In vitro detection of canine anti-human antibodies following intratumoral injection of the hu14.18-IL2 immunocytokine in spontaneous canine melanoma

Background 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. Objectives Characterize CAHA in sera from dogs with spontaneous melanoma receiving radiotherapy and intratumoral immunocytokine (IT-IC) with humanized 14.18-IL2. Methods 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. Results 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. Conclusions This study advances CAHA detection strategies and reports the kinetics of CAHA following IT-IC in dogs with spontaneous melanoma.