Patent Application: US-15900602-A

Abstract:
recombinant antibody proteins are provided that specifically bind fibroblast activation protein alpha and comprise framework modifications resulting in the improved producibility in host cells . the invention also relates to the use of said antibodies for diagnostic and therapeutic purposes and methods of producing said antibodies .

Description:
the chimeric f19 ( cf19 ) antibody was designed to have the mouse f19 v l and v h regions linked to human kappa and gamma - 1 constant regions , respectively . pcr primers were used to modify the 5 ′- and 3 ′- sequences flanking the cdna sequences coding for the mouse f19 v l and v h regions ( table 1 ). pcr primers specific for f19 light chain v - region were designed . these adapted mouse f19 variable regions were then subcloned into mammalian cell expression vectors already containing the human kappa ( pkn100 vector ) or gamma - 1 ( pg1d105 vector ) constant regions ( fig2 ). these vectors employ the human cytomegalovirus ( hcmv ) promoter / enhancer to efficiently transcribe the light and heavy chains . the vectors also contain the sv40 origin of replication to permit efficient dna replication and subsequent protein expression in cos cells . the expression vectors were designed to have the variable regions inserted as hindiii - bamhii dna fragments . pcr primers were designed to introduce these restrictions sites at the 5 ′-( hindiii ) and 3 ′-( bamhi ) ends of the cdnas coding for the v - regions . in addition the pcr primers were designed to introduce the kozak sequence ( gccgccacc ) at the 5 ′- ends of both the light and heavy chain cdnas to allow efficient translation ( kozak , m ., “ at least six nucleotides preceding the aug initiator codon enhance translation in mammalian cells ,” j . mol . biol . 196 : 947 ( 1987 )), and to introduce splice donor sites at the 3 ′- ends of both the light and heavy chain cdnas for the variable regions to be spliced to the constant regions . the pcr primers used in the construction of the chimeric f19 light and heavy chains are shown in table 1 . the dna and amino acid sequences of the mouse f19 v l and v h regions as adapted for use in the construction of chimeric f19 light and heavy chains are shown in fig2 and 25 . the dna sequences of mouse f19 light and heavy chains cloned into the eukaryotic expression vectors pkn100 and pg1d105 , respectively , are shown in fig2 and 27 . the two plasmid dnas coding for the chimeric f19 light and heavy chains ( see example 1 ) were co - transfected into cos cells to look for transient expression of chimeric f19 antibody as described below . after incubation for 72 hours , the medium was collected , centrifuged to remove cellular debris , and analyzed by elisa for the production of a human igg1 - like antibody . the cos cell supernatant containing the chimeric f19 antibody was analyzed for its ability to bind to ht 1080 cells ( see example 13 ) expressing the fap antigen on their surface . the mammalian expression vectors pg1d105 and pkn100 containing the chimeric or reshaped human heavy and light chains versions , respectively , were tested in cos cells to look for transient expression of f19 antibodies . cos - 7 cells were passaged routinely in dmem ( gibco brl cat . # 41966 ) containing penicillin ( 50 iu / ml ), streptomycin ( 50 mg / ml ), l - glutamine and 10 % heat - inactivated gamma globulin - free foetal calf serum ( fcs , harlan sera - lab cat . # d0001 ). the dna was introduced into the cos cells by electroporation using the gene pulsar apparatus ( biorad ). dna ( 10 mg of each vector ) was added to a 0 . 8 ml aliquot of 1 × 10 7 cells / ml in phosphate - buffered saline ( pbs , ca 2 + and mg 2 + free ). a pulse was delivered at 1 , 900 volts , 25 mf capacitance . after a 10 minutes recovery period at ambient temperature the electroporated cells were added to 8 ml of dmem containing 5 % fcs . after incubation at 37 ° c . for 72 hours , the medium was collected , centrifuged to remove cellular debris , and stored under sterile conditions at 4 ° c . for short periods of time , or at − 20 ° c . for longer periods . elisa method for measuring assembled igg1 / kappa antibody concentrations in cos cell supernatants samples of antibodies produced in transfected cos cells were assayed by elisa to determine how much chimeric or reshaped human antibody had been produced . for the detection of antibody , plates were coated with goat anti - human igg ( fcg fragment specific ) antibody ( jackson immunoresearch laboratories inc ., # 109 - 005 - 098 ). the samples from cos cells were serially diluted and added to each well . after incubation for 1 h at 37 ° c . and washing , horseradish peroxidase conjugated goat anti - human kappa light chain ( sigma , a - 7164 ) was added . after incubation for 30 minutes at 37 ° c . and washing , k - blue substrate ( a mixture of 3 , 3 ′, 5 , 5 ′ tetramethylbenzidine and hydrogen peroxide , bionostics limited , # kb 175 ) was added for 30 minutes at room temperature . the reaction was stopped using red stop solution ( bionostics limited , # rs20 ) and the optical density read on a microplate reader at 650 nm . purified human igg1 / kappa antibody ( sigma , i - 3889 ) of known concentration was used as a standard . the expression of chimeric f19 antibody in cos cells was poor ( table 2 ), between 10 and 60 ng / ml , which is at least 10 fold less than most antibodies . in an attempt to increase expression levels of the chimeric f19 antibody , the leader sequence of f19 v l region was changed by substitution of leucine to proline at position - 9 . this single change in amino acid in the leader sequence resulted in at least doubling the amount of chimeric antibody produced in cos cells . cell binding results show that chimeric f19 binds specifically and with the expected avidity to the fap target . the construction of the first version of reshaped human f19 v l region ( l a ) was carried out using overlapping pcr fragments in a method similar to that described by daugherty b . l ., et al ., “ polymerase chain reaction ( pcr ) facilitates the cloning , cdr - grafting , and rapid expression of a murine monoclonal antibody directed against the cd18 component of leukocyte integrins ,” nucl . acids res . 19 : 2471 ( 1991 ). ten oligonucleotides were synthesized that consisted of five primer pairs , apcr1 - vla1 , vla2 - vla3 , vla4 - vla5 , vla6 - vla7 , and vla8 - apcr4 ( table 3 and fig2 ). there was an overlapping sequence of at least 21 bases between adjacent pairs ( fig2 ). apcr1 and apcr4 hybridized to the flanking puc19 vector sequences . the mutagenic primers were designed such that their 5 ′ end immediately followed the wobble position of a codon . this strategy was used to counteract the gratuitous addition of one nucleotide to the 3 ′ end of the strand complementary to the mutagenic primer by the dna polymerase during pcr ( sharrocks , a . d ., and shaw , p . e ., “ improved primer design for pcr - based , site - directed mutagenesis ,” nucl . acids res . 20 : 1147 ( 1992 )). the appropriate primer pairs ( 0 . 