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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 May 20;106(23):9221–9225. doi: 10.1073/pnas.0904140106

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

Ekaterina O Serebrovskaya 1, Eveline F Edelweiss 1, Oleg A Stremovskiy 1, Konstantin A Lukyanov 1, Dmitry M Chudakov 1, Sergey M Deyev 1,1
PMCID: PMC2695119  PMID: 19458251

Abstract

Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185HER-2-ECD-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.

Keywords: reactive oxygen species, phototoxicity, KillerRed

Photodynamic therapy (PDT) is a promising approach to cancer treatment because of the absence of systemic toxicity of the drug in the absence of light irradiation, the possibility to irradiate the tumor selectively, the opportunity of treating multiple lesions simultaneously, and the ability to retreat a tumor to improve the response (1). Upon light irradiation in an oxygen-rich environment, photosensitizers produce short-lived but highly reactive oxygen species (ROS), such as singlet oxygen 1O2, or radical species, such as OH·, O2·, which effectively oxidize the cellular components at locations where they have been produced (2). To achieve the desired therapeutic effect, this procedure should leave the surrounding healthy tissue unharmed, and thus production of ROS should be spatially limited to the tumor tissue. PDT provides two opportunities of controlling the selectivity of treatment: (i) by preferential accumulation of photosensitizer molecules in a tumor and (ii) by spatially limited application of light. Many systemically administered photosensitizers are known to accumulate preferentially in the tumor compared with normal tissue. However, this selectivity may not be sufficient because even successfully treated patients often suffer from long-term skin sensitivity caused by retention of photosensitizer in the skin and subsequent exposure to ambient light (3).

Targeted macromolecular conjugates that employ cell type-specific binding by ligand–receptor or antibody–antigen recognition provide higher selectivity. In the latter approach, photosensitizers are attached to monoclonal antibodies by chemical conjugation (4–6). In particular, focusing on the suppression of p185HER-2-ECD-overexpressing cancer cells, different anti-p185HER-2-ECD antibodies have been used for the targeting of photosensitizers (7, 8). The main drawback of the chemical approach is the limited reproducibility of conjugate synthesis (9), which often results in loss of activity of either the monoclonal antibody (mAb) (reduced affinity) or the photosensitizer (altered photophysical properties leading to poor PDT efficiency) (10, 11). Moreover, the main disadvantage of chemical antibody-photosensitizer chemical conjugates may be the impossibility of intracellular production of the molecule with subsequent secretion into the bloodstream [as presented by Wang et al. (12)], thus generating an intraorganism photosensitizer-producing “factory.”

Recently, a phototoxic red fluorescent protein KillerRed has been reported (13). KillerRed can be used as a fully genetically encoded photosensitizer to kill bacterial and eukaryotic cells. For mammalian cells, the most efficient cell death was observed for plasma membrane-targeted KillerRed (14). The discovery of KillerRed gave rise to the idea of constructing a recombinant immunophotosensitizer that would be devoid of the disadvantages characteristic of photosensitizer-antibody chemical conjugates.

4D5 full-size humanized monoclonal antibody (15) is used widely in clinical practice (under commercial name Herceptin) as a “gold standard” in immunotherapy of HER2/neu-overexpressing tumors. A recombinant single-chain fragment of this humanized monoclonal antibody, 4D5scFv, has been confirmed to be an effective targeting moiety for use in various applications, such as the verification of therapeutic principles: immunotoxins, immuno-RNases, etc. (16, 17). Generally, scFvs are considered promising for medical applications because of superior tissue penetration, the absence of side reactions involving the constant domains, and facile engineering of fusion proteins, such as scFv-coupled toxins, enzymes for prodrug activation (ADEPT), or the creation of multivalent or bispecific proteins (18). Specifically, 4D5scFv is known to be highly thermodynamically stable and well folding; therefore, the 4D5 framework has been used repeatedly for antigen-binding region grafting to gain various antibody specificities along with good folding (19).

