Abstract Monoclonal antibodies (mAbs) targeting immune checkpoints, such as PD-1, PD-L1, and CTLA4, have provided unprecedented clinical benefits to human cancer patients. Novel strategies such as combination therapy with OX40 or 4-1BB agonists are currently under investigation in various pre-clinical studies to enhance the therapeutic benefits of immune checkpoint inhibitors. While syngeneic mouse tumor xenograft models offer valuable insights in pre-clinical testing, they fall short in capturing the nuanced heterogeneity within and between tumors and the dynamic tumor-immune interface. In contrast, canine patients with spontaneous neoplasms possess a closely analogous and intact immune system. These canine patients, who receive sophisticated and advanced medical therapy like their human counterparts, can serve as a vital intermediate pre-clinical model system between rodent studies and human clinical trials, potentially reducing clinical trial failures. In this study, we have developed a novel bispecific fusion protein, denoted BsOXPD, that combines OX40 agonism and PD-1 blockade into a single therapeutic. Our bispecific fusion protein employs an anti-PD-1 nanobody (Nb) to bind and block the PD1 pathway and the extracellular domain (ECD) of the OX40 ligand to stimulate the OX40 receptor. We have developed murinized, caninized, and humanized versions of this protein. All versions of the bispecific fusion protein retain their biochemical and functional properties following the murinization, caninization, and humanization processes. Importantly, all versions effectively disrupt the PD-1/PD-L1 axis in murine, canine, and human systems while concurrently acting as potent OX40 agonists. To further improve the humanized version of the BsOXPD, we partially humanized the anti-PD1 Nb by introducing amino acid substitutions in various framework regions. The humanized anti-PD1 Nb still completely inhibited the binding of human PD-L1 to the PD-1 receptor. The caninized version of BsOXPD exhibits no toxicities or immune-related adverse events (irAEs) following intravenous injection at doses of 1 mg/kg or 3 mg/kg body weight in healthy beagle dogs. In summary, we have designed a cross-species Nb-based fusion protein for PD1 blockade and OX40 agonism. Our next goal is to evaluate the therapeutic efficacy of caninized BsOXPD in pet dogs with oral melanoma in neoadjuvant settings. Therefore, our cross-species approach will offer comprehensive insights into the safety, pharmacokinetic (PK) profile, and clinical benefits associated with the concurrent application of PD1 blockade and OX40 agonism. Citation Format: Maninder Sandey, Damien Ruiz, Jonathan Marable, Deepa Bedi, Payal Agarwal. Bridging the gaps: A cross-species approach to PD-1 and OX40 combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3736.

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.

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.