Patent Application: US-201415033568-A

Abstract:
a glycan having the structure galβ1 - 3glcnacβ1 - 3galβ1 - 4glcnac which is attached to a lipid or protein backbone , and isolated binding members capable of binding thereto .

Description:
the invention will now be described further in the following non - limiting examples and accompanying drawings . 1 × 10 5 cancer cells were incubated with 50 μl of primary antibodies at 4 ° c . for 1 hr . cells were washed with 200 μl of rpmi 10 % new born calf serum ( nbcs : sigma , poole , uk ) and spun at 1 , 000 rpm for 5 min . supernatant was discarded and 50 μl of fitc conjugated anti - mouse igg fc specific mab ( sigma ; 1 / 100 in rpmi 10 % nbcs ) was used as secondary antibody . cells were incubated at 4 ° c . in dark for 1 hr then washed with 200 μl rpmi 10 % nbcs and spun at 1 , 000 rpm for 5 min . after discarding supernatant , 0 . 4 % formaldehyde was used to fix the cells . samples were analysed on a beckman coulter fc - 500 flow cytometer ( beckman coulter , high wycombe , uk ). to analyse and plot raw data , winmdi 2 . 9 software was used . 50 μl of healthy donor blood was incubated with 50 μl primary antibody at 4 ° c . for 1 hr . the blood was washed with 150 μl of rpmi 10 % nbcs and spun at 1 , 000 rpm for 5 min . supernatant was discarded and 50 μl fitc conjugated anti - mouse igg fc specific mab ( 1 / 100 in rpmi 10 % nbcs ) was used as the secondary antibody . cells were incubated at 4 ° c . in the dark for 1 hr then washed with 150 μl rpmi 10 % nbcs and spun at 1 , 000 rpm for 5 min . after discarding the supernatant , 50 μl / well cal - lyse ( invitrogen , paisley , uk ) was used followed by 500 μl / well distilled water to lyse red blood cells . the blood was subsequently spun at 1 , 000 rpm for 5 min . supernatant was discarded and 0 . 4 % formaldehyde was used to fix the cells . samples were analysed on a fc - 500 flow cytometer ( beckman coulter ). to analyse and plot raw data , winmdi 2 . 9 software was used . healthy donor erythrocytes were washed 3 times in pbs and then resuspended in 10 times the packed cell volume of pbs . 50 μl of washed erythrocytes were then incubated with 50 μl primary antibodies at 37 ° c . for 1 hr . cells were washed with 150 μl of pbs and spun at 2 , 000 rpm for 5 min . supernatant was discarded and cells resuspended in 50 μl fitc - conjugated anti - mouse igg fc - specific secondary antibody ( sigma , 1 / 100 dilution in pbs , 1 % bsa ). cells were incubated at 37 ° c . in the dark for 1 hr then washed with 150 μl pbs and spun at 2 , 000 rpm for 5 min . supernatant was discarded and cells were resuspended in 500 μl pbs . samples were analysed on a macsq flow cytometer ( miltenyi biotech , bisley , uk ) using macsq software . colo205 cell pellet ( 5 × 10 7 cells ) was resuspended in 500 μl of mannitol / hepes buffer ( 50 mm mannitol , 5 mm hepes , ph7 . 2 , both sigma ) and passed through 3 needles ( 23g , 25g , 27g ) each with 30 pulses . 5 μl of 1m cacl 2 was added to the cells and passed through 3 needles each with 30 pulses as above . sheared cells were incubated on ice for 20 min then spun at 3 , 000 g for 15 min at room temperature . supernatant was collected and spun at 48 , 000 g for 30 min at 4 ° c . and the supernatant was discarded . the pellet was resuspended in 1 ml methanol followed by 1 ml chloroform and incubated with rolling for 30 min at room temperature . the sample was then spun at 1 , 200 g for 10 min to remove precipitated protein . the supernatant , containing plasma membrane glycolipids , was collected and stored at − 20 ° c . lipid samples were blotted onto silica plates and developed in chloroform / methanol / distilled water ( 60 : 30 : 5 by volume ) twice followed by hexane : diethyl ether : acetic acid ( 80 : 20 : 1 . 5 by volume ) twice . the dried plates were sprayed with 0 . 1 % polyisobutylmethacrylate ( sigma ) in acetone . after drying in air , the plates were blocked with pbs 2 % bsa for 1 hr at room temperature . the plates were then incubated overnight at 4 ° c . with primary antibodies diluted in pbs 2 % bsa . the plates were then washed 3 times with pbs and incubated with biotin - conjugated anti - mouse igg fc specific secondary antibody ( sigma ) diluted 1 / 1000 in pbs 2 % bsa for 1 hr at room temperature . the plates were subsequently washed again in pbs before incubating with irdye 800cw streptavidin ( licor biosciences , cambridge , uk ) diluted 1 / 1000 in pbs 2 % bsa for 1 hr at room temperature in the dark . the plates were washed a further 3 times with pbs and air dried in the dark . lipid bands were visualized using a licor odyssey scanner . to clarify the fine specificities of the fg88 mabs further , the antibodies were fitc labelled and sent to the consortium for functional glycomics where they were screened against ≧ 600 natural and synthetic glycans . briefly , synthetic and mammalian glycans with amino linkers were printed onto n - hydroxysuccinimide ( nhs )- activated glass microscope slides , forming amide linkages . printed slides were incubated with 1 μg / ml of antibody for 1 hr before the binding was detected with alexa488 - conjugated goat anti - mouse igg . slides were then dried , scanned and the screening data compared to the consortium for functional glycomics database . surface plasmon resonance ( spr , biacore x , ge healthcare ) analysis was used to investigate real - time binding kinetics of the fg88 mabs . polyvalent le a - hsa was coupled onto a cm5 biosensor chip according to the manufacturer &# 39 ; s instructions and a reference cell was treated in a similar manner , but omitting the le a conjugate . fg88 mabs diluted in hbs - p buffer ( 10 mmol / l hepes , ph 7 . 4 , 150 mmol / l nacl , 0 . 005 % ( v / v ) surfactant p20 ) were run across the chip at a flow rate of 30 μl / min and biaevaluation software 4 . 1 was used to determine the kinetic binding parameters from which affinities are calculated . briefly , 1 × 10 5 or 10 6 cell equivalents of colo205 cell lysate , plasma membrane , total lipid extract , plasma membrane lipid extract or c170 cell lysates were analysed for fg88 binding . tumour cell total and plasma membrane lipid extracts and cell lysates were reduced with dithiothreitol ( dtt ; pierce biotechnology , thermofisher , loughborough , uk ) and subjected to sds - page using novex 4 % to 12 % bis - tris gels ( invitrogen ), and transferred to hybond - p pvdf membranes ( ge healthcare , amersham , uk ) using lx transfer buffer ( 20 ×, invitrogen ) and 20 % ( v / v ) methanol at 30v for 1 hr . membranes were blocked with 5 % ( w / v ) non - fat dry milk in 0 . 