https://orcid.org/0000-0002-6149-4927
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ABSTRACT
PD-1 checkpoint inhibitors have revolutionized the treatment of patients with different cancer histologies including melanoma, renal cell carcinoma, and non-small cell lung carcinoma. However, only a subset of patients show a dramatic clinical response to treatment. Despite intense biomarker discovery efforts, no single robust, prognostic correlation has emerged as a valid outcome predictor. Immune competent, pet dogs develop spontaneous tumors that share similar features to human cancers including chromosome aberrations, molecular subtypes, immune signatures, tumor heterogeneity, metastatic behavior, and chemotherapeutic response. As such, they represent a valuable parallel patient population in which to investigate predictive biomarkers of checkpoint inhibition. However, the lack of a validated, non-immunogenic, canine anti-PD-1 antibody for pre-clinical use hinders this comparative approach and prevents potential clinical benefits of PD-1 blockade being realized in the veterinary clinic. To address this, fully canine single-chain variable fragments (scFvs) that bind canine (c)PD-1 were isolated from a comprehensive canine scFv phage display library. Lead candidates were identified that bound with high affinity to cPD-1 and inhibited its interaction with canine PD-L1 (cPD-L1). The lead scFv candidate re-formatted into a fully canine IgGD reversed the inhibitory effects of cPD-1:cPD-L1 interaction on canine chimeric antigen receptor (CAR) T cell function. In vivo administration showed no toxicity and revealed favorable pharmacokinetics for a reasonable dosing schedule. These results pave the way for clinical trials with anti-cPD-1 in canine cancer patients to investigate predictive biomarkers and combination regimens to inform human clinical trials and bring a promising checkpoint inhibitor into the veterinary armamentarium.
Abbreviations
| ADA | = | anti-drug antibodies |
| AE | = | adverse events |
| AUCinf | = | area under the serum concentration vs. time curve extrapolated to time infinity |
| AUClast | = | area under the serum concentration vs. time curve from time 0 to 21 days |
| CAR | = | chimeric antigen receptor |
| CL | = | clearance |
| Cmax | = | maximum observed serum concentration |
| CMS | = | carboxymethyl surface |
| cPBMCs | = | canine peripheral blood mononuclear cells |
| CTLA4 | = | cytotoxic T lymphocyte antigen 4 |
| CTV | = | cell trace violet |
| DCs | = | dendritic cells |
| ELISA | = | enzyme linked immunosorbent assay |
| FDA | = | Federal Drug Administration |
| GLP | = | good laboratory practice |
| HA | = | hemagglutinin |
| HIS | = | 6x histidine tag |
| HNSTD | = | highest non severely toxic dose |
| IACUC | = | institutional animal care and use committee |
| ICI | = | immune checkpoint inhibitor |
| NCA | = | non-compartmental analysis |
| NOAEL | = | No Observed Adverse Event Level |
| LAG3 | = | lymphocyte-activation gene 3 |
| MERS | = | Middle Eastern Respiratory Syndrome |
| MFI | = | mean fluorescence intensity |
| MSI-H/MMRD | = | microsatellite instability-high/mis-match repair deficiency |
| MTD | = | maximum tolerated dose |
| PALS | = | periarteriolar lymphoid sheaths |
| PD-1 | = | programmed cell death protein 1 |
| PD-L1 | = | programmed cell death ligand 1 |
| PK | = | pharmacokinetics |
| RO | = | receptor occupancy |
| scFv | = | single chain variable fragment |
| SDS-PAGE | = | Sodium dodecyl-sulfate polyacrylamide gel electrophoresis |
| SEC-HPLC | = | Size exclusion-high-performance liquid chromatography |
| t1/2 | = | terminal log-linear half-life |
| TIGIT | = | T cell immunoreceptor with immunoglobulin and ITIM domain |
| TILs | = | tumor infiltrating lymphocytes |
| TIM-3 | = | T cell immunoglobulin and mucin-domain containing-3 |
| TMB | = | tumor mutational burden |
| TMDD | = | Target Mediated Drug Disposition |
| VH | = | variable heavy |
| VL | = | variable light |
| Vss | = | volume of distribution |
Acknowledgments
We would like to thank Joel Cassel and the Wistar Molecular Screening and Protein Expression facility for their assistance and expertise with SPR performance and analysis. We are also grateful for the support of the Penn Vet Comparative Pathology Core, which is part of the Abramson Cancer Center Support Grant (P30 CA016520); the Aperio Versa 200 scanner used for imaging was acquired through an NIH Shared Instrumentation Grant (S10 OD023465-01A1); the Leica BOND RXm instrument used for IHC and ISH was acquired through the Penn Vet IIZD Core pilot grant opportunity 2022. Special thanks to Amiko Saito Yoshimoto for assistance with high-resolution figures.
Disclosure statement
Authors NJM, DLS, HW, and MB have equity in Vetigenics LLC.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2287250.