We have constructed a recombinant vaccinia virus, v-p97NY, which expresses the human melanomaassociated glycoprotein p97. Immunization with v-p97NY could induce humoral and cell-mediated immunity to p97, including delayed-type hypersensitivity, in mice and in two of two monkeys (Macaca fascicularis). The fact that an immune response was induced also in monkeys is important because normal cells from monkeys, but not from mice, express a low level of cross-reactive p97. Mice immunized with v-p97NY rejected transplants of syngeneic mouse melanoma expressing p97. A rejection response could be detected also when immunization was started 2 days after tumor transplantation, irrespective of whether the transplanted cells grew subcutaneously or as lung metastases. Evidence was obtained that melanoma cells lacking p97 may be killed as "bystanders" at the site of an immune response to melanoma cells expressing p97.

The gene for the human melanoma-associated antigen p97 has been introduced by cDNA transfection into cells from clone M2 of the K1735 mouse melanoma, which metastasizes to the lung when injected iv into syngeneic C3H/HeN mice. Tumor clones were established from the transfected cells and found to differ in the level of p97 expression. Their outgrowth in immunocompetent syngeneic mice was shown to inversely correlate with p97 antigen expression, and lines that express higher p97 levels elicited a stronger delayed-type hypersensitivity response when injected into the footpads of mice immune to p97. Five clones which expressed very high levels of p97 failed to grow in immunocompetent C3H/HeN mice while they formed tumors in nude (nu/nu) mice. The highest expressing clone, 2A, grew slightly faster than any of the other clones when cultured in vitro. Since several of the transfected clones were found to express a stable level of p97 and have consistent in vivo growth behavior, they provide a useful model for various forms of antigen-specific active and passive immunotherapy with the same agents as those intended for human application.

p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.

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