05 % ( v / v ) tween - pbs for 1 hr then probed with primary antibodies diluted in tween - pbs , 2 % bsa for 1 hr . primary antibody binding was detected using biotin - conjugated anti - mouse igg fc specific secondary antibody ( sigma ; 1 / 2000 dilution in tween - pbs , 2 % bsa ) for 1 hr , and visualized using irdye 800cw streptavidin ( licor biosciences , uk ; 1 / 1000 in tween - pbs 2 % bsa ). approximately 5 × 10 6 cells from hybridomas fg88 . 2 and fg88 . 7 were taken from tissue culture , washed once in pbs , and the cell pellet treated with 5000 trizol ( invitrogen ). after the cells had been dispersed in the reagent , they were stored at − 80 ° c . until rna was prepared following manufacturer &# 39 ; s protocol . rna concentration and purity were determined by nanodrop . prior to cdna synthesis , rna was dnase i treated to remove genomic dna contamination ( dnase i recombinant , rnase - free , roche diagnostics , burgess hill , uk ) following manufacturer &# 39 ; s recommendations . first - strand cdna was prepared from 3 μg of total rna using a first - strand cdna synthesis kit and amv reverse transcriptase following manufacturer &# 39 ; s protocol ( roche diagnostics ). after cdna synthesis , reverse transcriptase activity was destroyed by incubation at 90 ° c . for 10 mins and cdna stored at − 20 ° c . gapdh pcr to assess cdna quality : a pcr was used to assess cdna quality ; primers specific for the mouse gapdh house - keeping gene ( 5 ′- ttagcacccctggccaagg - 3 ′ and 5 ′- cttactcccttggaggccatg - 3 ′) were used with a hot - start taq polymerase ( neb , hitchen , uk ) for 35 cycles ( 95 ° c ., 3 mins followed by 35 cycles of 94 ° c ./ 30 secs , 55 ° c ./ 30 secs , 72 ° c ./ 1 min ; final polishing step of 10 mins at 72 ° c .). amplified products were assessed by agarose gel electrophoresis . primers were designed to amplify the heavy and light chain variable regions based upon the pcr product sequence data . primers were designed to allow cloning of the relevant chain into unique restriction enzyme sites in the higg1 / kappa double expression vector pdcorig - higg1 . each 5 ′ primer was targeted to the starting codon and leader peptide of the defined variable region , with a kozak consensus immediately 5 ′ of the starting codon . each 3 ′ primer was designed to be complementary to the joining region of the antibody sequence , to maintain reading frame after cloning of the chain , and to preserve the amino acid sequence usually found at the joining region / constant region junction . all primers were purchased from eurofins mwg . immunoglobulin heavy chain variable region usage was determined using pcr with a previously published set of primers [ 60 ]. previous results using a mouse mab isotyping test kit ( serotec , oxford , uk ) had indicated that fg88 . 2 and fg88 . 7 were both mouse igg3 antibodies . appropriate constant region reverse primers were therefore used to amplify from the constant regions . pcr amplification was carried out with 12 mouse vh region - specific 5 ′ primers and 3 ′ primers specific for previously determined antibody subclass with a hot - start taq polymerase for 35 cycles ( 94 ° c ., 5 min followed by 35 cycles of 94 ° c ./ 1 min , 60 ° c ./ 1 min , 72 ° c ./ 2 min ; final polishing step of 20 min at 72 ° c .). amplified products were assessed by agarose gel electrophoresis . positive amplifications resulted for the vh4 primer . immunoglobulin light chain variable region usage was determined using pcr with a previously published set of primers [ 60 ]. previous results using a mouse mab isotyping test kit had indicated that both fg88 . 2 and fg88 . 7 used κ light chains . pcr amplification was carried out with mouse vκ region - specific 5 ′ and 3 ′ mouse cκ specific primers with a hot - start taq polymerase for 35 cycles ( 94 ° c ., 5 mins followed by 35 cycles of 94 ° c ./ 1 min , 60 ° c ./ 1 min , 72 ° c ./ 2 mins ; final polishing step of 20 mins at 72 ° c .). amplification products were assessed by agarose gel electrophoresis . positive amplifications resulted with the vκ1 and vκ2 primers for both fg88 . 2 and fg88 . 7 . pcr products were purified using a qiaquick pcr purification kit ( qiagen , crawley , uk ). the concentration of the resulting dna was determined by nanodrop and the purity assessed by agarose gel electrophoresis . pcr products were sequenced using the originating 5 ′ and 3 ′ pcr primers at the university of nottingham dna sequencing facility ( http :// www . nottingham . ac . uk / life - sciences / facilities / dna - sequencing / index . aspx ). sequences were analysed ( v region identification , junction analysis ) using the imgt database search facility ( http :// www . imgt . org / imgt_vquest / vquest ? livret = 0 & amp ; option = mouseig ). sequencing indicated that fg88 . 2 and fg88 . 7 shared near identical heavy and light chain variable regions ( heavy chain ; ighv6 - 6 * 01 , ighj1 * 01 , light chain ; igkv12 - 41 * 01 , igkj1 * 01 ). sufficient residual constant region was present in the heavy chain sequences to confirm that fg88 . 2 and fg88 . 7 were of the migg3 subclass , indicating clearly that the two clones had come from two independent splenocyte - nso fusion events . direct cloning of the pcr products into the pdcorig - higg1 vector using the restriction sites incorporated into the pcr primers was known to be relatively inefficient from previous scancell experience . a dual cloning strategy was therefore adopted ; the pcr product generated using a proof - reading polymerase was cloned into both pdcorig - higg1 and a ta vector ( pcr2 . 1 ; invitrogen ) simultaneously , with the ta vector - cloned product acting as an easily expanded backup source of material for cloning should the initial pdcorig - higg1 cloning fail . pcr amplification was carried out using a proof - reading polymerase ( phusion ; neb ) and the cloning primers described above using the fg88 . 7 cdna template previously described for 35 cycles ( 98 ° c ., 3 min followed by 35 cycles of 98 ° c ./ 30 sec , 58 ° c ./ 30 sec , 72 ° c ./ 45 sec ; final polishing step of 3 min at 72 ° c .). successful amplification was confirmed by agarose gel electrophoresis . amplified fg88 . 