Patent Application: US-79705210-A

Abstract:
disclosed are antibodies that bind to epithelial cell adhesion molecule and display certain advantages over known antibodies which bind to epcam , for example , the antibodies of the invention show good affinity , good cross - reactivity profiles and excellent adcc and cdcc activity . antibodies comprising specific heavy and light chain cdrs are disclosed . the invention thus relates to these antibodies and all uses thereof , in particular in the treatment of cancer . the present invention thus provides new antibody - based compositions , methods and combined protocols for treating cancer . advantageous immunoconjugate compositions and methods using the new anti - epcam antibodies are also provided .

Description:
the invention will now be described in more detail in the following non - limited examples with reference to the figures in which : fig1 shows the nucleotide and amino acid sequences of the heavy ( vh ) and light ( vl ) chain variable region of an scfv form of clone 3 - 171 . the scfv genes were cloned via nco / noti site into phog21 plasmid vector ( 3 . 7 kb ). the restriction sites used for initial gene cloning ( ncoi , hindiii , miui and noti ) are underlined . the linker sequence between vh and vl is shown in italics . fig2 shows flow cytometric analysis of 3 - 17i igg , moc31 igg and mt201 igg binding to the naturally epcam + kato iii cell line . anti - green fluorescent protein ( gfp ) antibody is used as a negative control . mfi = median fluorescence intensity . fig3 a , 3 b and 3 c show western blot binding analysis of 3 - 17i igg ( fig3 a ), mt201 igg ( fig3 b ) and moc31 igg ( fig3 c ) binding to human ( hu ) and cynomolgus ( cy ) epcam - fc variants under non - reducing and reducing conditions . m = molecular weight markers . fig4 a , 4 b and 4 c show elisa binding analysis of 3 - 17i igg ( fig4 a ), mt201 igg ( fig4 b ) and moc31 igg ( fig4 c ) to human and cynomolgus epcam - fc variants . maxi - sorb plates were coated with human and cynomolgus epcam - fc at a concentration of 1 μg / ml . the antibody was added in 2 - fold dilutions from 133 nm to 32 fm . fig5 a and 5b show biacore analysis of 3 - 17i igg binding to recombinant human epcam antigen ( fig5 a ) or to recombinant cynomolgus epcam antigen ( fig5 b ). the shown binding curves correspond to 3 - 17i igg concentrations of 7 . 8 , 3 . 9 , 2 . 0 , 0 . 98 , 0 . 49 and 0 . 24 nm . fig6 a , 6 b and 6 c show that 3 - 17i igg induces adcc in mda - mb - 453 ( fig6 a ), mda - mb - 231 ( fig6 b ) and bt - 474 ( fig6 c ) cells in the presence of human pbmcs . furthermore , these data demonstrate that 3 - 1711 g possesses superior adcc activity over the positive control antibody , mt201 igg . fig7 a and 7b show that 3 - 17i igg induces cdc in the cell lines kato iii ( fig7 a ) and mt - 3 ( fig7 b ) in the presence of human serum . furthermore , these data clearly demonstrate the superiority of 3 - 17i igg over positive control antibody , mt201 igg , in inducing cdc . fig8 a and 8b show the scfv expression vector . fig8 a shows the scfv expression vector phog21 . apr , ampicillin resistance gene ; coie1 , origin of dna replication ; fiig , intergenic region of phage f1 ; c - myc , sequence encoding the epitope recognized by the monoclonal antibody 9e10 ; his 6 , sequence encoding six histidine residues ; peib , sequence encoding signal peptide of bacterial pectate lyase ; p / o , wild type iac promoter operator . fig8 b shows the nucleotide and amino acid sequences of the c - terminal coding region . fig9 shows elisa binding analysis of 12 - c15 igg , 16 - g5 igg and 17 - c20 igg to human epcam - fc . maxi - sorb plates were coated with human epcam - fc at a concentration of 1 μg / ml . the antibody was added in 2 - fold dilutions from 133 nm to 65 μm . fig1 a and 10b show elisa binding analysis of 3 - 17i scfv , 7 - f17 scfv , 17 - c20 scfv and 24 - g6 scfv to human epcam - fc ( fig1 a ) and to cynomolgus epcam - fc ( fig1 b ). maxi - sorb plates were coated with human or cynomolgus epcam - fc at a concentration of 5 μg / ml . the antibody was added in 2 - fold dilutions from 667 nm to 3 . 