antibodies

Abstract

IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti-Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.

Keywords: Bacterial infections; Immunoglobulins; Infectious disease; Pharmacology; Therapeutics.

Figure 1. Cam-003 peptide fusions bind to human and murine pIgR.

Binding of C-terminal peptide antibody fusions to pIgR was determined by ELISA. Cam-003 alone showed no binding to either human (A) or murine (B) pIgR at the highest concentration tested (200 μg/ml). Cam-003–QRN and its derivative Cam-003–KLKL had improved binding to pIgR compared with the natural pIgR ligand dimeric IgA, whereas another peptide fusion Cam-003–SAM exhibited poorer binding (n = 4). Data are representative of at least 2 independent experiments.

Figure 2. Transcytosis of Cam-003 peptide fusions in pIgR-transfected MDCK cells.

Antibodies were added to the bottom chamber (basolateral) of transwells containing either untransfected, h-pIgR–, or m-pIgR–transfected Madin-Darby canine kidney (MDCK) monolayers (A). At 24 hours, antibodies in the top chamber (apical) were quantified by ELISA. Cam-003 levels were similar in all 3 cell lines, whereas Cam-003–QRN and Cam-003–KLKL, as well as human dimeric IgA, had increased accumulation in the apical chamber (B). Data are representative of at least 2 independent experiments (n = 10 for Cam-003 antibodies, n = 6 for dIgA). Significance was determined using 2-way ANOVA (with Tukey’s post hoc test; ****P < 0.0001).

Figure 3. Increased levels of Cam-003 variants in bronchoalveolar lavage and similar levels in serum.

(A) Serum levels of Cam-003, Cam-003–QRN, and Cam-003–KLKL were quantified from BALB/c mice 24 and 48 hours after initial i.v. injection. Serum levels did not differ significantly at 24 or 48 hours. (B) Cam-003–QRN and Cam-003–KLKL levels in BAL (assessed at 48 hours) were elevated from those of Cam-003 alone (P = 0.195 and 0.023, respectively; 1-way ANOVA with Tukey’s post hoc test; *P < 0.05). Data are representative of at least 2 independent experiments; n = 4 per group.

Figure 4. pIgR-binding Cam-003 fusions elicit opsonophagocytic killing and improved protection against lethal challenge in a P. aeruginosa acute pneumonia model.

(A) Opsonophagocytosis assay with luminescent P. aeruginosa serogroup 05 strain PAO1 (PAO1.lux). OPK with Cam-003–QRN, Cam-003–KLKL, and Cam-003 are shown with the isotype control IgG R347. (B–D) Survival of BALB/c mice prophylactically immunized with an IgG isotype control R347, Cam-003, and either Cam-003–QRN (B) or Cam-003–KLKL (C and D) prior to i.n. infection with P. aeruginosa strain 6077. Results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the log-rank test (Mantel-Cox). Data are representative of at least 2 independent experiments; n = 10 per group for survival experiments. (B) Cam-003 and Cam-003–QRN had significantly better protection than the isotype control at both doses tested (P < 0.0001 for both 5 and 10 mg/kg doses; 4.5 × 10⁵ CFU inculcation). Cam-003–QRN survival was significantly improved compared with Cam-003, as well (P = 0.0014 for 5 mg/kg doses and P = 0.0098 for 10 mg/kg doses). (C) At 5 × 10⁵ CFU, Both Cam-003 and Cam-003–KLKL were protective compared with R347 at both doses (P = 0.017, 2 mg/kg Cam-003; P = 0.0004, 6 mg/kg Cam-003; P < 0.0001, 2 and 6 mg/kg doses of Cam-003–KLKL). Cam-003–KLKL survival was superior to Cam-003 when comparing both the 2 mg/kg doses (P = 0.0042) and 6 mg/kg doses (P = 0.017). (D) At an 8 × 10⁵ CFU inoculum dose, Cam-003 and Cam-003–KLKL survival was again enhanced compared with R347 (P < 0.0001, 2 mg/kg Cam-003; P = 0.0002, 6 mg/kg Cam-003; P < 0.0001, Cam-003–KLKL), with Cam-003–KLKL survival also enhanced compared with Cam-003 (P = 0.017, 6 mg/kg; not significant, 2 mg/kg).