2 μm of each ) were combined with 10 ng of version “ b ” of reshaped human l25vl region cdna , and 1 unit of amplitaq ( perkin elmer cetus ) dna polymerase in 50 μl of pcr buffer containing 10 mm tris - hcl ( ph 8 . 3 ), 50 mm kcl , 200 μm dntps , and 1 . 5 mm mgcl 2 . this was overlaid with mineral oil and pcr was performed for 25 cycles , each cycle consisting of a denaturation step at 94 ° c . for 1 minute , a primer annealing step at 55 ° c . for 1 minute , and an extension step at 72 ° c . for 2 minutes . this was followed by a single cycle consisting of a further elongation step at 72 ° c . for 10 - minutes followed by cooling to 4 ° c . the ramp time between the primer - annealing and extension steps was 2 . 5 minutes . the pcr products of the five reactions ( a , b , c , d and e ) were then purified by gel electrophoresis followed by dna elution using wizard pcr preps ( promega ). pcr products a , b , c , d , and e were assembled by their complementarity to one another . in the second set of pcr reactions , pcr products b and c , and d and e , ( 50 ng of each ) were added to 50 ml pcr reactions ( as described above ) each containing 1 unit of amplitaq ( perkin elmer cetus ) dna polymerase . the reactions were cycled for 20 cycles as described above with the exception that the annealing temperature was raised to 60 ° c . in the third set of pcr reactions , pcr products f and g were pcr - amplified using 1 ml of each prior pcr reaction and the appropriate pair of pcr primers ( vla2 - vla5 or vla6 - apcr4 ). the pcr reactions contained 1 unit of amplitaq dna polymerase in 50 ml pcr reaction ( as described above ) and were amplified for 25 cycles as in the first stage . in the fourth set of pcr reactions , the pcr product h was pcr - amplified using 1 ml of each prior pcr reaction and the vla2 - apcr4 pair of pcr primers . finally , pcr products a and h were assembled by their own complementarity in a two step - pcr reaction similar to that described above using rsp and up as the terminal primers . the fully assembled fragment representing the entire reshaped human f19 v l region including a leader sequence was digested with hindiii and bamhi and cloned into puc19 for sequencing . a clone having the correct dna sequence was designated reshf19la ( fig2 ) and was then subcloned into the eukaryotic expression vector pkn100 . the dna sequence of reshf19la cloned into pkn100 is shown in fig3 . the second version of reshaped human f19 v l region ( l b ) was constructed using the same scheme as that described for la but where vla4 and vla7 primers were substituted by vlb4 and vlb7 respectively ( table 3 ). the dna sequence of l b is shown in fig2 . the third version of reshaped human f19 v l region ( l c ) was constructed using the quikchange ™ site - directed mutagenesis kit from stratagene . the quikchange site - directed mutagenesis method was performed according to the manufacturer &# 39 ; s instructions , using reshf19la in pkn100 vector as double stranded dna template . the mutagenic oligonucleotide primers f19lc - sense and f19lc - antisense ( table 3 ) for use in this protocol were designed according to the manufacturer &# 39 ; s instructions . briefly , both the mutagenic primers contained the desired point mutation ( codon ttt at kabat residue position 49 ( phe ) changed to tat coding for tyr ) and annealed to the same sequence on opposite strands of la in pkn100 vector . the point mutation was verified by dna sequencing the entire v l region . the dna sequence of l c is shown in fig2 . to eliminate the possibility that random mutations occurred in the pkn100 during the pcr reaction , the v l region was cut out of the pkn100 vector as an hindiii / bamhi fragment and re - subcloned into an unmodified pkn100 vector cut with the same two restriction enzymes beforehand . version “ a ” of reshaped human f19 v h regions ( h a ) was constructed using the same pcr methods as described for the construction of version “ a ” of reshaped human f19 v l region ( l a ) ( fig3 ). the template dna was version “ a ” of reshaped human 226 v h ( léger , o . j . p ., et al ., “ humanization of a mouse antibody against human alpha - 4 integrin : a potential therapeutic for the treatment of multiple sclerosis ,” hum . antibod . 8 : 3 ( 1997 )). six pcr primers were designed and synthesized for the construction of version “ a ” of reshaped human f19 v h region ( table 4 ). pcr products a , b , c , and d were obtained using apcr1 - vha1 , vha2 - vha3 , vha4 - vha5 and vha6 - apcr4 as pcr primer pairs , respectively . the pcr conditions were essentially as described for the construction of reshaped human f19 v l region . a clone having the correct dna sequence was designated reshf19ha ( fig3 ) and was then subcloned into the eukaryotic expression vector pg1d105 . the dna sequence of reshf19ha cloned into pg1d105 is shown in fig3 . the third version of reshaped human f19 v h region ( h c ) was constructed using the same scheme as that described for h a but where vha4 primer was substituted by vhc4 ( table 4 ). the dna sequence of h c is shown in fig3 . the second ( h b ) and fourth ( h d ) version of reshaped human f19 v h region were constructed based on the pcr - mutagenesis methods of kamman et al . ( kamman , m ., et al , “ rapid insertional mutagenesis of dna by polymerase chain reaction ( pcr ),” nucl . acids res . 17 : 5404 ( 1989 )). for h b and h d , a mutagenic primer f19vhbd6 ( tyr - 91 to phe - 91 , table 4 ) was used paired with apcr4 in pcr reactions with h a and h c as the template dna , respectively . the pcr products vhb and vhd were restriction enzyme digested with psti and bamhi and subcloned into reshf19ha and reshf19hc , respectively , previously digested with the same two restriction enzymes . the dna sequences of h b and h d are shown in fig3 . version “ e ” of reshaped human f19 v h region ( h e ) was constructed based on the pcr - mutagenesis methods of kamman et al . ( 1989 ) already mentioned above : for reshf19he mutagenic primer f19mscihe ( table 5 ) was used paired with primer f19hindiii ( table 5 ) in pcr reactions with h c cloned in pg1d105 mammalian expression vector as the template dna . the appropriate primer pairs ( 0 . 2 mm of each ) were combined with 10 ng of cdna of version “ a ” of reshaped human 226 vh region in 100 ml of pcr buffer containing 10 mm kcl , 10 mm ( nh 4 ) 2so 4 , 20 mm tris - hcl ( ph 8 . 8 ) 2 mm mgso 4 , 0 . 1 % triton x - 100 and 200 mm dntps . reaction mixtures were overlaid with mineral oil and kept at 94 ° c . for 5 minutes . then 1 unit of deep vent dna polymerase ( new england biolabs ) was added (“ hot start ” pcr ; chou q ., russell , m ., et al ., “ prevention of pre - pcr mis - priming and primer dimerization improves low - copy - number amplifications ,” nucl . acids res . 20 : 1717 ( 1992 )) and pcr was performed for 25 cycles on a trio - thermoblock thermal cycler ( biometra , göttingen , germany ). each cycle consisting of a denaturation step at 94 ° c . for 1 minute , a primer - annealing step at 70 ° c . for 1 minute , and an extension step at 72 ° c . for 2 minutes . this was followed by a single cycle consisting of a further elongation step at 72 ° c . for 10 minutes followed by cooling at 4 ° c . the pcr products were then extracted and purified from a tae 1 . 