NIR Fluorescence Imaging of Colon Cancer With cRGD-ZW800-1—Response
Authors: Cornelis F.M. Sier, Alexander L. Vahrmeijer
Journal: Clinical Cancer Research (2021)

We thank Kossatz and Notni for their interest in our article about the use of fluorescent cRGD-ZW800-1 for visualization of tumor tissue in humans during surgery (1). Their concern seems the specificity of the tracer for integrin αvβ6, which is lower than for αvβ3, and the relation with our hypothesis. The latter seems clearly defined: “Documentation of the first clinical experience with cRGD-ZW800-1, particularly in patients with colonic cancer.” The specificity of the tracer for αvβ6 was clearly of secondary importance, indicated by the absence in title and abstract. Furthermore, we have stated that the cRGD peptide is applicable for many integrins: “cRGD is a clinically well-known peptide that binds to a wide range of integrins, including αvβ1, αvβ3, αvβ5, αvβ6, and αvb8.”Concerning the affinity of cRGD-ZW800-1: in our (pre)clinical experience, there is no “threshold” affinity for effective in vivo imaging in the complex microenvironment of a malignant tumor. Many variables contribute to a tumor-to-background ratio of fluorescent signal, including number of targets on the cells, ligand on/off-rate, ligand-induced endocytosis, presence of various cell types, and intratumoral pressure. The Kapp paper mentions that the affinity (the ratio of off-rate to on-rate) of our cRGDyK sequence for αvβ6 is 86 nmol/L (2), a low but respectable affinity for effective in vivo imaging, especially for target concentrations. Suppose that the αvβ6 copy number on malignant cells is 10-fold higher than for αvβ3, but the cRGD affinity is 10-fold lower; the resulting net moles of ligand-bound-per-cell would be exactly the same! We previously showed that cells without detectable αvβ3 on their membrane are perfectly visualized by cRGD-ZW800-1, due to the presence of other integrins with relatively low cRGD affinity (3). Regarding the authors' remark: The statement “Uptake of cRGD-ZW800–1 by αvβ6-integrin expressing HT29 cells could not be blocked by competition (no significance)” is incorrect, referring to Fig. 1C where an in vivo competition experiment performed using HT-29 cells was statistically significant to P = 0.02 and P = 007, at, respectively, 4 and 24 hours after injection. The in vitro experiment had the identical trend, although not statistically significant.Our clinically oriented paper deliberately focused on the correlation of αvβ6 expression and cRGD-ZW800–1 signal strength, because αvβ3 has already been discussed ad nauseum. And indeed, the correlation existed, regardless whether it was dependent of αvβ3 coexpression or not. In terms of integrin biology and clinical perspective, the most important finding was the strong αvβ6 expression in positive lymph nodes, but absence in metastasis free nodes. This could be of key importance for clinical application. Therefore, we are eager to discuss and learn from experts of various disciplines to optimize our materials or models, but in the end the proof of the pudding is in the eating, established in the clinic.See the original Letter to the Editor, p. 4937No disclosures were reported.