Results

We report here a fully genetically encoded immunophotosensitizer consisting of a fusion of an anti-p185HER-2-ECD 4D5 single-chain Fv fragment (scFv) and KillerRed. scFv antibody fragments are ideal for use as targeting moieties in selective delivery applications and offer significant advantages over full-size antibodies because of their smaller size and rational design (20, 21). Using a rational approach to the recombinant protein construction, we studied molecular structures of both proteins. Noting the high structural similarity of fluorescent proteins, we used the structure of DsRed dimer [Protein Data Bank (PDB) ID code 1g7k] for the structure simulation. Data on the X-ray structure of the KillerRed confirm the high similarity of KillerRed architectonics to DsRed (Vladimir Pletnev, personal communication) and thus strongly support correctness of the modeling performed. We proposed that fusion to the KillerRed N terminus should be optimal for preserving the protein functions (Fig. 1A). On the one hand, this allows preservation of the natural N terminus of the antibody fragment 4D5scFv, which is important for proper antibody functioning (17).

Fig. 1.

Fig. 1.

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Genetically encoded immunophotosensitizer construction. (A) Molecular model (ribbon representation) of the 4D5 scFv-KillerRed dimer (PDB 1fve and 1g7k). The 4D5scFv-KillerRed construct starts with scFv in VL-linker-VH orientation (VL and VH, green; linker, gray), a 16-aa hinge-like linker (blue), KillerRed (red), and it terminates with a His5 tag (magenta). (B) Gene construct for expression of 4D5 scFv-KillerRed.

However, the N termini of the KillerRed β-barrels within a dimer are separated by sufficient distance (60 Å), whereas C termini are located in close proximity. The latter consideration can be important to avoid spatial constraints that could interrupt proper folding of fused 4D5scFv fragment. It is well known that conversion of scFv antibody fragments into multivalent format increases their functional affinity and decreases cell surface dissociation rates (20). Considering the intrinsic dimeric nature of KillerRed, we exploit it to design a bivalent supramolecular construct (Fig. 1A). As shown in Fig. 1A, a flexible hinge-like peptide linker was used to avoid sterical hindrance and enable simultaneous binding of two antigen molecules. Thus, anti-p185HER-2-ECD antibody fragment 4D5scFv was fused to the N terminus of KillerRed via a flexible 16-aa linker (Fig. 1B). The resulting 55-kDa 4D5scFv-KillerRed protein was expressed in Escherichia coli strain SB536 and purified by a combination of IMAC and molecular size exclusion chromatography, with a final purification yield of 0.3–0.5 mg per L of bacterial culture (see Fig. 2 A and B). Samples of purified proteins were either heated at 95 °C for 5 min (Fig. 2A, lane 1) or loaded directly onto the gel without heating (Fig. 2A, lane 2). Positions of molecular mass markers are shown on the Right (sizes in kDa). Upon heating of samples, red fluorescent proteins that carry DsRed-like chromophore often demonstrate partial fragmentation with a break point just before the chromophore (22, 23). This well-known effect is also observed for 4D5scFv-KillerRed and results in additional bands corresponding to 31-kDa (4D5scFv with KillerRed C-terminal part) and 24-kDa (KillerRed N-terminal part) fragments.

Fig. 2.

Fig. 2.

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Purification and characterization of the 4D5scFv-KillerRed fusion protein. (A) 4D5scFv-KillerRed after Ni2+–nitrilotriacetic acid chromatography, heated (Lane 1) and not heated (Lane 2) in sample buffer (SDS/PAGE, Coomassie staining). (B) Fluorescent visualization of the SDS/PAGE. Lane 1, KillerRed (26 kDa); Lane 2, 4D5scFv-KillerRed (55 kDa). (C) Excitation and emission spectra of 4D5scFv-KillerRed (solid line) and free KillerRed (dashed line), indicating that the fluorescent properties of KillerRed remain unaltered upon fusion to 4D5scFv.

Fluorescence spectroscopy showed that the excitation and emission spectra of scFv-conjugated KillerRed remained unaltered (Fig. 2C). The binding activity of 4D5scFv-KillerRed to the p185HER-2-ECD receptor was evaluated by fluorescence microscopy and flow cytometry. As shown in Fig. 3A, purified 4D5scFv-KillerRed effectively accumulated after 1-h incubation at 4 °C on the surface of the ovarian carcinoma SKOV-3 cell line, which is characterized by high expression levels of p185HER-2-ECD both in vitro and in vivo. At the same time, no binding activity was detected for the CHO cells that do not overexpress p185HER-2-ECD (Fig. 3C). KillerRed alone showed no significant binding activity on SKOV-3 cells (Fig. 3B). Anti-p185HER-2-ECD binding specificity was further verified by flow cytometry ex-periments (Fig. 3D), and ELISA with purified p185HER-2-ECD (Fig. 3 E and F). Binding of 4D5scFv-KillerRed was decreased by ≈4-fold upon addition of parental 4D5scFv antibody at the molar ratio of 1:2 (fusion protein:parental antibody).

