Photoimmunoconjugates for use in photodynamic therapy

Abstract

A compound comprising a photosensitizer covalently coupled to a protein selected from the group consisting of antibodies or their derivatives or fragments thereof, synthetic peptides such as scFv, mimotopes which bind CD antigens, cytokine receptors, interleukin receptors, hormone receptors, growth factor receptors, more particularly tyrosine kinase growth factor receptor of the ErbB family, wherein the photosensitizer is coupled to the binding protein via O6-alkylguanine-DNA alkyltransferase (hAGTm), a modified human DNA repair protein.

Claims

1. A compound comprising: a photosensitizer covalently coupled to a binding protein selected from the group consisting of an antibody or its antigen-binding fragment thereof; and wherein the binding protein binds a growth factor receptor, the photosensitizer is coupled to the binding protein via O6-alkylguanine-DNA alkyltransferase (hAGTm), a modified human DNA repair protein, and wherein the binding protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

2. The compound of claim 1, wherein the photosensitizer is coupled at the active site of the O6-alkylguanine-DNA alkyltransferase.

3. The compound of claim 1, wherein the photosensitizer is selected from the group consisting of porphyrins, chlorophylls and dyes.

4. The compound of claim 1, specifically targeting an internalizing and disease-specific cell surface receptor.

5. The compound of claim 1, wherein the binding protein is an scFv antibody.

6. A compound comprising: a binding protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the binding protein binds a growth factor receptor, and wherein the binding protein is covalently coupled to a modified human DNA repair protein called O6-alkylguanine-DNA alkyltransferase (hAGTm).

7. The compound of claim 6, wherein the binding protein is encoded by the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

8. A compound comprising: a photosensitizer covalently coupled to a binding protein comprising a protein encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 5; wherein the binding protein binds a growth factor receptor, and wherein the photosensitizer is coupled to the binding protein via O6-alkylguanine-DNA alkyltransferase (hAGTm), a modified human DNA repair protein.

9. A method for manufacturing the compound of claim 1 comprising the step of fusing O6-alkylguanine-DNA alkyltransferase (hAGTm) with the binding protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 to form a fusion protein.

10. The method of claim 9 wherein the binding protein encoded by the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is inserted into a SifI and NotI-digested site of eukaryotic expression vector pMS-SNAP providing an N-terminal binding ligand and a C-terminal SNAP-tag sequence.

11. The method of claim 9 wherein the fusion protein is expressed in human embryonic kidney cell line.

12. The method of claim 9 wherein the fusion protein is purified from cell-free supernatant by an affinity chromatography.

13. The compound of claim 1, wherein the photosensitizer is a porphyrin derivative of the formula ##STR00002##

14. A method of manufacturing the compound of claim 13, comprising reacting the carboxyl groups of the porphyrin derivative with an activated ester or a coupling agent, followed by reacting with O6-benzylguanine, O2-benzylcytosine or a coenzyme A (CoA).

15. The method of claim 14, wherein O6-benzylguanine, O2-benzylcytosine or CoA is coupled to a linker molecule and/or the activated ester is formed by succinimides, or the coupling agent is a carbodiimide.

16. A medicament comprising the compound of claim 1 and a pharmaceutically acceptable adjuvant for improving or rendering a pharmaceutical effect associated with photoimmunotherapy.

17. A method for treating cancer in a subject by photoimmunotherapy, comprising administering to the subject the compound of claim 1.

18. The compound of claim 1, wherein the binding protein is scFv.

19. The compound of claim 1, wherein the growth factor receptor is a tyrosine kinase growth factor receptor of the ErbB family.

20. The compound of claim 1, wherein the binding protein is encoded by the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

21. The compound of claim 6, wherein the binding protein is scFv.

22. The compound of claim 6, wherein the growth factor receptor is a tyrosine kinase growth factor receptor of the ErbB family.