7 light chain was digested sequentially with the restriction enzymes bsiwi and bamhi according to manufacturer &# 39 ; s instructions ( neb ). vector ( pdcorig - higg1 , containing v regions from a previously cloned antibody ) was simultaneously digested . vector dna was agarose gel purified using a qiaquick gel extraction kit ( qiagen ) and insert dna purified using a pcr purification kit . after dna quantification by nanodrop , vector dna was phosphatase treated according to manufacturer &# 39 ; s recommendations ( antarctic phosphatase , neb ) and light chain insert ligated into the vector ( t4 dna ligase , neb ). ligated dna was transformed into chemically competent top10f ′ cells ( invitrogen ) and spread on 35 μg / ml zeocin ( invitrogen , toulouse , france ) supplemented lb agar plates which were then incubated overnight at 37 ° c . amplified fg88 . 7 light chain was treated with taq polymerase ( neb ) for 15 min at 72 ° c . to add ‘ a ’ overhangs compatible with ta cloning . treated pcr product was incubated with the topo ta vector pcr2 . 1 ( invitrogen ) and transformed into chemically competent top10f ′ cells according to manufacturer &# 39 ; s instructions . transformed bacteria were spread on ampicillin ( 80 μg / ml ) supplemented lb agar plates which were then incubated overnight at 37 ° c . colonies were grown in liquid culture ( lb supplemented with 80 μg / ml ampicillin ) and plasmid dna prepared ( spin miniprep kit , qiagen ). presence of an insert was confirmed by sequential digestion with bsiwi and bamhi and agarose gel electrophoresis . sequencing was carried out on miniprep dna from colonies using t7 and m13rev primers . the dna insert from one such colony had the predicted fg88 . 7 light chain sequence ; a 300 ml bacterial lb / ampicillin culture was grown overnight and plasmid dna prepared by maxiprep ( plasmid maxi kit , qiagen ). maxiprep dna insert was confirmed by sequencing . amplified fg88 . 7 heavy chain was treated with taq polymerase ( neb ) for 15 mi at 72 ° c . to add ‘ a ’ overhangs . treated pcr product was incubated with the topo ta vector pcr2 . 1 and transformed into chemically competent top10f ′ cells as above . transformed bacteria were spread on ampicillin supplemented lb agar plates which were then incubated overnight at 37 ° c . colonies were grown in liquid culture ( lb / ampicillin ) and plasmid dna prepared ( spin miniprep kit ). presence of an insert was confirmed by digestion with hindiii and afei and agarose gel electrophoresis . sequencing was carried out on miniprep dna from a number of colonies using t7 and m13rev primers . the dna insert from one such colony had the predicted fg88 . 7 heavy chain sequence ; a 300 ml bacterial lb / ampicillin culture was grown overnight and plasmid dna prepared by maxiprep ( plasmid maxi kit , qiagen ). maxiprep dna insert was confirmed by sequencing . the fg88 . 7 light chain was digested from the topo vector pcr2 . 1 by sequential digestion with bsiwi and bamhi and the 400 bp insert dna agarose gel purified using a qiaquick gel extraction kit ( qiagen ) following manufacturer &# 39 ; s recommendations . this insert was ligated into previously prepared pdcorig - higg1 vector ( see above ) and transformed into chemically competent top10f ′ cells . transformations were spread on 35 μg / ml zeocin supplemented lb agar plates which were then incubated overnight at 37 ° c . colonies were grown in liquid culture ( lb supplemented with 35 μg / ml zeocin ) and plasmid dna prepared ( spin miniprep kit , qiagen ). sequencing was carried out on miniprep dna from all colonies using the p6 sequencing primer sited in the human kappa constant region . the dna insert from a colony had the predicted fg88 . 7 light chain sequence correctly inserted in pdcorig - higg1 ; a 300 ml bacterial lb / zeocin culture was grown overnight and plasmid dna prepared by maxiprep ( plasmid maxi kit , qiagen ). the fg88 . 7 heavy chain insert was digested from the topo vector pcr2 . 1 by digestion with hindiii and afei . vector ( pdcorig - higg1 - 27 . 18k ) containing the fg88 . 7 kappa light chain ( prepared above ) was also digested with hindiii and afei . the vector dna was then phosphatase treated according to manufacturer &# 39 ; s recommendations ( antarctic phosphatase , neb ). after agarose gel electrophoresis , the 6 . 5kb pdcorig - higg1 vector band and 400 bp fg88 . 7h insert band were isolated using a qiaquick gel extraction kit ( qiagen ) following manufacturer &# 39 ; s recommendations . the insert was ligated into the pdcorig - higg1 vector and transformed into chemically competent top10f ′ cells . transformations were spread on 35 μg / ml zeocin supplemented lb agar plates which were then incubated overnight at 37 ° c . colonies were grown in liquid culture ( lb supplemented with 35 μg / ml zeocin ) and plasmid dna prepared ( spin miniprep kit , qiagen ). presence of an insert was confirmed by digestion with hindiii and afei and agarose gel electrophoresis . sequencing was carried out on miniprep dna from a number of the colonies using the p3rev sequencing primer sited in the human igg1 constant region . the dna insert from one of the colonies had the predicted fg88 . 7 heavy chain sequence correctly inserted in pdcorig - higg1 ; a 300 ml bacterial lb / zeocin culture was grown overnight and plasmid dna prepared by maxiprep ( plasmid maxi kit , qiagen ). sequencing was used to confirm that both heavy and light chain loci . the methodology for the expression and purification of chimeric antibody described in the present invention can be achieved using methods well known in the art . briefly , antibodies can be purified from supernatant collected from transiently , or subsequently stable , transfected cells by protein a or protein g affinity chromatography based on standard protocols , for example sambrook et al . [ 61 ]. to determine the therapeutic value of fg88 , it was screened on gastric , ovarian , colorectal cancer tissue microarrays by immunohistochemistry ( ihc ). methodology : immunohistochemistry was performed using the standard avidin - biotin peroxidise method . paraffin embedded tissue sections were placed on a 60 ° c . hot block to melt the paraffin . tissue sections were deparaffinised with xylene and rehydrated through graded alcohol . the sections were then immersed in 500 ml of citrate buffer ( ph6 ) and heated for 20 min in a microwave ( whirlpool ) to retrieve antigens . endogenous peroxidase activity was blocked by incubating the tissue sections with endogenous peroxidase solution ( dako ltd , ely , uk ) for 5 min . normal swine serum ( nss ; vector labs , ca , usa ; 1 / 50 pbs ) was added to each section for 20 min to block non - specific primary antibody binding . all sections were incubated with avidin d / biotin blocking kit ( vector lab ) for 15 min each in order to block non - specific binding of avidin and biotin . the sections were re - blocked with nss ( 1 / 50 pbs ) for 5 mins . then tissue sections were incubated with primary antibody at room temperature for an hour . anti - β - 2 - microglobulin ( dako ltd ; 1 / 100 in pbs ) mab and pbs alone were used as positive and negative controls respectively . tissue sections were washed with pbs and incubated with biotinylated goat anti - mouse / rabbit immunoglobulin ( vector labs ; 1 / 50 in nss ) for 30 min at room temperature . tissue sections were washed with pbs and incubated with preformed 1 / 50 ( pbs ) streptavidin - biotin / horseradish peroxidase complex ( dako ltd ) for 30 min at room temperature . 3 , 3 ′- diaminobenzidine tetra hydrochloride ( dab ) was used as a substrate . each section was incubated twice with 100 μl of dab solution for 5 min . finally , sections were lightly counterstained with haematoxylin ( sigma - aldrich , poole dorset , uk ) before dehydrating in graded alcohols , cleaning by immersing in xylene and mounting the slides with distyrene , plasticiser , xylene ( dpx ) mountant ( sigma ). fg88 . 2 and fg88 . 7 mabs were labelled with alexa - 488 fluorophore ( a - fg88 . 2 and a - fg88 . 7 ) according to manufacturer &# 39 ; s protocol ( invitrogen ). 1 . 5 × 10 5 c170 cells were grown on sterile circular coverslips ( 22 mm diameter , 0 . 16 - 0 . 19 mm thick ) in 6 well plate for 24 hr in 5 % co 2 at 37 ° c . 24 hours later , cells on coverslips were treated with 5 μg / ml of a - fg88 . 2 and a - fg88 . 7 mabs for 2 hr at 37 ° c . in dark . 2 hours later , excess / unbound mabs were washed away using pbs . the cells were then fixed using 0 . 4 % paraformaldehyde for 20 min in dark . 0 . 4 % paraformaldehyde was washed away using pbs . the coverslips were mounted to slides with pbs : glycerol ( 1 : 1 ). the coverslip edge was sealed with clear nail varnish . localisation of the a - fg88 . 2 and a - fg88 . 7 mabs was visualised under a confocal microscope ( carl zeiss , jena , germany ). cells ( 5 × 10 3 ) were co - incubated with 100 μl of pbmcs , 10 % autologous serum or media alone or with mabs at a range of concentrations . spontaneous and maximum releases were evaluated by incubating the labeled cells with medium alone or with 10 % triton x - 100 , respectively . after 4 hr of incubation , 50 μl of supernatant from each well was transferred to 96 well lumaplates . plates were allow to dry overnight and counted on a topcount nxt counter ( perkin elmer , cambridge , uk ). the mean percentage lysis of target cells was calculated according to the following formula : cancer cells ( 1 × 10 3 / well ) were incubated on a 96 - well flat bottom microtitre plate for 24 hours to establish an adherent monolayer . next day , mabs were added 100 μl / well in quadruplicate ( 0 . 003 μg / ml to 3 μg / ml ) in the presence or absence of 20 μm of a pan - caspase inhibitor z - fmk - vad ( carbobenzoxy - valyl - alanyl - aspartyl -[ o - methyl ]- fluoromethylketone ; promega , eastleigh , uk ) for 48 hours . cells were then exposed to 0 . 5 μci / well of 3 h - thymidine during the final 24 hours of the 48 - hour period . the incorporation of 3 h - thymidine into cells of the culture was measured using a liquid scintillation counter ( microscint 0 liquid scintillant on a topcount nxt , both perkin elmer ). fg88 . 2 and fg88 . 7 were incubated with c170 cells and tested for uptake of the small molecular weight dye propidium iodide ( pi , sigma ) at various temperatures . tumour cells ( 5 × 10 4 ) were incubated on a 96 - well round bottom microtitre plate with 50 μl of primary antibodies at 37 ° c . or 4 ° c . for 2 hr . 1 μg of pi was added and cells were incubated at 37 ° c . or 4 ° c . for 30 min . 0 . 4 % formaldehyde was used to fix the cells . samples were analysed on a fc - 500 flow cytometer ( beckman coulter ). to analyse and plot raw data winmdi 2 . 9 software was used . for comparison , mab 505 / 4 which is known to induce membrane damage was also included . medium alone was included as a negative control . fg88 . 2 mab was incubated with c170 cells and tested for uptake of fluorochrome - labelled 3kda and 40kda molecular weight dextran ( invitrogen ) at 37 ° c . tumour cells ( 5 × 10 4 ) were incubated on a 96 - well flat bottom microtitre plate with 50 μl of primary antibodies at 37 ° c . for 2 hr . 1 μg of dextran was added and cells were incubated at 37 ° c . for 30 min . for comparison , 0 . 5 % h 2 o 2 and 0 . 4 % saponin , which are known to induce membrane damage , were also included . medium alone ( rpmi ) was included as a negative control . samples were analysed on a macsq flow cytometer ( miltenyi biotech ) using macsq software . c170 or jurkat cells ( 1 . 25 × 10 6 ) were incubated with 30 μg / ml of fg88 . 2 or fg88 . 7 or 0 . 5 μg / ml of anti - fas ( promega ) mabs in the presence or absence of the pan - caspase inhibitor z - fmk - vad ( 20 μm final concentration ) at 37 ° c . 20 hours later , cells were collected by centrifugation at 14 , 000 rpm for 5 min at room temperature . cell pellets were resuspended gently in 500 μl of lysis buffer ( 10 mm tris - hcl ph8 . 5 , 5 mm edta , 200 mm nacl , 0 . 5 % sds , all sigma ) and incubated at 60 ° c . for 5 min . rnase was added to each sample ( sigma ; final concentration of 4 μg / ml ) and incubated at 37 ° c . for 15 min . proteinase k was added to each sample ( active motif , la hulpe , belgium ; final concentration of 2 ng / ml ) and incubated at 60 ° c . for 1 hr . 350 μl of 5m nacl was added to each sample and incubated on ice for 5 min . samples were spun at 14 , 000 rpm for 15 min at 4 ° c . supernatants were collected and an equal volume of ice cold phenol : chloroform ( 1 : 1 v / v ) was added to each sample . samples were spun at 14 , 000 rpm for 5 min at 4 ° c . the aqueous phase of each sample was collected and 20 μl of sodium acetate , ph 5 . 2 ( sigma ; final concentration of 120 mm ) was added to each sample . 