Letter to Editor

March 12, 2013

Dear Editor-in-Chief

We want you to consider in detail the paper published in the January 2013 issue of Journal of Clinical Biochemistry and Nutrition, by Tomoki Aomatsu et al. entitled ”Neutralization of complement component C5 ameliorates the development of dextran sulfate sodium (DSS)-colitis in mice”.⁽¹⁾ The authors have evaluated the pathophysiological role of the complement system in a mice colitis model. To study the role of complement, they have used Eculizumab, an antibody directed towards the human complement protein C5. We have two strong issues against this study, both regarding the use of the C5-neutralizing antibody, Eculizumab.

1. The Eculizumab, whose CDRs are of mouse origin, is highly specific against human C5, and has shown not to bind C5 of any other tested species.⁽²⁾ Aomatsu et al. will not neutralize C5 in the mouse when giving Eculizumab, and the effect they report from the Eculizumab is thus complement independent. To verify that Eculizumab indeed not cross-react with mouse, and that our argue is valid, we tested the complement inhibitory effect of Eculizumab in mouse serum in vitro in our laboratory, and found no effect on mouse complement activity as evaluated in CH50 using adequate controls (Fig. 1).

Fig. 1

Mouse serum was preincubated with 20 µg/mL and 100 µg/mL each of Eculizumab, nonsense control antibody Rituximab and C5 neutralizing peptide OmCI. The complement inhibitory effect of each inhibitor was investigated in a mouse hemolytic assay. Percentage lysis is related to a serum control. Neither Eculizumab nor Rituximab has any inhibitory effect on the complement cascade whereas the OmCI peptide completely abolishes complement-mediated hemolysis.

2. A C5-neutralizing antibody must be given in a dose at least 100 times higher than what was used by Aomatsu et al. C5 circulates in mouse plasma (male) at a level of approximately 90 µg/mL (≈ 0.5 µM).⁽³⁾ Eculizumab was given in a dose of 1 µg/body every 48 h, which corresponds to a serum concentration of approximately 5 nM. The standard antibody for neutralizing mouse C5 is the IgG1 BB5.1. The supplier recommends a dose of 40 mg mAb/kg (approx 1 mg/body) 0–2 days followed by injection twice a week. To note, however, even if they had used a sufficient dose, it would have had no effect, due to the lack of specificity as mention under item 1.

We are surprised that this paper passed referees desk without noting this. We have sent two separate e- mails to the authors and asked for documentation that Eculizumab worked in their mice, but we have got no answer.

To summarize, the effect that Aomatsu et al. report in the intervention group is not an effect of neutralization of C5. The complement system is involved in several inflammatory disorders, likely also in colitis. The nature of complement involvement in colitis has to be carefully evaluated and not to be misled by this study. Therefore, we strongly suggest you to consider retraction of this paper.

Looking forward to hear your reply.

Sincerely,

  

Tom Eirik Mollnes, professor   

Per H. Nilsson, postdoc

References

• 1   Aomatsu  T,  Imaeda  H,  Takahashi  K, et al. Neutralization of complement component C5 ameliorates the development of dextran sulfate sodium (DSS)-colitis in mice. J Clin Biochem Nutr 2013; 52: 72–75.
• 2   Rother  RP,  Rollins  SA,  Mojcik  CF,  Brodsky  RA,  Bell  L. Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nat Biotechnol 2007; 25: 1256–1264.
• 3   Ashman  RB,  Kay  PH,  Lynch  DM,  Papadimitriou  JM. Murine candidiasis: sex differences in the severity of tissue lesions are not associated with levels of serum C3 and C5. Immunol Cell Biol 1991; 69: 7–10.