Description

LEGENDS OF THE FIGURES

(1) FIG. 1: Construction, expression and binding of the SNAP-tag fusion proteins. (a) Schematic diagram of the bicistronic eukaryotic expression cassettes for the recombinant SNAP-tag fusion protein. The pMS-scFV-425-SNAP vector encodes binding ligand (scFv-425) which is under the transcriptional control of the CMV promoter, and joined in-frame to the SNAP-tag. An immunoglobulin leader sequence (Ig--L) facilitates protein secretion, and a TGA stop codon is placed immediately after the C-terminal His.sub.6-tag. The expression cassettes for the control vector were the same as the PMS-scFv-425-SNAP but contain scFv-Ki4 instead of scFv-425 as a binding ligand. (b) Purification fractions of scFv-425-SNAP protein were separated by SDS-PAGE, and then stained with coomassie brilliant blue, (c) scFv-425-SNAP was incubated with BG-Vista green, proteins were visualized with UV light. M: Protein marker, 1: 3 l of eluted scFv-425-SNAP with 250 mM Imidazol, 2: 1.5 l of eluted scFv-425-SNAP with 250 mM Imidazol, 3: 10 l of eluted scFv-425-SNAP with 40 mM Imidazol, 4: eluted protein with 10 mM Imidazol, 5: Flow through, 6: HEK-293T cells supernatant. Binding analysis of scFv-425-SNAP and scFv-Ki4-SNAP were assessed by flow cytometry using EGFR.sup.+ A431 (d) and EGFR.sup. L540 cells (e). Filled gray curves represent untreated cells. Cells were incubated with 0.5 g/ml of the purified fusion protein scFv-425-SNAP (light gray curve) and Ki4-SNAP (black curve). As a secondary antibody a Penta-His Alexa Fluor 488 Conjugate (dilution 1/500) (Qiagen) was used. To exclude nonspecific staining of the anti-His Alexa Fluor 488 detection antibody, omission of the His-tagged fusion protein served as control (dotted black curves).

(2) FIG. 2: Analysis of Ce6 photosensitizer by mass spectrometry before and after modification with benzylguanine (BG). (a) ESI mass spectrum of Ce6, BG-PEG24-NH.sub.2, and BG-PEG24-Ce6. The top panel represents Ce6 (597.215 Da), the middle panel represents BG-PEG24-NH.sub.2 (1398.761 Da) and the bottom panel represents BG-PEG24-Ce6 (1979.004 Da). (b) Coupling of BG-PEG24-Ce6 to scFv-425-SNAP. M, protein marker; 1, scFv-425-SNAP incubated with a 1.5-fold molar excess of BG-VistaGreen; 2, scFv-425-SNAP blocked with a 3-fold molar excess of bromothenylpteridine (BTP), incubated with BG-Ce6 for 2 h, and finally mixed with BG-VistaGreen; 3, scFv-425-SNAP incubated with a 1.5-fold molar excess of BG-Ce6 for 2 h, then a 1.5-fold molar excess of BG-VistaGreen. Coupled proteins were separated by SDS-PAGE and visualized with the CRi Maestro Imaging System. The different dye spectra were unmixed using Maestro software and the corresponding gel was stained with Coomassie Brilliant Blue (c).

(3) FIG. 3: The binding activities of scFv-425-SNAP-VistaGreen and scFv-425-SNAP-Ce6, which specifically recognize EGFR.sup.+ cells. Flow cytometry analysis was carried out after incubating 410.sup.5 cells with each fusion protein for 20 min at 37 C. in PBS. (a) The scFv-425-SNAP-VistaGreen (light gray curve) was tested against A431, MDA-MB-468, MDA-MB-231, SiHa, L540, and CHO-K1 cells (filled gray curve). As a control, scFv-Ki4-SNAP was labeled with BG-VistaGreen (black curve) and its binding activity was tested against A431, L540 and CHO-K1 cells (filled gray curves). (b) The binding efficiency of scFv-425-SNAP-Ce6 (light gray curve) was tested against A431, MDA-MB-468, MDA-MB-231, SiHa, L540 and CHO-K1 cells (filled gray curves). As a control, scFv-Ki4-SNAP labeled with BG-Ce6 (black curve) was tested against A431, L540 and CHO-K1 cells (filled gray curves).

