A chemical cross-linking strategy was developed to couple two complementary molecular imaging agents onto a disulfide-rich knottin peptide that has been engineered to bind to αv
integrins with high affinity. The folding of cystine knot peptides is highly dependent on primary structure (22
). We observed that simple N-terminal acetylation or biotinylation of EETI-II severely affected its ability to properly fold, consistent with another study on permutants of the cyclotide kalata B1 (23
). These stringent folding requirements dictated that all conjugation chemistry be performed in solution after the knottin was folded, instead of using on-bead labeling of side-chain protected linear peptides immediately following synthesis. Therefore, we used a cross-linking strategy that selectively and sequentially exposed reactive groups for precise, stoichiometric coupling of both an optical imaging dye and a radiochelator to the knottin N-terminus. The method we developed is flexible, and can be used to accommodate a variety of imaging labels, therapeutics, or pharmacokinetic modulators for diverse biomedical applications. Importantly, we demonstrated that attachment of several chemical groups to the N-terminus of the engineered EETI-II knottin peptide did not interfere with its ability to bind to integrin receptors with high affinity.
Stability studies showed that the 64
Cu-DOTA-Lys(Cy5.5)-Gly-Gly-Tyr-2.5D probe is relatively labile, both in high concentrations of serum and after injection into the body. This breakdown is most likely due to proteolysis of the cross-linking moiety as we previously showed that knottin peptide 64
Cu-DOTA-2.5D exhibited only minimal fragmentation after incubation in serum for 24 h, and was recovered intact from the blood, tumor, and kidneys 1 hr after injection into mice (15
). Given that similar maximal tumor uptake levels were observed from 64
Cu-DOTA-2.5D and 64
Cu-DOTA-Lys(Cy5.5)-Gly-Gly-Tyr-2.5D, this proteolysis did not appear to affect its rapid tumor accumulation. However, degradation could have influenced kidney uptake and retention of dye molecules or metals. Cross-linkers composed of non-natural amino acids or peptoids may facilitate a more stable linkage (24
). In addition, the use of D-amino acids has been shown to stabilize a variety of imaging peptides such as somatostatin (25
). We are currently using these strategies to create more stable cross-linkers for the development of theranostic agents that will apply molecular imaging to quantify the delivery of cytotoxic drugs to tumors.
The use of a dual-labeled agent has distinct advantages as opposed to co-administering separate single-labeled agents. Having both labels on the same affinity ligand allows pharmacokinetic quantification of a single compound. This is beneficial because, as our study showed, the hydrophobic Cy5.5 dye strongly influenced the pharmacokinetics and tissue biodistribution of the radionuclide imaging agent. In addition, dual labeling allows quantitative cross validation and direct comparison between nuclear and optical imaging using an affinity ligand (26
). Several groups have developed dual-labeled NIRF/SPECT probes using near-infrared dyes and 111
In or 99m
Tc radiolabels (26
); these probes are specific to a variety of disease targets and have been recently reviewed (32
). Studies describing chemically-defined, dual-labeled NIRF/PET probes have been more limited. NIRF/PET probes have been developed by decorating quantum dots with tumor targeting moieties and radiolabeling the resulting nanoparticles with 64
). Compared to our knottin peptides, these nanoparticles exhibited extremely high uptake in the liver, spleen, lymph nodes, and bone marrow.
Addition of Cy5.5 had both a positive and negative effect on the pharmacokinetics and biodistribution of the probe. In comparison to 64
Cu-DOTA-2.5D, which rapidly washed out of the tumor, 64
Cu-DOTA-Lys(Cy5.5)-Gly-Gly-Tyr-2.5D was significantly retained by the tumor at later time points (). This tumor uptake was specific and mediated by integrin receptors as demonstrated by blocking experiments with c(RGDyK), which interacts with the same integrin binding site as the engineered knottin peptides (), as well as the lower levels of tumor uptake in MDA-MB-435 tumors, which express lower levels of integrin receptors compared to U87MG tumors (). Moreover, in previous studies we used knottin peptides containing a scrambled sequence to validate the integrin binding specificity of 64
Cu-DOTA- and Cy5.5-labeled knottin probes (13
Cy5.5 rapidly accumulated in the kidneys over the course of the imaging experiments ( and ), consistent with the high levels of kidney uptake and retention we observed with knottin peptide 2.5D labeled only with Cy5.5 (15
). In our previous studies, the kidney uptake for 64
Cu-DOTA-2.5D was 8.08 ± 0.67, 2.69 ± 0.48, and 1.25 ± 0.11 %ID/g at 1, 4, and 24 hr post injection, respectively (15
). In contrast, kidney uptake for 64
Cu-DOTA-Lys(Cy5.5)-Gly-Gly-Tyr-2.5D was 78.4 ± 13.9 and 80.9 ± 14.9 %ID/g for 1 hr and 4 hr post injection; however, after 24 hr the radioactivity in the kidneys decreased to 32.2 ± 2.2 %ID/g, suggesting that 64
Cu metabolites are cleared by the kidneys at later time points. This decrease in kidney signal is not due to the short half-life of 64
Cu (12.7 hr), as the data has been normalized to account for radioactive decay. Liver uptake (~ 4-7 %ID/g at 1-24 hr post injection) was also higher then previously observed for 64
Cu-DOTA-2.5D, in which values of ~1-2 %ID/g were observed 1 to 4 hr post injection. Increased liver and kidney uptake may preclude the use of this probe to identify lesions located in the abdomen; however, the spatial resolution of PET imaging, which is in the millimeter range, may allow identification of small lesions in close proximity to these organs. In addition, while kidney uptake and retention was high from Cy5.5-labeled probes, in future studies the use of alternate fluorescent dyes with more favorable biodistribution properties may help address this issue. Small amounts of probe are used for imaging applications, thus, we do not expect this probe to be immunogenic. However, as with any new compound intended for clinical use, this will have to be determined empirically.
In summary, the cross-linking strategy we describe here has broad applicability for site-specific coupling of two different imaging labels, or an imaging label and a therapeutic moiety, to knottin peptides for cancer diagnosis or therapy, or both. Conjugation of a tumor-specific knottin peptide to both a PET imaging agent and a chemotherapeutic agent could allow simultaneous and quantitative measurement of biodistribution and pharmacokinetics, drug delivery to tumors, and tumor response during the course of treatment. Moreover, different combinations of imaging labels may yield additional or unique information during a multimodal scan. The molecular tools developed here will allow research in these areas to be explored.