2 nm . given the need for further tumor specific antibodies which can be used as cancer therapeutics , a human antibody has been identified which specifically recognizes colon cancer cell lines such as ht29 . the antibody can specifically bind to epcam . a single chain form of the antibody was cloned in the phog21 plasmid ( fig8 ) which contains a c - myc and 6 × his tag epitopes . tg1 bacteria were transformed and the scfv was expressed upon iptg induction . the binding of the purified scfv was confirmed by flow cytometry using an easycyte flow cytometer . the nucleotide sequences of the heavy and light chain of the antibody producing clone were sequenced . the antibody is designated as 3 - 17i ( scfv ). the nucleotide sequence and amino acid sequence of the light and heavy chain of 3 - 17i ( scfv ) are shown in fig1 and table 1 . the cdr regions of the light and heavy chains of 3 - 171 are shown in table 1 . the igg form of this antibody has also been made . the igg form is of the igg1 isotype and it comprises two heavy chains and two light chains . each heavy chain comprises a vh domain of seq id no : 3 and an igg1 constant region . each light chain comprises a vl domain of seq id no : 4 and a kappa light constant region . the components of the igg form of the antibody were cloned into vectors based on a vector published by lars norderhaug et al , jim 204 ( 1997 ) 77 - 87 and using the method described in this citation . the vector contains a standard cmv promoter . an igg leader sequence ( mgwsciilflvatatgvhs ) was introduced into each vector . the vh and vl domains were cloned into separate vectors containing genomic copies ( introns + exons ) of the human igg1 and kappa genes , respectively into bsmi and bsiwi sites which had been introduced by pcr . the igg1 vector contains a hygromycin resistance gene , whereas the kappa vector contains a neomycin resistance gene . hek293 / t cells were transiently transfected and after 5 - 6 days , the igg was purified via protein - a followed by size exclusion to isolate the monomeric igg fraction . the amino acid sequences of the complete heavy and light chains of the 3 - 17i igg antibody are shown below . twelve related antibodies which bind to epcam and which have an identical heavy chain sequence to 3 - 17i but a different light chain sequence were also identified . the sequences of five of these related antibodies ( 7 - f17 , 12 - c15 , 16 - g5 , 17 - c20 and 24 - g6 ) are set out in tables 2 - 6 . flow cytometric analysis was performed to analyze binding of the 3 - 17i igg to epcam - positive cells . as positive controls , anti - epcam chimeric ( mouse variable / human constant domains ) and fully human antibodies , moc31 and mt201 , respectively , were used . the amino acid sequences of the complete heavy and light chains of the mt201 igg and moc31 igg antibodies used are shown below . the constant regions are in bold italics . these sequences were deduced from the amino acid sequences for mt201 , which is also referred to as hd69 , as given in wo98 / 46645 and raum et al ., cancer immunol immunother ( 2001 ) 50 : 141 - 150 . these sequences were deduced from the moc31 amino acid sequence as given in beiboer et al ., journal of molecular biology , volume 296 , issue 3 , 25 feb . 2000 , pages 833 - 849 . the anti - green fluorescent protein ( gfp ) antibody was used as a negative control . for flow cytometry , kato iii cells ( atcc number ( gastric carcinoma , atcc number htb - 103 )) were grown under standard conditions , harvested from the culture flasks , washed 2 times with pbs , re - suspended in pbs with 0 . 2 % bsa and 0 . 09 % nan 3 and finally distributed at 1 × 10 5 cells per well into v - shaped 96 - well plates ( greiner bio - one , frickenhausen , germany ). cells were centrifuged at 400 × g for 5 min and then incubated at 4 ° c . for 45 min with 50 μl of each different antibody dilution ( all dilutions made in pbs with 0 . 2 % bsa and 0 . 09 % nan 3 ). after washing in pbs with 0 . 2 % bsa and 0 . 09 % nan 3 , the cells were stained with 10 μg / ml of rpe - conjugated goat anti - human igg ( abdserotec , düsseldorf , germany ) for 30 min at 4 ° c . the stained cells were washed , re - suspended in 200 μl pbs with 0 . 2 % bsa and 0 . 