(4) FIG. 4: Internalization of fusion proteins analyzed by confocal microscopy. Confocal images were obtained for the EGFR.sup.+ cell lines A431, MDA-MB-468, MDA-MB-231 and SiHa, and for the EGFR.sup. cell lines L540 and CHO-K1 incubated with 0.5 g scFv-425-SNAP-Ce6 for 30 min at 4 C. (a) or for 60 min at 37 C. (b). (1) Ce6 fluorescence signal; (2) transmitted light; (3) overlay of fluorescence signal and transmitted light.

(5) FIG. 5: Evaluation of photodynamic therapeutic efficiency. Cell proliferation and apoptosis assays were carried out using the scFv-425-SNAP-Ce6. The cytotoxicity of scFv-425-BG-Ce6 was determined against cell lines A431 (.square-solid.), MDA-MB-468 (.box-tangle-solidup.), MDA-MB-231 (.diamond-solid.), SiHa (.circle-solid.) and CHO-K1 (.Math.) using the XTT assay on (a) irradiated cells and (b) non-irradiated cells. The cytotoxicity scFv-Ki4-SNAP-Ce6 against A431 cells (x) was tested as a control. The same cells were treated with different concentrations of BG-Ce6 and cell viability was analyzed with (c) and without (d) light activation. (e) Apoptosis was evaluated using the Apo-ONE Homogeneous Caspase-3/7 Assay, with 50 nM BG-Ce6, 200 nM scFv-SNAP-Ce6 and 200 nM scFv-Ki4-SNAP-Ce6. (f) The generation of reactive oxygen species by illuminating photosensitized A431 cells, detected using the dichlorofluorescein derivative carboxy-H2DCFDA.

(6) Photodynamic therapy (PDT) is a minimally invasive treatment that uses nontoxic photosensitizers and harmless visible light in combination with oxygen to produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis and/or necrosis.sup.12. Many different photosensitizers have been developed, but Ce6 has been chosen as a model because it has been evaluated extensively in PDT studies and also has advantageous physical and chemical properties. Ce6 has an absorption maximum at 664 nm, which is a good compromise between photon efficacy and cell penetration.sup.13, and the presence of carboxyl groups allows further functionalization.sup.5.

(7) The use of SNAP-tag technology of the present invention provides a unique conjugation site on the antibody, allowing the production of a homogeneous conjugate preparation. The construct of the invention in which the coding sequence of an scFv antibody that binds specifically to EGFR was genetically fused to the hAGT cassette, endows the antibody with a SNAP-tag and therefore allows site-specific conjugation BG-modified substrates, in particular Ce6. This conjugation method can be applied to any antibody-photosensitizer combination as long as the antibody carries the SNAP-tag and the substrate is modified with a BG group.

(8) The conjugation reaction was efficient, allowing the preparation of homogeneous samples of scFv-425-SNAP-Ce6 and scFv-Ki4-SNAP-Ce6. These preparations were tested for their ability to kill tumor cells specifically. It has been found that scFv-425-SNAP-Ce6 selectively killed EGFR.sup.+ cells in four human tumor-derived cell lines representing epidermal, breast and cervical carcinomas (A431, MDA-MB-231, MDA-MB468 and SiHa) after exposure to light. The phototoxicity of scFv-425-SNAP-Ce6 was dependent on the presence of EGFR and light, and toxicity was most potent in A431 and MDA-MB468 cells, which express the largest amount of the receptor (1-1.310.sup.6 receptors/cell).sup.14,15. The other cells lines expressed less EGFR (1.310.sup.5 receptors/cell for MDA-MB-231 and 210.sup.4-210.sup.5 receptors/cell for SiHa).sup.15,16, and the toxicity of scFv-425-SNAP-Ce6 was concomitantly reduced, although not to the point where the fusion protein would be therapeutically ineffective. This means that scFv-425-SNAP-Ce6 can target a wide range of EGFR.sup.+ cells not only those with the highest expression levels. No toxicity was observed when EGFR.sup. cells (CHO-K1) were exposed to scFv-425-SNAP-Ce6.

