Patent Application: US-201214381386-A

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 , a modified human dna repair protein .

Description:
the present invention is further exemplary described in greater detail using ce6 as photosensitizer and tyrosine kinase growth factor receptor of the erbb family as the binding . fig1 : 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 κ leader sequence ( ig - κ - l ) facilitates protein secretion , and a tga stop codon is placed immediately after the c - terminal his 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 μl of eluted scfv - 425 - snap with 250 mm imidazol , 2 : 1 . 5 μl of eluted scfv - 425 - snap with 250 mm imidazol , 3 : 10 μl 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 + a431 ( d ) and egfr − l540 cells ( e ). filled gray curves represent untreated cells . cells were incubated with 0 . 5 μg / 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 ). fig2 : analysis of ce6 photosensitizer by mass spectrometry before and after modification with benzylguanine ( bg ). ( a ) esi mass spectrum of ce6 , bg - peg24 - nh 2 , and bg - peg24 - ce6 . the top panel represents ce6 ( 597 . 215 da ), the middle panel represents bg - peg24 - nh 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 ). fig3 : the binding activities of scfv - 425 - snap - vistagreen and scfv - 425 - snap - ce6 , which specifically recognize egfr + cells . flow cytometry analysis was carried out after incubating 4 × 10 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 ). fig4 : internalization of fusion proteins analyzed by confocal microscopy . confocal images were obtained for the egfr + cell lines a431 , mda - mb - 468 , mda - mb - 231 and siha , and for the egfr − cell lines l540 and cho - k1 incubated with 0 . 5 μg 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 . fig5 : 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 (▪), mda - mb - 468 (▴), mda - mb - 231 (♦), siha () and cho - k1 (▾) 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 . 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 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 13 , and the presence of carboxyl groups allows further functionalization 5 . 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 . 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 + 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 . 3 × 10 6 receptors / cell ) 14 , 15 . the other cells lines expressed less egfr ( 1 . 3 × 10 5 receptors / cell for mda - mb - 231 and 2 × 10 4 - 2 × 10 5 receptors / cell for siha ) 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 + cells not only those with the highest expression levels . no toxicity was observed when egfr − cells ( cho - k1 ) were exposed to scfv - 425 - snap - ce6 . 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 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 . the coding sequences for the egfr - specific scfv - 425 antibody fragment 10 and a control fragment ( scfv - ki4 ) 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 ( fig1 a ). 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 6 tag ) and the final yield was 18 mg / l of protein in the supernatant ( fig1 b ). 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 ( fig1 c ). 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 5 alexa 488 antibody . flow cytometry data confirmed the rapid and efficient binding of scfv - 425 - snap specifically to egfr + target cells ( fig1 d ), whereas scfv - ki4 / snap bound only to the cd30 + l540 cells ( fig1 e ). 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 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 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 ( fig2 a ) 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 ( fig2 b , c ). 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 ( fig3 ). 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 ( fig4 a ). 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 . ( fig4 b ). in contrast , no signal was detected when the egfr − cell lines l540 and cho - k1 were incubated with scfv - 425 - snap - ce6 under the same conditions ( fig4 a , b ). 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 + 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 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 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 ( fig5 a , c ). 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 ( fig5 b , d ). 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 ( fig5 e ). 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 2 dcfda ). 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 ( fig5 f ). all cell lines were of human origin , including the egfr + a431 , mda - mb - 231 , mda - mb468 and siha cells , and the egfr − 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 2 atmosphere . all media and additives were obtained from invitrogen , darmstadt , germany . 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 6 tag sequence . his 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 2 po 4 , 1 . 2 m nacl , 40 mm imidazole , ph 8 ). bound his - tagged proteins were eluted in 50 mm nah 2 po 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 . 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 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 μm ( 4 . 6 × 50 mm ) water xbridge ™ ostc 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 μl 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 . 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 , münchen , germany ) or a cri maestro imaging system ( cri , woburn , mass ., usa ) using the blue and yellow filter sets . the binding efficiency of the labeled and unlabeled fusion proteins was determined by flow cytometry using a facscalibur ( becton & amp ; dickinson , heidelberg , germany ) and cellquest software . egfr + cell lines a431 , mda - mb - 231 , mda - mb468 and siha were used to test the binding efficiency of scfv - 425 - snap , and egfr − 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 4 × 10 5 cells were incubated in 200 μl pbs containing 0 . 5 μg 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 . 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 . aliquots of a431 , mda - mb - 231 , mda - mb468 , siha and cho - k1 cells ( 2 × 10 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 μg / ml zeocin instead of the photosensitizer . the cells were then irradiated with 24 j / cm 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 , müllheim , germany ) and incubated for a further 24 h at 37 ° c . in a 5 % co 2 atmosphere . 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 ). 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 μl 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 , 2 × 10 4 cells were incubated in the presence of 50 nm ce6 or 200 nm scfv - 425 - snap - ce6 and 10 μm h 2 dcfda 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 . statistical analysis and curve fitting were performed with graphpad prism software ( graphpad , san siego , calif .). data are presented as the mean ± mes . student &# 39 ; s t test and two - 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