09 % nan 3 and transferred to a u - shaped 96 - well plate ( corning , schiphol - rijk , the netherlands ) for analysis on easycyte flow cytometer ( guava technologies , hayward , calif ., usa ). the results shown in fig2 clearly demonstrate that the antibody 3 - 17i specifically interacts with the naturally epcam + kato iii cell line with an affinity comparable to that of moc31 . in contrast , the antibody mt201 demonstrated significantly weaker binding to the epcam - positive kato iii cells . to determine the ability of 3 - 17i to bind to epcam , western blot analysis , elisa and biacore assays were performed . to analyze the binding specificity and cross - reactivity of antibodies 3 - 17i , mt201 and moc31 to human and cynomolgus epcam , sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) and western blot analysis were performed under reducing and non - reducing conditions . all the antibodies were in igg format and were produced in transiently transfected hek - 293 cells . the antibodies were then purified on protein a sepharose ( ge healthcare , uppsala , sweden ) followed by fractionation on two serial coupled size exclusion columns , superdex 200 and superdex 75 . the resultant igg preparations appeared to be monomeric with & gt ; 95 % purity . the recombinant human and cynomolgus epcam antigens were also produced in hek - 293 cells and purified in one step on a protein a sepharose column . the determined purity of the antigen preparation was above 90 %. both epcam constructs consist of the extracellular domains of epcam fused to a human fc region from igg1 . the human epcam sequence was derived from ncbi databse np - 002345 and the cynomolgus from cs611076 . the amino acid sequences of the human epcam / fc fusion and the cynomolgus epcam / fc fusion are shown below : the samples of the recombinant human and cynomolgus epcam - fc antigens were either reduced or non - reduced before loading on sds - paa gels ( 1 μg / well ). the samples were run in duplicate . after electroblotting , the membranes were blocked with 5 % skimmed milk powder ( merck product no 1 . 15363 . 0500 ) in pbs ( block solution ) for 1 hr . the membrane was then incubated with the iggs 3 - 17i ( fig3 a ), mt201 ( fig3 b ) or moc 31 ( fig3 c ) at a concentration of 1 μg / ml diluted in block solution . to detect the membrane bound igg , the filter was incubated for 1 hr with a goat anti - human kappa - hrp antibody ( southern biotech , cat no 2060 - 05 ) diluted 1 : 5000 in block solution . between all incubations the membrane was washed with pbs - t ( pbs supplemented with 0 . 05 % tween - 20 ; medicago # 09 - 9410 - 100 ) 3 times for 5 min . all incubations were performed at room temperature for 1 hr if not otherwise indicated . the western blots were developed using dab ( thermo scientific # 1856090 ) substrate diluted according to the recommendations of the manufacturer . for control , an sds - paa gel was stained with coomassie instant blue ( expedeon # isbo1 l ), to verify the loaded amounts of the epcam variants . the successful transfer to the membrane was verified by detecting the epcam - fc on the membrane with a goat anti - human igg - hrp conjugate ( southern biotech cat no 2040 - 05 ) and dab staining ( data not shown ). the expected sizes for epcam - fc were approximately 110 kda and 55 kda under non - reducing and reducing conditions , respectively . western blot analysis demonstrated that antibody 3 - 17i bound equally well to both human and cynomolgus epcam under non - reducing conditions . in contrast , no binding of 3 - 17i igg was observed to any reduced epcam antigen . the antibody moc31 demonstrated different antigen - binding patterns . unlike 3 - 17i igg , it bound human epcam both under non - reducing and reducing conditions . it also recognized the cynomolgus antigen but only under non - reducing conditions . in addition , the weaker band intensity of the cynomolgus antigen staining indicated weaker binding to cynomolgus epcam than to the human antigen . the antibody mt201 igg only demonstrated binding to the human epcam under non - reducing conditions . no binding of this antibody to the cynomolgus antigen was detected . these results suggest