Introduction

The anti-CTLA4 checkpoint inhibitor ipilimumab, has revolutionized cancer patient treatment by promoting effector T cell function and eliminating intratumoral regulatory T cells. However, only a subset of patients respond clinically. Pet dogs with spontaneous tumors represent a valuable patient population to investigate predictive biomarkers and rational therapeutic checkpoint inhibitor combinations. Furthermore, dogs with immunogenic tumors might benefit clinically from CTLA4 inhibition. Here, we generate and validate a fully canine anti-CTLA4 monoclonal antibody for translational research and clinical veterinary use.

Methods

Fully canine single chain variable fragments (scFv) that bind canine CTLA4 (cCTLA4) were isolated from a comprehensive canine scFv phage display library. The scFv were evaluated for cCTLA4 binding by flow cytometry and surface plasmon resonance and by ELISA for their ability to inhibit cCTLA4:CD80/CD86 interaction. The effects of cCTLA4 blockade on mitogen-activated canine T cell function were evaluated in vitro and biodistribution studies were performed in mice.

Results

A selected fully canine anti-CTLA4 clone specifically bound cCTLA4 with subnanomolar binding affinity, inhibited cCTLA4 binding to CD80/CD86 and promoted T cell proliferation and effector function. The antibody exhibited linear kinetics in vivo in mice and produced no short-term clinical or hematological adverse effects.

Conclusion

This work paves the way for in vivo analysis of the first fully canine, anti-canine CTLA4 antibody to promote anti-tumor immunity in dogs with immune responsive cancers and provides an important comparative tool to investigate correlative biomarkers of response and mechanisms of resistance to CTLA4 checkpoint inhibition.

Funding Information

V Foundation for Cancer Research NCI/NIH/SBIR Contract 75N91018C000042.