Imaging the pharmacokinetics and therapeutic availability of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

Abstract

AbstractBackgroundCD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the absolute tumor uptake and the extracellular dose. To optimize their currently limited efficacy in solid tumors, increased understanding of their pharmacokinetics andin vivointernalization is needed.MethodsHere were studied the pharmacokinetics andin vivointernalization of CD3xTRP1, a fully murine Fc-inert bsAb, in endogenous TRP1-expressing immunocompetent male C57BL/6J mice bearing TRP1-positive and negative tumors over time. Matching bsAbs lacking TRP1- or CD3-binding capacity served as controls. BsAbs were radiolabeled with111In to investigate their pharmacokinetics, target binding, and biodistribution through SPECT/CT imaging andex vivobiodistribution analyses. Co-injection of111In- and125I-labeled bsAb was performed to investigate thein vivointernalization by comparing tissue concentrations of cellular residing111In versus effluxing125I. Anti-tumor therapy effects were evaluated by monitoring tumor growth and immunohistochemistry.ResultsSPECT/CT and biodistribution analyses showed that CD3xTRP1 specifically targeted TRP1-positive tumors and CD3-rich lymphoid organ and uptake peaked 24 hours pi (KPC3-TRP1: 37.7±5.3 %ID/g, spleen: 29.0±3.9 %ID/g). Studies with control bsAbs demonstrated that uptake of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues was primarily receptor-mediated. Together with CD3xTRP1 in the circulation being mainly unattached, this indicates that CD3+T cells are generally not traffickers of CD3-bsAbs to the tumor. Additionally, “antigen-sink” effects by TRP1-expressing melanocytes were not observed. We further demonstrated rapid internalization of CD3xTRP1 in KPC3-TRP1 tumors (24h pi: 54.9±2.3% internalized) and CD3-rich tissues (spleen, 24h pi: 79.7±0.9% internalized). Therapeutic effects by CD3xTRP1 were observed for TRP1-positive tumors and consisted of high tumor influx of CD8+T cells and neutrophils, which corresponded with increased necrosis and growth delay.ConclusionsWe show that CD3xTRP1 efficiently targets TRP1-positive tumors and CD3-rich tissues primarily through receptor-mediated targeting. We further demonstrate rapid receptor-mediated internalization of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues. Even though this significantly decreases the therapeutical available dose, CD3xTRP1 still induced effective anti-tumor T-cell responses and inhibited tumor growth. Together, our data on the pharmacokinetics and mechanism of action of CD3xTRP1 pave the way for further optimization of CD3-bsAb therapies.Graphical abstractImaging the pharmacokinetics and therapeutic availability of the bispecific CD3xTRPl antibody in syngeneic mouse tumor models