Figure 4. Radiolabeled B7-H3 nanobody has potent in vivo tumor targeting capacity. (A) Evaluation of binding kinetics of purified B7-H3 nanobody on immobilized recombinant human B7-H3 protein using SPR. The sensorgram represents binding and dissociation of a dilution series (1/2 dilution series ranging from 500 nM to 3.9 nM) of the nanobody. Binding kinetics were calculated using the Biacore T200 V.2.0 evaluation software to calculate the KD value. (B) Evaluation of binding kinetics of purified B7-H3 scFv on immobilized recombinant human B7-H3 protein using SPR. The sensorgram represents binding and dissociation of a dilution series (1/2 dilution series ranging from 500 nM to 1.9 nM) of the scFv. Binding kinetics were calculated using the Biacore T200 V.2.0 evaluation software to calculate the KD value. (C) Competition study of the B7-H3 nanobody and scFv showing that both bind a different epitope on human B7-H3, as determined by SPR. The absence of competition is shown by the increase in signal obtained by binding of the competitor in a second association step (right panel) as compared with the signal obtained by the individual components (left panel). (D) Representative SPECT/CT images of tumor-bearing mice 1 hour postinjection with radiolabeled B7-H3 or 5T2Id nanobodies. The images are representative for three mice. Yellow circles indicate tumor positioning. (E) Graph of percentages injected activity per gram (%IA/g) for isolated tumors as determined by ex vivo γ-counting. The graph summarizes the tumor uptake of the B7-H3 and 5T2Id nanobody in four mice. (F) Graph of percentages injected activity per gram (%IA/g) for all isolated organs and tissues as determined by ex vivo γ-counting. The graph summarizes the biodistribution of the B7-H3 and 5T2Id nanobody in four mice. Data represent mean±SD n=4 mice per group. *p≤0.05. Statistical significance in panel E was determined using an unpaired t-test. 5T2Id, 5T2 multiple myeloma idiotype; B7-H3, B7 homolog 3; KD, equilibrium dissociation constant; Nb, nanobody; scFv, single-chain variable fragment; SPECT, single photon emission computed tomography; SPR, surface plasmon resonance.
