The animals used in the present were 3–4-month-old A/Sn, C57Bl/6, CBA, C3H strain mice (22–25 g) bred at the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences (SB RAS), Novosibirsk, Russia. The mice were kept under standard conditions and received standard food and water according to the approved study protocol (EEC directive 86/609/EEC). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Administration of SB RAS (permit no. 8–2012), Novosibirsk, Russia. All the animal experiments were performed in compliance with the recommendations and requirements of the World Society for the Protection of Animals, and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986).

The hepatocarcinoma-1 (HA-1), hepatocarcinoma-29 (HA-29), lung adenocarcinoma (LA), Krebs-2 carcinoma, Lewis lung carcinoma (LLC) and Ehrlich carcinoma strains were obtained from the Institute of Cytology and Genetics, SB RAS (Novosibirsk, Russia) and maintained in the ascitic form by weekly intraperitoneal transplantations using the following mouse strains: A/Sn (HA-1, LA), C57 Black (Krebs-2 carcinoma), CBA (LLC, HA-29) and C3H (Ehrlich carcinoma).

For in vivo experiments, ascites tumor cells were resuspended in saline to a final concentration of 10⁶ cells/ml and 150 µl of suspension was injected subcutaneously into the back of each mouse. After the tumor nodes grew to 0.7–0.9 cm in diameter, the animals were used in the study experiments.

The HA-1 cells were cultured in vitro in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in the presence of 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM L-glutamine (Sigma-Aldrich; Merck Millipore), 250 mg/ml amphotericin В and 100 U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°С in an atmosphere containing 5% СО₂.

The В16 melanoma, human breast adenocarcinoma MDA-MB-231 and MCF-7 cell lines were obtained from the Russian Collection of Cell Cultures (Institute of Cytology, RAS, Saint Petersburg, Russia). В16 melanoma cells were cultured in vitro in DMEM (Sigma-Aldrich; Merck Millipore) in the presence of 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine (Sigma-Aldrich; Merck Millipore), 250 mg/ml amphotericin В and 100 U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°С in an atmosphere containing 5% СО_2. For the in vivo experiments, В16 melanoma cells were detached with 0.05% trypsin-EDTA, diluted to a final concentration of 10⁶ cells/ml, and 150 µl of the suspension was injected subcutaneously into the back of each mouse. After the tumor nodes grew to 0.5–0.6 cm in diameter, the animals were used in the study experiments.

MCF-7 cells were cultured in Iscove's modified Dulbecco's medium (Sigma-Aldrich; Merck Millipore) in the presence of 10% FBS, 2 mM L-glutamine, 250 mg/ml amphotericin В and 100 U/ml penicillin/streptomycin.

MDA-MB-231 cells were cultured in L15 in the presence of 10% FBS, 2 mM L-glutamine, 250 mg/ml amphotericin В and 100 U/ml penicillin/streptomycin at 37°С in an atmosphere containing 5% СО₂.

A mouse with a subcutaneously transplanted HA-1 tumor was administered, via tail vein injection, 0.5 ml of the phage-display peptide library (2×10⁹ PFU/ml; New England Biolabs, Inc., Ipswich, MA, USA) diluted in saline. The phage particles were allowed to circulate for 5 min, after which the mouse was sedated with 2.5% Avertin solution at a dose of 0.2 ml per 20 g of mouse body weight and the heart was perfused with 15 ml saline to remove the unbound phage particles from the bloodstream. The tumor was removed, washed with phosphate-buffered saline (PBS) and homogenized in 1 ml PBS containing 1 mM of the protease inhibitor phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 10,000 × g for 10 min at room temperature and then the supernatant was removed. Pellets were resuspended in 1 ml blocking buffer and centrifuged at 10,000 × g for 10 min at room temperature and then the supernatant was removed. Pellets were resuspended in 1 ml liquid E. сoli ER2738 culture in lysogeny broth (optical density at 600 nm, 0.2–0.3) to elute the bacteriophages that had bound to the tumor, and were incubated for 30 min at 37°С. The eluate containing the phage particles was centrifuged at 10,000 × g for 10 min at room temperature. The suspension containing the phage particles was then amplified and the amplified phage particles were used in further rounds of selection.

