Supplementary Figure 1. Production and Characterisation of Recombinant Hscr18-20

Supplementary Figure legends

Supplementary Figure 1. Production and characterisation of recombinant hSCR18-20.

(A)Pichia Pastoris culture supernatants were collected and SCR18-20 was purified by Ni-NTA chromatography. Supernatant (SN), flow through (FT) and elution fractions (E1 – E4) were analysed by SDS-PAGE and Western Blot using HRP-conjugated mouse anti-Hismonoclonal Ab. Recombinant SCR18-20 was detected in E2, which was further concentrated and dialyzed against PBS pH 7.0. (B) Recombinant hSCR16-17 and hSCR18-20 were applied to a HiTrap Heparin HP column as model to determine the binding activity to negatively charged surfaces of both fragments. Bound proteins were eluted by high salt (1M NaCl). Flow through (FT), wash (W1, W2) and elution fractions (E1, E2) were analysed by SDS-PAGE and Western Blot. SCR18-20 was detected in E1, which clearly indicates the heparin binding activity. In contrast, SCR16-17 appeared in the wash fraction, which confirms the lacking Heparin binding activity of the control fragment. (C) The heparin binding affinity of SCR18-20 was further studied by gradual elution of the protein with NaCl (0 – 1M) (dashed line). Protein was photometrically detected at an absorbance of 280nm. The elution profile of SCR18-20 (black line) shows it´s peak in elution fraction 13, which corresponds to a NaCl concentration of 300mM.

Supplementary Figure 2. Processing C3 fragments on the surface of CLL cells.

Processing C3 fragments on the surface of CLL cells in the presence or absence of hSCR18-20 was investigated by incubating the cells with NHS in the presence of RTX with or without SCR18-20 for different time points. As control hiNHS was used. Western blot analysis revealed that after 1 minute of incubation both 120 kDa -chain and 75 kDa -chain of C3 were clearly detected in all samples. After 5 minutes both 1-chain of inactive C3b (iC3b, 68 kDa) and C3dg/C3d were detectable in the presence of both RTX and RTX/SCR18-20 revealing on the activation and processing of C3 upon complement activation induced by RTX.

Supplementary Figure 3. Reproducibility of the CDC assay.

Five patient samples were tested more than once to analyse whether the lysis assay described above is robust and reproducible. Results are shown for one patient out of five. This representative experiment clearly indicates that the data obtained in three independent tests are highly comparable.

Supplementary Figure 4. Enhancement of RTX-mediated CDC by SCR18-20 is specific to B cells.

(A) CDC assays were performed were performed with isolated CD11b+ PMNs mixed with PBMCs from a CLL patient. Following the CDC assay, cells were co-stained with CD19 and CD11b monoclonal antibodies prior to PI staining. PI-negative viable cells were then counted in CD11b+ PMN and CD19+ B cell populations. Data represent three independent experiments. Error bars: SEM. CDC assay was also performed with erythrocytes (B), fibroblast cell line BHK-21 (C) and epithelial cell line Colo-699 (D) in the presence or absence of hSCR18-20. Data represent mean of two independent experiments.

Supplementary Figure 5. CD55 or CD59 expression levels on primary CLL cells did not differ between CDC responder and CDC non-responder patients.

(A) Patient cells were stained withmonoclonal mouse anti-human CD55ormonoclonal mouse anti-human CD59antibodies and secondary polyclonal goat anti-mouse IgG/FITCantibody to determine the expression of these RCAs on the cells. The MFI for both surface-bound proteins showed some variability among the 37 patient samples. (B) Comparison of CD55 and CD59 levels in responder and non-responder groups showed no significant difference in the expression level of either protein. Error bars: SEM.