UC Davis

Natural killer (NK) cells are key effectors in anti-tumor responses with great potential to extend the promise of cancer immunotherapy. Dogs develop spontaneous cancers with striking similarities to humans and can serve as a crucial link to inform NK biology, optimize NK immunotherapy for both dog and human patients, and identify potential biomarkers of response. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. We assessed NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product. Calculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment. Overall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs. To characterize the heterogeneity of canine NK cells regarding NK ontogeny, subset, and patterns of activation and inhibition we assessed canine NK cell populations by single cell RNA sequencing (scRNAseq) across blood and tissues, including canine soft tissue sarcoma, and canine and human lung, liver, spleen, and placenta. We observed tissue-specific NK cell signatures consistent with immature, stem-like NK cells in the placenta, mature and activated NK cells in the lung, and NK cells with a mixed activated and inhibited signature in the liver with significant cross-species homology. NK cells from both canine and human undifferentiated sarcoma exhibited an exhausted signature that most closely correlated with NK cells in the liver. We also analyzed NK cells in the peripheral blood of dogs on first-in-dog clinical trials undergoing three distinct NK-targeting regimens, observing that dogs with favorable response (good responders) demonstrated increased NK proportions post treatment. Genes upregulated in NK cells in the peripheral blood of good responders included genes associated with activated NK cells in the lung and revealed post-treatment gene expression changes in the blood as a better predictor of response than baseline NK gene expression. Together, our results point to heterogeneous canine NK populations highly comparable to human NK cells with effector functions adapted to their tissue of residence and dysregulated sarcoma infiltrating NK cells with features of both activation and inhibition. We provide a comprehensive atlas of canine NK cells across organs and sarcomas which will inform future cross-species NK studies and further substantiate the spontaneous canine model in immuno-oncology to optimize NK immunotherapy across species.