500 μl of 100 % ethanol ( pre - chilled to − 20 ° c .) was added to each sample and incubated at − 80 ° c . for 1 hr . samples were spun at 14 , 000 rpm for 10 min at 4 ° c . supernatants were discarded and pellets were washed with 500 μl of pre - chilled 70 % ethanol . samples were spun at 14 , 000 rpm for 10 min at 4 ° c . supernatants were removed , dna pellets were allowed to air dry , resuspended in 20 μl of 10 mm tris / hcl , ph8 . 5 buffer and analysed on a 0 . 8 % agarose gel . c170 cells ( 1 × 10 5 ) were grown on sterile circular coverslips ( 13 mm diameter , 0 . 2 mm thick ; thermanox , nunc , roskilde , denmark ) in a 6 well plate at 37 ° c ., 5 % co 2 . 24 hours later , cells on coverslips were treated with 30 μg / ml of fg88 . 2 and fg88 . 7 mabs , medium alone , 0 . 5 % h 2 o 2 and 0 . 4 % saponin ( sigma ) for 20 hours at 37 ° c . 20 hours later , cells were washed with pre - warmed 0 . 1m sodium cacodylate buffer ( ph7 . 4 ). then washed cells were fixed with pre - warmed glutaraldehyde ( final concentration of 12 . 5 % w / v ) for 24 hr . fixed cells were washed twice with 0 . 1m sodium cacodylate buffer and post - fixed with 1 % osmium tetroxide ( ph 7 . 4 ) for 45 min . subsequently , the cells were washed twice with deionised water . after the final wash , the cells were dehydrated in increasing concentration of ethanol from 40 % to 100 %. the prepared cells were exposed to critical point drying then sputtered with gold prior to sem analysis ( jsm - 840 sem , jeol ). the study was conducted under a uk home office licence . ncri guidelines for the welfare and use of animals in cancer research , lasa good practice guidelines and felas working group on pain and distress guidelines was also followed . endotoxin free (& lt ; 10 eu / ml ) fg88 mab was supplied in pre - formulated aliquots ready for dosing and stored at − 20 ° c . until use . age matched male mf - 1 nude mice were obtained from harlan laboratories ( bichester , uk ) with each group , fg88 , control mabs or the vehicle control , consisting of n & gt ; 8 animals . mice were implanted with c170hm2 dlux cells and monitored by optical imaging to determine tumour establishment and suitability to be entered into the study . mice were dosed with either fg88 or the positive control mab , 505 / 4 ( 1 mg / ml ) dosed at 0 . 1 mg 2 x weekly 1000 intravenously ( i . v .) until termination , or pbs , the vehicle control for the mab , 1000 2 × weekly i . v . until termination . weekly bioluminescent imaging was carried out on all mice to obtain pre and post dosing tumour measurements . in this way each mouse provided pre - dose control readings against which tumour growth could be compared . all measurements and readouts were transferred from the original dictation / notation to excel ( tab delineated ) format for data processing in spss v16 . 0 . data integrity was checked using explore and descriptive functions . erroneous points when identified were cross referenced against the original data and corrected accordingly . the data was screened for outliers and distribution profile ; data - points falling outside the 95 % confidence limit ( outliers ) were removed from analysis , but kept in the datasheet for reference purposes . mice were imaged weekly for bioluminescent tumour burden ( bli ) over the duration of the study as follows ; 60 mg / kg d - luciferin substrate was administered subcutaneously ( s . c . ), the mice were anesthetised and bli readings taken 15 mins post substrate administration on open filter block ( 2d ) and sequential emission filters ( for dlit , 3d reconstruction ). ventral and dorsal imaging was undertaken ; the optimum position for imaging was abdomen uppermost . bli was measured over the entire abdominal area , one region of interest ( roi ) for each mouse in order to include all lesions present . each mouse had a pre - dosing or baseline image taken to allow calculation of percentage tumour growth over time ; these data were averaged per group . bli readings were also taken after termination to identify tumours in pm tissue . fg88 was raised by immunisation with glycolipid antigens from the colorectal cell line , colo205 . analysis of antibody response to immunisations : antibody titres were initially monitored by lipid enzyme - linked immunosorbent assay ( elisa ). thin layer chromatography ( tlc ) analysis using colo205 total and plasma membrane lipid extracts , flow cytometry analysis ( facs ) using colo205 tumour cells and western blot using colo205 whole cell extract , total and plasma membrane lipid extracts were subsequently performed . the mouse considered to have the best response , compared to the pre - bleed serum control was boosted intravenously ( i . v .) with colo205 plasma membrane lipid extract prior to fusion . 8 days after fusion , supernatants were collected and screened against fresh colo205 tumour cells . hybridomas which demonstrated cell surface binding , using an indirect immunofluorescence assay , were harvested , washed in complete media and spread across 96 well plates at 0 . 3 cells per well to acquire a clone . the plate was then screened for positive wells and these grown on until a sufficient number of cells was obtained to spread across a 96 well plate at 0 . 3 cells per well for a second time . if the resulting number of colonies equalled ˜ 30 and all hybridomas were positive , the hybridoma was considered a clone . methods for clonal expansion , bulk culture and antibody purification of antibodies or antibody fragments are available using conventional techniques known to those skilled in the art . binding of fg88 hybridoma supernatant to colo205 cells : fg88 . 2 and fg88 . 7 were analysed for their ability to bind to colo205 cells by indirect immunofluorescence and facs analysis ( fig3 a ). both hybridomas bound with strong intensity to the cell surface of colo205 cells ( fg88 . 2 gm 4539 ; fg88 . 7 gm 2897 ) when compared to positive control mab anti - hla mab w6 / 32 ( ebioscience , ca , usa ), and the negative controls of and isotype control in contrast , fg88 . 2 and fg88 . 7 hybridoma supernatants did not bind to any normal blood cells ( fig3 b ). fg88 . 2 bound lipid antigens from c170 and colo205 but not those from ags ( atcc accession # crl - 1739 ; fig3 c ). to clarify the fine specificities of the fg88 mabs , they were screened against & gt ; 600 natural and synthetic glycans . binding of fg88 . 2 and fg88 . 7 mabs to the glycan array showed that both mabs bound to lecle x , le a le x , le x containing glycan , le a containing glycans , le a and di - le a ( fig4 a , b ). subtle differences were observed between the two antibodies with fg88 . 