4 % standard agarose gel using a qiaquick ™ gel extraction kit , following the protocol supplied by the manufacturer ( qiagen ltd ., uk ). the pcr product v h e was then restriction enzyme digested with msci and hindiii and ligated into reshf19hc cloned in pg1d105 previously digested with the same two restriction enzymes . the msci restriction recognition site is unique to all the reshaped human f19 v h region versions and is not present in the pg1d105 expression vector . the hindiii restriction recognition site is a unique site in pg1d105 for cloning of v h immunoglobulin genes . electroporation - competent xl - 1 blue e . coli cells were transformed with 1 μl of the ligated dna and plated on agarose plates containing ampicillin . colonies were then screened for the presence and correct size of inserts by direct pcr on colonies ( guissow , d ., and clackson , t ., “ direct clone characterization from plaques and colonies by the polymerase chain reaction ,” nucl . acids res . 17 : 4000 ( 1989 )) with primers hcmi and hucg1 hybridizing to the flanking pg1d105 vector sequences ( table 5 ). dna from positive colonies was prepared using a plasmid midi kit , following the protocol supplied by the manufacturer ( qiagen ltd ., uk ). dna sequencing was performed by the dideoxy chain termination method ( sanger , f ., et al ., “ dna sequencing with chain - terminating inhibitors ,” proc . natl . acad . sci . u . s . a . 74 : 5463 ( 1977 )) directly from circular vector dna using conventional heat denaturation ( andersen , a ., et al ., “ a fast and simple technique for sequencing plasmid dna with sequenase using heat denaturation ,” biotechniques 13 : 678 ( 1992 )) and sequenase 2 . 0 ( usb , cleveland , ohio ). the dna sequences of reshf19he is shown in fig3 . [ 0154 ] table 5 pcr primer for the construction of reshaped human f19 heavy chain variable regions version e 1 . primer for the synthesis of version “ e ” f19msclhe ( 65 mer , anti - sense ): 5 ′ cctttggccaggggcctgtctaacccagtgtatggtgtattcagtgaaggtgtatccactagtttccactagttt 3 ′ msci 2 . primers hybridizing to the flanking pg1d105 mammalian expression vector sequences hcmi ( 28 mer , sense ): 5 ′ gtcaccgtccttgacacgcgtctcggga 3 ′ hucg1 ( 17 mer , anti - sense ): 5 ′ ttggaggagggtgccag 3 ′ cos cells were transfected with one pair of a series of reshaped human f19 antibody constructs and the human antibody concentration was measured using the igg1 / kappa elisa as described in example 2 . [ 0156 ] table 7 reshaped human f19 antibody concentrations in cos cell supernatants transfected antibody components human γ1 / k heavy chain kappa light chain concentration [ μg / ml ] h a l a 2 . 00 h a l c 2 . 50 h c l a 2 . 90 h c l c 3 . 00 h e l a 2 . 80 h e l c 3 . 50 rna splicing events required for the expression of immunoglobulin genes in mammalian cells both mammalian expression vectors pkn100 and pg1d105 have an intron between the variable and the constant regions which is removed during the process of gene expression to give rise to an messenger rna . the splicing event which consists of a dna recombination between the heavy or light chain splice donor sites and the immunoglobulin splice acceptor site is described in fig3 . flow cytometric analysis of the binding of cf19 and l a h c to fap - expressing human cells the ability of l a h c to bind to both recombinant and endogenously expressed fap on cell surface was tested . the example was conducted to determine the binding of l a h c to cellular fap . both naturally fap expressing mf - sh human tumor cells ( shirasuma , k ., et al ., cancer 55 : 2521 - 2532 ( 1985 )) and fap - transfected human tumor cell lines were used as cellular targets . l a h c was studied in cytofluorometric assays evaluating direct binding to target cells as well as by the inhibitory effect on the binding of either murine f19 or chimeric cf19 anti - fap antibodies . antibodies and cell lines used were f19 ( murine monoclonal anti - human fap antibody , igg1 subclass ), migg ( murine immunoglobulin , igg class ), cf19 ( chimeric monoclonal anti - human fap antibody , igg1 subclass ), l a h c ( reshaped monoclonal anti - human fap antibody , igg1 subclass ), higg1 ( human immunoglobulin , igg1 subclass ), mf - sh ( human malignant fibrous histiocytoma cell line ), ht - 1080 ( human fibrosarcoma cell line ), ht - 1080fap clone 33 ( ht - 1080 cell line transfected with cdna encoding human fap ). antibodies were biotinylated as described in examples 8 and 12 . direct binding of l a h c to fap on the surface of human tumor cell lines 5 × 10 5 cells of the tumor cell line under investigation were incubated with the indicated concentration of test or control antibody in a total volume of 0 . 2 ml phosphate - buffered saline ( pbs ) supplemented with 1 % bovine serum albumin ( bsa ) for 30 minutes on ice . subsequently , cells were washed twice with 2 ml of pbs , resuspended in 0 . 2 ml of pbs supplemented with 1 % bsa , a 1 : 20 dilution of mouse anti - human igg fitc - labelled ( dianova ) as secondary reagent was added and incubated for another 30 minutes on ice . alternatively , 5 × 10 5 cells of the tumor cell line under investigation were incubated with the indicated concentration of biotin - labelled cf19 in a total volume of 0 . 2 ml pbs supplemented with 1 % bsa for 30 minutes on ice . subsequently , cells were washed twice with 2 ml of pbs , resuspended in 0 . 2 ml of pbs supplemented with 1 % bsa , and incubated for another 30 minutes on ice with 1 : 40 dilution of streptavidin - fitc ( dianova ) as secondary reagent . cells were again washed twice with 2 ml of pbs , resuspended in a total volume of 0 . 5 ml of pbs supplemented with 1 % paraformaldehyde ( pfa ) and kept on ice . single cell fluorescence was determined cytofluorometrically by analysing the cellular green fluorescence at 488 nm in an epics xl ( coulter ) fluorescence - activated cell analyzer . competition of l a h c for binding of biotinylated cf19 to cell - surface fap on fap - expressing human cell lines 5 × 10 5 cells of the tumor cell line under investigation were incubated with the indicated amounts of unlabeled test or control antibody added together with 1 μg / ml biotin - labelled cf19 antibody . subsequently , cells were washed twice with 2 ml of pbs , resuspended in 0 . 2 ml of pbs supplemented with 1 % bsa , 1 : 40 diluted streptavidin - fitc ( dianova ) as secondary reagent and incubated for another 30 minutes on ice . cells were then washed twice with 2 ml of pbs , resuspended in a total volume of 0 . 