Human IgG1 Monoclonal Antibody against Human Collagen 17 Noncollagenous 16A Domain Induces Blisters via Complement Activation in Experimental Bullous Pemphigoid Model (1)

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease. Circulating autoantibodies target human type XVII collagen (human collagen 17 [hCOL17]), also known as BP Ag 2 (BPAG2) or BP180, which is a major component in hemidesmosome-anchoring filament complexes at the epidermal basement membrane zone (BMZ). The major pathogenic epitope of BP autoantibodies is present at the extracellular noncollagenous 16A (NC16A) domain, which has distinctive diversity among different species. Deposition of anti-COL17 autoantibodies at the BMZ triggers sequential inflammatory cascades, including complement activation, degranulation of dermal mast cells, infiltration of eosinophils and neutrophils, and subepidermal blister formation elicited by proteinases derived from the inflammatory cells.

ELISA analysis showed that all the supernatants from the three clones (3.B6, 1.F5, and 7.H8) recognized the rhCOL17 NC16A protein (respective index values [mean ± SD]: 82.99 ± 4.32, 40.18 ± 2.53, and 30.79 ± 1.61). The IgG subclasses of the three clones are IgG1, IgG1, and IgG4, respectively. IIF analysis using normal human skin revealed that the supernatants of both 3.B6 and 1.F5 showed linear IgG deposition at the BMZ. In contrast, the supernatant of clone 7.H8 failed to recognize the BMZ.  

Epitope mapping by Western blot analysis using the rNC16A subpeptides revealed that the mAb 3.B6 specifically reacted with the full-length hNC16A, hNC16A 1–3, hNC16A 2, and hNC16A 2.5. Binding inhibition assays demonstrated that two peptides (hNC16A 2 and hNC16A 2.5) sharing seven amino acids inhibited the binding of mAb 3.B6 to the hNC16A in a dose-dependent manner.

All the mutated mAbs demonstrated significantly reduced binding ability to C1q compared with that of the nonmutated mAb. Alanine substitution at position P331 or P329 P331 seemed to demonstrate the lowest C1q-binding capacity of any of the mutated mAbs.

The CDC activity of the two mutated mAbs (E318A and K320A) was nearly half that of the nonmutated mAb, followed in decreasing order by K322A, E318A K320A K322A, and P329A. When the human serum complement (HSC) concentration was increased, the percentage of CDC activity increased in the mutants E318A, K320A, K322A, P329A, and E318A K320A K322A. No obvious change was observed in the CDC activity for P331A or P329A P331A, even at the highest HSC concentration. Similar results were shown when mouse serum complement was used instead of HSC.

The mutated mAbs (E318A, K320A, K322A, and E318A K320A K322A) exhibited ADCC activity similar to that of the nonmutated mAb. The substitutions at P329 or P329 P331 almost completely eliminated the ADCC activity of the mAbs. Alanine substitution at the P331 site partially decreased the ADCC activity. Altogether, these results suggest that P329 is a critical residue for IgG1–FcγR interaction in vitro.

Forty-eight hours after i.p. injection of the nonmutated IgG1 mAb (200 μg/g body weight), eight of nine neonatal mice became erythematous and showed BP-like skin blistering by gentle skin friction. Administration of the lower dose of the nonmutated IgG1 mAb (100, 50, or 25 μg/g body weight) resulted in a lower frequency of phenotypic changes (five of seven, four of seven, or zero of five mice, respectively).

DIF examination revealed the linear deposition of human IgG at the BMZ, as well as of mouse C1q and C3. 

In human, IgG1 and IgG3 are the most effective complement activators, whereas IgG4 is not capable of fixing complements. Previous clinical studies demonstrated that IgG1 and IgG4 autoantibodies are major IgG subclasses of BP patient autoantibodies and that IgG2 and IgG3 are minor subclasses of BP patient autoantibodies. Inflammatory cells, such as mast cells, neutrophils, and lymphocytes, are observed at the dermis. It has been demonstrated that C3 binding to complement receptors stimulates activation and chemotaxis of neutrophils and/or mast cells. Mast cells can produce various mediators, such as leukotrienes, platelet-activating factor, and cytokines, that contribute directly or indirectly to neutrophil recruitment. The neutrophils are recruited into skin then release elastase and gelatinase B, damaging the BMZ.  IgG1 Abs may directly recruit and activate effector cells via the interaction between the Fc region and reciprocal FcγRs.  The monoclonal Fab Ab lacked pathogenic activity when administered to neonatal COL17-humanized (COL17m−/−, h+) mice. Complete IgG1 mAbs with mutation at the C1q binding sites of the Fc region would protect the pathogenic epitopes from attack by the autoantibodies not only in BP but also in other autoimmune diseases in which complement activation plays an essential role.

1. Q. Li, H. Ujiie, A. Shibaki, G. Wang, R. Moriuchi, H.-J. Qiao, H. Morioka, S. Shinkuma, K. Natsuga, H. A. Long, Others, Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model. The Journal of Immunology. 185, 7746–7755 (2010).