INDUCTION OF T-CELL IMMUNITY AGAINST RAS ONCOPROTEINS BY SOLUBLE-PROTEIN OR RAS-EXPRESSING ESCHERICHIA-COLI

Background: Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers. Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Pas has been reported, Purpose: The goal of this study was to determine if immunization of mice with two forms of mutant Pas protein can induce high levels of Pas mutation-specific T-cell immunity in vitro and tumor regression in vivo, Methods: Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Pas oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines, In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Pas proteins, Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Pas peptides. For some of the cytolytic activity experiments, the spleen cells mere grown under T(H)1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or T(H)2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response). The specificity of immunity was examined in vivo by challenge of Pas-immunized mice with syngeneic tumor cells expressing mutant Pas oncoproteins (HaBalb, i,e,, BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 [Arg 12 Pas]; C3HL61, i,e., C3H/HeJ mouse cells expressing Pas with leucine substituted at position 61 [Leu 61 Ras]), Ten mice per group were used in each experiment. Results: Proliferative and cytolytic T-cell responses directed against the Arg 12 Pas protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Pas and therapeutic benefit in mice bearing established tumors expressing this protein, In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Pas. Immunization with heat-killed E, coli genetically engineered to express Leu 61 Pas also led to the induction of anti-Pas T-cell immunity, T cells grown under T(H)1 conditions were cytolytic against Pas-transformed tumor cells, whereas those grown under T(H)2 conditions were not, Conclusions: Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.