5 ml pbs supplemented with 1 % pfa and kept on ice . single cell fluorescence was determined cytofluorometrically by analysing the cellular green fluorescence at 488 nm in an epics xl ( coulter ) fluorescence - activated cell analyzer . both , cf19 and l a h c bind in a concentration dependent manner specifically to to fap - transfected ht - 1080fap clone33 human tumor cells ( table 8 ). no binding to fap - negative ht - 1080 cells was detectable ( table 9 ). both cf19 and l a h c bound in a concentration dependent manner to human mf - sh cells endogenously expressing fap ( table 10 ). biotinylated cf19 bound to human ht - 1080fap clone 33 ( table 11 ) in a concentration dependent manner . no binding was detectable to fap - negative ht - 1080 cells ( table 12 ). binding of biotinylated cf19 to ht - 1080fap clone 33 cells was inhibited by both unlabelled cf19 and unlabelled l a h c ( table 13 ). chimeric anti - human fap monoclonal antibody cf19 as well as reshaped human anti - human fap monoclonal antibody l a h c ( example 10 ) were shown to bind directly to fap expressed on human cell lines either endogenously expressing this protein or transfected with cdna encoding for it . this binding was shown to be concentration dependent . binding of biotinylated cf19 could be inhibited by both unlabelled cf19 and unlabelled l a h c . using cytofluorometric technology , direct binding as well as inhibition of specifically binding reagents showed specificity of chimeric cf19 and reshaped l a h c human monoclonal antibodies to cell surface expressed fap . [ 0175 ] table 13 competition of anti - fap antibodies with the binding of biotinylated cf19 to ht - 1080fap clone 33 cells concentration of competitor antibody mean fluorescence competitor antibody [ μg / ml ] concentration no 0 . 00 11 . 2 higg1 1 . 00 9 . 0 higg1 3 . 16 11 . 3 higg1 10 . 00 9 . 8 higg1 31 . 66 10 . 3 cf19 1 . 00 7 . 5 cf19 3 . 16 4 . 8 cf19 10 . 00 1 . 3 cf19 31 . 66 1 . 2 l a h c 1 . 00 8 . 0 l a h c 3 . 16 5 . 5 l a h c 10 . 00 2 . 9 l a h c 31 . 66 1 . 7 in vitro immune effector functions of monoclonal antibody l a h c this experiment was conducted to determine the potential of the monoclonal antibody ( mab ) l a h c with specificity for fibroblast activation antigen ( fap ) to lyse fap - expressing targets in the presence of human complement or human mononuclear leukocytes , respectively . in particular , the ability of l a h c to mediate cytotoxic effects against ht - 1080fap clone 33 cells , which expressed human fap on the surface , was studied . cytotoxicity was determined in vitro using the following approach : 51 cr - labelled target cells were incubated in the presence of l a h c with human serum as source of complement or human mnc ( peripheral blood mononuclear cells ) as effectors . release of 51 cr was measured as measure of target - cell lysis . antibodies and cell lines used were l a h c ( reshaped human anti - human fap igg1 antibody ), higg1 ( human igg1 isotype control ), 3s193 ( murine monoclonal anti - lewis y igg3 antibody ), migg ( murine igg control ), ht - 1080 ( human fibrosarcoma ), ht - 1080fap clone 33 , ( ht1080 transfected with cdna encoding human fap ), mcf - 7 ( human breast adenocarcinoma cell line ). tumor cells were radiolabelled by incubation in rpmi 1640 medium with 100 μl - ci 51 cr ( nen ) at 37 ° c . for one hour . subsequently , cells were washed twice in 51 cr - free medium and resuspended at a concentration of 2 × 10 5 cells per ml . human serum as source of complement was freshly prepared from blood of different volunteers . blood was taken by puncturing the arm vein , remained at room temperature for one hour to allow clotting to occur , and was kept at 4 ° c . over night . serum was separated by centrifugation and taken off from the sediment . the antibody under study was diluted from the stock solution to the appropriate concentration in rpmi1640 cell culture medium . 1 × 10 5 radiolabelled tumor cells of the indicated cell line were incubated for 2 h at 37 ° c . in an incubator ( 95 % air and 5 % co 2 ) in the presence of different concentrations of test or control antibody and 25 % ( v / v ) human serum as the source of human complement . incubations were performed in u - shaped 96 - well plates in a total volume of 200 μl rpmi1640 and done in triplicate . after the incubation period , plates were centrifuged , 100 μl of the supernatant was removed and radioactivity was counted in a gamma - counter . the total amount of incorporated radioactivity was determined by measuring 10 4 target cells . spontaneous release was defined as activity released from the target cells in the absence of both antibody and complement during the described incubation period . specific   lysis ( in   % ) } = [ activity   sample ] - [ activity   spontaneous   release ] [ maximum   activity ] - [ activity   spontaneous   release ] × 100 tumor cells were radiolabelled by incubation in rpmi1640 medium with 100 μl - ci 51 cr at 37 ° c . for one hour . subsequently , cells were washed twice in 51 cr - free medium and resuspended at a concentration of 2 × 10 5 cells per ml . mnc ( peripheral blood mononuclear cells ) were prepared from peripheral blood taken by puncturing the arm vein of different healthy human volunteers . clotting was prevented by the addition of 20 % citrate buffer . mnc from 4 ml of this blood preparation were purified by centrifugation ( 30 minutes at 400 g and room temperature ) on 3 ml of lymphocyte preparation medium ( boehringer mannheim , germany ). mnc ( peripheral blood mononuclear cells ) were taken off from the gradient , washed three times and diluted with rpmi1640 to the appropriate concentration . lymphocyte activated killer ( lak ) cells were derived from mnc ( peripheral blood mononuclear cells ) by incubation for 5 days at 37 ° c . in an 95 % air and 5 % co 2 incubator at an initial density of 1 . 3 × 10 6 cells per ml in the presence of 100 u recombinant human interleukin - 2 ( il - 2 ). the antibody under study was diluted from the stock solution to the appropriate concentration in rpmi1640 cell culture medium . 1 × 10 4 radiolabelled tumor cells of the indicated cell line were incubated for 5 h at 37 ° c . and 5 % co 2 in the presence of different concentrations of test or control antibody and mnc . mnc were added in amounts to reach the indicated effector : target cell ratio . incubation was performed in u - shaped 96 - well plates in a total volume of 200 μl rpmi1640 and done in duplicate . after the incubation period , plates were centrifugated , 100 μl of the supernatant were taken off and radioactivity was determined in a gamma - counter . the total amount of incorporated radioactivity was determined by measuring 10 4 target cells . spontaneous release was defined as activity released from the target cells in the absence of both antibody and effector cells during the described incubation period . specific   lysis ( in   % ) } = [ activity   sample ] - [ activity   spontaneous   release ] [ maximum   activity ] - [ activity   spontaneous   release ] × 100 no l a h c - specific complement - mediated lysis ( above that seen with an isotype control ) was observed in ht - 1080fap clone 33 cells treated with l a h c at concentrations up to 50 μg / ml ( table 14 , table 15a ). lytic activity of human serum used as source of complement was shown by lysis of mcf - 7 human breast carcinoma cells in the presence of 12 . 