A mutant B7-1/Ig fusion protein that selectively binds to CTLA-4 ameliorates anti-tumor DNA vaccination and counters regulatory T cell activity.

We have shown that a plasmid encoding a B7-1/Ig fusion protein enhanced DNA vaccination against human carcinoembryonic antigen (CEA) more effectively than the plasmid encoding membrane-bound B7-1. However, it was not known if B7-1/Ig acted only by binding CD28 (amplifying a stimulatory signal) or by blocking CTLA-4 on T cells (removing inhibitory signals). Here, we aimed to determine this using a plasmid encoding mutant B7-1/Ig (B7-1wa/Ig), which binds only to CTLA-4 but not to CD28. Our results showed that both the B7-1/Ig and B7-1wa/Ig plasmids, when co-administered with a CEA plasmid, enhanced tumor rejection and the in vitro anti-CEA response. Therefore, B7-1wa/Ig ameliorates DNA vaccination, presumably by binding to CTLA-4. This could result from a number of non-exclusive mechanisms, such as a reduced threshold for T-cell activation, or blockade of CTLA-4/B7-mediated tolerogenic signals in DCs or T cells. We found that, in vitro, a significant fraction of CD3/CD28-activated T cells (in the absence of DCs) expressed CTLA-4 and B7-1. Primed T cells of CTLA-4(+)B7-1(+/-) phenotype acted as regulatory T cells by inhibiting IFNgamma production by re-stimulated CTLA-4(-)B7-1(-) cells, and this was reversed by antibodies against IL-10 or TGF-beta1. Both B7-1wa/Ig and CTLA-4/Ig, which bind to CTLA-4 and B7-1/B7-2 respectively, enhanced IFNgamma production, but not the proliferation or IL-4 release in mixed T-cell populations containing these two cell types. In contrast, CTLA-4(-)B7-1(-) T cells produced IFNgamma which was not affected by B7-1wa/Ig or CTLA-4/Ig. These results suggest that blocking of CTLA-4/B7-1 binding in T cell/T cell interactions blocks negative regulatory signals. This might be the mechanism, at least in part, of the enhancement of anti-tumor immunity by the B7-1wa/Ig and B7-1/Ig plasmids.

Intramuscular gene transfer of soluble B7.1/IgG(1) fusion cDNA induces potent antitumor immunity as an adjuvant for DNA vaccination.

Soluble B7.1/IgG Fc fusion protein, which has costimulatory effects, is an effective molecular adjuvant in tumor immune therapy. Here, we describe a nonviral intramuscular (i.m.) gene transfer method to deliver this therapeutic protein. Gene transfer was greatly enhanced by electroporation and highly efficient production of this protein was achieved. Serum levels reached up to 1 microg/ml with considerable length of expression and without apparent systemic adverse effects. Lymphocytes from mice coinjected with soluble B7.1/IgG(1) and carcinoembryonic antigen (CEA)-encoding plasmids showed significantly elevated CEA-stimulated proliferation, cytokine production, and cytotoxic T-lymphocyte (CTL) activity. These mice gained significant protection against a CEA-positive transplanted tumor, in terms of reduced tumor incidence and growth. The effects were superior when soluble B7.1/IgG(1) was expressed as compared to membrane-bound wild-type B7.1. Notably, expression of soluble B7.1/IgG(1) alone did not induce any protection against tumor, confirming its primary role as a costimulatory molecule rather than a direct antitumor agent. The plasmid encoding B7.1/IgG(1) did not have to be injected at the same site as the antigen-encoding plasmid to exert its adjuvant effect, indicating that circulating protein was sufficient. Muscle histopathology revealed minimal damage to DNA-injected muscles. Importantly, we show that, after gene transfer, muscle tissue can produce this protein in large quantity to exert its immune costimulatory effect for cancer therapy and it would be otherwise difficult and expensive to maintain this high a level of recombinant protein.