Abstract
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.
Highlights
Cancer is the leading cause of death in the geriatric dog population
The labeled canine CTLA4 protein was used for biopanning, and CTLA4 binders were enriched by magnetic-assisted cell sorting (MACS) using anti-AF647 or anti-biotin microbeads (Miltenyi)
To isolate single yeast clones, AF647+, HA488+ and Propidium Iodide− yeast cells were sorted from an enriched Nb library using fluorescence-activated cell sorting (FACS)
Summary
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. Abbreviations cHcAb Chimeric heavy chain only antibody CTLA4 Cytotoxic T lymphocyte antigen-4 DNA Deoxyribonucleic acid FACS Fluorescence-activated cell sorting HcAbs Heavy chain only antibodies ICB Immune checkpoint blockade ICIs Immune checkpoint inhibitors ICs Immune checkpoints IFN-γ Interferon-γ mAbs Monoclonal antibodies MACS Magnetic-assisted cell sorting MHC Major histocompatibility complex Nb Nanobody. Immune checkpoint molecules are critical for maintaining self-tolerance, minimizing collateral tissue damage, and developing immunity against pathogens[8] Tumor cells disrupt this balance by activating inhibitory immune checkpoints to create an immunosuppressive milieu, which favors immune evasion and tumor growth[8]. The blockade of CTLA4-B7 interaction by ipilimumab allows unrestrained CD28-mediated positive stimulation and activation of cytotoxic T cell responses[16,17,18]
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