5 μg / ml 3s193 , a murine monoclonal anti - lewis y antibody with known complement activating ability ( table 15b ). in the presence of l a h c at concentrations up to 10 μg / ml , no adcc ( antibody - dependent cellular toxicity ) mediated by human mnc ( table 16 ) or human lak cells ( lymphokine activated killer cells , table 17 ) of l a h c on ht - 1080fap clone 33 as measured by lysis was detectable above that seen with an isotype control at an effector : target ratio of 50 : 1 . in appropriate in vitro assays with either human complement or with human mnc as effector mechanisms , human anti - fap monoclonal antibody l a h c revealed no detectable cytotoxic effects above isotype controls on fap - expressing tumor cell line ht - 1080fap clone 33 . [ 0193 ] table 15a specific complement lysis ( in %) of ht - 1080fap clone 33 tumor cell targets mediated by human anti - fap monoclonal antibody l a h c source of human serum : ht - 1080 clone 33 : concentration of antibody higg1 l a h c a 10 . 00 μg / ml 2 1 a 2 . 50 μg / ml 2 2 a 0 . 60 μg / ml 1 1 a 0 . 15 μg / ml 1 2 a 0 . 00 μg / ml 2 2 b 10 . 00 μg / ml 2 2 b 2 . 50 μg / ml 2 2 b 0 . 60 μg / ml 2 2 b 0 . 15 μg / ml 2 2 b 0 . 00 μg / ml 2 2 c 10 . 00 μg / ml 2 2 c 2 . 50 μg / ml 1 1 c 0 . 60 μg / ml 1 1 c 0 . 15 μg / ml 2 1 c 0 . 00 μg / ml 3 3 [ 0194 ] table 15b specific complement lysis ( in %) of mcf - 7 tumor cell targets mediated by murine anti - lewis y monoclonal antibody 3s193 source of human serum : mcf - 7 : concentration of antibody migg 3s193 a 10 . 00 μg / ml 0 21 a 2 . 50 μg / ml 1 21 a 0 . 60 μg / ml 0 21 a 0 . 15 μg / ml 1 18 a 0 . 00 μg / ml 0 0 b 10 . 00 μg / ml 1 13 b 2 . 50 μg / ml 0 17 b 0 . 60 μg / ml 1 18 b 0 . 15 μg / ml 1 15 b 0 . 00 μg / ml 0 0 c 10 . 00 μg / ml 1 22 c 2 . 50 μg / ml 0 23 c 0 . 60 μg / ml 1 26 c 0 . 15 μg / ml 1 20 c 0 . 00 μg / ml 1 1 [ 0195 ] table 16 adcc ( antibody - dependant cellular cytotoxicity ) ( specific lysis in %) of ht - 1080fap clone 33 target cells by human mnc ( peripheral blood mononuclear cells ) mediated by l a h c ht - 1080fap clone 33 : concentration of antibody : ht - 1080fap clone 33 : [ in μg / ml ] higg1 l a h c 10 2 2 2 . 5 2 2 0 . 625 2 2 0 . 156 3 3 0 3 3 [ 0196 ] table 17 adcc ( antibody - dependent cellular cytotoxicity , specific lysis in %) of ht - 1080fap clone 33 target cells by lak cells ( lymphokine activated killer cells ) mediated by l a h c concentration of antibody : ht - 1080fap clone 33 : [ in μg / ml ] higg1 l a h c 10 12 14 2 . 5 14 17 0 . 625 14 21 0 . 156 15 21 0 14 14 immunohistochemical analysis of monoclonal antibody l a h c binding to normal and neoplastic human tissues this experiment was performed to determine the binding characteristics of the humanized mab l a h c to normal and neoplastic human tissues . the following antibodies were used : l a h c , cf19 , and the negative control higg1 were directly biotinylated according to methods of the state of the art and used at concentrations of 2 . 5 to 0 . 25 mg / ml in 2 % bsa / pbs ( bovine serum albumin in phosphate - buffered saline ). murine mab f19 was used as tissue culture supernatant of the f19 hybridoma , at dilutions of 1 : 5 to 1 : 10 in 2 % bsa / pbs . the following reagents were used for immunochemical assays : streptavidin peroxidase complex ( vector labs ., burlingame , calif ., usa ), avidin - biotin peroxidase complex ( vector labs . ), biotinylated horse anti - mouse ( vector labs . ), dab ( diaminobenzidine , sigma chemical co ., st . louis , mo ., usa ), harris &# 39 ; hematoxylin . fresh frozen tissue samples examined included the following : normal colon , breast , lung , stomach , pancreas , skin , larynx , urinary bladder , smooth and skeletal muscle . among the tumors tested were carcinomas from breast , colon , lung , esophagus , uterus , ovary , pancreas , stomach , and head and neck . an indirect immunoperoxidase method was carried out according to state of the art methods ( garin - chesa , p ., et al ., “ cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers ,” proc . natl . acad . sci . usa 87 : 7235 - 7239 ( 1990 )) on five micrometer thickness fresh frozen sections . dab was used as a substrate for the final reaction product . the sections were counterstained with harris &# 39 ; hematoxylin and examined for antigen expression . the normal tissues tested were negative for l a h c expression , except for the normal pancreas in which a subset of positive endocrine cells in the islets of langerhans ( a cells ) were identified with l a h c , cf19 and f19 . ( table 18 ). no immunoreactivity was observed with the hlgg1 ( human immunoglobulin igg1 subclass ) used as a negative control . in the tumor samples , l a h c , cf19 and f19 showed an indistinguishable pattern of expression in the tumor stromal fibroblasts . a strong and homogeneous expression was found in the majority of the cases examined , especially in the cancer samples derived from breast , colon , lung , pancreas and in the squamous cell carcinomas ( sqcc ) of the head and neck tested ( table 18 ). no immunoreactivity was observed with the hlgg1 used as negative control . l a h c , cf19 and f19 showed immunoreactivity with the tumor stromal fibroblasts in the epithelial cancer samples tested . no l a h c or f19 immuno - reactivity was seen with either the fibrocytes of the normal organ mesenchyme or the parenchymal cells of normal adult organs . anti - fap immunoreactivity was only observed in a subset of endocrine cells in the pancreatic islets , presumably glucagon - producing a cells , and in four of nine uterine samples tested , representing subsets of stromal fibroblasts in these tissues . immunohistochemical analysis of l a h c in normal human tissues and fap - expressing human carcinomas showed indistinguishable patterns of binding for l a h c , cf19 and murine mab f19 . the following fresh frozen tissue samples from mouse , rat , rabbit and cynomolgus were tested : brain , liver , lung , kidney , stomach , pancreas , intestine , thymus , skin , muscle , heart , spleen , ovary , uterus and testes . as positive control , sections from normal human pancreas and a breast carcinoma sample were included in every assay . an indirect immunoperoxidase method was carried out as described in the state of the art ( garin - chesa , p ., et al ., “ cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers ,” proc . natl . acad . sci . usa 87 : 7235 - 7239 ( 1990 )) on five micrometer thickness fresh frozen sections . the antibodies l a h c , cf19 and hlgg1 ( at 1 μg / ml ) were biotinylated according to the state of the art and were detected with streptavidin peroxidase complex . dab was used as a substrate for the final reaction product . the sections were counterstained with harris &# 39 ; hematoxylin and examined for antigen expression . the normal tissues tested did not react with either l a h c or cf19 in the experiments ( table 1 ). the normal human pancreas used as positive control showed l a h c and cf19 binding in a subset of endocrine cells in the islets of langerhans as previously described for f19 . in addition , binding of l a h c and cf19 was seen in the tumor stromal fibroblasts in the breast carcinoma sample . immunohistochemical analysis of normal tissues from mouse , rat , rabbit and cynomolgus failed to detect any binding of either l a h c or cf19 , in the experiments performed . the objective of this experiment was to demonstrate stable cell lines according to the invention expressing l a h c , l a h a , l b h b , l b h d and cf19 in cho dg44 cells . stable cell lines transfected with humanized or chimeric f19 antibodies were produced and their identity was confirmed