After the third and fourth rounds of selection, the phage particles were titrated to obtain individual phage colonies, which were used for the isolation of DNA according to the protocol described by the manufacturer of the phage-display peptide library.

The single-stranded DNAs were used in Sanger sequencing (−96 gIII sequencing primer; 5′-CCCTCATAGTTAGCGTAACG-3′; New England Biolabs, Inc.). The sequencing reaction products were determined using an ABI 310 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.) (located at the Genomics Core Facility of SB RA, Novosibirsk, Russia). The nucleotide sequences of the inserts encoding the peptides were analyzed using MEGA 4.0 software (10).

The RL2 sequence was amplified from plasmid pGSDI/RL2 (4) using RL2_F and c1_RL2_R primers (Table I) with the following cycling conditions: 96°C for 5 min, followed by 35 cycles of 96°C for 30 sec, 66°C for 30 sec, 72°C for 30 sec and a final step of 72°C for 10 min. At the first stage, to produce a fusion DNA sequence that encodes a selected peptide and RL2, the sequences that encode the peptides Т1 and Т2 and the region of overlap with the RL2 sequence were produced. The Т1 sequence was amplified from ClonN1_target as a template using c1_RL2_F and c1_R primers with the following cycling conditions: 96°C for 5 min, followed by 35 cycles of 96°C for 30 sec, 65°C for 30 sec, 72°C for 15 sec and a final step of 72°C for 10 min. The T2 sequence was amplified from ClonN2_target as a template using c2_RL2_F and c2_R primers with the following cycling conditions: 96°C for 5 min, followed by 35 cycles of 96°C for 30 sec, 65°C for 30 sec, 72°C for 15 sec and a final step of 72°C for 10 min. At the second stage, the fusion T1_RL2 and T2_RL2 DNA sequences encoding the tumor-targeting peptide and RL2 were produced by polymerase chain reaction using the primer pairs c1_RL2_F, c1_RL2_R and c2_RL2_F, c1_RL2_R with the following cycling conditions: 96°C for 5 min, followed by 35 cycles of 96°C for 30 sec, 65°C for 35 sec, 72°C for 35 sec and a final step of 72°C for 10 min. The amplicons produced at the first stage and the RL2 amplicon were used as templates. All the amplicons were separated on an agarose gel and extracted from the gel according to the manufacturer's instructions (Qiagen, Inc., Valencia, CA, USA).

DNA fragments encoding the fusion proteins T1_RL2 and T2_RL2 were inserted in plasmid pET-15b (Novagen; Merck Millipore) using the BamHI and NcoI restriction sites. The recombinant plasmids were cloned in E. coli TOP10 (Life Technologies; Thermo Fisher Scientific, Inc.).

The sequencing reaction was run using a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the reaction products were purified using a DyeEx Spin kit (Qiagen, Inc.) at the Genomics Core Facility of SB RAS. For the sequencing reaction, Т7seq_F 5′-TAATACGACTCACTATAGGG-3′ and Т7seq_R 5′-GCTAGTTATTGCTCAGCGGT-3′ were used as primers. The purified products were sequenced using an ABI 310 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). The nucleotide sequences of interest were analyzed using MEGA 4.0 software.

The recombinant plasmids pET-15b_Т1_RL and pET-15b_Т2_RL, which encode the fusion proteins T1_RL and T2_RL, respectively, were expressed in E. coli BL21 (DE3) (New England Biolabs, Inc., Ipswich, MA, USA).

The cytotoxic effects of the recombinant lactaptin analogs RL2, T1_RL and T2_RL on mouse and human tumor cells were investigated using the МТТ assay (Sigma-Aldrich; Merck Millipore) according to a protocol described previously (6). The cells that had reached 30% confluence in a 96-well plate were incubated for 48 h with protein preparations at various concentrations (0.1–0.4 mg/ml). After incubation, the supernatant was removed and 200 µl MTT solution in RPMI 1640 medium (0.5 mg/ml) was added to each well and incubated for 4 h at 37°С. The formazan crystals were dissolved in 150 µl dimethyl sulfoxide. The optical density of the formazan solutions was measured using an Apollo LB912 photometer (Berthold Technologies, Oak Ridge, TN, USA) at a wavelength of 570 nm. Cell viability was determined relative to the viability of the control cells (100%) ± standard deviation in three independent experiments.