(9) It has been previously shown that scFv-425-SNAP accumulates directly in mouse kidneys after injection, and is subsequently detected in the bladder, indicating clearance by renal filtration.sup.10. Despite the rapid clearance, the accumulation and retention of scFv-425-SNAP in tumor tissue was evidently sufficient to yield very high tumor to background ratio 10 h post-injection.

(10) Expression, Purification and Functional Analysis of scFv/SNAP-Tag Fusion Proteins

(11) The coding sequences for the EGFR-specific scFv-425 antibody fragment.sup.10 and a control fragment (scFv-Ki4).sup.17 that binds to a different antigen (CD30) were transferred to the pMS-SNAP bicistronic vector to generate the complete scFv-425-SNAP and scFv-Ki4-SNAP cassettes, as shown in (FIG. 1a). The constructs were introduced into HEK-293T cells by transfection and stably transformed cells were identified by selection on zeocin and by monitoring green fluorescent protein (GFP) activity. The fusion proteins were isolated from the culture supernatant to a final purity of 90% by affinity chromatography (using the C-terminal His.sub.6 tag) and the final yield was 18 mg/L of protein in the supernatant (FIG. 1b).

(12) The activity of the SNAP-tag was confirmed in each of the fusion proteins by mixing the unprocessed culture supernatant, the flow through fraction and the eluate from the chromatography step with BG-modified Vista Green (FIG. 1c). The binding activity of the scFv-425-SNAP protein was confirmed by flow cytometry using one target cell line expressing EGFR (A431), and one control cell line lacking this antigen but expressing CD30 (L540). Binding was detected with a secondary anti-His.sub.5 Alexa 488 antibody. Flow cytometry data confirmed the rapid and efficient binding of scFv-425-SNAP specifically to EGFR.sup.+ target cells (FIG. 1d), whereas scFv-Ki4/SNAP bound only to the CD30.sup.+ L540 cells (FIG. 1e).

(13) Modification of the Photosensitizer Chlorin e6 with Benzylguanine

(14) The photosensitizer chlorin e6 (Ce6) was modified successfully using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), the sodium salt of hydroxysulfosuccinimide (sulfo-NHS) and a BG-PEG24-NH.sub.2 linker. Ce6 carboxyl groups were modified to BG groups, and the efficiency of the reaction was determined by HPLC (data not shown). The high purity of BG-PEG24-Ce6 was confirmed by mass spectrometry. The accurate masses of Ce6, BG-PEG24-NH.sub.2 and BG-PEG24-Ce6 were detected on a Micromass QTOFII mass spectrometer, which confirmed that purified BG-PEG24-Ce6 had the same mass as the theoretical mass calculated for coupled Ce6 and BG-PEG24-NH2 (FIG. 2a)

(15) Protein Labeling with BG-Modified Fluorophores and Ce6

(16) The functionality of the SNAP-tag was tested by coupling to BG-modified fluorescent dye, which revealed a labeling efficiency of 85-90% after a 2-h incubation at room temperature (data not shown). The reaction was repeated using BG-modified Ce6. The photosensitizer reacted solely with the active SNAP-tag in the fusion proteins and the reaction could be irreversibly blocked with the bromothenylpteridine (BTP), as shown by post-incubation with a 1.5-fold molar excess of BG-Vista Green. Analysis with the CRi Maestro imaging system showed no fluorescence associated with the previously blocked fusion protein (FIG. 2b,c).