2 binding most strongly to lecle x and le a le x , followed le a containing glycan , le a , di - le a and le x containing glycan . fg88 . 7 bound most strongly to lecle x and di - le a , followed by le a containing glycans , le a le x and le x containing glycan . additionally , the mabs bound simple le a on the array but not le c or le x . this was corroborated by competition experiments where preincubation of both mabs with a le a - hsa conjugate , but not a le x - hsa conjugate , abolished colo205 binding ( data not shown ). the le a - hsa binding kinetics of the mabs was examined using spr ( biacore x ). fitting of the binding curves revealed strong apparent functional affinity ( kd ˜ 10 − 10 m ) with fast association (˜ 10 5 1 / ms ) and slow dissociation (˜ 10 − 5 l / s ) rates for both mabs . to confirm that these sugars were expressed on proteins from tumour cells , fg88 mabs were screened for binding to glycoproteins by sds - page / western blotting ( fig5 ). fg88 . 2 and fg88 . 7 recognise low , intermediate ( mw between 10 - 230 kda ) and high ( molecules that do not enter the separation gel ) molecular weight molecules by western blot analysis of colo205 whole cell extract , c170 whole cell extract , colo205 plasma membrane , colo205 plasma membrane lipid and total lipid extracts . fg88 . 2 and fg88 . 7 also recognised a band at the dye front in colo205 total lipid extract and colo205 plasma membrane lipid extract lanes which is presumed to be glycolipid . the mab 505 / 4 recognising sialyl - di - le a was included as positive control and demonstrated a similar blotting pattern to fg88 . 2 and fg88 . 7 , recognising high , intermediate and low molecular weight proteins and the glycolipid band at the dye front . to confirm that lecle x , le a le x , le a containing glycans , le a and di - le a glycans are expressed on lipids , the mabs were screened for binding to tumour associated lipids by thin layer chromatography ( tlc ). fg88 . 2 mab bound lipid antigens from colo205 , mkn45 ( atcc accession # ccl - 171 ) and c170 but not those from ags ( fig6 ). two glycolipids were stained by fg88 . 2 in colo205 cells ( r f = 0 . 21 and 0 . 14 ). in addition , fg88 . 2 stained another glycolipid with less polarity ( r f = 0 . 46 ). in contrast , although fg88 . 7 also bound the same tumour cell lines as fg88 . 2 , one of the glycolipids stained in the colo205 sample demonstrated an intermediate mobility ( r f = 0 . 19 ). fg88 . 7 also stained an extra glycolipid with less polarity ( r f = 0 . 29 ). to determine the therapeutic value of fg88 , it was screened on colorectal , gastric , pancreatic , lung , ovarian and breast tumour tissue microarrays ( tmas ) by immunohistochemistry ( ihc ). to assess the binding of fg88 to human tissues , a number of tumour tmas were stained ; 69 % of colorectal ( 142 / 208 ), 56 % of gastric ( 52 / 93 ), 74 % of pancreatic ( 658 / 890 ), 23 % of lung ( 62 / 275 ), 31 % of ovarian ( 58 / 186 ) and 27 % of breast ( 241 / 902 ) tumour tissues were stained ( table 2 ). whilst fg88 recognised only 27 % of the 902 breast tumour tissues stained , 34 % were triple negative breast cancer ( tnbc ) and 32 % of tumours with a basal phenotype stained . further , the staining of the er negative breast tma using fg88 . 2 at 0 . 3 ug / ml ( staining for fg88 . 7 not determined ) showed 25 % positive staining ( 84 / 338 ). stained er negative breast tissues correlated to all basal type significantly . with tnbc being such a challenging disease with the poorest prognosis of all breast cancer subtypes , and currently cytotoxic chemotherapy is the only systemic treatment option available , fg88 could provide a valued immunotherapeutic agent for this group of patients to assess the possible toxicity of mabs fg88 . 2 and fg88 . 7 , human and cynologous monkey normal tissue tmas were stained . for human normal tissue tma , fg88 . 2 did not stain placenta , rectum , skin , adipose , heart , skeletal , bladder , spleen , brain , stomach , breast , kidney , testis , cerebellum , cervix , lung , ovary , diaphragm , uterus , duodenum and thyroid . staining was seen against oesophagus ( moderate squamous epithelium staining ), gall bladder ( strong columnar epithelium staining ), ileum ( strong columnar mucosa staining ), jejunum ( weak columnar mucosa staining ), liver ( strong bile duct staining ), thymus ( weak staining ), colon ( strong glandular epithelium staining ), tonsil ( moderate squamous epithelium staining ) and pancreas ( moderate staining ) ( table 3 ). fg88 . 7 showed the same staining pattern as fg88 . 2 except that it also stained normal rectum ( weak glandular epithelium stainin . for the monkey normal tissue tma , staining was seen against small intestine , skin , colon , stomach , ovary , liver and thymus for both fg88 . 2 and fg88 . 7 ( data not shown ). the term “ chimeric antibody ” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species , such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody . chimeric ( or humanised ) antibodies of the present invention can be prepared based on the sequence of a murine mab prepared as described above . the amino acid and nucleotide sequence for the variable region of the heavy ( fig1 a ) and light chains ( fig1 b ) of the fg88 . 2 mab and the amino acid and nucleotide sequence for the variable region of the heavy ( fig2 a ) and light chains ( fig2 b ) of the fg88 . 7 mab are shown in fig1 and 2 . numbers refer to the standardised imgt system for the numbering of antibody sequences [ 59 ]. the cdr1 , cdr 2 and cdr 3 regions are indicated . fg88 . 2 and fg88 . 7 both belong to the ighv6 - 6 * 01 heavy chain and igkv12 - 41 * 01 gene families . fg88 . 2 has 10 mutations from ighv6 - 6 * 01 and fg88 . 7 eight . fg88 . 2 has 11 mutations from igkv12 - 41 * 01 and fg88 . 7 twelve . fg88 . 2 and fg88 . 7 heavy and light chain variable regions were cloned into human igg1 expression vector . this was transfected into cho - s cells and human antibody purified on protein g . the chimeric mabs ch88 . 2 and ch88 . 7 bound to the colorectal cell line , colo205 ( fig1 c and 2 c and fig7 ). the amino acid and nucleotide sequence for the heavy ( fig1 d ) and light chains ( fig1 e ) of the human fg88 . 2 mab and the amino acid and nucleotide sequence for the human heavy ( fig2 d ) and light chains ( fig2 e ) of the fg88 . 7 mab are shown in fig1 and 2 . fg88 . 2 and fg88 . 