T cell costimulation has great therapeutic potential if it can be optimized and controlled. To achieve this, we engineered T cell-activating fusion proteins and immunocytokines that specifically attach to viral antigens of a virus-infected tumor vaccine. We employed the avian Newcastle Disease Virus because this agent is highly efficient for human tumor cell infection, and leads to introduction of viral hemagglutinin-neuraminidase (HN) molecules at the tumor cell surface. Here, we demonstrated the strong potentiation of the T cell stimulatory activity of such a vaccine upon attachment of bispecific or trispecific fusion proteins which bind with one arm to viral HN molecules of the vaccine, and with the other arm either to CD3 (signal 1), to CD28 (costimulatory signal 2a), or to interleukin-2 receptor (costimulatory signal 2b) on T cells. A vaccine with a combination of all three signals triggered the strongest activation of naïve human T cells, thereby inducing the most durable bystander antitumor activity in vitro. Adoptive transfer of such polyclonally activated cells into immunodeficient mice bearing human breast carcinoma caused tumor regression. Furthermore, tumor-reactive memory T cells from draining lymph nodes of carcinoma patients could be efficiently reactivated in a short-term ELISpot assay using an autologous tumor vaccine with optimized signals 1 and 2, but not with a similarly modified vaccine from an unrelated tumor cell line. Our data describe new bioactive molecules which in combination with an established virus-modified tumor vaccine greatly augments the antitumor activity of T cells from healthy donors and cancer patients.

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