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Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer’s safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6.

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WITH THEIR ENRICHED EXPRESSION and activity of an apical vacuolar (V-type) H ϩ -ATPase (2, 52, 86), the principal cells of renal (Malpighian) tubules in the mosquito (Aedes aegypti) are similar to acid-secreting cells of the mammalian renal collecting tubule (85), mammalian male reproductive tract (10), turtle and amphibian urinary bladders (9), amphibian skin (17), and mammalian bone (76). In these acid-secreting cells, the apical V-type H ϩ -ATPase is often opposed by a basolateral Cl/HCO 3 anion exchanger of the solute-linked carrier 4 (SLC4) superfamily of bicarbonate transporters. The anion exchanger mediates the basolateral extrusion of intracellular HCO 3 Ϫ , thereby removing the intracellular HCO 3 Ϫ that was generated during the ionization of carbonic acid (H 2 CO 3 ).Disulfonic stilbene derivates, such as SITS and DIDS, are potent inhibitors of almost all SLC4 transporters (61). These negatively charged compounds are thought to interact with positively charged lysine residues on the external surface of SLC4 proteins (47, 83), perhaps blocking a conduit for anion translocation. Pharmacological studies using stilbene derivatives suggest the presence of Cl/HCO 3 exchangers in Malpighian tubules of some insects. For example, the addition of SITS to the peritubular bath of isolated Aedes Malpighian tubules lowers the unstimulated rates of fluid secretion (25), and both peritubular SITS and DIDS inhibit the serotoninstimulated rates of fluid secretion in isolated Malpighian tubules of Rhodnius prolixus (23,29). In isolated Malpighian tubules of the cricket (Teleogryllus oceanicus), SITS inhibits unstimulated rates of fluid secretion in the distal and main segments (89); DIDS also inhibits secretion in the distal segment, but it paradoxically stimulates secretion in the main segment (89). Finally, X-ray microanalysis on larval Drosophila Malpighian tubules indicate that DIDS decreases the intracellular Cl Ϫ concentration ([Cl Ϫ ]) in the basal cytoplasm of principal cells, which is consistent with the presence of a Cl/HCO 3 exchanger (88).To date, only one SLC4-like transporter has been cloned and characterized from any insect, i.e., the Na ϩ

The isolation of apolipoprotein A-I-containing lipoproteins [Lp(A-I)] by selected-affinity immunosorption minimizes the loss of associated proteins that occurs during the isolation of high-density lipoproteins (HDL) by sequential ultracentrifugation. We have used two-dimensional gel electrophoretic analysis to separate the proteins associated with Lp(A-I). Using a combination of amino acid sequencing of transblotted proteins and Western blotting with specific antisera, we have identified a number of associated proteins. The positions of the apolipoproteins (apo) A-I, A-II, A-IV, C-III, D, and E were located on the gels. Lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein were identified in association with Lp(A-I) to a greater extent than found associated with HDL after centrifugation. In addition to those proteins previously identified in association with HDL, we detected a number of plasma proteins associated with Lp(A-I), namely, fibrinogen, haptoglobin, proline-rich protein (C4b-binding protein), and apolipoprotein J (SP40,40 sulfated glycoprotein). The co-isolation of these proteins with Lp(A-I) does not appear to be an artifact in that they have very low affinity for a sham column containing covalently bound preimmune goat IgG in place of the anti-apoA-I IgG. These findings suggest that in addition to apolipoproteins that exist largely in association with lipoproteins there is another class of proteins which exist in lipoprotein-associated form and in the dispersed state. Detection and identification of these lipoprotein-associated proteins may aid in the mechanistic determination of a number of observed functions attributed to HDL.