Exploration of serum biomarkers in dogs with malignant melanoma receiving anti-PD-L1 therapy and potential of COX-2 inhibition for combination therapy

Immune checkpoint inhibitors (ICIs) such as anti-PD-L1 antibodies are widely used to treat human cancers, and growing evidence suggests that ICIs are promising treatments for canine malignancies. However, only some canine oral malignant melanoma (OMM) cases respond to ICIs. To explore biomarkers predictive of survival in dogs with pulmonary metastatic OMM receiving the anti-PD-L1 antibody c4G12 (n = 27), serum concentrations of prostaglandin E2 (PGE(2)), cytokines, chemokines, and growth factors were measured prior to treatment initiation. Among 12 factors tested, PGE(2), interleukin (IL)-12p40, IL-8, monocyte chemotactic protein-1 (MCP-1), and stem cell factor (SCF) were higher in OMM dogs compared to healthy dogs (n = 8). Further, lower baseline serum PGE(2), MCP-1, and vascular endothelial growth factor (VEGF)-A concentrations as well as higher IL-2, IL-12, and SCF concentrations predicted prolonged overall survival. These observations suggest that PGE(2) confers resistance against anti-PD-L1 therapy through immunosuppression and thus is a candidate target for combination therapy. Indeed, PGE(2) suppressed IL-2 and interferon (IFN)-gamma production by stimulated canine peripheral blood mononuclear cells (PBMCs), while inhibition of PGE(2) biosynthesis using the COX-2 inhibitor meloxicam in combination with c4G12 enhanced Th1 cytokine production by PBMCs. Thus, serum PGE(2) may be predictive of c4G12 treatment response, and concomitant use of COX-2 inhibitors may enhance ICI antitumor efficacy

Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-gamma stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors

PD-L1 expression in feline macrophage and mammary adenocarcinoma cell lines.

(a) Binding of the anti-feline PD-L1 monoclonal antibody CL1Mab-7 to fePD-L1–EGFP-expressing cells. Cells were transfected with EGFP (mock) or fePD-L1–EGFP expression vector and analyzed by flow cytometry at 24 h post transfection. (b) PD-L1 expression in the feline macrophage cell line Fcwf-4. (c) PD-L1 expression in feline mammary adenocarcinoma cell lines FKNp, FMCp, FMCm, FYMp, FONp, and FONm. Cells were incubated with 100 ng/mL of recombinant feline IFN-γ for 24 h where indicated. Mouse IgG1 (mIgG1) was used as an isotype-matched negative control antibody.</p

Characterization of feline PD-1/PD-L1/PD-L2 proteins.

(a) Preparation of fePD-1–EGFP- and fePD-L1–EGFP-expressing cells. Cells were harvested and observed under a fluorescent microscope at 24 h post transfection. (b) Preparation of fePD-1–Ig, fePD-L1–Ig, and fePD-L2–Ig. The purified Fc-fusion proteins were separated under reducing (left) or non-reducing (right) conditions by SDS-PAGE. The proteins were visualized by CBB staining. (c) Binding of Fc-fusion proteins to fePD-1–EGFP- and fePD-L1–EGFP-expressing cells. Cells were incubated with 10 μg/mL of each Fc-fusion protein and their binding was detected using anti-rabbit IgG secondary antibody. Rabbit IgG was used as a negative control.</p

Identification of feline <i>PD-L2</i> mRNA sequence.

(a) Multiple sequence alignment of PD-L2 amino acid sequences in various mammalian species. The SP, IgSF domains, and TM domain are indicated with numbers representing the positions of amino acid residues. (b) Phylogenetic tree (inferred as described in Fig 1) of the feline PD-L2 amino acid sequence in relation to those of other mammalian species. Numbers next to the branches indicate the bootstrap percentage. The scale bar indicates the divergence time.</p

Summary of PD-L1 immunohistochemistry in feline malignant tumors.

Summary of PD-L1 immunohistochemistry in feline malignant tumors.</p

PCR primer sequences.

PCR primer sequences.</p

The original uncropped and unadjusted images for Fig 3b.

(TIF)</p