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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 vitro characterization of five Cy5-labeled ACPPs containing RTRYED, GMMYRS, RWRTNF, RLQLKL, and rlqlkl at the protease cleavage site. Uppercase and lowercase letters denote l- and d-amino acids, respectively. For each reaction, 5 μm ACPP was incubated with either 2% tumor or 2% liver/kidney tissue extracts at 37 °C for 2 h (compared with 16 h; Table 1). Cleavage was detected by electrophoresis on 16% Tricine acrylamide gels. RLQLKL, RTRYED, and RWRTNF ACPPs were more efficiently cleaved by tumor extract versus liver/kidney extracts (see ratio of cleaved to uncleaved). Of these, RWRTNF showed the greatest differential cleavage by tumor versus liver/kidney extracts. As expected, the all-d-rlqlkl peptide showed no cleavage under any of the tested conditions. B, in vivo characterization of tumor uptake on mice injected with Cy5-labeled ACPPs. PyMT tumor-bearing mice were imaged 6 h after injection with 100 μl of a 100 μm concentration of either the protease-cleavable ACPP NH2-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or the uncleavable all d-amino acid control peptide NH2-e9-(ahx)-rlqlkl-r9-c(Cy5)-NH2. Images with skin removed are shown because they highlight the variability of the labeling throughout the tumor. Skin-off images also eliminate artifacts that sometime appear due to incomplete shaving of the animal or nicks that occur during shaving. Skin-on images are shown for comparison in supplemental Fig. 2. Tissues were removed post mortem for SUV determination. C, nude mice with MDA-MB-435 M4A4 xenografts expressing GFP were injected with either RLQLKL ACPP (far left) or control rlqlkl peptide (middle right), 100 μl of 100 μm in each case. The RLQLKL ACPP gave visible contrast in tumors that were confirmed by imaging GFP fluorescence. Mice injected with rlqlkl peptide showed no tumor contrast in the Cy5 channel (middle right), although the tumor was clearly visible by GFP (far right). All images were taken 6 h after injection, in live animals with skin on, using identical settings on the Maestro imager. Images for rlqlkl peptides were brightened 3-fold to illustrate the lack of contrast for tumor compared with surrounding tissue for the uncleavable control.
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Figure 2: A, in vitro characterization of five Cy5-labeled ACPPs containing RTRYED, GMMYRS, RWRTNF, RLQLKL, and rlqlkl at the protease cleavage site. Uppercase and lowercase letters denote l- and d-amino acids, respectively. For each reaction, 5 μm ACPP was incubated with either 2% tumor or 2% liver/kidney tissue extracts at 37 °C for 2 h (compared with 16 h; Table 1). Cleavage was detected by electrophoresis on 16% Tricine acrylamide gels. RLQLKL, RTRYED, and RWRTNF ACPPs were more efficiently cleaved by tumor extract versus liver/kidney extracts (see ratio of cleaved to uncleaved). Of these, RWRTNF showed the greatest differential cleavage by tumor versus liver/kidney extracts. As expected, the all-d-rlqlkl peptide showed no cleavage under any of the tested conditions. B, in vivo characterization of tumor uptake on mice injected with Cy5-labeled ACPPs. PyMT tumor-bearing mice were imaged 6 h after injection with 100 μl of a 100 μm concentration of either the protease-cleavable ACPP NH2-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or the uncleavable all d-amino acid control peptide NH2-e9-(ahx)-rlqlkl-r9-c(Cy5)-NH2. Images with skin removed are shown because they highlight the variability of the labeling throughout the tumor. Skin-off images also eliminate artifacts that sometime appear due to incomplete shaving of the animal or nicks that occur during shaving. Skin-on images are shown for comparison in supplemental Fig. 2. Tissues were removed post mortem for SUV determination. C, nude mice with MDA-MB-435 M4A4 xenografts expressing GFP were injected with either RLQLKL ACPP (far left) or control rlqlkl peptide (middle right), 100 μl of 100 μm in each case. The RLQLKL ACPP gave visible contrast in tumors that were confirmed by imaging GFP fluorescence. Mice injected with rlqlkl peptide showed no tumor contrast in the Cy5 channel (middle right), although the tumor was clearly visible by GFP (far right). All images were taken 6 h after injection, in live animals with skin on, using identical settings on the Maestro imager. Images for rlqlkl peptides were brightened 3-fold to illustrate the lack of contrast for tumor compared with surrounding tissue for the uncleavable control.

Mentions: These top four peptides were resynthesized in the format H2N-e9-(ahx)-X6-r9-(Cy5-labeled d-cysteinamide), followed by purification to greater than 95% for in vitro and in vivo testing. Each of these peptides was tested for differential cleavage by tumor versus mixed liver/kidney tissue extracts after a 2-h digestion compared with the 16-h digestion shown in Table 1 (Fig. 2A). All four peptides were cleaved >50% by tumor tissue extract, with RLQLKL being the most rapidly cleaved, >90% after 4 h (full time course not shown). Each peptide except for GMMYRS showed increased cleavage by tumor extract relative to mixed liver/kidney extracts. A control peptide with all d-amino acids, X6 = rlqlkl, was not cleaved by any of the tissue extracts.


