American Association for Cancer Research
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Supplementary Figures 1-9 from Construction of an Immunotoxin, D2C7-(scdsFv)-PE38KDEL, Targeting EGFRwt and EGFRvIII for Brain Tumor Therapy

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posted on 2023-03-31, 17:20 authored by Vidyalakshmi Chandramohan, Xuhui Bao, Stephen T. Keir, Charles N. Pegram, Scott E. Szafranski, Hailan Piao, Carol J. Wikstrand, Roger E. McLendon, Chien-Tsun Kuan, Ira H. Pastan, Darell D. Bigner

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)



Purpose: The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.Experimental Design: The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models.Results: The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6 × 10−9 mol/L and 1.3 × 10−9 mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P = 0.006), 28% (P = 0.002), and 166% (P = 0.001), respectively.Conclusions: In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both. Clin Cancer Res; 19(17); 4717–27. ©2013 AACR.