Abstract We have identified a highly tumor specific cell surface antigen, ALPPL2 or ALPG, that is not expressed in any normal tissue except for the placenta. ALPPL2 is expressed in a number of cancers including mesothelioma, ovarian, lung, gastric, pancreatic, endometrioma, and testicular cancer. ALPPL2 is one of those rare cell surface antigens that are genuinely tumor specific as opposed to tumor associated. The exquisite tissue specificity makes ALPPL2 an attractive target for developing cancer therapies that require a high degree of specificity. We propose to develop an ALPPL2-targeted bispecific T cell engager for treatment of ALPPL2 positive cancers. Target specificity is the hallmark of our new therapy development effort. In addition, we will engineer the CD3 arm: (1) a low affinity CD3 arm has the potential of reducing target independent cytokine release and thus gaining a wider therapeutic window; (2) tumor killing and cytokine release are mediated by two thresholds that can be differentially impacted by CD3 arm engineering. A mild or minimal on-target cytokine release is desired. Another important variable that affects developability and therapeutic window is the molecular architecture on which the bispecific is constructed. In this proposal, we will address the three critical issues, i.e., target specificity, CD3 arm optimization, and molecular architecture, and use a system engineering approach to identify an optimized T cell engager with matching components and forms that impart it with an excellent developability and a wide therapeutic window, enabling translation to clinical testing. In Aim 1, we will generate ALPPL2 BiTEs with engineered CD3 arm variants in forms compatible with clinical translation. In Aim 2, we will study tumor-specific cytotoxicity and cytokine release of system-engineered BiTEs targeting the true tumor specific antigen ALPPL2. In Aim 3, we will determine efficacy in vivo and identify a lead ALPPL2 BiTE for clinical translation. Impact: Successful completion of the proposed study will lead directly to clinical translation of a novel bispecific T cell engager with unprecedented tumor targeting specificity in multiple incurable cancer types with dire clinical needs.