{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:47:16Z","timestamp":1782280036355,"version":"3.54.5"},"posted":{"date-parts":[[2025,3,25]]},"group-title":"Immunology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,25]],"date-time":"2025-03-25T00:00:00Z","timestamp":1742860800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Canine and human malignant melanoma are naturally occurring cancers with many similarities, making the dog an important parallel patient population to study both diseases. However, development of canine anti-human antibodies (CAHA) needs to be considered when evaluating humanized biotherapeutics in dogs.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Objectives<\/jats:title>\n                  <jats:p>Characterize CAHA in sera from dogs with spontaneous melanoma receiving radiotherapy and intratumoral immunocytokine (IT-IC) with humanized 14.18-IL2.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Serum samples were obtained pre-treatment and at several post-treatment times from 12 dogs with locally advanced or metastatic melanoma treated with radiotherapy to the primary site and regional lymph nodes (when clinically involved) followed by IT-IC of humanized 14.18-IL2. Two CAHA assays were developed. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against the humanized IgG component of hu14.18-IL2. A flow cytometry assay was developed to determine the ability of CAHA to inhibit binding of a mouse anti-GD2 monoclonal antibody to its target.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Post-treatment sera from 7 of 12 dogs developed CAHA levels over pre-treatment that were identified by ELISA as significant increases at Day 30 and\/or Day 60. Day 10, Day 30, and Day 60 post-treatment sera from 10 of 12 dogs significantly inhibited the binding of anti-GD2 monoclonal antibody to its target compared to pre-treatment. Significant binding inhibition was also detected in 2 of 12 dogs after local RT but before IT-IC (Day 1). Normal canine sera did not mediate binding inhibition.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>This study advances CAHA detection strategies and reports the kinetics of CAHA following IT-IC in dogs with spontaneous melanoma.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.21.644578","type":"posted-content","created":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T03:10:25Z","timestamp":1742958625000},"source":"Crossref","is-referenced-by-count":0,"title":["In vitro detection of canine anti-human antibodies following intratumoral injection of the hu14.18-IL2 immunocytokine in spontaneous canine melanoma"],"prefix":"10.64898","author":[{"given":"Andrew T.","family":"Kosharek","sequence":"first","affiliation":[{"name":"University of Wisconsin School of Medicine and Public Health, Madison, WI, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2672-3840","authenticated-orcid":false,"given":"Cindy L.","family":"Zuleger","sequence":"additional","affiliation":[{"name":"University of Wisconsin School of Medicine and Public Health, Department of Medicine, Madison, WI, United States of America"},{"name":"University of Wisconsin Carbone Cancer Center, Madison, WI, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William S.","family":"Glass","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Madison, Madison, WI, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jens","family":"Eickhoff","sequence":"additional","affiliation":[{"name":"University of Wisconsin Carbone Cancer Center, Madison, WI, United States of America"},{"name":"University of Wisconsin School of Medicine and Public Health, Department of Biostatistics & Medical Informatics, Madison, WI, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul M.","family":"Sondel","sequence":"additional","affiliation":[{"name":"University of Wisconsin Carbone Cancer Center, Madison, WI, United States of America"},{"name":"University of Wisconsin School of Medicine and Public Health, Department of Human Oncology, Madison, WI, United States of America"},{"name":"University of Wisconsin School of Medicine and Public Health, Department of Pediatrics, Madison, WI, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David M.","family":"Vail","sequence":"additional","affiliation":[{"name":"University of Wisconsin Carbone Cancer Center, Madison, WI, United States of America"},{"name":"University of Wisconsin School of Veterinary Medicine, Department of Medical Sciences, Madison, WI, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark R.","family":"Albertini","sequence":"additional","affiliation":[{"name":"University of Wisconsin School of Medicine and Public Health, Department of Medicine, Madison, WI, United States of America"},{"name":"University of Wisconsin Carbone Cancer Center, Madison, WI, United States of America"},{"name":"University of Wisconsin School of Medicine and Public Health, Department of Dermatology, Madison, WI, United States of America"},{"name":"The Medical Service, William S. 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