Fig. 3.

Fig. 3.

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4D5scFv-KillerRed binding properties. (A–C) Bright-field images (Left) and red fluorescence images (Right). (A) SKOV-3 cells incubated with 4D5scFv-KillerRed. (B) SKOV-3 cells incubated with unconjugated KillerRed, control. (C) p185HER-2-ECD-nonoverexpressing CHO cells incubated with 4D5scFv-KillerRed, control. (D) Detection of 4D5scFv-KillerRed binding to p185HER-2-ECD-overexpressing SKOV-3 cells by flow cytometry. SKOV-3 cells were incubated with 4D5scFv-KillerRed (filled peak). Cells treated with PBS alone (solid line peak) were used as controls. (E) Competitive ELISA with fixed concentration of 4D5scFv-KillerRed and rising concentration of free 4D5scFv. (Data in D–F are represented as mean ± SD.) (F) ELISA detection of 4D5scFv-KillerRed binding to p185HER-2-ECD at various dilutions.

To evaluate the specific cytotoxicity of 4D5scFv-KillerRed on living cells we performed an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric cell proliferation assay on the SKOV-3 cell line. Decreasing concentrations of 4D5scFv-KillerRed were added to the target cells, and proliferation of these cells after a 1-h incubation at 4 °C and irradiation with bright white light for 10 min (≈1 W/cm2, i.e., ≈0.2 W/cm2 of KillerRed-activating green light, within the efficient KillerRed absorption range) was determined compared with untreated cells. As shown in Fig. 4A, 4D5scFv-KillerRed specifically destroyed the SKOV-3 cells with an IC50 of 0.5 μM. In contrast, the p185HER-2-ECD-nonoverexpressing cell line CHO was not affected at immunophotosensitizer concentrations up to 3.4 μM (Fig. 4A). Free 4D5scFv and free KillerRed also did not affect cell viability (Fig. 4B), indicating the high specificity of the 4D5scFv-KillerRed phototoxic effect.

Fig. 4.

Fig. 4.

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In vitro cytotoxicity analysis of 4D5scFv-KillerRed. (A) Relative cell viability of SKOV-3 cells after treatment with 4D5scFv-KillerRed with or without light irradiation. (B) Relative cell viability of SKOV-3 cells after treatment with free 4D5scFv and KillerRed, with or without irradiation (negative control). (C) Relative viability of SKOV-3 and CHO cells after combined treatment with 4D5scFv-KillerRed and cisplatin. (D) Relative cell viability of SKBR-3 cells after treatment with 4D5scFv-KillerRed in H2O and D2O-based buffers. (Data are represented as mean ± SD.)

To test 4D5scFv-KillerRed photochemistry further, we compared cytotoxicity of the immunophotosensitizer in water and D2O. Deuterium oxide is known to extend a lifetime of 1O2 and thus increases efficiency of singlet oxygen-generating photosensitizers (25, 26). SKOV-3 cell survival was analyzed after incubation and illumination with 1.8 μM 4D5scFv-KillerRed (as described above) in either water or D2O-based buffers. Unexpectedly, cell viability in the D2O sample was ≈2-fold higher compared with the control sample in water (Fig. 4D). We thus concluded that 1O2 is not involved in the cytotoxic effect of 4D5scFv-KillerRed.

Further, we investigated the phototoxic activity of 4D5scFv-KillerRed on living cells in combination with the commonly used antitumor agent, cisplatin. Several concentrations of cisplatin were tested in toxicity experiments, and treatment with 0.5 μg/mL cisplatin was found to cause ≈60% cell death. This concentration was further used in toxicity experiments. The cells were treated with 0.5 μg/mL cisplatin and serial dilutions of 4D5scFv-KillerRed, starting from 0.35 μM. SKOV-3 and CHO (control) cells were treated with 4D5scFv-KillerRed and cisplatin alone and in combination, and the cell viability was measured by MTT assay. As shown in Fig. 4C, the viability of the p185HER-2-ECD-overexpressing cell line treated with the combination of targeted and traditional cytotoxic agents was lower than with either single agent treatment. At the same time, the CHO cell line showed no decrease in cell viability after treatment with the combination of cytotoxic agents. Thus, combined treatment makes it possible to use lower concentrations of either of the toxic agents to reach the same level of cell death.