that both moc31 and mt201 bind to different epitopes on human epcam than 3 - 171 . this evidence is particularly strong when comparing 3 - 171 and moc - 31 . to determine the specificity and affinity of 3 - 17i , mt201 and moc31 to human and cynomolgus epcam , a set of elisa experiments was performed . all antibodies in igg1 format as well as the human and cynomolgus epcam antigens were produced and purified as previously indicated ( see examples 1 and 2 and earlier in example 3 ). the specificity of antibody clones 12 - c15 igg , 16 - g5 igg and 17 - c20 igg for human epcam were determined in elisa experiments . the specificity and affinity of antibody clones 7 - f17 , 17 - c20 and 24 - g6 in scfv format for human and cynomolgus epcam were determined in elisa experiments . for elisa of antibodies in igg format , the maxi - sorb plate was coated with 1 μg / ml ( 100 μl per well ) of human or cynomolgus epcam - fc in pbs overnight at 4 ° c . the plates were blocked with 3 % bsa in pbs for 2 hrs . the iggs to be tested were added in 2 - fold dilutions from 133 nm to 32 fm and incubated for 1 hr at room temperature . for detection of bound igg , a goat anti - human kappa - hrp antibody ( southern biotech , cat no 2060 - 05 ) was added in 1 : 5000 dilution in block solution and incubated for 1 hr at room temperature . for elisa of antibodies in scfv format , the maxi - sorb plate was coated with 5 μg / ml ( 100 μl per well ) of human or cynomolgus epcam - fc in pbs overnight at 4 ° c . the plates were blocked with 4 % skimmed milk ( merck product no 1 . 15363 . 0500 ) in pbs for 2 hrs . the scfvs to be tested were added in 2 - fold dilutions from 667 nm to 3 . 2 nm and incubated with 0 . 125 μg mouse anti - cmyc igg ( clone 9e10 diatec ) for 1 hr at room temperature . for detection of bound scfv , a rabbit anti - mouse - hrp antibody ( dako , cat no p0260 ) was added in 1 : 3000 dilution in block solution and incubated for 1 hr at room temperature . between all incubations the plates were washed with pbs - t ( 0 . 05 % tween - 20 , medicago # 09 - 9410 - 100 ) three times . all incubations were performed at room temperature for one hour unless otherwise indicated . the elisa of antibodies in igg format was developed using a 1 - step abts kit ( thermo scientific prod # 37615 ) and a 25 min incubation time . the absorbance was measured on a slt spectra plate reader . the software prism ( graphpad , san diego , calif .) was used to estimate the affinities using a non - linear fit of one - site model on the retrieved elisa data . the elisa of antibodies in scfv format was developed using a tmb substrate kit ( thermo scientific , prod # 34021 ) and a 10 min incubation time . the reaction was stopped by adding h2s04 . the absorbance was measured on a slt spectra plate reader . the software prism ( graphpad , san diego , calif .) was used to estimate the affinities using a non - linear fit of one - site model on the retrieved elisa data . the elisa data demonstrated comparably good binding of 3 - 17i igg to both human and cynomolgus epcam ( fig4 a ). in contrast , moc31 antibody showed a significant difference in binding patterns to human and cynomolgus antigens ( fig4 c ), thus indicating a significantly lower affinity to cynomolgus epcam . interestingly , the antibody mt201 showed only relatively weak binding to human epcam and no binding of this antibody to the cynomolgus antigen was detected ( fig4 b ). the igg format of antibody clones 12 - c15 , 16 - g5 and 17 - c20 also bound to human epcam ( fig9 ). the scfv format of antibody clones 7 - f17 , 17 - c20 and 24 - g6 bound to human and cynomolgus epcam and displayed affinities for both human and cynomolgus epcam which were comparable to the affinity of the scfv format of 3 - 17i for both human and cynomolgus epcam ( fig1 a and 10b ). binding of 3 - 17i to human and cynomolgus monkey epcam was analyzed using surface plasmon resonance ( spr ) on a biacore t100 ( biacore , inc , piscataway , n . j .). the extracellular domains of human or cynomolgus monkey epcam fused to human fc ( igg1 ), were coupled to a cm5 chip ( biacore , inc , piscataway , n . j .) at density of 100 ru . the spr studies were performed