Patent Application: US-201515512276-A

Abstract:
there is provided a novel conjugate that binds to the cell surface receptor upa . the conjugate is based on a fluorescence - labeled peptide useful as a diagnostic probe to the surfaces of cells expressing upar . the conjugate is capable of carrying a suitable detectable and imageable label that will allow qualitative detection and also quantitation of upar levels in vitro and in vivo . this renders the surgical resection of tumors more optimal .

Description:
concerning the synthesis of the peptides used in the present invention reference is made to u . s . pat . no . 7 , 026 , 282 . the peptide / chelate conjugates of the invention are labelled by reacting the conjugate with radionuclide , e . g . as a metal salt , preferably water soluble . the reaction is carried out by known methods in the art . the peptide ae105 ( asp - cha - phe - ser - arg - tyr - leu - trp - ser - oh ) was synthesized by standard solid - phase peptide chemistry . the peptide ae105 was conjugated to icg ( 4 -( 2 -(( 1e , 3e , 5e , 7z )- 7 -( 3 ( 5 - carboxypentyl )- 1 , 1 - dimethyl - 1h - benzo [ e ] indol - 2 ( 3 )- ydlidene ) hepta - 1 , 3 , 5 - trienyl )- 1 , 1dimethyl - 1h - benzo -[ e ] indolium - 3 - yl ) butane - 1 - sulfonate ) with two glutamic acids as linker ( icg - glu - glu - ae105 ); see fig1 . the probe has a final weight of 2197 . 55 g / mol . for in vivo injection icg - glu - glu - ae105 was dissolved in ( 2 - hydroxypropyl )- β - cyclodextrin with 2 % dsmo . human glioblastoma cell line u87mg was purchased from the american type culture collection and culture media was obtained from invitrogen . u87mg was cultured in dmem added 10 % fbs and 1 % penstrep . when the cells reached 70 - 80 % confluency the cells were harvested . all animal experiments were performed under a protocol approved by the animal research committee of the danish ministry of justice . 5 * 10 6 u87mg cells were suspended in 200 ul pbs and inoculated on both flanks of the mouse . when the tumours reached an appropriate size the mice were imaged with ae105 - glu - glu - icg . after harvesting of cells were washed in buffer and stained with either an in - house produced antibody ( 3 μg / ml ), igg isotype ( 3 g / ml ; 14 - 4714 ebioscience ) or blank control for 1 hr in 4 ° c . on a shaking table . the cells were washed 3 times with buffer and then stained with a secondary antibody ( goat - anti - mouse - pe 1 / 500 ;) for 30 min in 4 ° c . on a shaking table . the result was analysed on the bd facscanto cell analyser . tumours were homogenised and a suspension containing the tumor lysate were stored at − 80 ° c . the plate was coated with an anti upar antibody r2 ( 3 μg / ml ) overnight at 4 ° c . after this incubation 2 % bsa was added for 5 min and the plate was washed with buffer . upar standard ( 10 ng / ml ) or tumor lysate ( diluted 1 : 20 ) was added and incubated for 2 hr in rt and washed with buffer . a primary antibody ( rabbit - anti - upar , 1 μg / ml ) was added to the well and incubated for 30 min in rt and washed . a secondary hrp conjugated anti - rabbit antibody was added ( diluted 1 : 2500 ) and incubated for 30 min in rt and washed . the bound hrp conjugated antibody was quantified by adding 4 opd tablets ( dako # s2045 ) in 12 ml water and 10 μl h 2 o 2 . the reaction was stopped with 1m h 2 so 4 when the proper coloration of the well was present . an elisa reader was used to analyze the plate at 490 nm and 650 nm as reference . the mice were injected with 10 nmol of ae105 - glu - glu - icg or icg i . v ., and imaged 15 hr post injection . before scan the mice were anaesthetized with 2 % isofluran and positioned in a prone position . for imaging the ivis lumina xr and the acquisition software living image were used . the excitation filter was set to 710 nm and the emission filter was set in the icg position . acquisition was set to auto - settings to achieve the best acquisition as possible . after imaging with ivis lumina xr the mouse were moved to a fluobeam setup and imaged with appropriate acquisition time . the tbr values were calculated by drawing a roi over each tumor and place the background roi in an area with constant background signal . in the production of the novel upar targeted fluorescence probe of the present invention two glutamic acids were introduced as linkers to partly reduce a potential interaction between icg and the binding affinity of ae105 toward upar . the results indeed revealed a reduction in the binding affinity towards purified upar for icg - glu - glu - ae105 ( ic 50 ≈ 80 nm ) compared to ae105 ( ic 50 ≈ 10 nm ), however the probe surprisingly retained sufficient affinity for guided surgical procedures . before any in vivo experiments were initiated , with u87mg cancer cells the expression of upar was measured in vitro by flowcytometry . the staining with rabbit - anti - upar showed a clear rightshift in fluorescence compared to the control , thus confirming high level of upar expression ( fig2 a ). the expression of upar was also investigated on histological samples from tumors grown for 5 weeks in vivo using ihc staining ( fig2 b ). an intense staining for upar expression was found , thus confirming the result from flowcytometry . a group of mice were scanned 15 hr post injection with icg - glu - glu - ae105 in the ivis lumina xr . a high uptake in the tumor was observed ( fig3 ) and quantitative analysis of the tumor and background uptake , resulted in a tumor - to - background ( tbr ) ratio of 3 . 52 ± 0 . 167 ( n = 10 ) ( fig4 a ). the max radiance for the tumors was in the range 3 . 43e + 08 ± 0 . 34e + 08 radiance efficiency . next , a group of mice were imaged with only icg in order to validate the specificity of the new probe . no specific uptake was seen in the tumor . tbr for icg was 1 . 04 ± 0 . 04 ( n = 10 ) ( the max radiance for the tumors were in the range 7 . 51e + 06 ± 3 . 