The cells were incubated in 6-well culture plates and used in the experiments upon reaching 80–90% confluence. The recombinant lactaptin analogs RL2, Т1_RL and T2_RL (0.6 mg/ml) were added to culture medium and incubated for 18 and 24 h. After incubation, the cells detached from the support were pooled with the cells detached by trypsinization (PBS, 0.5 mg/ml trypsin and 0.4 mg/ml EDTA). The pooled cells were precipitated by centrifugation at 400 × g for 5 min and washed with PBS. The exposure of phosphatidylserine (PS) at the outer surface of the cell membrane, and the activation of caspase-3 and −7 were analyzed by flow cytofluorometry with fluorescein isothiocyanate Annexin V Apoptosis Detection kit I (BD Pharmingen, San Jose, CA, USA) and the Vybrant FAM Caspase-3 and −7 assay kit (Life Technologies; Thermo Fisher Scientific, Inc.) using a FACSCanto II cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA).

The recombinant lactaptin analogs RL2, Т1_RL and Т2_RL were isolated from E. coli and purified according to a protocol for the isolation of RL2 (5).

To label RL2, T1_RL and T2_RL with sulfo-Cy5, 0.2 mM of each protein was incubated with 1.25 mM of the fluorescent dye sulfo-Cy5 maleimide (Ex/Em=646/662; Lumiprobe GmbH, Hannover, Germany) in Tris-HCl buffer (pH 5.5) for 4 h at 25°С. The purification of the conjugates RL2-Cy5, T1_RL-Cy5 and T2_RL-Cy5 was performed by reversed-phase high-performance liquid chromatography on a С18 column using a Milichrom A-02 chromatograph (EcoNova, Novosibirsk, Russia) with acetonitrile-water as a mobile phase.

Mice with subcutaneously transplanted HA-1 tumors were administered with 80 µg of the conjugates RL2-Cy5, T1_RL-Cy5 and T2_RL-Cy5 in 200 µl of PBS via tail vein injection. The conjugates were allowed to circulate for 10 min, after which the tumors were removed and analyzed using a Kodak In Vivo FX Professional Imaging system. The excitation (650 nm) and emission (700 nm) filters were used to detect the fluorescence of Cy5. The fluorescence intensity was recorded as total photons per second per square centimeter (mean ± standard deviation).

The data from the mouse experiments were statistically processed using one-way analysis of variance. Post-hoc testing was performed using Fisher's least significant differences (LSD). P<0.05 was considered to indicate a statistically significant difference. STATISTICA v10.0 was used to perform the statistical analyses (StatSoft, Inc., Tulsa, OK, USA).

To isolate tumor-specific peptides, four rounds of selection were performed in vivo on A/Sn strain mice with subcutaneously transplanted HA-1 tumors. After the third round of selection, 37 displayed sequences were identified and analyzed, and after the fourth round, 38 sequences were identified (Table II).

After the third round of in vivo selection on the HA-1 tumor model, the most frequent outputs were two displayed peptides, GLHTSATNLYLH (35.1%) and SGVYKVAYDWQH (21.6%). After the fourth round of in vivo selection, the frequency of the peptide GLHTSATNLYLH increased to 63.2%, while that of the peptide SGVYKVAYDWQH remained unchanged.

As the displayed peptides GLHTSATNLYLH and SGVYKVAYDWQH occurred with the highest frequency in the in vivo experiments, those clones were used in further experiments.

The ability of the displayed peptides GLHTSATNLYLH and SGVYKVAYDWQH to specifically bind to tumors in vivo was studied using HA-1, LA, В16 melanoma, Krebs-2 carcinoma, LLC, Ehrlich carcinoma and HA-29 tumors.

The individual phage clones displaying the selected peptides were administered to mice via tail vein injection. At 24 h post-administration, the tumor and the control organs (lung and liver) were examined for bacteriophages (PFU/mg tissue) by titrating on E. сoli ER2738 culture cells (Fig. 1).

As shown from the data presented in Fig. 1, the selected peptides were significantly bound not only to the selection-specific tumor (HA-1), but also to other tumors (LA, В16 melanoma, Krebs-2 carcinoma and LLC) (P<0.05). The highest amount of binding was for HA-1. At 24 h post-intravenous administration, the concentration of the control bacteriophage (without any displayed peptide) in the tumor remained relatively low (just two or three times the value in the liver and lung). This is consistent with literature data, according to which the wild-type bacteriophage has a low capacity for retention and accumulation in tumor tissue, as the vascular system in tumors is poorly organized (11).

