Novel non-immunogenic scaffold-based polypeptides have drawn a lot of attention for the development of molecular imaging probes because of easy synthesis, site-specific labeling, stable structure and high versatility targeting various cancer biomarkers. In our recent work, an anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule, engineered from one IgG binding domain of protein A and ~1/20 size of an antibody, was site-specifically labeled with 18
F. The resulting probe demonstrated great potential for PET imaging of the expression of HER2 in the clinic (16
). HER2 Affibody molecules have also been labeled with many other radioisotopes and organic dyes for preclinical and clinical imaging (17
). These progressions in scaffold protein based molecular probe development have motivated us to explore other scaffold protein as molecular platforms targeting different biomarkers.
The cystine knot miniprotein family represents a unique peptide scaffold of ~ 3 kDa with three interwoven disulfide bridges. Engineered knottin peptides, such as the integrin-binding peptide 2.5D, have been identified and their potential for in vivo
integrin molecular imaging has recently been evaluated as a 64
Cu-labeled PET tracer (12
). Preliminary evaluation of 64
Cu-labeled knottin peptides showed excellent tumor imaging properties in living mice. To facilitate the translation of knottin peptides as clinical imaging agents, we next tested an 18
F-labeled version of knottin peptide 2.5D in tumor-bearing mouse models in this research. With these studies, we wanted to determine whether there are any advantages and disadvantages of using knottin scaffolds as imaging agents as well as to further enrich our understanding of the in vivo
behavior of knottin peptides when they are coupled with different radionuclides.
In this research, 18
F-SFB was used as a prosthetic group for site-specifically labeling knottin 2.5D (). Methods to couple 18
F-SFB have been well-established in our program (14
). The non-radioactive version, 19
F-FB-2.5D, competes with 125
I-echistatin for binding cell surface integrins with an IC50
of 13.2 ± 5.4 nM, which is similar to that of the unmodified knottin peptide 2.5D (20.3 ± 7.3 nM). These data suggest that the N terminal amine of the knottin peptide tolerates modification, and further in vivo
evaluation of its radioactive counterpart confirmed this finding. Radiofluorination of 2.5D was efficiently achieved through site-specific conjugation of the radiosynthon, 18
F-SFB, with the N terminus amino group under mild conditions (60 °C, 1 h). 18
F-FB-2.5D was prepared in a reasonable yield (final yield, 28.3%) and high specific activity (~100 GBq/μmol) at EOS, which bode well for clinical applications.
Considering the short physical half life of 18
F as well as short biological blood half life of the knottin peptide, our biodistribution and imaging data of 18
F-FB-2.5D were acquired in a relatively short time frame (up to 1 h p.i.). Consistent with our expectation, we observed good tumor uptake resulting in good image quality. These data are attributable to peptide's tumor targeting ability and rapid clearance from normal tissues (). As early as 0.5 h p.i., probe uptake in the tumor was 1.90 ± 1.15 %ID/g. Liver uptake remained low at around 1.4 ± 0.5 %ID/g at 0.5 h, which was similar compared with its 64
Cu labeled knottin counterpart (2.3 ± 0.8%ID/g) (12
). Overall, microPET imaging data correlated well with the data derived from biodistribution studies. The U87MG tumor is clearly delineated from normal tissue at both 0.5 and 1 h p.i.. Quantification of microPET images showed that 56% of the activity remained in the tumor 1 h p.i., compared to the 0.5 h time point. Dynamic PET scans showed that the majority of 18
F-FB-2.5D was quickly cleared through renal system, and the blood radioactivity concentration dropped dramatically in the first 35 min p.i. These pharmacokinetic properties suggest translational potential.
expression in vivo
has extensively been imaged by small, backbone cyclized peptidomimetics [c(RGDyK), c(RGDfK), etc.] (20
F-FB labeled c(RGDyK) [abbreviated as FB-RGD] was reported to moderately target U87MG tumors in vivo
(ca 3 and 2.5 %ID/g uptake at 0.5 and 1 h, respectively) (21
F-FB-2.5D demonstrated lower liver uptake (1.5 %ID/g at 0.5 h p.i.) compared to 18
F-FB-RGD (ca 2.5 and 1 %ID/g at 0.5). But 18
F-FB-2.5D has lower tumor uptake (ca 2 % ID/g at 0.5 h p.i.) and the kidney uptake is slightly higher than 18
F-FB-RGD (4.5 vs.
3.5 %ID/g at 0.5 h p.i.). The findings presented here are different from our previous experience. In our recent report, 64
Cu labeled DOTA-2.5D did show significant advantages over 64
). This difference indicates that various labeling methods impact in vivo
performance of knottins. Moreover, it suggests that further optimization of 18
F-FB-2.5D may lead to a radiofluorinated knottin probe with improved properties. It was reported that PEGylation enhanced the tumor uptake and prolonged the retention of 18
F labeled c(RGDyK) (ca 5 %ID/g at 0.5 h and 2.5 %ID/g at 2 h p.i.), while the liver uptake remained the same (ca 2.6 %ID/g at 0.5 h) (24
). Similar strategies could be adapted to improve the tumor uptake and retention of 18
F-labeled knottins and increase the tumor-to-liver ratios.
As an important target for cancer therapy, αv
integrin is highly overexpressed on tumor cells and the tumor neovasculature of many cancer including melanoma and glioblastoma (13
). An optimized 18
F labeled 2.5D probe could be used for diagnosis of αv
-positive tumors, both at primary and metastatic sites. 18
F-labeled 2.5D could also be used for stratification of patients, detection of cancer recurrence, and for monitoring therapeutic efficacy of integrin-targeted drugs. In Conclusion, radiolabeled knottin peptide 18
F-FB-2.5D allows integrin-specific PET imaging of U87MG tumors with good contrast, and is mainly cleared from the body through the renal system. Knottins are excellent peptide scaffolds for the development of PET probes for clinical translation.