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 vitro characterization of five Cy5-labeled ACPPs containing RTRYED, GMMYRS, RWRTNF, RLQLKL, and rlqlkl at the protease cleavage site. Uppercase and lowercase letters denote l- and d-amino acids, respectively. For each reaction, 5 μm ACPP was incubated with either 2% tumor or 2% liver/kidney tissue extracts at 37 °C for 2 h (compared with 16 h; Table 1). Cleavage was detected by electrophoresis on 16% Tricine acrylamide gels. RLQLKL, RTRYED, and RWRTNF ACPPs were more efficiently cleaved by tumor extract versus liver/kidney extracts (see ratio of cleaved to uncleaved). Of these, RWRTNF showed the greatest differential cleavage by tumor versus liver/kidney extracts. As expected, the all-d-rlqlkl peptide showed no cleavage under any of the tested conditions. B, in vivo characterization of tumor uptake on mice injected with Cy5-labeled ACPPs. PyMT tumor-bearing mice were imaged 6 h after injection with 100 μl of a 100 μm concentration of either the protease-cleavable ACPP NH2-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or the uncleavable all d-amino acid control peptide NH2-e9-(ahx)-rlqlkl-r9-c(Cy5)-NH2. Images with skin removed are shown because they highlight the variability of the labeling throughout the tumor. Skin-off images also eliminate artifacts that sometime appear due to incomplete shaving of the animal or nicks that occur during shaving. Skin-on images are shown for comparison in supplemental Fig. 2. Tissues were removed post mortem for SUV determination. C, nude mice with MDA-MB-435 M4A4 xenografts expressing GFP were injected with either RLQLKL ACPP (far left) or control rlqlkl peptide (middle right), 100 μl of 100 μm in each case. The RLQLKL ACPP gave visible contrast in tumors that were confirmed by imaging GFP fluorescence. Mice injected with rlqlkl peptide showed no tumor contrast in the Cy5 channel (middle right), although the tumor was clearly visible by GFP (far right). All images were taken 6 h after injection, in live animals with skin on, using identical settings on the Maestro imager. Images for rlqlkl peptides were brightened 3-fold to illustrate the lack of contrast for tumor compared with surrounding tissue for the uncleavable control.
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Related In: Results  -  Collection

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Figure 2: A, in vitro characterization of five Cy5-labeled ACPPs containing RTRYED, GMMYRS, RWRTNF, RLQLKL, and rlqlkl at the protease cleavage site. Uppercase and lowercase letters denote l- and d-amino acids, respectively. For each reaction, 5 μm ACPP was incubated with either 2% tumor or 2% liver/kidney tissue extracts at 37 °C for 2 h (compared with 16 h; Table 1). Cleavage was detected by electrophoresis on 16% Tricine acrylamide gels. RLQLKL, RTRYED, and RWRTNF ACPPs were more efficiently cleaved by tumor extract versus liver/kidney extracts (see ratio of cleaved to uncleaved). Of these, RWRTNF showed the greatest differential cleavage by tumor versus liver/kidney extracts. As expected, the all-d-rlqlkl peptide showed no cleavage under any of the tested conditions. B, in vivo characterization of tumor uptake on mice injected with Cy5-labeled ACPPs. PyMT tumor-bearing mice were imaged 6 h after injection with 100 μl of a 100 μm concentration of either the protease-cleavable ACPP NH2-e9-(ahx)-RLQLKL-r9-c-(Cy5)-NH2 (left) or the uncleavable all d-amino acid control peptide NH2-e9-(ahx)-rlqlkl-r9-c(Cy5)-NH2. Images with skin removed are shown because they highlight the variability of the labeling throughout the tumor. Skin-off images also eliminate artifacts that sometime appear due to incomplete shaving of the animal or nicks that occur during shaving. Skin-on images are shown for comparison in supplemental Fig. 2. Tissues were removed post mortem for SUV determination. C, nude mice with MDA-MB-435 M4A4 xenografts expressing GFP were injected with either RLQLKL ACPP (far left) or control rlqlkl peptide (middle right), 100 μl of 100 μm in each case. The RLQLKL ACPP gave visible contrast in tumors that were confirmed by imaging GFP fluorescence. Mice injected with rlqlkl peptide showed no tumor contrast in the Cy5 channel (middle right), although the tumor was clearly visible by GFP (far right). All images were taken 6 h after injection, in live animals with skin on, using identical settings on the Maestro imager. Images for rlqlkl peptides were brightened 3-fold to illustrate the lack of contrast for tumor compared with surrounding tissue for the uncleavable control.
Mentions: These top four peptides were resynthesized in the format H2N-e9-(ahx)-X6-r9-(Cy5-labeled d-cysteinamide), followed by purification to greater than 95% for in vitro and in vivo testing. Each of these peptides was tested for differential cleavage by tumor versus mixed liver/kidney tissue extracts after a 2-h digestion compared with the 16-h digestion shown in Table 1 (Fig. 2A). All four peptides were cleaved >50% by tumor tissue extract, with RLQLKL being the most rapidly cleaved, >90% after 4 h (full time course not shown). Each peptide except for GMMYRS showed increased cleavage by tumor extract relative to mixed liver/kidney extracts. A control peptide with all d-amino acids, X6 = rlqlkl, was not cleaved by any of the tissue extracts.

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