Discussion

Overexpression of p185HER-2-ECD in human ovarian and breast tumors correlates with poor patient prognosis (24), making it an attractive target for tumor therapy. A number of mAbs have been produced against the external domain of the p185HER-2-ECD that showed potent inhibition of in vitro and in vivo growth in a wide range of tumors (24). However, because p185HER-2-ECD is also expressed in epithelial tissues like skin, liver, and vascular endothelium, side effects to normal tissue occur with the use of anti-p185HER-2-ECD-immunotoxins.

The immunophotosensitizer 4D5scFv-KillerRed fusion is devoid of such side effects because its cytotoxic effect is limited to the area of illumination. In contrast to chemically conjugated immunotoxins, 4D5scFv-KillerRed displays defined ligand–toxin junction, precise photosensitizer:antibody ratio, and simple production of the ready-to-use photosensitizer in bacteria.

Generally, photosensitizers can act via photodynamic reactions of type I or type II (25). In type I reactions, a photoinduced electron transfer from appropriate molecule to photosensitizer occurs as a primary step followed by interaction of the oxidized photosensitizer with molecular oxygen and formation of superoxide anion radical O˙̄2. Type II reactions consist of direct energy transfer from excited photosensitizer to oxygen resulting in generation of singlet oxygen. The latter is a useful indicator allowing discriminating between type I and II reactions.

To check whether 4D5scFv-KillerRed undergoes photodynamic reaction of type I or type II, we performed a widely used test on comparison of phototoxicity in H2O and D2O. A greatly increased phototoxicity in D2O is expected for photosensitizers, and it generates singlet oxygen because of its longer lifetime in this solvent photosensitizer (25, 26). Surprisingly, phototoxicity of 4D5scFv-KillerRed in D2O was even lower compared with water. These data show that cytotoxicity of 4D5scFv-KillerRed is mediated by ROS other than singlet oxygen. Decreased phototoxicity in D2O indicates that 4D5scFv-KillerRed photoreaction involves proton transfer, which is slowed down in deuterated water because of an isotopic effect (27, 28). These data allow us to conclude that in the present model, 4D5scFv-KillerRed acts as a type I photosensitizer.

According to Wentworth et al. (29), antibodies are capable of catalyzing the reaction of oxidation of water to hydrogen peroxide by singlet oxygen molecules. Thus, in the 4D5scFv-KillerRed, a type II photosensitizer, the synergism between the 4D5scFv and KillerRed could be expected, although, according to the data presented, a type II reaction is not likely for the 4D5scFv-KillerRed protein. Besides, the catalytic mechanism of water oxidation reaction was not shown for scFv, as it was for other antibody formats (30). Therefore, we believe that the phototoxic effect observed could be attributed solely to the KillerRed phototoxicity, not to the KillerRed and scFv synergism.

Compared with chemical photosensitizers, KillerRed has a lower phototoxicity (13). Thus, a higher light doze is required to achieve reliable cell death. This is perhaps the main drawback of the present approach, although improved KillerRed variants with enhanced phototoxicity might be developed in the future. At the same time, the lower level of ROS production can be advantageous in some respects. For instance, preparation of antibody-photosensitizer conjugates should be performed in subdued light and in the absence of oxygen, otherwise the integrity of the antibodies can be affected by ROS (5). In contrast, purification and storage of KillerRed fusions can be performed under normal light and oxygen conditions. Also, very high phototoxicity of a photosensitizer may result in such side effects as undesirable surrounding tissue necrosis. In addition, skin phototoxicity is often observed, so patients have to avoid sunlight for a long time after treatment (5). Such problems are not expected for KillerRed because only rather strong light can induce considerable damage of cells in this case.

Combined treatment with conventional chemotherapeutic agent cisplatin and genetically encoded photosensitizer is likely to employ different mechanisms of cell death, thus increasing the probability of successful killing of a given tumor cell. Cisplatin-induced cell death is known to proceed either by necrosis or by apoptosis pathway depending on the extracellular and intracellular conditions, such as energy availability (31). However, in both cases the main initiation mechanism of cisplatin-induced cell death is DNA and protein damage by platination. However, phototoxicity of KillerRed presumably involves production of singlet oxygen (13), as shown by experiments with singlet oxygen quenchers. Also, KillerRed-mediated phototoxicity is likely to proceed by apoptosis, as shown by caspase inhibition experiments. Thus, treatment with a combination of two toxic agents with different mechanisms of toxicity may lower the probability of resistance.