(17) Flow Cytometry and Confocal Microscopy

(18) To determine the activity of labeled scFv-425-SNAP fusion proteins, flow cytometry analysis was carried out using proteins that had been labeled with either BG-Vista Green or BG-Ce6. All the labeled proteins showed a strong fluorescence signal on the corresponding target cell line (A431, MDA-MB-231, MDA-MB-468 and SiHa) but not on control cells (L540 and CHO-K1) after a 30-min incubation on ice. As expected, labeled scFv-Ki4-SNAP showed a strong fluorescence signal on L540 but not on A431 and CHO-K1 cells (FIG. 3).

(19) Confocal microscopy revealed strong, specific and homogeneous membrane staining on A431, MDA-MB-231, MDA-MB468 and SiHa cells incubated with scFv-425-SNAP-Ce6 (FIG. 4a). The labeled fusion protein was specifically and efficiently taken up into A431, MDA-MB-231, MDA-MB468 and SiHa cells after a 30-min incubation at 37 C. but not at 4 C. (FIG. 4b). In contrast, no signal was detected when the EGFR.sup. cell lines L540 and CHO-K1 were incubated with scFv-425-SNAP-Ce6 under the same conditions (FIG. 4a,b).

(20) Photocytotoxicity of scFv-425-SNAP-Ce6

(21) The concentration-dependent cytotoxic effects of scFv-425-SNAP-Ce6 and unconjugated BG-Ce6 were evaluated using an XTT-based colorimetric cell proliferation assay with the four EGFR.sup.+ cell lines and CHO-K1 as a negative controls. The viability of A431, MDA-MB-231, MDA-MB-468 and SiHa cells treated with scFv-425-SNAP-Ce6 was reduced significantly, in a concentration-dependent manner, after a 24-h incubation followed the light activation. The IC.sub.50 values were 48 nM (A431), 200 nM (MDA-MB-231), 38 nM (MDA-MB-468) and 218 nM (SiHa). CHO-K1 cells remained unaffected even when exposed to 800 nM of the conjugated fusion proteins, and the control construct scFv-Ki4-SNAP-Ce6 had a negligible effect in both A431 and CHO-K1 cells. In contrast, unconjugated Ce6 was toxic towards all the cell lines, with IC.sub.50 values of 16 nM (A431), 22 nM (MDA-MB231), 22 nM (MDA-MB-468), 26 nM (SiHa) and 18 nM (CHO-K1). These data are shown in (FIG. 5a,c).

(22) Both the conjugated and unconjugated forms of Ce6 were toxic only after light activation, as confirmed by carrying out parallel experiments without the light activation step. No significant reduction in viability was observed in any of the cell lines (FIG. 5b,d).

(23) To determine whether scFv-425-SNAP-Ce6 selectively induced programmed cell death in target cells by triggering the apoptotic pathway, the activity of caspase-3 and caspase-7 has been analyzed in A431, MDA-MB-231, MDA-MB468, SiHa and CHO-K1 cells 24 h after light activation. Both scFv-425-SNAP-Ce6 (200 nM) and unconjugated Ce6 (50 nM) increased the levels of caspase-3 and caspase-7, whereas no significant increase was observed in A431 cells treated with 200 nM scFv-Ki4-SNAP-Ce6 (FIG. 5e).

(24) The production of ROS in photoactivated A431 cells was investigated by measuring the 485/535-nm fluorescence of DCF, produced by the oxidation and deacetylation of 6-carboxy-20,70-dichlorodihydrofluoresceindiacetatedi-(acetoxy-methyl)ester (H.sub.2DCFDA). It has been found that a burst of ROS synthesis follows light activation in the presence of 200 nM of the conjugated Ce6 and 50 nM of the unconjugated Ce6, but there was only a small increase in ROS levels in non-irradiated cells, barely above the background level observed in cells that were not treated with the photosensitizer (FIG. 5f).

