LAUSR

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved anti-tumor potency of anti-CD20-interferon alpha (IFNα) fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency and receptor distribution. Here, we used positron emission tomography (immunoPET) to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it to the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 h) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic (2 h p.i.) and static (4, 24 and 72 h) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 {plus minus} 1.0 %ID/g at 75 h p.i.), which was significantly lower than 89Zr-Rit (38.4 {plus minus} 9.9 %ID/g, p<0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared to mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the non-invasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.