{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T12:13:53Z","timestamp":1774440833634,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,23]]},"group-title":"Cancer Biology","reference-count":25,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T00:00:00Z","timestamp":1774224000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["HL07899"],"award-info":[{"award-number":["HL07899"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["TR00375"],"award-info":[{"award-number":["TR00375"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["GM008692"],"award-info":[{"award-number":["GM008692"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["CA009135"],"award-info":[{"award-number":["CA009135"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Czar's Promise"},{"name":"St. Baldrick\u2019s Foundation Empowering Immunotherapies for Childhood Cancer"},{"name":"Midwest Athletes Against Childhood Cancer Fund"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Companion canines need advances in therapeutic options for solid tumor malignancies. Prior studies established feasibility of autologous natural killer (NK) cell infusions in canines with solid tumors; however, autologous products are limited by dysfunctional immunity and a manufacturing process that delays care. Allogeneic NK cells offer the possibility of \u201coff-the-shelf\u201d therapy to be administered from healthy donors.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Peripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15. Additional experiments included IL-12 in the expansions. In vitro potency was assessed via co-culture with the D17-mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Flow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days of expansion. Residual T cell numbers varied based on donor. The addition of IL-12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic\/refractory solid tumors were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>Canine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro, and safety in vivo. Further studies will optimize the NK cell product and escalate dosing to reach the maximal tolerable dose.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.19.712729","type":"posted-content","created":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T09:45:20Z","timestamp":1774259120000},"source":"Crossref","is-referenced-by-count":0,"title":["Safety and Feasibility of Infusing\n                  <i>Ex Vivo<\/i>\n                  Expanded Allogeneic Canine Natural Killer Cells for the Treatment of Metastatic Solid Tumors"],"prefix":"10.64898","author":[{"given":"Allison M.","family":"Weisnicht","sequence":"first","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernanda","family":"Szewc","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monica Moonkyung","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Han-Yun Hannah","family":"Cheng","sequence":"additional","affiliation":[{"name":"Department of Medical Sciences, University of Wisconsin School of Veterinary Medicine, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Swetha","family":"Ganesh","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lauren","family":"Mahoney","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kathryn","family":"Fox","sequence":"additional","affiliation":[{"name":"Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Portia R.","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mallery","family":"Olsen","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rebecca M.","family":"Richards","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"},{"name":"Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David M.","family":"Vail","sequence":"additional","affiliation":[{"name":"Department of Medical Sciences, University of Wisconsin School of Veterinary Medicine, Madison, WI, USA"},{"name":"Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2276-6731","authenticated-orcid":false,"given":"Christian M","family":"Capitini","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"},{"name":"Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026032502050740000_2026.03.19.712729v1.1","doi-asserted-by":"publisher","DOI":"10.1186\/s13028-017-0341-9"},{"key":"2026032502050740000_2026.03.19.712729v1.2","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.34163"},{"key":"2026032502050740000_2026.03.19.712729v1.3","doi-asserted-by":"publisher","DOI":"10.1002\/pbc.24429"},{"key":"2026032502050740000_2026.03.19.712729v1.4","doi-asserted-by":"publisher","DOI":"10.1002\/pbc.28352"},{"key":"2026032502050740000_2026.03.19.712729v1.5","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-030-43032-0_12"},{"key":"2026032502050740000_2026.03.19.712729v1.6","doi-asserted-by":"publisher","DOI":"10.1172\/JCI31405"},{"issue":"5","key":"2026032502050740000_2026.03.19.712729v1.7","first-page":"291","article-title":"Activating and inhibitory receptors and their role in natural killer cell function","volume":"40","year":"2003","journal-title":"Indian J 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