SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1. Characterization of hu recombinant Ab constructs for retargeting of T cells to AML blasts. (a) Comparison of the schematic structure of the components of the modular system and the conventional reference bsAb CD33-CD3. For humanization of the scFvs anti-CD3(MT-301), anti-CD33(DRB2) and anti-La(5B9) murine complementarity determining regions were grafted onto human frame work regions. In order to generate the effector module hu single-chain bispecific tandem variable fragment (scBsTaFv) CD3-La(5B9), the sequence of the hu scFv La(5B9) (VH-(G4S)3-VL) was cloned downstream of the previously described hu scFv CD3 (VH-G4S-VL).23 The anti-La(5B9) mAb recognizes the epitope E5B9, which is localized in the N-domain of the nuclear La protein.21,23,25 The sequence of this epitope was cloned in frame downstream of the hu scFv CD33 (VL-(G4S)2GASGA(G4S)2-VH) to generate the target module hu scFv CD33-E5B9. For construction of the reference bsAb CD33-CD3 hu scFvs anti-CD33 and anti-CD3 were connected in row. Repetitions of glycine-serine (G4S) linkers served as connecting elements in all recombinant molecules. Each Ab component was endowed N-terminally with an Igk leader sequence as signal peptide (SP) for protein secretion and C-terminally with myc-tag and 6xhistidine (his)-tag for protein purification and detection. (b) After eukaryotic expression and Ni-NTA-based protein purification, recombinant Abs were analyzed by SDS-Page and subsequently stained with Coomassie-Brilliant Blue (bi). After immunoblotting recombinant Abs were detected via their C-terminal his-tag (bii). (c) To determine the binding properties of newly generated recombinant Abs, preactivated PBMCs (ci), La-decorated CD33+ MOLM-13 cells (cii), or CD33+ MOLM-13 cells (ciii) were stained with 20 ng/µl of the effector module, target module or bsAb CD33-CD3. Specific binding was detected by a FITC-conjugated anti-myc mAb (grey graph). To investigate the accessibility of the E5B9-epitope after binding of the target module on CD33+ MOLM-13 cells, the anti-La(5B9) mAb and a PE-conjugated anti-mouse-IgG mAb were used as detection Abs (ciii, right panel). Mean fluorescence intensity (MFI) and percentage of positive cells in M1 region are shown. Marker was adjusted to appropriate negative controls (transparent graph).
Supplemental Figure 2. Ab stability in human serum. Freshly isolated T cells and MOLM-13 cells were incubated at an e:t ratio of 5:1 in RPMI 1640 supplemented with 10% human serum (HS) (left and middle panel) or 10% fetal calf serum (FCS) (right panel). Co-cultivation occurred in the absence or presence of 5 pmol/ml of each Ab component. Recombinant Abs were either pre-incubated at 37°C for 24 hours in RPMIHS (left panel) or stored at 4°C in PBS (middle, right panel). CD25 and CD69 expression on gated CD3+ T cells are shown in dot plots for one donor.
Supplemental Figure 3. Redirection of T cells to normal myeloid cells via CD33-specific Ab-based systems. PBMCs were incubated with or without 30 pmol/ml of indicated Ab components for 24 hours. (a) CD69 and CD25 expression levels of CD3+ gated T cells are shown. Data are presented as mean ± SD of three independent, healthy donors (***p<0.001, *p<0.05; one-way ANOVA with Bonferroni multiple comparison test). (b) Concentration of pro-inflammatory cytokines IFN-g and TNF in co-culture supernatants were determined for three individual donors. (c) To analyze elimination of normal myeloid cells within PBMCs, cells were stained with fluorescently labeled CD3, CD13, CD33 and CD45 Abs. Percentage of CD45+CD3-CD13+ myeloid cells and CD45+CD3+CD13- cells of one representative donor are depicted in dot plots (left panel). To summarize data of three individual donors, percentage of surviving CD45+CD3-CD13+ cells within PBMCs were normalized to control without Ab (right panel). Data are presented as mean ± SD.
Supplemental Figure 4. Efficient killing of PSCA+ tumor cells via the modular system. (a) In order to humanize the previously published anti-PSCA(7F5)10 scFv, the murine complementarity determining regions were grafted onto human frame work regions. Based on this fully humanized anti-PSCA scFv, we generated a PSCA-directed target module by cloning the E5B9 peptide epitope downstream of the scFv. (b) In a chromium-release assay pre-stimulated PBMCs and PC3-PSCA cells were incubated with 30 pmol/ml of each recombinant Ab for 20 hours. Killing efficiency of the PSCA-specific modular system was compared to previously published murine scBsDb CD3xPSCA(7F5).10 Specific lysis and SD for one representative donor are shown.