7 were screened by indirect immunofluorescence staining and flow cytometric analysis for binding to the cell surface of a panel of tumour cell lines table 4 . fg88 . 2 bound strongly ( gm & gt ; 500 ) to c170 , colo205 , colo201 , st16 , du4475 and panc - 1 , moderately ( gm 100 - 500 ) to ht29 , h69 and ovcar - 3 , weakly ( gm & lt ; 100 ) to ags , ovcar - 4 and oaw42 and failed to bind mkn45 , aspc - 1 , ovca433 , mcf - 7 and mda - mb - 231 cell lines . fg88 . 7 showed a similar binding pattern as fg88 . 2 , except that it bound moderately to mkn45 . in order to establish whether binding was tumour cell specific and not cross reactive with normal blood cells , the fg88 . 2 , fg88 . 7 , ch88 . 2 and ch88 . 7 mabs were incubated with healthy normal donor whole blood . neither the murine ( fg88 . 2 and fg88 . 7 ) nor the chimeric mabs ( ch88 . 2 and ch88 . 7 ) bound to peripheral blood mononuclear cells ( pbmcs , lower left quadrant ) or granulocytes ( upper left quadrant ) ( fig8 a ). the need to determine normal red blood cell binding was further necessitated as within the literature , there is an indication that le a antigens found in the secretions of various tissue types have the capability of adsorbing to the surface of erythrocytes . the term abh secretor refers to the secretion of abo blood group antigens into the individual &# 39 ; s body fluids . among lewis antigen positive individuals , abh secretors are always le a − b + whereas abh non - secretors are always le a + b − . in caucasians , it was reported that approximately 80 % are of secretor status and 20 % are non - secretors . the secretor status of nine healthy human donors was determined by saliva sandwich elisa ( fig8 b ), followed by binding analysis of the fg88 mabs to erythrocytes from a le a - positive donor . neither fg88 mab bound to the erythrocytes ( fig8 c ). fg88 mabs were analysed for cellular internalisation via confocal microscopy . they were labelled with alexa - 488 fluorophore following the manufacturer &# 39 ; s protocol and the labelling efficiency checked via direct flow cytometric analysis of the mab binding to the c170 cell surface . confocal microscopy was then used to follow the cellular internalisation of fg88 mabs by c170 cells over a two - hour incubation period . cross - sectional images were obtained at 0 . 8 μm intervals and showed efficient internalisation after a two - hour incubation period . in addition , clustering of fg88 mabs on c170 cell surface was observed , suggesting the heterogeneous distribution of the antigen in the c170 plasma membrane ( fig9 a ). a more quantitative analysis was performed using direct flow cytometry on colo205 cells after acid wash and fitc - labelled murine fg88 mabs . the results showed that wash buffer at ph2 . 0 strips any surface - remaining antibody ( as seen by the near complete removal of epcam pe fluorescence ), but fg88 - fitc labelled cells remain fluorescent after acid wash at ph 2 . 0 , indicating the internalisation of the fitc - labelled fg88 mabs ( and thus protection from the acid wash ). colo205 cells internalised fg88 . 7 and fg88 . 2 to a similar degree at 37 ° c . and 4 ° c . ( fig9 b ). over time , internalised fg88 mabs co - localised with lysosomal compartments ( fig9 c ). similar results were obtained with fg88 . 7 ( data not shown ). importantly , internalization was validated through toxicity of fab - zap - fg88 immune complexes containing saporin . internalization of the fab - zap - fg88 . 2 and fab - zap - fg88 . 7 complexes , but not the fab - zap alone or the fab - zap preincubated with a control mab ( data not shown ), led to a dose - dependent decrease in cell viability of the high glyco - epitope expressing c170 , panc1 and st16 cells ( fig9 d ). the moderately binding ht29 cells were more refractory . in summary , colo205 , c170 panc 1 and st16 cells efficiently internalise the murine fg88 antibodies and this may be linked to their direct cell killing ability . the ability of murine and chimeric fg88 mabs to induce tumour cell death through adcc was screened . human pbmcs were used as the source of effector cells while colo205 cells served as target cells . the number of cells killed by mabs fg88 . 2 , fg88 . 7 , ch88 . 2 and ch88 . 7 was measured after 18 hr incubation at 37 ° c . as shown in fig1 a , colo205 cells were susceptible to fg88 . 2 , fg88 . 7 , ch88 . 2 and ch88 . 7 mabs killing showing a maximum of 57 %, 56 %, 64 % and 59 % lysis respectively . a range of tumor cell lines were analyzed for their susceptibility to fg88 - mediated adcc . the fg88 mabs significantly lysed the high glyco - epitope expressing colo205 , c170 , st16 and panc1 cells above the killing observed with pbmcs alone ( fig1 b ). the mab 791t / 36 , a murine igg2b that cannot bind human cd16 ( 32 ), showed no significant killing over the background observed with pbmcs alone . pbmc killing in the absence of fg88 mabs was highest for cell lines lacking mhci such as c170 , st16 , panc1 and ags and probably reflects nk killing . noticeably less immune - mediated killing was seen with the fg88 mabs on the moderate - binding ht29 and dms79 cells even at high mab concentration of 10 μg / ml ; the weak - binding ovcar3 and ags were refractory . cdc is known to be an important mechanism involved in eliminating tumour cells in vivo . the capacity of the c170 cells to be killed by cdc induced by mabs fg88 . 2 and fg88 . 7 in the presence or absence of human serum as source of complement at 37 ° c . for 18 hr was assayed . fg88 . 2 and fg88 . 7 showed a maximum of 80 % and 91 % lysis respectively ( fig1 c ). the fg88 mabs displayed significant cdc activity against colo205 and panc1 cells and to a lesser degree st16 and dms79 cells ( fig1 d ). no or little cdc was seen on the low - to moderate - binding cell lines ht29 , ovcar3 and ags ( data not shown ). the low level of cdc killing of the high - binding st16 cells could be due to higher levels of membrane complement regulatory proteins ( mcrps ) ( 33 ). additionally , the efficient fg88 - mediated adcc of st16 cells under the same conditions , rules out the possibility that the reduced complement activation was due to suboptimal mab binding . in summary , fg88 strongly induced adcc using human pbmcs as effector cells as well as significant cdc with human serum as a complement source . fg88 . 2 and fg88 . 7 induced membrane damage resulting in the uptake of the small molecular weight dye propidium iodide ( pi ; fig1 a ). at 37 ° c ., fg88 . 