(25) Methods

(26) Cell Culture

(27) All cell lines were of human origin, including the EGFR.sup.+ A431, MDA-MB-231, MDA-MB468 and SiHa cells, and the EGFR.sup. L540, CHO-K1 and HEK-293T cells. A431, L540, CHO-K1 and HEK-293T cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. MDA-MB-231, MDA-MB468 and SiHa cells were cultured in DMEM with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. All cells were incubated at 37 C. in a 5% CO.sub.2 atmosphere. All media and additives were obtained from Invitrogen, Darmstadt, Germany.

(28) Protein Expression and Purification.

(29) The sequence for each scFv was inserted into an expression cassette providing an N-terminal binding ligand (scFv-425 or scFv-Ki4) and a C-terminal O6-alkylguanine-DNA alkyltransferase (SNAP-tag) sequence. The TGA stop codon is generated immediately after His.sub.6 tag sequence. His.sub.6-tagged fusion proteins were purified from cell-free supernatants by Ni-NTA metal affinity chromatography. Larger volumes were purified on an Akta FLPC system with a 5-mL Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany) after equilibration with 4 buffer (200 mM NaH.sub.2PO.sub.4, 1.2 M NaCl, 40 mM imidazole, pH 8). Bound His-tagged proteins were eluted in 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM imidazole, pH 8). After elution, proteins were dialyzed at 4 C. overnight against phosphate-buffered saline (PBS) containing 1 mM dithioerythritol (Carl Roth GmBH, Karlsruhe, Germany). Ectoine cryopreservative was added to a final concentration of 50 mM, and aliquots were stored at 20 C.

(30) Modification of Ce6 with Benzylguanine

(31) The carboxyl groups of Ce6 (Porphyrin Products, Logan, Utah), were modified with benzylguanine by mixing 2 mg Ce6 in dimethylformamide for 30 min at room temperature with a five-fold molar excess of EDC and sulfo-NHS (Sigma-Aldrich, St Louis, Mo.). The activated mixture was then mixed with a four-fold molar excess of the benzylguanine linker BG-PEG24-NH.sub.2 (Covalys Biosciences AG, Witterswil, Switzerland) in the dark at room temperature overnight. The modified Ce6 was purified by HPLC using a Shimadzu Prominence HPLC system, and a 2.5 m (4.650 mm) Water XBridge OSTC.sub.18 column (Waters, Milford, Mass.) at a flow rate was 1 mL/min. Separations were carried out using a 20-min gradient from 100% 0.1 M TEAA to 100% acetonitrile, monitored at 280 and 410 nm. The masses of Ce6, BG-PEG24-NH2 and BG-PEG24-Ce6 were confirmed using a Micromass QTOFII mass spectrometer with an electrospray ion source Advion Nanomate (Advion, Ithaca, N.Y., USA) 7 l sample volume, 1.4 kV. Accurate masses were derived from mass spectra in the range 300-2500 m/z using the MaxEnt3 algorithm (Micromass) in the range of 400-2000 Da.

(32) Protein Labeling

(33) The purified SNAP-tag fusion proteins were conjugated with BG-modified dyes (Covalys Biosciences AG, Witterswil, Switzerland) or BG-modified Ce6 by incubation in the dark with a 1.5-3-fold molar excess of dye for 2 h at room temperature. Residual dye was removed by gel filtration chromatography using zeba spin desalting columns, 7K MWCO (Thermo Fisher Scientific, Rockford, Ill.). Coupling efficiency was determined photometrically using the extinction coefficients of the corresponding dyes and the theoretical extinction coefficient of the fusion proteins. Labeled proteins were visualized after separation by SDS-PAGE with either a UV transilluminator Gel Doc XR gel documentation (Bio-Rad Laboratories, Mnchen, Germany) or a CRi Maestro imaging system (CRi, Woburn, Mass., USA) using the blue and yellow filter sets.

