C1-inhibitor treatment in patients with severe complement-mediated autoimmune hemolytic anemia

Abstract

Complement-mediated (CM) autoimmune hemolytic anemia (AIHA) is characterized by the destruction of red blood cells (RBCs) by autoantibodies that activate the classical complement pathway. These antibodies also reduce transfusion efficacy via the lysis of donor RBCs. Because C1-inhibitor (C1-INH) is an endogenous regulator of the classical complement pathway, we hypothesized that peritransfusional C1-INH in patients with severe CM-AIHA reduces complement activation and hemolysis, and thus enhances RBC transfusion efficacy. We conducted a prospective, single-center, phase 2, open-label trial (EudraCT2012-003710-13). Patients with confirmed CM-AIHA and indication for the transfusion of 2 RBC units were eligible for inclusion. Four IV C1-INH doses (6000, 3000, 2000, and 1000 U) were administered with 12-hour intervals around RBC transfusion. Serial blood samples were analyzed for hemolytic activity, RBC opsonization, complement activation, and inflammation markers. Ten patients were included in the study. C1-INH administration increased plasma C1-INH antigen and activity, peaking at 48 hours after the first dose and accompanied by a significant reduction of RBC C3d deposition. Hemoglobin levels increased briefly after transfusion but returned to baseline within 48 hours. Overall, markers of hemolysis, inflammation, and complement activation remained unchanged. Five grade 3 and 1 grade 4 adverse event occurred but were considered unrelated to the study medication. In conclusion, peritransfusional C1-INH temporarily reduced complement activation. However, C1-INH failed to halt hemolytic activity in severe transfusion-dependent-CM-AIHA. We cannot exclude that posttransfusional hemolytic activity would have been even higher without C1-INH. The potential of complement inhibition on transfusion efficacy in severe CM-AIHA remains to be determined.

Graphical abstract

Figure 1.

Study design.

Figure 2.

C1-INH antigen and activity levels increase upon Cinryze administration. Both C1-INH antigen (A) and activity (B) levels increase upon administration of four C1-INH doses in the first 36 h, reaching maximum levels at 48 h after first administration respectively. Red area indicates normal healthy levels of C1-INH antigen and activity. Data expressed as median + IQR, significance tested using Wilcoxon matched-pairs signed rank test. ∗P ≤ 0.05.

Figure 3.

Hemolytic parameters remain unaffected by C1-INH treatment. (A) Hb significantly increased after 12 and 24 hours as well as after 7 and 14 days. (B,C) No significant change in LDH and total bilirubin levels was found. (A-C) Red area indicates normal values. Data shown as median with interquartile range (IQR), significance tested using mixed-effect analysis with Geisser-Greenhouse correction and Holm-Šídák multiple comparisons test comparing each timepoint with t = 0.

Figure 4.

Complement deposition and antibody binding on RBCs at baseline and after treatment. (A-D) Baseline deposition of C3, IgG, IgM, and IgA on RBCs as analyzed by flow cytometry. (A-C) C3 deposition and IgG and IgM binding is significantly increased in patients with AIHA compared with HCs. (D) IgA binding is not increased compared with the HCs, which were comparable to background geometric mean fluorescence intensity (MFI) except for 1 individual. (E) C3 deposition on RBCs is significantly reduced at 12, 24, and 48 hours compared with baseline in the trial participants. All data are expressed as a scatter plot with median + IQR. Significance was tested using the Mann-Whitney test (A-D) and 1-sample Wilcoxon test (E). ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001. Ctrl, control; ns, not significant.

Figure 5.

Complement activity in plasma is hardly affected by C1-INH treatment. (A-D) Complement activation as measured by C4b/c and C3b/c detection in EDTA plasma by ELISA. (A) C4b/c levels at baseline do not differ between patients with AIHA and HCs. (B) C4b/c is at low levels, similar to HCs, throughout the trial. (C-D) C3b/c levels at baseline are all significantly increased compared with HCs and are reduced significantly at t = 36 hours only. (E) C3 levels are at the lower end of normal values (red area). (F-G) C4 and C2 levels are below normal values (red area) at most timepoints, indicating high consumption of C2 and C4. Data expressed as median + IQR. Significance was tested using the Mann-Whitney test (A,C), Wilcoxon matched-pairs signed rank test (B,D), and mixed-effects analysis with Geisser-Greenhouse correction (E-G). ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001.

Figure 6.

Inflammatory parameters are increased in patients with AIHA and are unaffected by C1-INH treatment. (A,C) Nucleosome and EA complex levels are increased in patients with AIHA compared with HCs, indicating inflammation and neutrophil activation. (B,D) Nucleosome and EA complexes are unaffected by C1-INH treatment. Data expressed as median + IQR, significance tested using Mann-Whitney test (A,C) and Wilcoxon matched-pairs signed rank test (B,D). ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001.