89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFN alpha 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 immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFN alpha 1 fusion protein (Rit-mIFN alpha) and compared it with the parental mAb (rituximab, Rit). Rit-mIFN alpha and Rit were radiolabeled with zirconium-89 (Zr-89, t(1/2) 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both Zr-89-Rit-mIFN alpha and Zr-89-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. Zr-89-Rit-mIFN alpha showed specific uptake in tumors (7.6 +/- 1.0 %ID/g at 75 hours p.i.), which was significantly lower than Zr-89-Rit (38.4 +/- 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, Zr-89-Rit-mIFN alpha showed rapid blood clearance and high accumulation in the liver compared with Zr-89-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single Zr-89-Rit-mIFN alpha dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving Zr-89-Rit. Mice receiving Zr-89-Rit-mIFN alpha had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFN alpha. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.