using standard techniques at 37 ° c . with a flow rate of 50 μl / min . the data were analyzed using the 1 : 1 langmuir binding model of the biacore software . the results from the kinetic analysis demonstrated that 3 - 17i igg bound both human and cynomolgus epcam with almost the same on - rates ( k on ) and off - rates ( k off ) values thus providing the equilibrium constants ( k d ) close to 1 nm ( see fig5 a and fig5 b and table 8 ). thus , the binding affinity of the 3 - 17i igg antibody of the invention as measured by biacore analysis is approximately 1 nm for binding to both human and monkey epcam . the binding affinity of moc31 igg for human epcam as measured by biacore is described in the art as being similar , i . e . k d = 3 nm ( roovers et al ., 1998 , brit . j . cancer , 78 : 1407 - 1416 ). it can however be seen from the elisa data above , that the binding affinity of moc31 igg for monkey epcam is significantly lower than that of 3 - 171 . the binding affinity of mt201 igg for human epcam as measured by biacore is described in the art as being much lower , i . e . k d = 175 nm ( naundorf et al ., 2002 , int . j . cancer , 100 : 101 - 110 ). functional assays were carried out in order to determine the ability of 3 - 17i igg to mediate target cell killing via adcc and / or cdc . results from these studies show that 3 - 17i igg does mediate adcc and cdc . in addition , these data clearly show the superiority of 3 - 17i igg over mt201 igg in inducing both adcc and cdc . the ability of 3 - 17i igg to induce adcc was analyzed using three different breast cancer cell lines mda - mb - 231 , mda - 453 and bt - 474 which cover a range of more than 100 - fold difference in surface density of epcam ( prang et al ., 2005 ). mt201 igg was used as a positive control antibody as it was reported by prang et al to induce adcc on all these cell lines . the mda - mb - 453 ( breast mammary gland , atcc number htb - 131 ), mda - mb - 231 ( breast mammary gland , atcc number htb - 26 ) and bt - 474 ( breast mammary gland , atcc number htb - 20 ) cell lines were obtained from the american type culture collection ( atcc , rockville , md .). the mda - mb - 453 and bt - 474 cells were maintained in rpmi - 1640 culture medium and the mda - mb - 231 cells were maintained in leibovitz &# 39 ; s culture medium . all media were supplemented with 10 % fetal calf serum ( fcs ), penicillin and streptomycin . all cell media and supplements were obtained from paa ( pasching , austria ). the target cells , cultivated under regular conditions , were harvested by trypsin - edta , sedimented by centrifugation and resuspended twice in rpmi - 1640 culture medium . 1 ml containing 2 . 5 × 10 6 cells was mixed with calcein - am ( invitrogen , carlsbad , calif .) to a final concentration of 10 μm and then incubated at 37 ° c . for 30 min on a vertical rotating wheel ( 7 rpm ). the cells were washed three times in rpmi - 1640 with 10 % fcs and the cell density was adjusted to 3 × 10 5 per ml . the peripheral blood mononuclear cells ( pbmc ) were prepared from blood of a single healthy volunteer by ficoll - hypaque gradient centrifugation . the isolated pbmc were washed in rpmi - 1640 with 10 % fcs and resuspended at 6 × 10 6 cells per ml . 50 μl of each target and effector cell suspension were added to the same wells in a 96 - well microtiter plate providing a ratio of effector ( e ) to target ( t ) cells ( e : t ) of 20 : 1 . the antibody dilutions were added in a volume of 20 μl in quadruplicates for each concentration . the microtiter plate was then incubated for 4 hrs at 37 ° c ., and 20 μl 0 . 9 % tritonx - 100 was added to some of the wells after 3 hrs 45 minutes to achieve complete lysis of the target cells . 100 μl of the supernatant of each sample was then transferred to a black microtiter plate and the fluorescence ( excitation : 488 nm , emission : 518 nm ) was analyzed in a tecan m200 plate reader . the fluorescence intensity in the samples with no antibodies was subtracted from the intensity of all other samples . the percentage of lysis in samples with antibodies was estimated on the basis of fluorescence intensity in the samples with 100 % cell lysis after treatment with tritonx - 100 . the dose - response curves were generated by nonlinear regression analysis using a three - parameter fit model of software prism ( graphpad , san diego , calif ., usa ). the results shown in fig6 a , 6 b and 6 c clearly demonstrate that 3 - 17i igg induces adcc in all the three cell lines mda - mb - 453 ( fig6 a ), mda - mb - 231 ( fig6 b ) and bt - 474 ( fig6 c ) in the presence of human pbmcs . ec 50 values were estimated to be 0 . 