Adoptive immunotherapy mediates effective regression of metastatic melanoma in patients. To target other cancers, we transduced T-cells to express chimeric antigen receptors (CARs) endowing them with activity against the tumor-associated-antigen, erbB2. Our current standard CAR, incorporating CD28-CD3ζ signaling domains, although promising, mediates lower activity compared to endogenous TCR responses against foreign antigens. Therefore, we hypothesized that utilizing CAR containing multiple alternate signaling domains may result in significantly enhanced anti-tumor responses. To test this, we generated a library of approximately 30,000 signaling chains that incorporated 14 different signaling molecules randomly ligated together directionally and in-frame flanked by SfiI sites and inserted into an anti-erbB2 CAR retroviral vector. The library was expressed in the Jurkat T-cell line and screened for high CD69 expression or IL-2 secretion in response to anti-erbB2 stimulation. The CAR DNA of selected clones was incorporated into the pSAMEN vector to transduce human peripheral blood mononuclear cells. A diverse range of CARs containing different signaling chains were identified. One CAR with the unique signaling domains, ICOS-LAT-TRIM showed promising antigen specific activity in vitro. Another CAR, containing the domains DAP10-CD3ζ-CD27 endowed human T-cells with the ability to mediate significantly enhanced killing of erbB2+ tumor cells in vitro compared to the CD28-CD3ζ CAR (p< 0.05). Strikingly, adoptive transfer of human T-cells expressing this novel receptor induced significantly increased anti-tumor effects in vivo (p< 0.01). In conclusion, it is anticipated that the generation of CARs with novel combinations of signaling domains will lead to stronger T-cells for the effective treatment of cancer.