Synthesis and characterization of a high-affinity v 6-specific ligand for in vitro and in vivo applications

Division of Translational Research, Department of Internal Medicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9185, USA.
Molecular Cancer Therapeutics (Impact Factor: 5.68). 06/2009; 8(5):1239-49. DOI: 10.1158/1535-7163.MCT-08-1098
Source: PubMed


The α(v)β(6) integrin is an attractive therapeutic target for several cancers due to its role in metastasis and its negligible expression in normal tissues. We previously identified a peptide from a phage-displayed peptide library that binds specifically to α(v)β(6). The tetrameric version of the peptide has higher affinity for its cellular targets than the corresponding monomers. However, the inefficient synthesis limits its clinical potential. We report here a convergent synthesis producing the tetrameric peptide in high yield and purity. The ease of the synthesis allows for rapid optimization of the peptide. We have optimized this α(v)β(6) integrin-binding peptide, determining the minimal binding domain and valency. Importantly, the half-maximal binding affinity of the optimal peptide for its target cell is in the 40 to 60 pmol/L range, rivaling the affinity of commonly used antibody-targeting reagents. This peptide mediates cell-specific uptake, is functional in diagnostic formats, is stable in sera, and can home to a tumor in an animal. We anticipate that this high-affinity ligand for α(v)β(6) will find clinical use as a diagnostic and therapeutic reagent.

  • Source
    • "Deleting this region removes the cysteine residue which must remain protected to avoid intra- and inter-branch aggregation. Similarly, a fully functional 10-mer form of H2009.1 peptide was employed16. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.
    Full-text · Article · Mar 2014 · Scientific Reports
  • Source
    • "Similar effects have been observed with other targeted nanoparticles in which the targeting ligand does not affect overall tumor accumulation but increases cellular uptake compared to the nontargeted counterpart [60]. We previously demonstrated that the H2009.1 peptide triggers αvβ6-mediated cellular uptake [54] and, that H2009.1 modified liposomes are internalized into αvβ6-expressing cells in vitro [39]. While the H2009.1 peptide mediates rapid cellular uptake, unfortunately a similar increase in therapeutic efficacy for H2009.1-liposomes is not observed. "
    [Show abstract] [Hide abstract]
    ABSTRACT: One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for αvβ6-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to αvβ6. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.
    Full-text · Article · Aug 2013 · PLoS ONE
  • Source
    • "The H2009.1 peptide was selected by biopanning a phage displayed-peptide library on intact H2009 NSCLC cells. Subsequently, we determined that the H2009.1 peptide binds with high binding affinity and selectivity to αvβ6+ 8, 20. Not surprisingly, the selected peptide sequence contains an Arginine-Glycine-Aspartate (RGD) tripeptide sequence, a well-known ligand for many integrins 41, 42. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The integrin αvβ6 is an emerging biomarker for non-small cell lung cancer (NSCLC). An αvβ6- binding peptide was previously selected from a phage-displayed peptide library. Here, we utilize a multivalent design to develop a peptidic probe for positron emission tomography (PET) imaging of αvβ6+NSCLC tumors.Multimeric presentation of this peptide, RGDLATLRQL, on a bifunctional copper chelator was achieved using two approaches: dimerization of the peptide followed by conjugation to the chelator (H2-D10) and direct presentation of two copies of the peptide on the chelator scaffold (H2-(M10)2). Binding affinities of the divalent peptide conjugates are four-fold higher than their monovalent counterpart (H2-M10), suggestive of multivalent binding. PET imaging using the bivalent 64Cu-labeled conjugates showed rapid and persistent accumulation in αvβ6+ tumors. By contrast, no significant accumulation was observed in αvβ6- tumors. Irrespective of the dimerization approach, all divalent probes showed three-fold higher tumor uptake than the monovalent probe, indicating the role of valency in signal enhancement. However, the divalent probes have elevated uptake in non-target organs, especially the kidneys. To abrogate nonspecific uptake, the peptide’s N-terminus was acetylated. The resultant bivalent probe, 64Cu- AcD10, showed drastic decrease of kidney accumulation while maintaining tumor uptake. In conclusion, we developed an αvβ6-integrin specific probe with optimized biodistribution for noninvasive PET imaging of NSCLC. Further, we have demonstrated that use of multivalent scaffolds is a plausible method to improve library selected peptides, which would be suboptimal or useless otherwise, for imaging probe development.
    Full-text · Article · Jan 2013 · Theranostics
Show more