(34) Flow Cytometry

(35) The binding efficiency of the labeled and unlabeled fusion proteins was determined by flow cytometry using a FACSCalibur (Becton & Dickinson, Heidelberg, Germany) and CellQuest software. EGFR.sup.+ cell lines A431, MDA-MB-231, MDA-MB468 and SiHa were used to test the binding efficiency of scFv-425-SNAP, and EGFR.sup. cell lines L540 and CHO-K1 were used as negative controls. The control fusion protein scFv-Ki4-SNAP recognizes the antigen CD30 and should therefore bind to L540 cells but not to the other cell lines. Approximately 410.sup.5 cells were incubated in 200 L PBS containing 0.5 g of labeled protein for 20 min on ice. The cells were then washed twice with 1.8 mL PBS in a conventional cell washer and analyzed by flow cytometry.

(36) Confocal Microscopy

(37) Images were visualized with a TCS SP5 confocal microscope (LEICA Microsystem, Wetzlar, Germany). Cells were prepared as described above for flow cytometry. Binding efficiency was determined by incubating cells with the labeled fusion proteins for 30 min on ice. Internalization was monitored by incubating cells with the labeled fusion proteins for 30 min at 37 C.

(38) Phototoxicity of scFv-425-SNAP-Ce6

(39) Aliquots of A431, MDA-MB-231, MDA-MB468, SiHa and CHO-K1 cells (210.sup.4) cultured as described above were washed twice in PBS and then treated with increasing concentrations of either Ce6, scFv-425-SNAP-Ce6 or Ki4-scFv/SNAP-Ce6 followed by incubation for 3 h at 37 C. Control cultures were incubated with 500 g/ml zeocin instead of the photosensitizer. The cells were then irradiated with 24 J/cm.sup.2 broadband visible/near infrared light using Hydrosun type 505, 7-mm water cuvette and orange filter OG590, spectrum in the range 580-1400 nm (Hydrosun Medizintechnik GmbH, Mllheim, Germany) and incubated for a further 24 h at 37 C. in a 5% CO.sub.2 atmosphere.

(40) Cell viability was determined using the XTT cell proliferation kit II (Roche, Mannheim Germany), 24 h after light activation. Cells were incubated with 2,3-bis(2-methoxy-4-nitro-5sulphonyl)-5[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide reagent (1 mg/ml), and incubated for 2 h at 37 C. Reduction of XTT to formazan by viable tumor cells was monitored colorimetrically at an absorbance wavelength of 450 nm and a reference wavelength of 630 nm using an ELISA plate reader Elisareader ELx808 (Bio-TEK, Bad Friedrichsahll, Germany).

(41) Caspase-3/7 activity in cell lysates was determined using the Apo-ONE Caspase-3/7 assay (Promega, Mannheim, Germany) 24 h after light activation. Briefly, 100 l of Apo-ONE reagent was added to the cells, and they were incubated for 6 h before fluorescence readings were taken with an ELISA plate reader Elisareader ELx808 (Bio-TEK, Bad Friedrichsahll, Germany) using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The concentration of ROS was determined by measuring the 485/535 nm fluorescence ratio of H2DCFDA (Invitrogen, Darmstadt, Germany). Briefly, 210.sup.4 cells were incubated in the presence of 50 nM Ce6 or 200 nM scFv-425-SNAP-Ce6 and 10 M H.sub.2DCFDA for 30 min in PBS containing 1% FCS. The cells were washed twice with warm PBS containing 2.5% FCS, cultured for 2 h in RPMI-160 medium and illuminated as described above. Fluorescence readings were taken directly after illumination. A blank probe (cells and medium) reading was used as the background and subtracted from all the sample readings.

(42) Data Analysis

(43) Statistical analysis and curve fitting were performed with GraphPad Prism software (GraphPad, San Siego, Calif.). Data are presented as the meanMES. Student's t test and two-way analysis of variance were used to assess the significance of independent experiments. The criterion p<0.05 was used to determine statistical significance.

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