Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

Abstract

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

Keywords: 44G4; CD105; anti-endoglin monoclonal antibody; anti-tumor therapy; cancer therapy; endoglin; recombinant musarmin.

Figure 1

Molecular characterization of rMU1. Left panel: Superdex 75 chromatography of rMU1 and SDS-PAGE analysis of the preparation (inset); the horizontal bar indicates the collected factions. Right panel: N-glycosidase of rMU1 on rabbit reticulocyte ribosomes; A: control; B: soluble rMU1; C: rMU1 from solubilized inclusion bodies; D: Saporin-S5; (+) indicates the treatment of the lysates with acid aniline and (*) indicates the Endo fragment; numbers on the left indicate base pairs and on the right the rRNA type.

Figure 2

Purification (upper panel) and SDS-PAGE analysis (lower panel) of the rMU1-44G4 immunotoxin. Upper panel: The reaction mixtures were carried out as indicated in Materials and Methods and purified by gel filtration chromatography on Superdex 200 HiLoad. Letters in the chromatogram of the upper panel indicate peaks analyzed by SDS-PAGE; the fractions collected in each peak were: B (high molecular weight aggregates of 44G4 and RMU1), 135–140 mL; C1 (medium molecular weight conjugates rMU1-44G4), 150–155 mL; C2, (low molecular weight conjugates rMU1-44G4), 165–170 mL; D1 (low molecular weight conjugates rMU1-44G4), 170–175 mL; D2 (low molecular weight conjugates rMU1-44G4), 175–180 mL; E (rMU1 tetramer), 185–190 mL; F (rMU1 trimer), 192–207 mL; G (rMU1 dimer), 207–230 mL; H (rMU1), 235–260 mL. Both C1 and C2 were mixed and used as immunotoxin; Lower panel: Proteins contained in the indicated peaks were analyzed by SDS-PAGE using three different concentrations of acrylamide: 7.5% (w/v) (A); 10% (w/v) (B); 18% (w/v) (C); numbers on the right indicate the apparent Mr; numbers in parentheses indicate the estimated size. The amount of protein loaded into each well was: 30 micrograms in lanes B, C1, C2, D1, D2; four micrograms in lane H; 10 micrograms in lanes G, F and E. Numbers on the right indicate the corresponding apparent Mr values of the standards.

Figure 3

Analysis of rMU1-44G4 IT by SDS-polyacrylamide gel electrophoresis in the absence (A) or presence (B) of 2-mercaptoethanol (2-ME). (A) Electrophoresis in the absence of 2-ME was carried out with 7.5% (w/v) polyacrylamide gels; the rMU1/44G4 ratio is indicated on the left of the picture; (B): Electrophoresis in the presence of 2-ME was carried out with 18% (w/v) polyacrylamide gels. The numbers on the right of both gels indicated the apparent Mr of the markers used. The numbers on the left indicate the RIP/mAb stoichiometries.

Figure 4

Cytotoxicity of the rMU1-44G4 immunotoxin on L929 expressing the short form of human endoglin (upper panel) and parental L929 mouse fibroblasts (lower panel). Growing cultures of transfected L929-hCD105+ and parental L929 cells were incubated for 3 h, either with rMU1, immunotoxin or 44G4 and the viability of cells was assayed with the WST-1 reagent as indicated in the Materials and Methods Section. Vertical bars indicate the ±SEM (n = 3).