Induction of antitumor immunity via intratumoral tetra- costimulator protein transfer

Our group recently described a novel two-step Fc(gamma1) fusion protein tra nsfer method, which entails the docking of Fc(gamma1) fusion proteins onto cells precoated with chemically palmitated protein A (pal-prot A). In the p resent study, we have adapted this protein transfer method, originally used in an ex vivo context, for in situ tumor cell engineering, and in so doing , we have evaluated its utility for the induction of antitumor immunity via combinatorial costimulator protein transfer on to tumor cell surfaces. The feasibility of "painting" cells with preformed conjugates of a murine B7-1 costimulator derivative, B7-1.Fc(gamma1), and pal-prot A in a single step was first established ex vivo. Next, B7-1.Fc(gamma1):pal-prot A transfer wa s accomplished in vivo by directly injecting the preformed conjugates into highly aggressive L5178Y-R lymphomas grown intradermally in syngeneic mice. The presence of cell surface-associated B7-1 epitopes on cells of the inje cted tumors was documented by flow cytometric analysis of cells recovered s ubsequently from the injected tumors. B7-1.Fc(gamma1), along with Fc(gamma1 ) fusion protein derivatives of three additional costimulators (Fc(gamma1). 4-1BBL, CD48.Fc(gamma1), and Fc(gamma1).CD40L) geared toward a variety of i mmune effectors, were together preconjugated with pal-prot A and injected d irectly into tumor beds. Significantly, this "tetra-costimulator" combinati on, delivered intratumorally, induced complete tumor regression in similar to 45% of treated mice, whereas control injections of pal-prot A alone had no therapeutic effect. Furthermore, there was evidence for systemic antitum or immunity in that tumor-specific CTLs were detected in spleens recovered from cured mice, and these mice were uniformly protected against tumor rech allenge at distant tumor sites. Hence, combinatorial costimulator transfer, coupled to intratumoral delivery, may have special advantages for the indu ction of antitumor immunity.