Supplemental Figure1:Dose dependent killing of CD33 positive cell lines
KG-1 and U937 cells were found to express 55 000 and 20 000 CD33 molecules per cell surface, respectively, and thereby qualified as target cells lines for in vitro assays. Target cells were loaded with 51Cr and incubated for 19 h with enriched CD8+ T-cells from healthy donors at an E:T ratio of 1:1. AMG330 or a Control BiTE® antibody that is solely sharing the CD3-binding domain with AMG330 were added to co-cultures at concentrations ranging from 1 pg/ml to 10 ng/ml. Both cell lines showed significant AMG330-specific lysis at concentrations as low as 0.1 ng/ml (1.8 pM) and reached maximal lysis at a concentration of 1 ng/ml. No lysis was observed in the presence of the T cell-binding Control BiTE®.
Supplemental Figure 2:CD33 expression of different myeloid subpopulations in clinical samples from 17 AML patients.CD33 expression was analyzed by flow cytometry. MFI, mean fluorescence intensity.
Supplemental Figure 3: Flow cytometry gating strategy.For evaluation of BiTE® efficacy, myeloid populations were gated as shown above. CD45+ CD13+ CD34+ cells were considered as AML blasts. CD34 staining was substituted by CD117 when patients were diagnosed with CD117+ CD34- AML.
SupplementalFigure 4: The effect of AMG330 on specific secretion of cytokines in primary AML cultures.Cell culture supernatants from autologous AML MNC were analyzed for different cytokines after 24, 48, 72 and 144h, respectively by FlowCytomix technique (eBioscience, San Diego, USA). Besides low levels of TNF, secretion of cytokines could only be detected in cultures containing CD33 specific BiTEs®.
Supplemental Figure 5: CD8+ Subpopulations
CD3+ CD8+ T cells subpopulations were analysed over 144 h of culture. AML patients showed lower levels of the naïve T cell subpopulation compared to (not age matched) healthy donors. N=10. TemRA, CD45RA positive effector memory T cells; Teff, effector T cells; Tem, effector memory T cells; Tcm, central memory T cells.
Age (y) / Sex / Cytogenetics / FLT3-ITD / NPM1 / Material / % T Cells64 / m / structural changes on chromosome 17, LOH p53 / neg / neg / PB / 18.1
79 / m / trisomy 13, trisomy 14 / pos / neg / PB / 6.5
55 / f / trisomy 4 / pos / pos / PB / 3.1
69 / f / normal / pos / neg / BM / 4.6
75 / f / normal / pos / neg / PB / 10.9
67 / f / t(8;21), structural changes on chromosome 3 / neg / neg / BM / 2.0
73 / m / partial trisomy 6 / n.d. / n.d. / PB / 4.5
45 / m / normal / pos / neg / PB / 3.7
20 / m / normal / neg / neg / PB / 1.6
54 / m / normal / neg / neg / PB / 6.7
73 / m / n.d. / neg / neg / BM / 9.6
47 / f / normal / pos / pos / BM / 2.6
75 / f / normal / n.d. / n.d. / PB / 6.2
66 / m / complex aberrant caryotype / neg / neg / PB / 4.9
66 / f / normal / neg / neg / PB / 7.7
50 / m / t(8;21), trisomy 15 / neg / neg / BM / 5.7
69 / f / n.d. / n.d. / n.d. / PB / 3.1
58 / f / inv(16), trisomy 8 / neg / neg / BM / 8.9
31 / f / normal / neg / pos / PB / 16.5
44 / f / normal / pos / pos / PB / 2.5
44 / f / inv(16) / neg / neg / PB / 4.9
74 / m / normal / neg / neg / BM / 7.2
69 / m / normal / pos / pos / PB / 11.1
Supplemental Table 1: AML Patient Characteristics
Abbreviations used are: y, years; m, male; f, female; n.d., not determined; PB, peripheral blood; BM, bone marrow, FLT3-ITD, fms-like tyrosine kinase 3 internal tandem duplication; NPM1, mutation in nucleophosmin; % T Cells, % of CD3+ cells of all live, single cells.
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