Background: Bavituximab is a chimeric monoclonal antibody that targets the membrane phospholipid phosphatidylserine (PS) exposed on endothelial cells and cancer cells in solid tumors. Bavituximab blocks PS-mediated immune suppression in the tumor microenvironment. Methods: Fresh tumor tissues from consented patients with adenocarcinoma of the lung extracted at the time of surgical resection were utilized in a proprietary 3D ex vivo tumor miscrosphere assay to assess the immunomodulatory effects of bavituximab and potential immunosuppressive mechanisms such as expression of PD-1, CTLA-4, LAG3, TIM3, BTLA, and adenosine A2A receptor on the CD4+ and CD8+ tumor infiltrating T-cells. 3D tumor microspheres were prepared and cells were treated ex vivo with f(ab)’₂version of bavituximab, bavituximab, docetaxel, and a combination of bavituximab and docetaxel for 36 hours within an intact tumor microenvironment. Flow cytometry analysis evaluated treatment-mediated activation of TILs and changes in CD4, CD8 and Treg (CD25+/CD127-) subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines. Additionally, a NanoString platform containing probes to quantitate 770 immune function genes was used to determine potential positive or negative associations between expression of immune function genes and TIL activation by bavituximab. Results: Bavituximab induces activation of TILs in 3D ex vivo tumor microsphere model of lung cancer, as evaluated by a significant increase in IFNgamma, TNF-a, and GM-CSF secretion. Flow cytometry analysis revealed that this effect was associated with low PD-1 expression on CD8 cells, but did not correlate with expression of other immune inhibitory molecules. Conclusions: Our preliminary data support the use of bavituximab as an immunomodulatory treatment in adenocarcinoma of the lung by enhancing the activation of CD8+ TIL that correlates with increased cytokine production by lymphoid and myeloid cells. We identified PD-1 expression as a potential biomarker of response to bavituximab treatment, suggesting that the interruption of the PD-1/PD-L1 axis may enhance the bavituximab effect in lung cancer.