Conditional intracellular immunization against Ras inhibits proliferation of transformed human endothelial cells

E Radtke PhD^a,a, C Goemans PhD^c, B Feldhaus PhD^a, T Gronemeyer^e, C Grote-Westrick^a, J Kuhlmann PhD^d, A Eggert MD^b, R Heumann PhD^a

^aDept. of Biochemistry II, Ruhr-University, Bochum, Germany, ^bPediatric Oncology, University Hospital, Essen, Germany, ^cDept. of Biochemistry, University of Cambridge, Cambridge, United Kingdom, ^dBiophysical Analysis, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany, ^eEcole polytechnique fédérale de Lausanne, Institut de chimie moléculaire et biologique, Lausanne, Switzerland

AIM Oncogenic mutations of Ras are found in several tumor types. The aim of this study was to analyse the possible function of endogenous ras activity for the proliferation of transformed human umbilical vein endothelial cells (T-HUVEC). In transfected T-HUVECs the effects of cytoplasmic expression of a Ras-function blocking recombinant single chain antibody (anti-ras-scFv) were examined for in vitro proliferation and tumor growth after implantation into nude mice. METHODS The variable regions of the light and heavy chains, respectively of the monoclonal anti-ras-antibody Y13-259 were cloned from hybridoma cells for constructing a recombinant anti-ras single chain (anti-ras-scFv) antibody. Transgenic cell lines (T-HUVEC-anti-ras-scFv) expressing an inducible anti-ras-scFv (TetOff-System, BD Clontech) were generated by stable transfection. The binding affinity of purified recombinant anti-ras-scFv to its Ras target was determined by surface plasmon resonance (Biacore) analysis. Induction of cellular expression of anti-ras-scFv was revealed by Western-blot analyses and immunohistochemical stainings. Ras-binding activity of intracellular anti-ras-scFv was demonstrated by immunoprecipitations while proliferation was measured by BrdU incorporation into cultured cells. T-HUVEC-anti-ras-scFv were injected subcutaneously into nude mice and the size of the tumor was monitored in each mouse. Three groups of mice were examined (each n=6): i) without anti-ras-scFv expression (control group), ii) with anti-ras-scFv expression after injection (prevention group) and iii) with anti-ras-scFv expression after manifestation of solid tumor (treatment group). Sections of tumors were investigated for expression of anti-ras-scFv, CD31 and VEGF by immunohistochemical staining, by ELISA and by Western-blot analysis. RESULTS Biacore-analysis demonstrated high affinity binding of anti-ras-scFv to Ras protein (KD=0,4 nM). Cytoplasmic expression of anti-ras-scFv inhibited vascular endothelial growth factor (VEGF)-induced cell division of cultured T-HUVEC-anti-ras-scFv. In the xenograft tumor model, mean tumor growth rate was strongly decelerated in the prevention and treatment group as compared to the control group. Control group: 6/6 showed fast tumor growth. Prevention group: 2/6 did not form tumors, 2/6 showed stabilization or reduction of tumor volume, 2/6 were non-responders. Treatment group: 2/6 showed reduction of the initial tumor, 1/6 showed tumor stabilization, 1/6 decelerated growth, 2/6 were non-responders. CONCLUSIONS Ras regulates proliferation of endothelial cells. Intracellular expression of anti-ras-scFv reduces proliferation of transformed HUVEC and decelerates growth rates of T-HUVEC derived tumors. Our results show that intracellular immunization against Ras might provide a tool for targeted tumor therapy, especially in combination with other targeted therapies.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 998 (Signaling pathways - Part III).