Additional file 1 of A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death
Creators: name: Rosato, Francesca
name: Pasupuleti, Rajeev
name: Tomisch, Jana
name: Meléndez, Ana Valeria
name: Kolanovic, Dajana
name: Makshakova, Olga N.
name: Wiltschi, Birgit
name: Römer, Winfried
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. Expression, purification and characterization of Stx1B K9AzK. Coomassie stained SDS gels showed. a Expression of Stx1B K9AzK with the addition of AzK at a final concentration of 5 mM during induction with IPTG. b No expression of the target protein was detected when the culture medium was not supplemented with AzK during induction. Expression of Stx1B K9AzK with the addition of AzK at a final concentration of 5 mM during induction. M, molecular weight marker, the sizes of the marker bands are indicated; lane 1, clarified lysate; lane 2, flow through after loading clarified lysate on a zinc-charged sepharose column; lane 3, column wash; lanes 4 to 11, elution fractions. c 1.5 µg purified Stx1B K9AzK were subjected to native PAGE, a silver-stained 3–12% PA gel is shown. M, native PAGE marker, marker band sizes indicated in kDa; lane 1, Stx1B K9AzK (MWcalc ~ 45 kDa) in its native pentamer conformation. The black arrows indicate the protein bands of interest. Figure S2. Electrospray ionization mass spectrometry (ESI–MS) measurements of intact a Stx1B, b Stx1B K9AzK, c scFv OKT3 and d scFv OKT3 E129AzK. The proteins identified from the peaks are detailed in the Additional file 1: Table S1. Figure S3. Isothermal titration calorimetry (ITC) analysis of the interaction of a Stx1B and b Stx1B K9AzK with globotriaose. Raw data (top panel) and the binding isotherms obtained by plotting integrated data from titrations (bottom panel) are shown. Figure S4. Size analysis of the Stx1B-scFv OKT3 conjugate. a Representative size exclusion chromatogram of the IEDDA reaction mixture. b Protein standards aprotinin (Apr)- 6500 Da, ovalbumin (O)- 43000 Da, conalbumin (C)- 75000 Da, aldolase (Ald)- 158000 Da and ferritin (F)- 440000 Da were run on the S200 Increase 10/300 GL column for calibration. The column void volume (V0) 7.88 mL was determined by Blue dextran 2000. The gel-phase distribution coefficient (Kav) was calculated using the formula Kav = (Ve-Vo)/(Vb-Vo) where Ve is elution volume and Vb is the column bed volume (24 mL). A calibration curve was plotted with Kav versus the logarithm of molecular weight. The calibration curve calculated from molecular weight standards is a straight line with a coefficient of determination (R2) of 0.9972. The red circle represents the Stx1B-scFv OKT3 conjugate and the green circle unreacted scFv OKT3 E129TCO. The straight-line equation Kav = (-0.3208) x (MW) + 1.9415 deduced from the calibration curve was used to determine the experimental molecular weights of the unknown as stated in Additional file 1: Table S3. Figure S5. Comparative analysis of Stx1B wild-type and mutant proteins binding to tumor cells. Representative histograms of flow cytometry analysis of gated living Ramos and Namalwa cells, incubated with a Stx1B wild-type or b Stx1B K9AzK for 30 min on ice. a Histograms of fluorescence intensity of Gb3+ Ramos and Gb3− Namalwa cells incubated with Stx1B wt produced in this study (dotted, gray: negative control; light blue: 2.6 nM). b Histograms of fluorescence intensity of Ramos and Namalwa cells incubated with Stx1B K9AzK (dotted, gray: negative control; light blue: 2.6 nM; blue: 13 nM; dark blue: 26 nM). Stx1B K9AzK exhibited a similar binding pattern to Gb3 in comparison to the wild-type protein at the cell surface of Gb3+ Ramos cells. Fluorescence intensity did not change following incubation of AzK-incorporating Stx1B with Gb3− cells, excluding unspecific binding of the protein to the cell surface, even at higher Stx1B K9AzK concentrations (13 nM and 26 nM). All treated cells were stained with anti-6-His epitope tag AF647 antibody to detect the presence of wild-type and mutant proteins at the plasma membrane. The number of cells within the live population (y-axis) is plotted against the fluorescence intensity of tested proteins (x-axis). Figure S6. Binding of the Stx1B-scFv OKT3 lectibody to tumor and effector cells after 24 and 48 h co-incubation. Representative histograms of flow cytometry analysis of gated living a Ramos, and b PBMCs from healthy donors co-incubated in presence of Stx1B-scFv OKT3 for 30 min on ice (t = 0 h) or for 24 and 48 h at 37 °C (t = 24 h, t = 48 h). a Histograms of fluorescence intensity of Gb3+ Ramos incubated with Stx1B-scFv OKT3 for different time points (dotted, gray: negative control; light blue: 3.5 nM). Histograms depict a stable lectibody binding to Ramos cells after 24 h (right plot). b Histograms of fluorescence intensity of PBMCs incubated with Stx1B-scFv OKT3 for different time points (dotted, gray: negative control; blue: 35.6 nM). Histograms display lectibody binding to CD3 receptors on treated cells. At t = 24 h and t = 48 h following incubation with Gb3+ Ramos and Stx1B-scFv OKT3, the lectibody could still be partly detected at the membrane of effector cells. The number of cells within the live population (y-axis) is plotted against the fluorescence intensity of Stx1B-scFv OKT3 (x-axis). Figure S7. Comparative analysis of Stx1B wild-type and mutant proteins binding to solid tumor cells. Representative histograms of flow cytometry analysis of gated living HT-29 and LS-174 cells, incubated with a Stx1B wild-type or b Stx1B K9AzK for 30 min on ice. a Histograms of fluorescence intensity of HT-29 and LS-174 cells incubated with Stx1B wt produced in this study (dotted, gray: negative control; light blue: 2.6 nM; blue: 26 nM, red: 65 nM). b Histograms of fluorescence intensity of HT-29 and LS-174 cells incubated with Stx1B K9AzK (dotted, gray: negative control; light blue: 2.6 nM; blue: 26 nM; red: 65 nM). Stx1B K9AzK exhibited a similar binding pattern to Gb3 in comparison to the wild-type protein at the cell surface of HT-29 and LS-174 cells. All treated cells were stained with anti-6-His epitope tag AF647 antibody to detect the presence of wild-type and mutant proteins at the plasma membrane. The number of cells within the live population (y-axis) is plotted against the fluorescence intensity of tested proteins (x-axis). Figure S8. Flow cytometry analysis of PPMP-treated HT-29 cells. Representative histograms of gated living HT-29 cells incubated with Stx1B-Cy5 for 30 min on ice without (left panel) or after (right panel) Gb3 depletion. At 72 h post-treatment with 2 µM PPMP, Stx1B no longer bound to HT-29 cells confirming Gb3 depletion from the cell surface. Table S1. Analysis of ESI–MS spectral data. Table S2. Stx1B-scFv OKT3 conjugate identified by mass analysis. Table S3. Experimental molecular weights calculated from the calibration curve shown in Additional file 1: Fig. S4.