Our results demonstrate efficient elimination of the ovarian carcinoma cell line SKOV-3 after incubation with the recombinant anti-p185HER-2-ECD-immunotoxin 4D5scFv-KillerRed and subsequent irradiation with white light. This report describes the generation of a fully genetically encoded recombinant immunophotosensitizer.

Obviously, the targeted phototoxic agent intended for systemic administration needs to be evaluated on tumor xenograft models. However, systemic treatment for remote metastases elimination is not the only perspective mode of advanced cancer treatment. For instance, i.p. chemotherapy versus conventional systemic chemotherapy is currently considered an option for patients with stage III ovarian cancer (32). It is performed during or after so-called “second-look” laparoscopic surgery in patients who appear to be clinically free of disease after primary cytoreductive surgery but who actually harbor persistent cancer. Even before further in vivo experiments, it appears that our photosensitizer-antibody fusion protein might be effective in this model for postsurgical elimination of remaining malignant cells.

Furthermore, in perspective, the development of virus-based systems of gene delivery into tumors (33) should enable intratumoral production of genetically encoded immunophotosensitisers. Such an approach is principally impossible for antibody-photosensitizer chemical conjugates.

Methods

Construction and Expression of 4D5scFv-KillerRed.

Genetic engineering manipulations, cell culture, and cell lysis followed the standard protocols. The DNA fragment encoding the KillerRed protein was amplified from a pKillerRed-N plasmid containing the KillerRed gene (Evrogen) by using primers 5′-ATCTATGGCGCGCTGCCTATGGGTTCAGAGGGCGG-3′ (BssHI recognition sequence in bold) and 5′-ATCATAGGCGCGCCATCCTCGTCGCTACCGATG-3′ (BssHI and AscI recognition sequence in bold). The amplification product was digested with BssHI and was cloned into the AscI sites of pSD-4D5scFv-barnase (17), replacing the barnase gene part of the genetic construct. To produce the recombinant protein, E. coli SB536 was transformed with pSD-4D5scFv-KillerRed and was grown in LB broth. At an OD550 of 1.2 the culture was induced with 0.5 mM IPTG and incubated at 27 °C for 24 h.

Purification of 4D5scFv-KillerRed Fusion Protein.

The cells were harvested, washed with PBS, centrifuged, and the pellet was resuspended in lysis buffer [0.01 M Tris·HCl (pH 8.3), containing 0.01 M Na2B4O7, 0.01 M K2HPO4, 0.5 M NaCl, and 10% (vol/vol) glycerol] and sonicated on ice, followed by the addition of Tween 20 to 0.5%. The lysate was then centrifuged at 22,000 × g for 20 min at 4 °C. The supernatant was used for the purification of His-tagged proteins on Ni2+–nitrilotriacetic acid–Sepharose. Proteins were eluted with 200 mM imidazole, concentrated using Microcon concentrators (Millipore) with a molecular mass cutoff of 10 kDa, and verified by Western blot analysis. The 4D5scFv, 4D5scFv-barnase-His5, and 4D5scFv-dibarnase-His5 fusion proteins produced according to Deyev et al. (17) were used as molecular mass markers (29, 42, and 54 kDa, respectively). SDS/PAGE analyses were performed according to the standard protocols using 12% polyacrylamide gels. Immunoblots on Immobilon-P transfer membrane (Millipore) were carried out according to the manufacturer's instructions using rabbit anti-4D5scFv antibody, produced in our laboratory, followed by a goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma) for detection. The blots were visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate.

Fluorescence Microscopy and Flow Cytometry Analysis.

The Chinese hamster ovary cell line CHO and human ovarian carcinoma cell line SKOV-3 were maintained in RPMI medium 1640 (PanEco), supplemented with 10% FCS (HyClone) and 2 mM l-glutamine (Flow Laboratories) in culture flasks. For binding experiments, cells were plated in 96-well plates (Corning) at density of 4 × 103 cells per well and cultured overnight at 37 °C in a 5% CO2 atmosphere.

