Background CD3 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... Show moreBackground CD3 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 tumor uptake and the extracellular dose. To optimize their currently limited efficacy in solid tumors, increased understanding of their pharmacokinetics and in vivo internalization is needed.Methods Here, were studied the pharmacokinetics and in vivo internalization 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-binding or CD3-binding capacity served as controls. BsAbs were radiolabeled with 111In to investigate their pharmacokinetics, target binding, and biodistribution through SPECT/CT imaging and ex vivo biodistribution analyses. Co-injection of 111In- and 125I-labeled bsAb was performed to investigate the in vivo internalization by comparing tissue concentrations of cellular residing 111In versus effluxing 125I. Antitumor therapy effects were evaluated by monitoring tumor growth and immunohistochemistry.Results SPECT/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%ID/g±5.3%ID/g, spleen: 29.0%ID/g±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, target-mediated clearance by TRP1-expressing melanocytes was not observed. We further demonstrated rapid internalization of CD3xTRP1 in KPC3-TRP1 tumors (24 hours pi: 54.9%±2.3% internalized) and CD3-rich tissues (spleen, 24 hours 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.Conclusions We 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 antitumor 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. Show less