To find a new way for gene therapy against tumors with weak immunogenicity, the effect of mB7-1 costimulation alone, or combined with IL-6, in inducing antitumor immunityin vitro was investigated. It was found that mB7-1 cD-NA transfected B16 cells (B16-mB7-1) induced the expansion of effector lymphocytes and the generation of specific lytic activity more effectively than wild type B16 melanoma cells (B16-wt) or mock-transfected B16 cells (B16-neo) did. (P < 0. 01), IL-6 could effectively stimulate lymphocytes proliferation, but failed to enhance its cytotoxicity, while the combination of mB7-1 and IL-6 increased both lymphocyte proliferative response and T-cell-mediated cytotoxicity more significantly than B7-1 or IL-6 did alone (P<0. 01). It was inferred that the costimulatory molecule B7-1 is required for the activation and proliferation of T lymphocytes; the expression of mB7-1 in tumor cells could increase their immunogenicity and induce effective antitumor immune response, and the combination of B7-1 and IL-6 could induce more effective antitumor immunity, indicating that cooperation of IL-6 and mB7-1 plays a role in T lymphocyte activation.
Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-EH8S-K516A-F530A were cloned inB. Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be degraded into two peptides of 37ku and 21ku.