Supplementary Figures 1-9 from Construction of an Immunotoxin, D2C7-(scdsFv)-PE38KDEL, Targeting EGFRwt and EGFRvIII for Brain Tumor Therapy
Supplementary Figures 1-9 - Supplementary Figures 1-9 - PDF file 516K, Supplementary Figure S1. Deduced amino acid sequence of the D2C7 scFv cloned from D2C7 hybridoma. VH (A) and VL (B) antigen-binding regions of the D2C7 scFv. Amino acid numbering and CDR (underlined) delimitation were determined according to the Kabat database. Supplementary Figure S2. Purified D2C7-(scdsFv)-PE38KDEL immunotoxin. For this analysis, 200 ng of the purified D2C7-(scdsFv)-PE38KDEL immunotoxin was run on NuPAGE 4-12% Bis-Tris gel (Invitrogen), along with a standard molecular weight protein marker. Supplementary Figure S3. Surface plasmon resonance analysis of D2C7-(scdsFv)-PE38KDEL. Binding kinetics and affinity constants of D2C7-(scdsFv)-PE38KDEL for EGFRwt (A) and EGFRvIII (B) were determined by surface plasmon resonance against bacterially expressed recombinant EGFRwt or EGFRvIII extracellular domain (ECD) proteins. The association and dissociation rates from the sensogram were KA = 6.3 �~ 108 M-1 and KD = 1.6 �~ 10-9 M against EGFRwt and KA = 7.8 �~ 108 M-1 and KD = 1.3 �~ 10-9M against EGFRvIII. Supplementary Figure S4. Flow cytometric analysis of CD133 and D2C7 mAb on 43 and D2159MG glioblastoma xenograft cells. FACS analysis demonstrates the double staining of CD133 and D2C7 (percentage of cells in Q2) on 71% (A) and 86% (B) of cells freshly isolated from glioblastoma xenografts 43 and D2159MG, respectively. Q1 represents the percentage of cells positive for D2C7 only; Q4 represents the percentage of cells positive for CD133 only and Q3 represents the percentage of cells negative for both CD133 and D2C7. Supplementary Figure S5. Stability of D2C7-(scdsFv)-PE38KDEL. D2C7-(scdsFv)-PE38KDEL was incubated with 0.2% BSA/PBS at 40 ��g/mL (A) and 7.5 ��g/mL (B), over a 7 day period at 37{degree sign}C and then assayed for cytotoxic activity on NR6M cells. Cytotoxicity data are given as IC50 value versus radioactive leucine incorporation. Supplementary Figure S6. Flow cytometric analysis of D2C7 mAb and mouse EGFR antibody on normal mousekidney fibroblast cells (499) or avian sarcoma virus transformed mouse glioma cells (539). FACS analysis demonstrates the reactivity of mouse EGFR (msEGFR) antibody (pink peaks) to EGFR expressing 499 (A) and 539 (C) cells and the absence of D2C7 (pink peaks) reactivity to both 499 (B) and 539 (D) cells, confirming that D2C7 is human specific and does not react with murine EGFR. Isotype-specific antibody (IgG1) was used as control (filled purple peaks). Supplementary Figure S7. Survival of mice injected intracranially with 43 xenograft cells (A), NR6M cells (B), or D270MG xenograft cells (C). Mice were injected intracranially with 1x105 43, NR6M, or D270MG cells and were checked daily for survival. Data are presented as the survival time in days versus percentage of mice surviving. Supplementary Figure S8. Toxicity of D2C7-(scdsFv)-PE38KDEL immunotoxin administered to NSG mice. Different doses of D2C7-(scdsFv)-PE38KDEL were delivered intracranially over a 7 day period to NSG mice (5 mice/group). Animals were monitored for toxicity related death. Data are expressed as percentage of mice surviving versus time. Supplementary Figure S9. Immunohistochemical detection of D2C7-(scdsFv)-PE38KDEL distribution in D270MG orthotopic model after immunotoxin treatment. Acetone-fixed frozen D270MG-sham (negative control) (a) and D270MG-D2C7-(scdsFv)-PE38KDEL group (test) (c) sections were stained with 1.0 ��g of mouse-anti-PE38KDEL antibodies. Tumor sections from D270MG-D2C7-(scdsFv)-PE38KDEL group, pre-stained with D2C7-(scdsFv)-PE38KDEL was used as positive control (b)