After a brief wash with PBS the cells were incubated with 1.8 μM 4D5scFv-KillerRed or KillerRed (negative control) for 1 h at 4 °C. For flow cytometry analysis, the adherent SKOV-3 cells were carefully detached with PBS (pH 8.0) containing 5 mM EDTA. To avoid enzymatic cleavage of cell surface receptors, no trypsin was used. After a brief wash with cold PBS the cells were incubated with 20 nM 4D5scFv-KillerRed for 1 h at 4 °C. Cells were analyzed by using an inverted fluorescence microscope Axiovert 200 (Zeiss) and measured on an EPICS XL-MCL flow cytometer (Beckman–Coulter) at 488-nm excitation (argon laser). Micrographs were captured by using a CCD camera (AxioCam HRc; Zeiss) and AxioVision software (Zeiss).

ELISA.

To assay binding of 4D5scFv-KillerRed fusion protein to p185HER-2-ECD by ELISA, 96-well plates (Corning) were coated overnight at 4 °C with p185HER-2-ECD (200 ng/mL) in carbonate–bicarbonate buffer (pH 9.6). Plates were washed twice in PBS (pH 7.4) and incubated for 1 h at room temperature in 5% nonfat dry milk in PBS and then washed again. Then, plates were incubated for 1.5 h at room temperature with 4D5scFv-KillerRed fusion protein diluted in PBS to the concentrations shown on Fig. 3E. After this step, plates were washed 3 times with PBS and 0.1% Tween 20 and incubated for 1.5 h at room temperature with rabbit anti-4D5scFv antibodies (1:250 in PBS and 1% dry milk). Then plates were washed again, and horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:4,000 in PBS and 1% milk) (Amersham) were added for 1 h at room temperature. After final washing, plates were developed by 20-min room temperature incubation with 0.04% o-phenylenediamine in 0.05 M citrate buffer (pH 5.5) and 0.06% hydrogen peroxide, and the absorbance at 492 nm was determined by using a plate reader (MR 580 Microelisa Autoreader; Dynatech).

Binding Specificity.

The binding specificity of 4D5scFv-KillerRed was tested by ELISA following the protocol described above with the parental 4D5scFv for competition. Briefly, ELISA 96-well plates (Corning) were coated overnight at 4 °C with p185HER-2-ECD (200 ng/mL) in carbonate-bicarbonate buffer (pH 9.6). After blocking, the plates were incubated with a fixed concentration (1.5 μg/mL) of recombinant immunotoxin 4D5scFv-KillerRed. Competition experiments were performed in the presence or absence of different concentrations (0.8 and 1.5 μg/mL) of 4D5scFv. Binding of 4D5scFv-KillerRed was detected by using anti-KillerRed antibodies (Evrogen) and peroxidase-labeled goat anti-rabbit antibodies (Amersham) after incubation with 0.04% o-phenylenediamine in 0.05 M citrate buffer (pH 5.5) and 0.06% hydrogen peroxide.

Cell Treatment and Cell Viability Assay.

Light-induced cytotoxicity of 4D5scFv-KillerRed immunophotosensitizer on the SKOV-3 cell line was determined by the MTT cell viability assay as described in ref. 34. Briefly, SKOV-3 cells were seeded at a density of ≈4.0 × 103 cells per well in a 96-well plate, and CHO cells at a density of ≈6.0 × 103 and were allowed to attach overnight. The medium was replaced, and cells were incubated with PBS containing different concentrations of 4D5scFv-KillerRed at 4 °C for 1 h. The cells were then irradiated with white light (≈1 W/cm2) for 10 min, PBS was substituted for the RPMI medium 1640, and the cells were incubated for an additional 72 h at 37 °C in a 5% CO2 atmosphere. After 72 h, cells were washed in PBS and then incubated with serum-free medium containing 0.5 mg/mL MTT for 1 h. Formazan formed from the reduction of MTT by mitochondrial dehydrogenases was dissolved in DMSO, and the absorbance was measured spectrophotometrically at 540 nm. The cell viability was expressed as a percentage of the optical density of untreated cells from two experiments carried out in triplicate. For cumulative cytotoxicity experiments, cisplatin was added in RPMI medium 1640 after the illumination to the final concentration of 0.5 mg/L. 4D5scFv-KillerRed was added in two-times dilutions starting from 0.06 g/L.

Acknowledgments.

This work was supported by Molecular and Cellular Biology Program Russian Academy of Sciences, Russian Foundation for Basic Research Grants 07-02-00649-a and 09-04-01201, Russian Federal Agency for Science and Innovation and Howard Hughes Medical Institute Grant 55005618.

Footnotes

The authors declare no conflict of interest.

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