Osteopontin (OPN), a circulating protein with pleiotropic functions, is highly expressed in many cancers and is known to have significant tumor-promoting activity. Thrombin cleaves OPN at arginine(Arg)153 and exposes a previously cryptic α4β1 and α9β1 integrin-binding site at the new C-terminus in the N-terminal thrombin-cleaved OPN fragment, OPN-Arg (OPN-R). OPN-R can be further processed to OPN-Leu (OPN-L). We have shown elevated OPN-R and OPN-L levels in arthritis joint fluid, and cerebral spinal fluid in glioblastoma, demonstrating that these cleavages occur in vivo. To define the role of thrombin cleavage of OPN in cancer biology, we created a thrombin cleavage-resistant OPN-knock in (KI) mouse in which Arg153 is substituted with alanine. In a murine B16 melanoma model, OPN-KI mice showed significant suppression of tumor growth and pulmonary metastasis. Tumor suppression was eliminated in severely immune deficient NOG-OPN-KI mice and was abolished by macrophage depletion in OPN-KI mice. Thrombin inhibition by the direct oral thrombin inhibitor, dabigatran, replicated the B16 tumor suppression in wild type (WT) mice. Tumor-associated macrophages (TAMs) were increased in tumors from OPN-KO and OPN-KI mice, with a switch from M2 macrophages to TAMs with a new activation profile. We also showed suppression of murine ovarian tumor in the OPN-KI mouse. Thus, thrombin cleavage of OPN initiates OPN’s tumor-promoting activity by suppressing the host-antitumor immune response mediated by modulation of the TAMs. We hypothesize that an anti-OPN monoclonal antibody (mAb) that blocks OPN cleavage by thrombin would be a novel adjunctive immuno-oncologic therapeutic that enhances the host- antitumor immune response mediated by TAMs. We have generated rabbit mAbs targeting the thrombin cleavage site of OPN that inhibit thrombin cleavage of human and mouse OPN. We converted the rabbit mAb A6 to a chimeric murine/rabbit mAb, and showed that chimeric mAb A6 suppressed B16 tumor growth and metastasis in WT mice, without any associated anticoagulant side effects, thus demonstrating proof-of-concept that A6 can be a lead therapeutic candidate. A6 has been humanized, and mAb hA6v3 binds human and mouse OPN and prevents thrombin cleavage of OPN. In collaboration with CIEA (Japan), we have created NOG-OPN- KI and NOG-OPN-KO mice, and have reconstituted the NOGhIL3/GM-CSF mice with human cord blood CD34+ cells. We have also generated a series of mAbs against OPN-R, the OPN fragment that may play the key role in suppressing the host-antitumor immune response. Anti-OPN-R mAb may be as effective as A6 with a major advantage over A6 in that the circulating pool of OPN-R is much smaller than OPN, and therefore the drug requirement as a therapeutic will be much less. We have identified a lead anti-OPN-R mAb C6r and it is a co- lead for this translation project. Our goal is to convert the lead mAbs into a clinical candidate with the target of filing an IND such that it will ...