by pcr amplification of heavy and light variable regions using genomic dna derived from each transfectant as template . cho dg44 cells maintained under serum - free conditions in sfm - ii medium . lipofectin and sfm - ii serum - free medium were obtained from gibco / brl . geneticin and all restriction enzymes were obtained from boehringer mannheim . pfu polymerase was obtained from stratagene . dna for transfections was purified from e . coli cells using qiafilter maxi cartridges ( qiagen ) as directed by the manufacturer . all dna preparations were examined by restriction enzyme digestion . sequences of l a h c variable regions in their respective vectors were confirmed using an abi prism 310 sequencer ( perkin - elmer ). further information regarding the vectors and dna sequences employed is available in the prior examples . cells in logarithmic growth were plated into 6 well plates containing 1 ml fresh sfm - ii medium . plasmids encoding heavy and light chains of humanized or chimeric f19 versions were cotransfected into cho dg44 cells using liposomal transfection . liposomes were prepared using 6 μl lipofectin reagent and 0 . 5 μg of each vector ( one for the desired heavy chain and one for the light ) as described for lipofectamine transfections except that sfm - ii medium was used to dilute all reagents . twenty - four hours later , cells were diluted 1 : 10 into sfm - ii medium containing 300 μg / ml geneticin . after the initial phase of cell killing was over ( 10 - 14 days ), the concentration of geneticin was reduced to 200 mg / ml and methotrexate was added to a final concentration of 5 nm . methotrexate concentrations were increased after 10 - 14 days to a final concentration of 20 nm . 10 7 cho dg44 cells were centrifuged in an eppendorf microcentrifuge briefly at full speed , washed once with pbs , and pelleted once again . genomic dna was prepared by ethanol precipitation after sds lysis and proteinase k treatment of the cell pellets . a mixture containing one of the following primer pairs , dntps , buffer , and pfu polymerase was used to amplify either the heavy or light chain variable region using genomic dna as template . the resulting pcr products were digested with the appropriate restriction enzyme and analyzed by agarose gel electrophoresis to confirm their identity . light chain primer set : 5 ′- gag aca ttg tga ccc aat ctc c - 3 ′ pkn 1690 5 ′- gac agt cat aaa ctg cca cat ctt c - 3 ′ pkn . 1930 . r heavy chain primer set : 5 ′- ttg aca cgc gtc tcg gga agc tt - 3 ′ pg 5863 5 ′- ggc gca gag gat cca ctc acc t - 3 ′ pg 6332 . r the undigested heavy chain pcr product has a predicted size of 469 bp while the light chain pcr product has a predicted size of 286 bp . verification of identity was determined by restriction enzyme digest with bsteii ( heavy chain ) or nlaiv ( light chain ). cho cell lines were transfected with l a h c , l a h a , l b h b and l b h d , as well as cf19 . geneticin - resistant cells were obtained and these cells were further selected for resistance to methotrexate . pcr amplification , followed by restriction enzyme cleavage of the light and heavy chain dna produced the expected bands and confirmed the identity of l a h c , l b h b , l a h a and l b h d transfectants . the cells described were maintained under serum - free conditions at all times and were not treated with animal - derived products such as trypsin . producer cell lines transfected with expressing monoclonal l a h c , l a h a , l b h b , l b h d and cf19 antibodies were produced . their identities were confirmed using pcr amplification and restriction enzyme cleavage of the resulting pcr products of both their heavy and light chain variable regions . expression of antibody proteins in chinese hamster ovary dg 44 cells and their purification the objective of this experiment was to express and purify l a h c , l a h a , l b h b , and l b h d mabs to enable their characterization . other goals included the establishment of a quantitative elisa to permit measurement of antibody concentrations in both crude media samples as well as purified ig samples and determination of relative expression levels of various humanized f19 constructs using this assay . serum - free cho dg44 cells and usp - grade methotrexate were obtained from the biotechnical production unit of the dr . karl thomae gmbh , biberach , germany ; both products are also commercially available . cells were maintained under serum - free conditions at all times . sfm - ii serum - free medium was obtained from gibco / brl . protein a agarose was from pierce chemical ( indianapolis , ind ., usa ). human igg1 standards ( cat . no . 13889 ), p - nitrophenyl phosphate tablets ( n 2640 ), bovine serum albumin ( bsa ) ( a 7906 ), and goat anti - human kappa chain specific alkaline phosphatase - conjugated antibody ( a 3813 ) were obtained from sigma chemical ( st . louis , mo ., usa ). goat anti - human gamma - chain specific alkaline phosphatase - conjugated antibody was obtained from jackson immunoresearch laboratories ( through stratech scientific ). tris - buffered saline ( tbs ) consisted of 150 mm nacl , 50 mm tris , ph 7 . 5 . cells were cultured and maintained in t - 175 flasks in sfm - ii serum - free medium without agitation . the medium contained 200 μg / ml geneticin and 20 nm methotrexate without antibiotics . cells were passaged by dilution , were not adherent , and grew in small clusters . when the cells reached stationary phase , the medium was collected and centrifuged to remove cells and frozen at − 20 ° c . until needed . all purification steps were carried out at 4 ° c . a c10 / 10 column ( pharmacia fine chemicals ) was packed with protein a agarose ( 3 ml bed volume ). the column was washed with tbs and preeluted once with 0 . 1 m na citrate , ph 3 . 0 to insure that no loosely bound material remained on the column . the column was then immediately reequilibrated with tbs and stored at 4 ° c . spent culture supernatants were thawed and centrifuged at 10 , 000 × g for 30 minutes prior to protein a chromatography to remove debris and diluted with an equal volume of tbs . this material was loaded onto the protein a column at 0 . 5 ml / minute using a p - 1 peristaltic pump ( pharmacia ) and washed with tbs until the absorbance at 280 nm was undetectable . elution of the antibody was initiated with 0 . 1 m na citrate ph 3 . 0 at approximately 0 . 2 ml / minute . the elution was monitored at 280 nm and one ml fractions of the eluted material were collected into tubes containing sufficient tris base ph 9 to neutralize the citrate buffer . protein - containing fractions were pooled and concentrated using an amicon filtration apparatus with a ym - 30 filter and dialyzed against pbs . the column was immediately regenerated with tbs . protein dye - binding assays were performed with the biorad ( hercules , calif .) protein determination kit , according to the manufacturer &# 39 ; s instructions , using bovine serum albumin as a standard . elisa plates were coated overnight with 100 μl of goat anti - human gamma - chain specific alkaline phosphatase - conjugated antibody at 0 . 