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Parallel in vivo and in vitro selection using phage display identifies protease-dependent tumor-targeting peptides.

Whitney M, Crisp JL, Olson ES, Aguilera TA, Gross LA, Ellies LG, Tsien RY - J. Biol. Chem. (2010)

Bottom Line: Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts.The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast.The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

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A, in vivo characterization of tumor uptake on mice injected with Cy5-labeled RLQLK(Ac)L ACPP and albumin-reactive Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 ACPP compared with parent RLQLKL ACPP. PyMT tumor-bearing mice were imaged 6 h after injection with 10 nmol of either H2N-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or H2N-e9-(ahx)-RLQLK(Ac)L-r9-c-(Cy5)-NH2 PyMT tumor-bearing mouse imaged 48 h after injection with 3 nmol of Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 (right) (complete structures diagramed in supplemental Fig. 1). Skin-off images are shown for free peptide (left), and skin-off/skin-on images are shown for albumin-reactive peptide (right). Tissues were removed post mortem for SUV determination. B, detection of PyMT lung metastases and seeded lung tumors with various ACPPs. PyMT tumor mice were injected with free peptide RLQLKL ACPP. Lungs were removed and imaged 6 h postinjection. Lung metastases were seen as distinct fluorescent spots compared with adjacent tissue, within the lungs (top). Fluorescent spots were verified to be metastasis by hematoxylin/eosin staining (supplemental Fig. 4). Lungs from animals injected with uncleavable control rlqlkl ACPP show much less distinct fluorescent spots (bottom). Metastases were confirmed to be of similar size. C, lungs from mice that had been generated by seeding 4T1 tumor cells into lungs 10 days prior to injection of contrast agent. Mice were injected with either Mal-RLQLK(Ac)L ACPP (top row) or an uncleavable Mal-mpeg-ACPP (bottom row) and imaged. Lungs were then sectioned, and serial sections were either imaged for Cy5 fluorescence or used for hematoxylin/eosin staining. Bright fluorescence in the top center was from a single metastatic nodule.
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Figure 5: A, in vivo characterization of tumor uptake on mice injected with Cy5-labeled RLQLK(Ac)L ACPP and albumin-reactive Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 ACPP compared with parent RLQLKL ACPP. PyMT tumor-bearing mice were imaged 6 h after injection with 10 nmol of either H2N-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or H2N-e9-(ahx)-RLQLK(Ac)L-r9-c-(Cy5)-NH2 PyMT tumor-bearing mouse imaged 48 h after injection with 3 nmol of Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 (right) (complete structures diagramed in supplemental Fig. 1). Skin-off images are shown for free peptide (left), and skin-off/skin-on images are shown for albumin-reactive peptide (right). Tissues were removed post mortem for SUV determination. B, detection of PyMT lung metastases and seeded lung tumors with various ACPPs. PyMT tumor mice were injected with free peptide RLQLKL ACPP. Lungs were removed and imaged 6 h postinjection. Lung metastases were seen as distinct fluorescent spots compared with adjacent tissue, within the lungs (top). Fluorescent spots were verified to be metastasis by hematoxylin/eosin staining (supplemental Fig. 4). Lungs from animals injected with uncleavable control rlqlkl ACPP show much less distinct fluorescent spots (bottom). Metastases were confirmed to be of similar size. C, lungs from mice that had been generated by seeding 4T1 tumor cells into lungs 10 days prior to injection of contrast agent. Mice were injected with either Mal-RLQLK(Ac)L ACPP (top row) or an uncleavable Mal-mpeg-ACPP (bottom row) and imaged. Lungs were then sectioned, and serial sections were either imaged for Cy5 fluorescence or used for hematoxylin/eosin staining. Bright fluorescence in the top center was from a single metastatic nodule.

Mentions: To test in vivo uptake, Cy5-labeled RLQLK(Ac)L ACPP was injected into PyMT tumor-bearing mice. This optimized ACPP was taken up in tumors to levels equal to or slightly greater than the unmodified parent RLQLKL ACPP (SUV = 0.39 ± 0.14 (n = 8) for RLQLKL and 0.48 ± 0.04 (n = 3) for RLQLK(Ac)L, p = 0.33), supporting the hypothesis that elastase but not plasmin could be responsible for tumor uptake in the PyMT model (Fig. 5A, top left). In addition to improved specificity, acetylation of the RLQLKL ACPP simplifies attachment of dyes or other functional groups to the N-terminal amine of the peptide by blocking cross-reactivity to the lysine within the protease cleavage site.


Parallel in vivo and in vitro selection using phage display identifies protease-dependent tumor-targeting peptides.

