Discovery and characterization of a high-affinity and high-specificity peptide ligand LXY30 for in vivo targeting of α3 integrin-expressing human tumors

Abstract

Backgroundα3β1 integrin is overexpressed in several types of human cancer and is associated with poor prognosis, metastasis, and resistance to cancer treatment. We previously identified a cyclic peptide ligand LXY1 that specifically binds to the α3β1 integrin on human glioblastoma U-87MG cells. Here, we optimized LXY1 through one-bead one-compound combinatorial library screening and site-specific modifications to improve its in vivo binding property.MethodsThree bead libraries were synthesized and whole-cell binding assays were performed. The binding capacity of individual peptide ligands against different tumor cells was determined by flow cytometry and confirmed by optical imaging. A complex joining biotinylated ligand with streptavidin-Cy5.5 was used for in vivo target imaging in both subcutaneous and orthotopic U-87MG xenograft mouse models.ResultsLXY30, a cyclic peptide with the sequence cdG-Phe(3,5-diF)-G-Hyp-NcR, emerged as the most potent and selective ligand for the α3 subunit of α3β1 integrin with improved in vitro and in vivo tumor-targeting effects compared to LXY1 in U-87MG cells. LXY30 is considerably stable in plasma as demonstrated in an in vitro stability study in 90 % human plasma. LXY30 also binds to several other known α3β1 integrin-expressing glioblastoma, lung, and breast cancer cell lines with various affinities.ConclusionsOur data support further investigating the role of LXY30 as a human tumor-targeting peptide ligand for systemic and intracranial delivery of imaging agents and cancer therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0165-z) contains supplementary material, which is available to authorized users.

Targeting Tumor‐Associated Exosomes with Integrin‐Binding Peptides

“…As previously reported, SKOV-3 cells exhibited high affinity for LXY30-Fl but only minimal affinity for the scrambled analogue (Figure 2c, left panel ). [34] Next, SKOV-3 exosomes were pre-mixed with either LXY30-Fl or scr-LXY30-Fl overnight. After purification from unbound ligand by centrifugal filtration, exosomes were mixed with the Latex beads (Figure 2b).…”

“…This generated the highly potent cyclic disulfide-containing peptide: LXY30 [cdG-Phe(3,5-diF)-G-Hyp-NcR]. [34] LXY30 showed increased in vitro and in vivo tumor targeting across a panel of ovarian, breast, brain, and non-small cell lung carcinoma (NSCLC) cells. Furthermore, the binding specificity of LXY30 to the α 3 integrin subunit was confirmed, since binding to α 3 -expressing U-87MG cells could be inhibited by pre-incubation of the cells with anti-α 3 antibody.…”

“…Furthermore, the binding specificity of LXY30 to the α 3 integrin subunit was confirmed, since binding to α 3 -expressing U-87MG cells could be inhibited by pre-incubation of the cells with anti-α 3 antibody. [34] Finally, a scrambled version of LXY30 (scr-LXY30: [cGd-Hyp-Phe(3,5-diF)-G-NcR]), featuring switched positions of some amino acids in the peptide sequence, did not significantly bind to α 3 β 1 integrin-expressing cells, demonstrating the sequence specificity of LXY30. [34] …”

“…[34] Finally, a scrambled version of LXY30 (scr-LXY30: [cGd-Hyp-Phe(3,5-diF)-G-NcR]), featuring switched positions of some amino acids in the peptide sequence, did not significantly bind to α 3 β 1 integrin-expressing cells, demonstrating the sequence specificity of LXY30. [34] …”

Dominant Suppression of β1 Integrin by Ectopic CD98-ICD Inhibits Hepatocellular Carcinoma Progression

“…Recently, a high-affinity and high-specificity peptide ligand, LXY30, was used for in vivo targeting of α3-integrin-expressing human tumors [35]. Previously, a tripeptidic RGD integrin-recognition motif was able to block tumor invasion and angiogenesis [36,37].…”