Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients
Autor: | Jonathan Marable, Bruce F. Smith, Damien Ruiz, Ritankar Bhattacharya, Robert J. Pantazes, Amarjit Mishra, Payal Agarwal, Anil Kumar Jaiswal, Deepa Bedi, Maninder Sandey, Amol Suryawanshi |
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Rok vydání: | 2021 |
Předmět: |
medicine.drug_class
Science Population chemical and pharmacologic phenomena Monoclonal antibody Article Dogs Neoplasms medicine Animals Cytotoxic T cell CTLA-4 Antigen Dog Diseases Cancer models education Immune Checkpoint Inhibitors education.field_of_study Multidisciplinary biology Chemistry Single-Domain Antibodies Cell sorting Isotype Fragment crystallizable region Immune checkpoint Cancer research biology.protein Medicine Antibody therapy Antibody |
Zdroj: | Scientific Reports Scientific Reports, Vol 11, Iss 1, Pp 1-12 (2021) |
ISSN: | 2045-2322 |
Popis: | Background 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.Methods 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.Results 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 FcƳ 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 canine PBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control.Conclusions This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients. Furthermore, this approach provides a critical proof-of-concept for developing nanobody-based humanized anti-CTLA4 therapy for advanced stages of cancers. |
Databáze: | OpenAIRE |
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