Whitney M, Crisp JL, Olson ES, Aguilera TA, Gross LA, Ellies LG, Tsien RY - J. Biol. Chem. (2010)

A, in vivo characterization of tumor uptake on mice injected with Cy5-labeled RLQLK(Ac)L ACPP and albumin-reactive Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 ACPP compared with parent RLQLKL ACPP. PyMT tumor-bearing mice were imaged 6 h after injection with 10 nmol of either H2N-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or H2N-e9-(ahx)-RLQLK(Ac)L-r9-c-(Cy5)-NH2 PyMT tumor-bearing mouse imaged 48 h after injection with 3 nmol of Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 (right) (complete structures diagramed in supplemental Fig. 1). Skin-off images are shown for free peptide (left), and skin-off/skin-on images are shown for albumin-reactive peptide (right). Tissues were removed post mortem for SUV determination. B, detection of PyMT lung metastases and seeded lung tumors with various ACPPs. PyMT tumor mice were injected with free peptide RLQLKL ACPP. Lungs were removed and imaged 6 h postinjection. Lung metastases were seen as distinct fluorescent spots compared with adjacent tissue, within the lungs (top). Fluorescent spots were verified to be metastasis by hematoxylin/eosin staining (supplemental Fig. 4). Lungs from animals injected with uncleavable control rlqlkl ACPP show much less distinct fluorescent spots (bottom). Metastases were confirmed to be of similar size. C, lungs from mice that had been generated by seeding 4T1 tumor cells into lungs 10 days prior to injection of contrast agent. Mice were injected with either Mal-RLQLK(Ac)L ACPP (top row) or an uncleavable Mal-mpeg-ACPP (bottom row) and imaged. Lungs were then sectioned, and serial sections were either imaged for Cy5 fluorescence or used for hematoxylin/eosin staining. Bright fluorescence in the top center was from a single metastatic nodule.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2903386&req=5

Figure 5: A, in vivo characterization of tumor uptake on mice injected with Cy5-labeled RLQLK(Ac)L ACPP and albumin-reactive Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 ACPP compared with parent RLQLKL ACPP. PyMT tumor-bearing mice were imaged 6 h after injection with 10 nmol of either H2N-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or H2N-e9-(ahx)-RLQLK(Ac)L-r9-c-(Cy5)-NH2 PyMT tumor-bearing mouse imaged 48 h after injection with 3 nmol of Mal-e9-(ahx)-RLQLK(Ac)L-r9-c(Cy5)-NH2 (right) (complete structures diagramed in supplemental Fig. 1). Skin-off images are shown for free peptide (left), and skin-off/skin-on images are shown for albumin-reactive peptide (right). Tissues were removed post mortem for SUV determination. B, detection of PyMT lung metastases and seeded lung tumors with various ACPPs. PyMT tumor mice were injected with free peptide RLQLKL ACPP. Lungs were removed and imaged 6 h postinjection. Lung metastases were seen as distinct fluorescent spots compared with adjacent tissue, within the lungs (top). Fluorescent spots were verified to be metastasis by hematoxylin/eosin staining (supplemental Fig. 4). Lungs from animals injected with uncleavable control rlqlkl ACPP show much less distinct fluorescent spots (bottom). Metastases were confirmed to be of similar size. C, lungs from mice that had been generated by seeding 4T1 tumor cells into lungs 10 days prior to injection of contrast agent. Mice were injected with either Mal-RLQLK(Ac)L ACPP (top row) or an uncleavable Mal-mpeg-ACPP (bottom row) and imaged. Lungs were then sectioned, and serial sections were either imaged for Cy5 fluorescence or used for hematoxylin/eosin staining. Bright fluorescence in the top center was from a single metastatic nodule.
Mentions: To test in vivo uptake, Cy5-labeled RLQLK(Ac)L ACPP was injected into PyMT tumor-bearing mice. This optimized ACPP was taken up in tumors to levels equal to or slightly greater than the unmodified parent RLQLKL ACPP (SUV = 0.39 ± 0.14 (n = 8) for RLQLKL and 0.48 ± 0.04 (n = 3) for RLQLK(Ac)L, p = 0.33), supporting the hypothesis that elastase but not plasmin could be responsible for tumor uptake in the PyMT model (Fig. 5A, top left). In addition to improved specificity, acetylation of the RLQLKL ACPP simplifies attachment of dyes or other functional groups to the N-terminal amine of the peptide by blocking cross-reactivity to the lysine within the protease cleavage site.

Bottom Line: Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts.The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast.The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

Show MeSH
Related in: MedlinePlus