2 induced 76 % ( 20 μg / ml ) and 76 % ( 10 μg / ml ) and fg88 . 7 induced 82 % ( 20 μg / ml ) and 82 % ( 10 μg / ml ). cells incubated with medium alone showed 21 % pi uptake . interestingly even at 4 ° c ., fg88 . 2 induced 85 % ( 20 μg / ml ) and 85 % ( 10 μg / ml ) and fg88 . 7 induced 92 % ( 20 μg / ml ) and 86 % ( 10 μg / ml ) of the cells to take up pi . cells incubated with medium alone showed 13 % pi uptake . cells incubated with chimeric fg88 . 7 induced 54 % ( 30 μg / ml ) pi uptake ( fig1 b ). it has been shown that at temperature lower than 15 ° c ., apoptosis cannot occur . this would suggest that both fg88 . 2 and fg88 . 7 induced cell death independent of apoptotic mechanisms . further evidence for an alternative mechanism of apoptotic induced cell death comes from experiments with the caspase inhibitor z - fmk - vad which failed to prevent the direct cell killing of the colorectal cell line , c170 , at 4 ° c . ( data not shown although almost identical to those at 37 ° c .) or 37 ° c ., by the mab ( fig1 ). classical apoptotic cell death can be defined by certain morphological and biochemical characteristics which distinguish it from other forms of cell death . one of the hallmarks of apoptosis is dna fragmentation . in apoptotic cells , dna is fragmented by endonuclease activity . dna of c170 cells treated with fg88 mabs ( 30 μg / ml ) in the presence or absence of pan - caspase inhibitor ( z - fmk - vad ) were analysed using conventional agarose gel electrophoresis . jurkat cells treated with anti - fas mab ( 0 . 5 μg / ml ) in the presence or absence of z - fmk - vad were used as controls for apoptosis . anti - fas mab - treated jurkat cells showed strong dna fragmentation and z - fmk - vad was shown to inhibit apoptosis induced by anti - fas mab ( no dna fragmentation ). in contrast , neither fg88 . 2 or fg88 . 7 induced dna fragmentation with or without z - fmk - vad again suggesting that these mabs were not inducing apoptosis ( fig1 ). inhibition of c170 cell growth by fg88 . 2 and fg88 . 7 : pi uptake assays were performed on cells in suspension . to ensure that the mabs also inhibited growth of adherent cells , they were exposed to fg88 . 2 and fg88 . 7 mabs and cell growth was assessed by 3 h - thymidine incorporation ( fig1 ). both mabs significantly inhibited adherent cell growth with ic 50 &# 39 ; s of 3 μg / ml . similarly an anti - fas mab inhibited the growth of jurkat tumour cells ( fig1 a ). however , in contrast to anti - fas whose growth inhibition was abrogated by a pan - caspase inhibitor , the pan - caspase inhibitor z - fmk - vad ( carbobenzoxy - valyl - alanyl - aspartyl -[ o - methyl ]- fluoromethylketone ; promega ) failed to inhibit growth induced by the fg88 mabs suggesting they inhibited cell growth via a non - apoptotic mechanism ( fig1 b , c ). to confirm that the pi assay truly reflect cell death in growing cells , c170 cells ( day 0 : 1 × 10 5 cells / well ) were treated with fg88 . 2 and fg88 . 7 and were observed microscopically . c170 cells exhibited monolayer disruption , cell rounding and cell detachment within 30 min after the addition of fg88 . 2 , fg88 . 7 or 505 / 4 mabs . however , these phenomena did not develop when c170 cells were incubated with medium alone . as shown in fig1 ; fg88 . 2 , fg88 . 7 and 505 / 4 mabs inhibited c170 cell growth at day 1 , 2 and 3 . at day 4 , c170 cells treated with fg88 . 7 ( 1 . 9 × 10 5 cells ) and fg88 . 2 ( 9 . 4 × 10 4 cells ) mabs started to regrow . cells incubated with media alone did not show growth inhibition and achieved 80 % confluency at day 3 . to confirm the morphological changes observed under the light microscope , c170 cells were exposed to fg88 . 2 and fg88 . 7 mabs for 20 hr prior to analysis under a scanning electron microscope ( sem ). pronounced cell aggregation of c170 adherent cells after incubation with fg88 mabs was observed . these cell aggregates displayed a loss of surface microvilli , the formation of membrane blebs and surface wrinkles . most importantly , these clumped cells showed evidence of membrane pores which is reminiscent of oncosis . the sizes of these pores were heterogeneous with diameters ranged from 0 . 2 μm to 1 μm ( white arrows ) ( fig1 ). to further confirm that fg88 mabs induced oncosis , c170 cells were treated with fg88 . 2 mab and then the uptake of dextran of different molecular weights ( 3 kda and 40 kda ) was assessed . fg88 . 2 mab induced uptake of both 3 and 40kda molecular weight dextran in 2 hr ( fig1 ). to assess direct killing on cells with varying expression of glycans at 37 ° c . and 4 ° c ., pi uptake assay was carried out using fg88 . 2 and fg88 . 7 mabs with panc - 1 , ht29 and ovcar - 4 cells ( fig1 ). 505 / 4 and medium alone were included as positive and negative controls respectively . 7 - le and ca19 - 9 mabs were included for comparison . fg88 . 2 and fg88 . 7 mabs induced direct cell death on panc - 1 at both 37 ° c . and 4 ° c . whereas 505 / 4 induced panc - 1 cell death only at 37 ° c . suggesting fg88 . 2 and fg88 . 7 induced direct cell death independent of an apoptotic mechanism . interestingly , fg88 . 2 , fg88 . 7 and 505 / 4 mabs did not induce ht29 cell death despite binding moderately to ht29 cells . the lecle x related glycan negative cell line , ovcar - 4 , was not killed . these results revealed a correlation between killing efficiency and the level of lecle x related glycan expression with cells expressing moderate / low levels not being killed . chimeric fg88 . 2 also induced pi uptake in cells expressing high ( c170 , colo205 and panc1 ) but not low or negative density ( ht29 and ags ) antigen ( fig1 ). experiments with different antigen negative human colorectal tumour cells , whole blood ( pbmcs and granulocytes ) or erythrocytes from normal human donors displayed no binding and no direct killing activities for mouse and / or chimeric fg88 mabs . taken together the chimeric fg88 mab had similar potency and specificity when compared with parental mouse fg88 mab . examination of fg88 treated tumour cell surface by scanning electron microscopy ( sem ) revealed pore formation . this mechanism of cell death resembles that described for oncosis . comparison of the therapeutic effect of the mab fg88 in the c170hm2 dlux human hepatic metastasis model : the mouse c170hm2 dlux human hepatic metastasis tumour model was used to investigate the anti - 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