Abstract To continue efforts of Specific Aim 1 as stated in U01CA244314, a new mouse model recently commercialized by GenOway will be used to perform key experiments testing the potential therapeutic effects of anti-human CD3 Fab fragments derived from the monoclonal antibody OKT3 specific for the ectodomain of human CD3. The mouse model in the C57BL/6 background consists of a knock in manipulation to substitute murine genes encoding for the CD3, and subunits of the TCR/CD3 complex with chimeric genes encoding for the fusion of human ectodomains with murine transmembrane and cytoplasmic domains for each of these three CD3 subunits. The KI mice present normal development of functional mature T cells that are targetable with anti- human antibodies specific for the ectodomain of human CD3, such as the mAb OKT3. This model has been validated by others as T cell targetable when using anti-human CD3 specificities. Specifically, in this model B cell tumor cells can be killed by T cells when using bi-specific IgG reagents designed to bind and crosslink human CD3 ectodomain and tumor epitopes. Our current data generated with the support of our U01 award indicate that the OKT3 mAb derived reagent Mono-OKT3-Fab, a Fab fragment controlled for monovalence, possesses the capacity to promote anti-tumor immune responses against patient derived xenograft models of non-small cell lung carcinoma (NSCLC) in immunocompromised NSG mice when they carry human T cells autologous to the human NSCLC grafts. GenOway B6 hu-ms CD3 mice will be purchased to test anti-tumor effects of Mono-OKT3-Fab in the presence of intact immune system tolerant to autologous tumors with fully functional T cells expressing the human ectodomain of the CD3 subunit of the TCR/CD3 complex. The goal is to determine the potential therapeutic effects of Mono-OKT3-Fab against cancer when targeting functional T cells in tumor hosts that are immunocompetent and tolerant to the tumor. Positive results in the GenOway model, in conjunction with our current data in immunocompromised mice, would make a solid case to pursue further steps towards clinical translation of Mono-OKT3-Fab as a novel T cell targeted immunotherapy against cancer.