Abstract Adoptive cell therapy using T cells modified by gene transfer to express T cell receptors or synthetic chimeric antigen receptors (CARs) that specify T cell recognition of tumor associated antigens can be effective in patients with refractory malignancies. Clinical data has shown that tumor cells that express low levels of antigen or that have heterogeneous antigen expression can escape and cause relapse. To address the barrier of low antigen density on tumor cells, we have designed novel synthetic hybrid receptors based on principles of T cell receptor signaling that have superior properties and may be capable of eliminating tumor cells with low antigen levels. To address the barrier of heterogeneous antigen expression on tumor cells, we designed colocalization-dependent orthogonal protein switches that perform ‘AND’, ‘OR’, and ‘NOT’ Boolean logic at the cell surface. In principle, this approach can instruct T cells to eliminate heterogeneous tumor cell populations and spare normal antigen positive cells, extending the constellation of antigens that can be safely targeted. The studies in this application will advance these two classes of next generation synthetic receptors for multiple clinically relevant target antigens to achieve more effective and safe elimination of tumors with low and/or heterogeneous antigen expression. The specific aims are: Aim 1: To evaluate signaling and function of novel CAR/TCR hybrid receptors in primary T cells. Signaling, synapse formation, antigen sensitivity, and in vivo function and fate of T cells expressing CAR-TCR hybrid receptors introduced by lentiviral transduction or by knock-in to the T cell receptor alpha gene locus, will be compared to T cells expressing CD28/CD3z and 4-1BB/CD3z CARs targeting the same antigens. Aim 2: To evaluate chimeric costimulatory receptors for T cells engineered with CAR/TCRs. We will evaluate whether the addition of costimulation in CAR/TCR engineered T cells by co-expressing chimeric molecules that engage a tumor associated ligand will enhance antitumor function. Aim 3 To design OR and AND gated CARS specific for multiple myeloma (MM) antigens and evaluate their ability to prevent tumor escape. Multiple myeloma is responsive to T cell therapy targeting single antigens, but tumor cell heterogeneity allows escape. Co-localization dependent protein switches that can perform OR and AND logic gated recognition of heterogeneous tumor cells will be developed and evaluated for targeting multiple myeloma antigens.