08 ng / ml , 15 ng / ml and 0 . 12 ng / ml for these cell lines , respectively . the achieved maximum killing was 75 %, 92 % and 61 %, respectively . the control , antibody mt201 , demonstrated much inferior adcc activity with ec 50 values of 7 ng / ml and 382 ng / ml and maximum killing of 56 % and 33 % for the cell lines mda - mb - 453 and bt - 474 , respectively . no killing of mda - mb - 231 cells with lowest level of epcam expression was observed for antibody mt201 . these data clearly show the superiority of 3 - 17i igg over mt201 igg in inducing adcc . the ability of 3 - 17i igg to induce complement dependent cytotoxicity ( cdc ) was analyzed using two cell lines kato iii and mt - 3 . three related antibody clones , 12 - c15 igg , 16 - g5 igg and 17 - c20 igg were also tested for their ability to induce cdc using the kato iii cell line . mt201 igg was used as a positive control antibody as it was reported by prang et al ( 2005 ) to induce cdc on these cell lines . kato iii ( gastric carcinoma , atcc number htb - 103 ) and mt - 3 ( breast carcinoma , dsmz number acc 403 ) cell lines were obtained from the american type culture collection ( atcc , rockville , md .) and dsmz ( braunschweig , germany ), respectively . the kato iii and mt - 3 cells were maintained in rpmi - 1640 culture medium with the addition of fetal calf serum ( fcs ) at 20 % and 10 %, respectively . all media were supplemented with penicillin and streptomycin . all cell media and supplements were obtained from paa ( pasching , austria ). the target cells cultivated under regular conditions were sedimented by centrifugation and resuspended twice in rpmi - 1640 culture medium . 5 ml containing 12 . 5 × 10 6 cells were mixed with calcein - am ( invitrogen , carlsbad , calif .) to a final concentration of 10 μm and then incubated at 37 ° c . for 30 min on a vertical rotating wheel ( 7 rpm ). the cells were washed three times in rpmi - 1640 with 10 % heat inactivated fetal calf serum ( hifcs ) and the cell density was adjusted to 4 × 10 6 / ml . 25 μl of target cell suspension was mixed with 25 μl human serum and 50 μl antibody dilutions in rpmi - 1640 with 10 % hifcs . for experiments using the 3 - 17i igg antibody , the final antibody concentrations in the wells ranged from 0 . 8 ng / ml to 50 μg / ml . for experiments using antibody clones 12 - c15 igg , 16 - g5 igg and 17 - c20 igg , the final antibody concentration in the wells ranged from 0 . 19 μg / ml to 1 . 5 μg / ml . the assays were performed in quadruplicate . 20 μl of 0 . 9 % tritonx - 100 was added to some wells to achieve complete lysis of the target cells and the plate was then incubated for one hour at 37 ° c . 100 μl of rpmi - 1640 with 10 % hifcs was added to the wells to increase the volume and the cells were then sedimented by centrifugation . 100 μl of the supernatant was transferred to a black microtiter plate and the fluorescence ( excitation : 488 nm , emission : 518 nm ) was analyzed using tecan m200 plate reader . the fluorescence intensity in the samples with no antibodies was subtracted from the intensity of all other samples . the percentage of lysis in samples with antibodies was estimated on the basis of fluorescence intensity in the samples with 100 % cell lysis after treatment with tritonx - 100 . the dose - response curves were generated by nonlinear regression analysis using a three - parameter fit model of software prism ( graphpad , san diego , calif ., usa ). the results shown in fig7 a and 7b clearly demonstrate that 3 - 17i igg induces cdc in the cell lines kato iii ( fig7 a ) and mt - 3 ( fig7 b ) in the presence of human serum . cdc activity was also demonstrated for antibody clones 12 - 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