4 mg / ml in coating buffer at 4 ° c . coating antibody was removed and plates were blocked with 2 % bsa in pbs for 2 hours . all subsequent steps were performed at 37 ° c . blocking buffer was replaced with antibody samples or human igg1 standard diluted in dilution buffer , serially diluted in a 200 ml volume , and incubated for one hour . negative controls included dilution buffer and / or culture medium of nontransfected cells . wells were washed and 100 μl of goat anti - human kappa chain specific alkaline phosphatase - conjugated antibody diluted 1 : 5000 was added and incubated for one hour . wells were washed and 100 μl reaction buffer was added and incubated for 30 minutes . the reaction was stopped by addition of 1 m naoh and absorbance read at 405 nm in an elisa plate reader . results were analyzed by four - parameter iterative curve fitting . amino acid analysis was performed according to methods available in the state of the art . monoclonal antibody l a h c was produced and purified to homogeneity using protein a affinity chromatography . elisa assays using human igg1 as standard indicated l a h c recoveries exceeding 70 %. the purity of the material was estimated to be & gt ; 90 % by sds - polyacrylamide gel electrophoresis . representative expression data and typical purification yields are shown in table 20 . elisa plates were coated overnight with 100 μl of mouse anti - rat antibody ( sigma chemical r0761 ) at 1 : 2000 in coating buffer at 4 ° c . coating antibody was removed and plates were blocked with 2 % bsa in pbs for one hour . all subsequent steps were performed at room temperature . blocking buffer was replaced with 100 ml of 1 μg / ml rat anti - cd8 antibody ( pharmingen 01041d ) and incubated for one hour . plates were washed and 100 μl cd8 - fap culture supernatant ( see example 14 ) ( 1 : 2 in pbs ) was added and allowed to bind for one hour . plates were washed and antibody samples were added ( two - fold serial dilutions ) in a 100 μl volume and incubated for one hour . negative controls included human igg and / or culture medium of nontransfected cells . wells were washed and 100 μl of horse radish peroxidase ( hrp ) conjugated mouse anti - human igg1 antibody ( zymed 05 - 3320 ) diluted 1 : 500 in dilution buffer were added and incubated for one hour . wells were washed and 100 μl hrp substrate , ( azino - bis ( 3 - ethylbenzthiazoline 6 - sulfonic ) acid , sigma chemical a9941 ), were added and incubated for 60 minutes . the reaction was stopped by addition of 1 m naoh and absorbance read at 405 / 490 nm in an elisa plate reader . results were analyzed by four - parameter curve iterative curve fitting . alternatively , plates were coated directly with cf19 . fap ( recombinant human fap , see example 13 ) was allowed to bind to these plates as above and biotinylated l a h c (˜ 1 μg / ml ) was then added . antibody binding was detected with hrp - streptavidin conjugate as above . fap - expressing 293fap i / 2 cells or control 293 cells were washed with pbs and lysed with 1 % triton x - 114 in tris - buffered saline . nuclei and debris were removed by centrifugation at 10 , 000 × g . the supernatant was phase - partitioned ( estreicher , a ., et al ., “ characterization of the cellular binding site for the urokinase - type plasminogen activator ,” j . biol . chem . 264 : 1180 - 1189 ( 1989 )) to enrich membrane proteins . the detergent phase was collected and diluted in buffer containing 1 % empigen bb ( calbiochem ) to prevent reaggregation of the triton x - 114 . this material was subjected to concanavalin a agarose chromatography ( rettig , w . j ., et al ., “ regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin ,” cancer res 53 : 3327 - 3335 ( 1993 )). l a h c ( 1 - 2 mg ) was dialyzed against 50 mm bicarbonate buffer and biotinylated with a ten - fold molar excess of sulfosuccinimidyl - 6 - biotinamido hexanoate ( nhs - lc biotin , pierce chemical , rockford , ill ., usa ) for 2 hours at room temperature . unreacted product was removed by repeated microdialysis in a microconcentrator . cos - 7 cells ( american type tissue culture collection , reference number crl 1651 ) were cotransfected by electroporation with the heavy and light chain vectors encoding l a h c . anti - cd8 monoclonal antibody was immobilized onto microtiter plates . cd8 - fap from medium of insect cells infected with cd8 - fap baculovirus was allowed to bind to these plates . spent medium from cos - 7 cell cultures transiently transfected with two separate vectors encoding l a h c was serially diluted and added to the wells containing the immobilized cd8 - fap . l a h c bound to isolated immobilized cd8 - fap protein ( fig3 ). culture supernatants from mock - transfected cos - 7 cells failed to demonstrate binding . recombinant membrane - bound fap from detergent extracts of 293fap 1 / 2 cells or control extracts was serially diluted and immobilized via chimeric f19 monoclonal antibody bound to microtiter plates . biotinylated l a h c bound recombinant human fap immobilized with cf19 ( fig3 ) in a concentration - dependent manner . l a h c recognized isolated immobilized recombinant human fap carrying the epitope for murine f19 . l a h c bound to both cd8 - fap produced in insect cells , as well as fap protein produced in 293fap i / 2 cells . culture supernatants from cos - 7 cells transfected with either heavy and light chain vectors encoding l a h c or without dna ( control ) were collected three days posttransfection . cd8 - fap was immobilized via an anti - cd8 antibody as described in the text . serial dilutions of the cos - 7 supernatants were allowed to bind to the immobilized cd8 - fap and subsequently detected with an hrp - conjugated anti - human igg1 antibody . detergent extracts of fap - expressing 293fap i / 2 cells or control 293 cells were serially diluted and added to cf19 - coated microtiter plates . biotinylated l a h c was added and binding of biotinylated l a h c was detected with hrp - conjugated streptavidin . characterization of ht - 1080 fibrosarcoma cells and 293 human embryonic kidney cells transfected with cdna for human fap fibroblast activation protein ( fap ) is a cell - surface , membrane - bound protein which carries the f19 epitope and is expressed on tumor stromal fibroblasts . cell lines expressing recombinant fap protein and matched controls lacking fap were generated for the characterization of anti - fap monoclonal antibodies . cells used were ht - 1080 cells ( reference number ccl 121 ) and 293 human embryonic kidney cells ( reference number crl 1573 ) were obtained from the american type culture collection ( maryland , usa ). transfectam was obtained from promega ( madison , wis .). geneticin and all restriction enzymes were obtained from boehringer mannheim . dna for transfections was purified from e . coli cells using qiafilter maxi cartridges ( qiagen ) as directed by the manufacturer . all dna preparations were examined by restriction enzyme digestion . vector sequences were confirmed using an abi prism 310 sequencer . further information regarding the vectors and dna sequences employed has been described in scanlan , m . j ., et al ., “ molecular cloning of fibroblast activation protein alpha , a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers ,” proc . natl . acad . sci . usa 89 : 10832 - 10836 ( 1992 ). the fap cdna sequence has been deposited in genbank ( accession number hs09287 ). ht - 1080 cells were transfected with 1 mg dna using transfectam according to the manufacturer &# 39 ; s instructions . human embryonic kidney 293 cells were transfected by calcium phosphate transfection ( brann , m . r ., et al ., “ expression of cloned muscarinic receptor in a9 l cells ,” mol . pharmacol . 