Staudinger et al.: HM1.24-ETA´

Supplementary Information

Suppl. Figure 1

A

B

Suppl. Figure 1:Analysis of CD317 surface expression levels on primary multiple myeloma cells and MM cell lines. A) CD317 and CD20 surface expression was analyzed on MM cell lines (JK-6, RPMI8226,INA-6, L363), patient derived primary tumor cells (patient #1, 3, 6, and 7) and monocytes obtained from healthy donors. White histograms = isotype control antibody; Black histograms = specific antibody.B)CD317 expression was quantified using the QIFI kitand displayed as specific antibody-binding capacity (SABC).

Suppl. Figure 2

Suppl. Figure 2: HM1.24-ETA´ does not mediate growth inhibition of T-cell line Jurkat, expressing low surface levels of CD317 after interferon  stimulation.A) Expression of CD317 and CD7 on Jurkat cells cultivated in the presence or absence of IFN was analyzed by flow cytometry (black: HM1.24-IgG1 or CD7-IgG1, white: isotype control). B) After treatment with increasing concentrations of HM1.24-ETA´ (= with IFN = w/o IFN ) or positive control CD7-ETA´ (= with IFN = w/o IFN ), vital cell masswas measured using the MTT assay. Data represent mean values +/- SEM of threeindependent experiments.

Suppl. Figure 3

Suppl. Fig. 3: IFN  sensitizes monocytes for HM1.24-ETA´ induced apoptosis.

A) To analyze internalization of surface exposed CD317, equal amounts of plasma cell leukemia cells (cell line L363), endothelial cells (Huvec) as well as healthy donors monocytes enriched by MACS technology were stained with CD317 monoclonal antibody at saturating concentrations (30 µg/ml). An irrelevant antibody was used as isotype control (white). Unbound antibody was removed by washing with cold PBS. Cells were incubated at 37°C (grey) or 4°C as control (black). After four hours of cell culture, bound antibody was detected by flow cytometry using a FITC labeled anti-mouse IgG antibody. B) The relative decrease of fluorescence intensity after 4 hours of incubation at 37° C was calculated in relation to control cells (4°C). C) To evaluate the influence of cytokine activation on HM1.24-ETA´-mediated induction of apoptosis in isolated monocytes, 5 x 104 monocytes were cultivated in the presence () or absence () of IFN  (100 U/ml). Increasing concentrations of immunotoxin were added. At 72 h, cell viability was analyzed using a colorimetric MTT proliferation assay. D) As the surface expression of CD317 on monocytes may be influenced by IFN  mediated activation, isolated monocytes were cultured in medium with or without supplementation of interferon  (100 U/ml). Surface expression of HM1.24 was analyzed by flow cytometry using FITC coupled mouse monoclonal CD317 antibody (black; isotype control: white).

Supplementary Table 1: Patient characteristics

Pat.
# / Sex / Disease / Stage / M protein / Cytogenetics / Source
1 / F / MM in leukemic stage / III B / IgG kappa / t (4;14) / Blood
2 / m / MM / III B / IgA kappa / t (4;14), del 13q, del 17p, del 6q, del 11q / Pleural effusion
3 / f / Plasma Cell Leukemia / n.a. / t (14;16), Tris 8 / Blood
4 / f / MM / III A / IgG kappa / t (4;14), del 13q / Bone Marrow
5 / f / MM / Amyloidosis / I A / Lambda light chain / n.d. / Bone Marrow
6 / m / MM in leukemic stage / IIIA / IgA lambda / t (4;14), del 13q / Blood
7 / f / MM, extramedullary disease / IIA / IgG kappa / Complex aberrant / Pleural effusion

stage = staging according to Salmon and Durie

n.a.= not applicable

n.d.= not determined

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