13e + 05 ). all tumors from both groups of mice were subsequently resected after the last scan and the upar expression in the tumor lysate was analysed . upar expression level was identical in each group ( 3 . 19 ± 0 . 59 for icg and 2 . 64 ± 0 . 28 for icg - glu - glu - ae105 ) ( fig4 a ). finally , to delineate the translational use of this method , the group of mice injected with icg - glu - glu - ae105 was also imaged with the clinically approved camera fluobeam ® ( fig5 ). clear tumor identification was possible due to high uptake of icg - glu - glu - ae105 as seen in fig5 . this imaging modality gave similar tbr ( 3 . 58 ± 0 . 29 .) as the ivis lumina xr and thus confirms the translational potential of icg - glu - glu - ae105 . intraoperative optical imaging with nir is a new emerging technique that can help surgeons remove solid tumours with higher accuracy and decrease the number of patient with positive margins . in this study , the newly synthesised probe icg - glu - glu - ae105 was characterized in vitro and in vivo in a human glioblastoma xenograft mouse model . many designs of optical probes have been constructed . several groups have investigated probes targeting the egfr receptor [ 9 ], integrin α v β 3 [ 10 ] and her1 and her2 [ 11 ]. numerous probes are based on antibodies as targeting vectors because of the ease of conjugating them to fluorophors and the well - known high affinity for the target . however , a number of limitations in using antibodies for in vivo optical imaging are present . the size of an antibody influences the pharmacological profile , and result in a long plasma half - life which again results in a high background and decrease the potential tbr value . an acceptable tbr value is therefore only achievable 1 - 3 days after injection [ 9 , 12 ], thus limiting the clinical usefulness and thereby the translation potential . if smaller peptides are used an optimal imaging timepoint can get as low as 3 - 6 hours after injection as a result of faster clearing time . in the present study , a scan time 15 hrs post injection was found to be optimal for the peptide - based probe , thus providing a clinical useful application where a patient would be injected in the evening before planned surgery the next day . the conjugated fluorophor is also an important choice to make . there exist numerous fluorophors in the nir window with different properties . it was chosen to use icg since it is the most often - used fluorophor because of its long history in angiographies , it is fda approved and has a well - established safety profile , thus paving the way for a more easy clinical translation . the fluorescent properties of icg has been passed by other upcoming fluorophors such as irdye 800cw . this newer developed fluorophor exhibit features as higher brightness , easier conjugation and hydrophilicity . especially the hydrophobicity of icg seems to be an important feature considering the reduction in binding affinity found in this study due to conjugation of icg , where both the size and high hydrophobicity seems to be responsible for this observation . one potential solution to this observation could be to use a longer linker and / or a more hydrophilic linker such as peg . this approach has been done with success by others [ 13 ]. however , the limited safety profile and no clinical data for irdye 800cw in contrast to icg , makes any clinical translation difficult in near future . translation of a new probe from preclinical studies to the clinical bed is with an approved fluorophor as icg more advantageous . however the linker is not only for protection of the peptide . several studies [ 13 ] have shown that conjugation of icg to an antibody decrease the fluorescent signal from icg . a comparison of icg and icg - glu - glu - ae105 showed a 2 - fold decrease in fluorescence intensity for the conjugated probe ( data not shown ). a group have though shown that quenching of icg is eliminated when the probe interact with cells [ 11 ], due to internalization and degradation of the conjugated vector . the icg molecule is released and de - quenched . this property can be exploited in vivo where the non - internalized circulating probe has lower fluorescence intensity than the targeted internalized probe . icg have primarily been used for delineating malignant glioblastomas . however , icg has only been used in excessive doses were macroscopic colouration of the tissue have delineated the tumour and the fluorescent properties have been neglected . further , this delineation of the tumour is most likely a result of the epr effect and not a tumour specific accumulation . several targets for optical imaging in cancer detection have been investigated and both endogenous and exogenous fluorophors has shown great potential for clinical translation . conversion of 5 - ala to ppix , an endogenous fluorescent process , has been shown to occur in excess in glioblastomas and have reached clinical studies with convincing results . an advantage upar , as target , holds over 5 - ala is the information given regarding the tumors phenotype . upar has been correlated with a poor prognosis and aggressive metastatic behavior . further upar have shown to be expressed in the invasive front of the tumor and in the surrounding stroma . this makes upar an ideal target for nir intraoperative optical resection of solid tumors . in addition the receptor need to be over expressed on the surface of the cancer cells . this has been confirmed by flowcytometry for the glioblastoma cell line used in this human xenograft model . the main aim was to develop a targeted icg probe , with high affinity and specificity towards upar and high in vivo stability . results from this study have shown that the newly developed probe icg - glu - glu - ae105 possesses all these properties . conjugated to the clinical approved fluorophor icg the use of this probe in intra - 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