Blood and tissue biomarker analysis in dogs with osteosarcoma treated with palliative radiation and intra-tumoral autologous natural killer cell transfer

Abstract

We have previously reported radiation-induced sensitization of canine osteosarcoma (OSA) to natural killer (NK) therapy, including results from a first-in-dog clinical trial. Here, we report correlative analyses of blood and tissue specimens for signals of immune activation in trial subjects. Among 10 dogs treated with palliative radiotherapy (RT) and intra-tumoral adoptive NK transfer, we performed ELISA on serum cytokines, flow cytometry for immune phenotype of PBMCs, and PCR on tumor tissue for immune-related gene expression. We then queried The Cancer Genome Atlas (TCGA) to evaluate the association of cytotoxic/ immune-related gene expression with human sarcoma survival. Updated survival analysis revealed five 6-month survivors, including one dog who lived 17.9 months. Using feeder line co-culture for NK expansion, we observed maximal activation of dog NK cells on day 17-19 post isolation with near 100% expression of granzyme B and NKp46 and high cytotoxic function in the injected NK product. Among dogs on trial, we observed a trend for higher baseline serum IL-6 to predict worse lung metastasis-free and overall survival (P = 0.08). PCR analysis revealed low absolute gene expression of CD3, CD8, and NKG2D in untreated OSA. Among treated dogs, there was marked heterogeneity in the expression of immune-related genes pre-and post-treatment, but increases in CD3 and CD8 gene expression were higher among dogs that lived > 6 months compared to those who did not. Analysis of the TCGA

Approaches to Enhance Natural Killer Cell-Based Immunotherapy for Pediatric Solid Tumors

“…Similar to human NK cells, canine NK cells can be grown with irradiated feeder cell lines, such as genetically modified K562 cells or with cytokines including IL-2/12/15/18/21. Canine NK cells demonstrated cytotoxicity in vitro and show increased expression of Granzyme B [ 124 ].…”

“…Four dogs were euthanized related to progressive osteosarcoma and two died of unrelated causes. Four dogs (40%) remained alive at 18 months [ 123 , 124 ]. This demonstrates the feasibility of NK cell immunotherapy as a viable treatment option for canine osteosarcoma patients and adds to the evidence that NK cell therapy is a new treatment option for pediatric/AYA osteosarcoma patients.…”

“…These drugs kill tumor cells in an NK cell-dependent fashion by antibody-dependent cell-mediated cytotoxicity (ADCC) and NK cell independent-fashion by complement-mediated cytotoxicity (CMC). These effects can be augmented by coadministration with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) [ 124 ]. Naxitumab (formerly humanized 3F8), another anti-GD2 monoclonal antibody, mediated peripheral blood mononuclear cell (PBMC) ADCC against neuroblastoma, which produced significant evidence of this killing mechanism over previous studies implicating oxidative lytic PMN cytotoxicity against target cells [ 117 ].…”

Adoptive Natural Killer Cell Immunotherapy for Canine Osteosarcoma

“…One of the major strengths of clinical trials in dogs with spontaneously occurring cancers is the ability to do intensive longitudinal patient biospecimen sampling and clinical assessments, often more intensively than is possible in a comparable clinical trial in human patients. This is well-illustrated in the afore described first-in-dog clinical trial, where pre- and post-treatment serum cytokine concentrations were assessed by ELISA, tumor gene expression profiles by qRT-PCR, circulating immune cell phenotypes by flow cytometry, and intra-tumoral immune cell phenotypes by immunohistochemistry and qRT-PCR ( 59 ). Gradually increasing availability of new canine-specific reagents and application of new technologies to the dog, will further increase the number and power of the correlative studies that can be done and their translational relevance.…”

There and back again: Translating adoptive cell therapy to canine cancer and improving human treatment

“…After a pre‐conditioning regimen of four weekly radiation doses, they injected two doses of 7.5 × 10 6 NK cells/kg with 2500,000 IU/kg rhIL‐2 in 10 dogs 1 week apart. Follow up showed five patients that survived 6 months, including one dog that lived 17.9 months 40 . Of the 10 dogs, one dog obtained a grade 3 toxicity with symptoms consistent with IL‐2 toxicity.…”