Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP in vitro. A, cleavage of 5 μm Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 mm Ca-DTPA, 50 μm TPEN, 18 μm SB3CT (43), 50 nm GM6001 (also tested at 1 μm and no inhibition; data not shown), 0.26 μm prinomastat, 150 nm aprotinin, 10 μm E-64, 100 μm leupeptin, 1 μm pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity. B, cleavage of RLQLKL ACPP for 2 h with 50 nm MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen (supplemental Fig. 4). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in supplemental Fig. 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2903386&req=5

Figure 3: Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP in vitro. A, cleavage of 5 μm Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 mm Ca-DTPA, 50 μm TPEN, 18 μm SB3CT (43), 50 nm GM6001 (also tested at 1 μm and no inhibition; data not shown), 0.26 μm prinomastat, 150 nm aprotinin, 10 μm E-64, 100 μm leupeptin, 1 μm pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity. B, cleavage of RLQLKL ACPP for 2 h with 50 nm MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen (supplemental Fig. 4). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in supplemental Fig. 4.

Mentions: To characterize the protease activity responsible for tumor uptake, the effect of various protease inhibitors on tissue extract mediated cleavage of RLQLKL ACPP was tested. Unfortunately, cleavage of RLQLKL ACPP by crude tissue extracts is quite resistant to inhibition by protease inhibitors, which may be due to high protease activity in tissue extracts or the multiplicity of proteases released upon tissue disruption. We therefore switched to cystic fluid obtained from fluid-filled nodules of PyMT tumors because this fluid probably contained proteases that had been secreted by adjacent tumor tissue and was less likely than tumor extracts to be contaminated with intracellular proteases. The use of similar tumor cystic fluids (although obtained from pancreatic and not mammary tumors) for cancer protease profiling has been reported elsewhere (28, 29). Cystic fluid cleaved RLQLKL ACPP similarly to tumor tissue extracts, and this cleavage was partially inhibited by two serine protease inhibitors, aprotinin and leupeptin (Fig. 3A). This inhibitor profile supported the involvement of a serine protease in the in vivo cleavage and tumor uptake of RLQLKL ACPP.


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)

Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP in vitro. A, cleavage of 5 μm Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 mm Ca-DTPA, 50 μm TPEN, 18 μm SB3CT (43), 50 nm GM6001 (also tested at 1 μm and no inhibition; data not shown), 0.26 μm prinomastat, 150 nm aprotinin, 10 μm E-64, 100 μm leupeptin, 1 μm pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity. B, cleavage of RLQLKL ACPP for 2 h with 50 nm MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen (supplemental Fig. 4). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in supplemental Fig. 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effects of enzymes and inhibitors on cleavage of the RLQLKL ACPP in vitro. A, cleavage of 5 μm Cy5-labeled RLQLKL ACPP for 2 h with 2% cystic fluid obtained from PyMT tumors, in the absence of inhibitor or with 5 mm Ca-DTPA, 50 μm TPEN, 18 μm SB3CT (43), 50 nm GM6001 (also tested at 1 μm and no inhibition; data not shown), 0.26 μm prinomastat, 150 nm aprotinin, 10 μm E-64, 100 μm leupeptin, 1 μm pepstatin A, Calbiochem mixture III (diluted 1:1000), mixture III + DTPA. All inhibitors were used at the manufacturers' recommended concentrations, which should inhibit ≥95% of the target enzymes' activity. B, cleavage of RLQLKL ACPP for 2 h with 50 nm MMP-1, MMP-2, MMP-7, MMP-9, MMP-14, kallikrein 5, thrombin, plasmin, urokinase plasminogen activator, tissue plasminogen activator, factor VIIa, factor IXa, factor Xa, factor XIa, factor XIIa, trypsin (cathepsins B, G, L, and H), and neutrophil elastase. The percentage of cleavage measured with UVP software was 93.0 ± 2.9% for trypsin, 79.0 ± 2.8% for neutrophil elastase, 77.9 ± 2.1% for plasmin, 60% ± 2.3% for cathepsin G, 23.4 ± 2.5% for MMP7, 9.7 ± 1.1% for MMP1, and less than 3% for all other enzymes tested, including hepsin, enterokinase, and prostate-specific antigen (supplemental Fig. 4). MMP-8, MMP-13, matriptase, urokinase, and legumain also showed no cleavage (data not shown). Trypsin and trypsin-2 cleavage of various ACPP are also shown in supplemental Fig. 4.
Mentions: To characterize the protease activity responsible for tumor uptake, the effect of various protease inhibitors on tissue extract mediated cleavage of RLQLKL ACPP was tested. Unfortunately, cleavage of RLQLKL ACPP by crude tissue extracts is quite resistant to inhibition by protease inhibitors, which may be due to high protease activity in tissue extracts or the multiplicity of proteases released upon tissue disruption. We therefore switched to cystic fluid obtained from fluid-filled nodules of PyMT tumors because this fluid probably contained proteases that had been secreted by adjacent tumor tissue and was less likely than tumor extracts to be contaminated with intracellular proteases. The use of similar tumor cystic fluids (although obtained from pancreatic and not mammary tumors) for cancer protease profiling has been reported elsewhere (28, 29). Cystic fluid cleaved RLQLKL ACPP similarly to tumor tissue extracts, and this cleavage was partially inhibited by two serine protease inhibitors, aprotinin and leupeptin (Fig. 3A). This inhibitor profile supported the involvement of a serine protease in the in vivo cleavage and tumor uptake of RLQLKL ACPP.

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