ARTICLE ABSTRACTDespite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HAhigh) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER23+ primary breast tumors and approximately 40% of EGFR+ head and neck squamous cell carcinomas are HAhigh, and hypothesized that HAhigh tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HAhigh tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HAhigh tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HAhigh tumors, resulting in enhanced trastuzumab- and NK cell–mediated tumor growth inhibition in vivo. These results suggest that HAhigh matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HAhigh tumors. Mol Cancer Ther; 14(2); 523–32. ©2014 AACR.