Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma

Abstract

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to α v β 3 , α v β 5 , and α 5 β 1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α 5 β 1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F–Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F–Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5–Fc as targeted molecular probes for brain tumor imaging.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

“…Several small proteins have been explored as scaffolds that can be adapted to interact with structurally diverse protein-binding partners (11)(12)(13). The defined tertiary structures of such scaffolds allow them to present large binding surfaces that can engage large and complementary surfaces on target proteins.…”

Pacifastin-derived Peptides Target Tumors for Use in In Vivo Imaging

“…To investigate the biodistribution of the THP1, both with and without conjugation to AF647, we evaluated a further modified form, termed THP1* and containing a single-lysine (K18), by using whole-body autoradiography. THP1* was prepared with and without a single AF647 conjugate and radiolabeled by reductive-methylation using 14 C formaldehyde. Mice bearing RH28 human sarcoma flank tumors were dosed with 2 μCi of a radiolabeled form of THP1* and THP1*-AF647.…”

“…Confocal microscopy was done using the Zeiss LSM 780 LMO microscope on sections cut from live, unfixed RH28 flank tumors 30 min after IV injection of with 10 nmol THP1-AF647. 14 C methylated peptide. Peptides were labeled with 14 C methyl groups via reductive amination using 14 C labeled formaldehyde (25).…”

“…14 C methylated peptide. Peptides were labeled with 14 C methyl groups via reductive amination using 14 C labeled formaldehyde (25). Briefly, peptides were resuspended in water and 10x PBS was added followed by 1% 14 C formaldehyde (1 mCi 14 C formaldehyde per 6 mg of peptide).…”

“…Cystine-knotted peptides address these challenges and are of interest for tumor delivery (13). Examples include RGD-containing peptides, such as EETI 2.5F, and chlorotoxin, a variant of which is the targeting component of tozuleristide (14)(15)(16). Differences in protein expression between cancer cells lead to variable sensitivity to targeting agents (17,18).…”