Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

Abstract

Interruption of the programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway is an established and effective therapeutic strategy in human oncology and holds promise for veterinary oncology. We report the generation and characterization of monoclonal antibodies specific for canine PD-1 and PD-L1. Antibodies were initially assessed for their capacity to block the binding of recombinant canine PD-1 to recombinant canine PD-L1 and then ranked based on efficiency of binding as judged by flow cytometry. Selected antibodies were capable of detecting PD-1 and PD-L1 on canine tissues by flow cytometry and Western blot. Anti-PD-L1 worked for immunocytochemistry and anti-PD-1 worked for immunohistochemistry on formalin-fixed paraffin embedded canine tissues, suggesting the usage of this antibody with archived tissues. Additionally, anti-PD-L1 (JC071) revealed significantly increased PD-L1 expression on canine monocytes after stimulation with peptidoglycan or lipopolysaccharide. Together, these antibodies display specificity for the natural canine ligand using a variety of potential diagnostic applications. Importantly, multiple PD-L1-specific antibodies amplified IFN-γ production in a canine peripheral blood mononuclear cells (PBMC) concanavlin A (Con A) stimulation assay, demonstrating functional activity.

Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

“…Moreover, dog-specific anti-PD-1 or PD-L1 antibodies are also not widely available. Canine T cells have previously been shown to strongly upregulate PD-1 when assessed with specifically developed anti-canine PD-1 antibodies upon ConA activation [ 7 ]. Under these conditions we observed distinct PD-1 staining with nivolumab, while pembrolizumab provided only a small shift and staining was not detectable in case of cemiplimab by flow cytometry ( Figure 1 D).…”

“…Targeting the PD-1/PD-L1 axis has become the first choice for combination therapies in human clinical testing [ 3 ]. Most dog studies on ICIs investigated this axis and a number of attempts have been made to develop blocking antibodies specific for dogs [ 7 , 8 , 9 , 10 ]. These pioneering findings have showcased the importance of this axis also in dogs by studying peripheral blood mononuclear cells (PBMCs) from healthy and tumor-bearing dogs [ 7 , 11 , 12 ], tumor explant cultures [ 9 ] and tumor cell lines [ 8 , 10 , 13 ].…”

“…Most dog studies on ICIs investigated this axis and a number of attempts have been made to develop blocking antibodies specific for dogs [ 7 , 8 , 9 , 10 ]. These pioneering findings have showcased the importance of this axis also in dogs by studying peripheral blood mononuclear cells (PBMCs) from healthy and tumor-bearing dogs [ 7 , 11 , 12 ], tumor explant cultures [ 9 ] and tumor cell lines [ 8 , 10 , 13 ]. Moreover, increased expression of PD-1 has been shown on canine T cells [ 9 , 12 , 14 ], as well as PD-L1 on canine antigen presenting cells (APCs) [ 10 , 12 ], and tumor cells [ 10 , 12 , 13 , 15 ].…”

Dog leukocyte antigen‐88*034:01 presents nonamer peptides from canine distemper virus hemagglutinin, large polymerase, and matrix proteins

“…1‐8). Studies with CAR T cells and PD‐1/PD‐L1 blockade have been reported 9‐11 . When contrasted with tumor‐grafted murine models, spontaneous canine cancers exhibit genetic and epigenetic heterogeneity, complex tumor microenvironments, and immunoediting.…”

Spontaneously occurring canine cancer as a relevant animal model for developing novel treatments for human cancers

“…Although there are no antibody drugs for the treatment of canine tumors in the market, many groups, including academic laboratories and veterinary drug companies, are developing antibody drugs for canine tumors. In particular, several groups have reported the development of anti-dog programmed death-1 (PD-1) and anti-dog programmed death ligand1 (PD-L1) antibodies [86][87][88][89][90]. In each of these reports, it is clear that anti-dog PD-1 and anti-dog PD-L1 antibodies enhance T cell activation and that PD-L1 is expressed in a variety of canine tumors, such as malignant melanoma, nasal carcinoma, squamous cell carcinoma, osteosarcoma, mammary adenocarcinoma, and lymphoma [90][91][92].…”