Two genetic constructs, pET-15b_T1_RL and pET-15b_T2_RL, were made for the production of recombinant fusion proteins composed of RL2 and the selected tumor-targeting peptides that deliver RL2.

The sequence encoding the fusion protein was constructed so that the synthesis product appears as a polypeptide with the tumor-targeting peptide fused to the N-terminus (with methionine as the first amino acid) and RL2 fused to the C-terminus. The targeting peptide and RL2 are spaced by a glycine linker, which provides for the flexibility of the peptide (Fig. 2).

To produce the desired recombinant proteins, two E. сoli expression systems, BL21 (DE3)/pET-15b_Т1_RL and BL21 (DE3)/pET-15b_Т2_RL, were developed.

A comparative analysis of the effects of RL2 and the recombinant fusion proteins T1_RL and T2_RL on tumor cell viability was performed using HA-1, MDA-MB-231 and MCF-7 cell cultures. Changes in cell viability were assessed using the МТТ assay.

It was demonstrated that T1_RL and T2_RL reduce the viability of HA-1, MDA-MB-231 and MCF-7 cells in a dose-dependent manner, while the cytotoxic activity of the fusion proteins was practically the same as that of RL2 (Fig. 3).

As is known, no wild-type bacteriophage can infect eukaryotic cells or produce a cytotoxic effect on them (11). Additionally, the present study demonstrated that the incubation of MDA-MB-231 and MCF-7 tumor cells with the bacteriophages that display the peptides GLHTSATNLYLH and SGVYKVAYDWQH has no effect on cell viability (data not shown).

Data obtained strongly suggest that the cytotoxic activity of the fusion proteins is solely due to RL2, while the tumor-targeting peptide fused to the N-terminus of RL2 has no effect on its cytotoxic properties.

The cytotoxic activity of RL2 in relation to tumor cells is due to its ability to induce apoptosis in these cells (6,7). The current study assessed the ability of the fusion proteins T1_RL and T2_RL to induce apoptosis in MDA-MB-231 cells by cytometry. After the incubation of MDA-MB-231 cells with RL2 and T2_RL for 18 h, the population of apoptotic MDA-MB-231 cells increased to 27.9 and 35.6%, respectively, compared with the population of control cells (16.1%). The incubation of MDA-MB-231 cells with T1_RL for 18 h led to no change in the number of apoptotic cells as compared to that in the control cells (Fig. 4); however, incubation with T1_RL for a longer time, 24 h, led to an increase in the number of apoptotic cells (data not shown).

A key role in apoptosis is played by specific proteins, the caspases (cysteine proteases) (12). As is known, RL2-induced apoptosis is accompanied by the activation of effector caspase-3 and −7, and mitochondrial transmembrane potential dissipation (7,13). Activation of caspase-3 and −7 in MDA-MB-231 cells by the recombinant proteins RL2, T1_RL and T2_RL was analyzed by cytofluorometry in the present study. As shown in the data presented in Fig. 4B, following incubation with RL2, the percentage of cells expressing active forms of caspase-3 and −7 increased to 19.7, to 24.8% after incubation with T1_RL and to 18.3% after incubation with Т2_RL, while the increase in the control cells was only 7.7%.

The accumulation of the recombinant fusion proteins in tumor tissue was assessed using the mouse HA-1 tumor model. Mice with subcutaneously transplanted tumors were administered with the conjugates RL2-Cy5, T1_RL-Cy5 and T2_RL-Cy5 via tail vein injection. The Cy5-labeled conjugates were allowed to circulate for 10 min, after which the tumors were removed. The intensity of the fluorescent signal from the tumors was measured using a Kodak In Vivo FX Professional Imaging system (Fig. 5).

As observed in Fig. 5, subsequent to circulation of the recombinant proteins in the bloodstream for 10 min, the strongest fluorescent signal from the tumor was for T1_RL; its intensity was 5.08±1.15 times as high as that of the signal from the tumor with RL2 (P<0.05). The intensity of the fluorescent signal from the tumor with T2_RL was only 2.81±1.48 (P<0.05) times as high as that the signal from the tumor with RL2.