32 : 450 - 455 ( 1987 )) with 10 mg dna . twenty - four hours later , cells were diluted 1 : 10 into fresh medium containing 200 mg / ml geneticin . colonies were picked and examined by immunofluorescence for fap expression as described in rettig , w . j ., et al ., “ cell - surface glycoproteins of human sarcomas : differential expression in normal and malignant tissues and cultured cells ,” proc . natl . acad . sci . usa 85 : 3110 - 3114 ( 1988 ). immunoprecipitations with cf19 were performed with metabolically labelled cells as described in rettig , w . j ., et al ., “ regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin ,” cancer res . 53 : 3327 - 3335 ( 1993 ). ht - 1080 and 293 cells were tested for fap antigen expression in immunofluorescence assays with anti - fap antibodies and were found to be antigen - negative . transfection of these cells with fap . 38 vector resulted in the generation of geneticin - resistant colonies . isolated colonies were picked and analyzed by immunofluorescence for fap expression . two cell clones were identified , designated ht - 1080fap clone 33 and 293fap i / 2 , which express cell surface - bound fap protein , as recognized by cf19 antibody . staining of nonpermeabilized ht - 1080fap clone 33 cells and 293fap i / 2 with cf19 antibody confirmed the cell surface localization of the fap protein . immunoprecipitation of radiolabelled fap protein with cf19 from extracts of 35s - methionine labelled ht - 1080fap clone 33 cells or 293fap i / 2 cells resulted in the appearance of a 93 kilodalton band after autoradiography . this band is not detectable in immunoprecipitates of parental ht - 1080 or 293 cell extracts . two stably transfected cell lines , ht - 1080fap clone 33 and 293fap i / 2 , express fap on the cell surface as determined in immunological assays with anti - fap mabs . neither parental ht - 1080 cells nor parental 293 cells express detectable levels of fap . a soluble form of human fap ( fibroblast activation protein ) in the form of a cd8 - fap fusion protein was produced in insect cells for the characterization of l a h c containing the binding site for anti - fap mabs . murine cd8 was chosen to permit secretion of the protein and to provide an additional epitope tag . the cdna encoding the extracellular domain of cd8 , consisting of the first 189 amino acids of murine cd8α ( genbank m12825 ), was linked to that of the extracellular domain of fap ( amino acids 27 to 760 ), essentially as described by lane , et al . ( lane , p ., et al ., “ soluble cd40 ligand can replace the normal t cell - derived cd40 ligand signal to b cells in t cell - dependent activation ,” j . exp . med . 177 : 1209 - 1213 ( 1993 )) using standard pcr protocols . the authenticity of all clones was verified by dna sequencing . the resulting dna was inserted into the pvl1393 vector ( invitrogen ) and transfection of sf9 cells ( invitrogen ) with this vector and amplification of the resulting recombinant baculovirus were performed as described ( baculovirus expression vectors . a laboratory manual , o &# 39 ; reilly , d . r ., et al ., eds ., oxford university press :, new york ( 1994 )). the spent medium of high five ™ cells ( invitrogen ) infected with recombinant cd8 - fap baculovirus for four days was collected and cleared by ultracentrifugation . the cd8 - fap elisa ( enzyme - linked immunosorbent assay ) has been described above ( example 12 ). insect cell cultures infected with cd8 - fap virus secreted a fusion protein into the medium which carries the f19 epitope and is recognized by an anti - fap antibody ( fig1 ). neither the cell culture medium alone nor medium from insect cells infected with cd8 - cd40l fusion protein bound anti - fap antibody . soluble cd8 - fap protein carrying the f19 epitope was secreted into the medium of infected insect cell cultures . culture supernatant from cells infected with a control construct did not contain antigen bearing the f19 epitope . a soluble form of fap , cd8 - fap , was produced in insect cells and cd8 - fap was shown to carry the epitope recognized by cf19 . supernatants from insect cells infected with recombinant baculovirus encoding either cd8 - fap or cd8 - cd40l fusion protein were collected four days postinfection . cell culture medium without cells was used as an additional control ( medium ). serial dilutions of these materials were added to anti - cd8 antibody - coated microtiter plates and allowed to bind . cf19 ( 1 mg / ml ) was subsequently 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asp ser leu ala val ser leu gly glu arg ala thr ile asn cys lys ser ser gln ser leu leu tyr ser pro pro lys leu leu ile tyr trp ala ser thr arg glu ser gly val tyr phe ser tyr pro leu thr phe gly gln gly thr lys val glu ile gly ser ser gly asp ile val met thr gln ser pro asp ser leu ala val ser leu gly glu arg ala thr ile asn cys lys ser ser gln ser lys pro gly gln pro pro lys leu leu ile phe trp ala ser thr arg phe thr leu thr ile ser ser leu gln ala glu asp val ala val tyr pro pro ser asp glu gln leu lys ser gly thr ala ser val val cys leu leu asn asn phe tyr pro arg glu ala lys val gln trp lys val gln gly leu ser ser pro val thr lys ser phe asn arg gly glu cys thr ile his trp val arg gln ala pro gly gln arg leu glu trp ile met glu leu ser ser leu arg ser glu asp thr ala val tyr tyr cys thr ile his trp val arg gln ala pro gly gln arg leu glu trp ile met glu leu ser ser leu arg ser glu asp thr ala val tyr phe cys thr ile his trp val arg gln ala pro gly gln arg leu glu trp ile met glu leu ser ser leu arg ser glu asp thr ala val tyr tyr cys thr ile his trp val arg gln ala pro gly gln arg leu glu trp ile met glu leu ser ser leu arg ser glu asp thr ala val tyr tyr cys met asp trp thr trp arg val phe cys leu leu ala val ala pro gly pro gly ala ser val lys val ser cys lys thr ser arg tyr thr phe thr glu tyr thr ile his trp val arg gln ala pro gly gln arg leu ala met asp tyr trp gly gln gly thr leu val thr val ser ser ser ser gly gly thr ala ala leu gly cys leu val lys asp tyr phe pro his thr phe pro ala val leu gln ser ser gly leu tyr ser leu ser lys asp thr leu met ile ser arg thr pro glu val thr cys val val val asp val ser his glu asp pro glu val lys phe asn trp tyr val asp gly val glu val his asn ala lys thr lys pro arg glu glu gln asp trp leu asn gly lys glu tyr lys cys lys val ser asn lys ala lys asn gln val ser leu thr cys leu val lys gly phe tyr pro ser asp ile ala val glu trp glu ser asn gly gln pro glu asn asn tyr ser lys leu thr val asp lys ser arg trp gln gln gly asn val phe ser cys ser val met his glu ala leu his asn his tyr thr gln lys thr ile his trp val arg gln ala pro gly gln arg leu glu trp ile met glu leu ser ser leu arg ser glu asp thr ala val tyr phe cys