Afuco™ Anti-Human C5 ADCC Recombinant Antibody (IFX-1), ADCC Enhanced (CAT#: AFC-080CL)

Figure 1 Detection of C5a in human plasma and interference of eculizumab.

BD OptEIA™ Human C5a ELISA kit II and InflaRx in-house C5a ELISA were employed to test C5a levels in human plasma samples spiked with different concentrations of eculizumab. Data presented as mean ± SD.

Riedemann,N.C.,Habel, M.,Ziereisen, J.,Hermann, M.,Schneider, C., Wehling, C.,& Guo, R.(2017).Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition.Clinical Immunology,180,25-32.

Figure 2 Inhibition of the terminal complement pathway by eculizumab and IFX-1.

Complement system was activated in human plasma samples containing IFX-1 or eculizumab using Zymosan A. Samples were used to test concentration of C3a (A), C5a (B) and sC5b-9 (C). Data presented as mean ± SD. Tukey's multiple comparison test (comparison to saline). **P = 0.0038, ***P < 0.001, ****P < 0.0001.

Riedemann,N.C.,Habel, M.,Ziereisen, J.,Hermann, M.,Schneider, C., Wehling, C.,& Guo, R.(2017).Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition.Clinical Immunology,180,25-32.

Figure 3 Trypsin-induced cleavage of human C5. (A) Purified human C5 was incubated with trypsin in presence or absence of IFX-1 or eculizumab.

Samples were used to test C5a concentration. Data presented as mean ± SD. Tukey's multiple comparison test (comparison to C5 [50 nM] + trypsin [0.1 μg/ml]) ****P < 0.0001. (B) Eculizumab was incubated with trypsin and the binding of eculizumab to C5 was tested thereafter. Recovery of eculizumab spiked in trypsin is indicated with eculizumab spiked in HBSS as baseline. Data presented as mean ± SD. (C) Purified human C5 was incubated with trypsin in presence or absence of IFX-1 or eculizumab. Samples were used to test the biological activity of C5a and the blocking effects of the antibodies. The increase of CD11b mean fluorescence intensity compared to the HBSS sample is shown. Data presented as mean ± SD. Tukey's multiple comparison test (comparison to C5 [50 nM] + trypsin [0.1 μg/ml]) ***P = 0.0002, ****P < 0.0001. (D) Sequence analysis indicated that C5 after trypsinization contains a C5a fragment from Thr678 to Arg751.

Riedemann,N.C.,Habel, M.,Ziereisen, J.,Hermann, M.,Schneider, C., Wehling, C.,& Guo, R.(2017).Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition.Clinical Immunology,180,25-32.

Figure 4 Thrombin-induced cleavage of human C5. (A) Purified human C5 was incubated with thrombin in presence or absence of IFX-1 or eculizumab.

Samples were used to test C5a concentration. Data presented as mean ± SD. Tukey's multiple comparison test (comparison to C5 [50 nM] + thrombin [10 μg/ml]) ****P < 0.0001. (B) Eculizumab was incubated with trypsin and the binding of eculizumab to C5 was tested thereafter. Recovery of eculizumab spiked in thrombin is indicated with eculizumab spiked in HBSS as baseline. Data presented as mean ± SD. (C) Purified human C5 was incubated with thrombin in presence or absence of IFX-1 or eculizumab. Samples were used to test the biological activity of C5a and the blocking effects of the antibodies. The increase of CD11b mean fluorescence intensity compared to the HBSS sample is shown. Data presented as mean ± SD. Tukey's multiple comparison test (comparison to C5 [50 nM] + thrombin [10 μg/ml]) ****P < 0.0001.

Riedemann,N.C.,Habel, M.,Ziereisen, J.,Hermann, M.,Schneider, C., Wehling, C.,& Guo, R.(2017).Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition.Clinical Immunology,180,25-32.

Figure 5 Complement factors in patient treated with eculizumab.

(A) The course of C5a, CH50 and eculizumab in an aHUS patient was evaluated for up to nine months after start of treatment with eculizumab. Reference value for C5a in citrate plasma (17.08 ng/ml ± 6.96 ng/ml) is marked in grey. (B) The course of C5a, CH50 and eculizumab of four aHUS patients (patient 3, 4, 9, 11) and three C3GN patients (patient 18, 19, 21) is indicated at pre-dose, as well as 3 and 6 months after treatment with eculizumab. Reference values for C5a in EDTA plasma (24.59 ng/ml ± 10.90 ng/ml) and CH50 (65% to 135%) are marked in grey. Target concentration of eculizumab after treatment (70 μg/ml) is indicated as dashed line.

Riedemann,N.C.,Habel, M.,Ziereisen, J.,Hermann, M.,Schneider, C., Wehling, C.,& Guo, R.(2017).Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition.Clinical Immunology,180,25-32.

Figure 6 IFX-1 has no influence influence on the membrane membrane attack complex complex (MAC) C5b-9 formation formation in human plasma.

The total hemolytic complement activity activity (CH50) test was performed with the presence of various concentrations of IFX-1, and CH50 values were not affected by the presence of IFX-1 in the plasma samples.

Guo, R.,Habel,M.,Zenker,O.,Giamarellos-Bourboulis,E.J.,Guo,N.R.R.,Habel,M.,& Riedemann, N. IFX-1 blocking the anaphylatoxin C6a-an anti-inflammatory effect in.

Figure 7 Treatment response utilizing the HiSCR endpoint for the HS patients.

Guo, R.,Habel,M.,Zenker,O.,Giamarellos-Bourboulis,E.J.,Guo,N.R.R.,Habel,M.,& Riedemann, N. IFX-1 blocking the anaphylatoxin C6a-an anti-inflammatory effect in.