RESUMO
Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.
RESUMO
Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients.
