Patent Application: US-48776195-A

Abstract:
hybridoma cell lines producing monoclonal antibodies specific to the human epidermal growth factor receptor are disclosed . the antibodies are capable of inhibiting the growth of human tumor cells expressing human epidermal growth factor receptors . therapeutic uses of these monoclonal antibodies by themselves and in combination with anti - neoplastic agents are also disclosed .

Description:
balb / c mice were immunized by intraperitioneal injections of ch 71 cells or ch 71 cell membrane preparation . ch 71 cells are chinese hamster ovary cells which have been transfected with a plasmid bearing a truncated form ( deletion of most of the intracellular domain of the egf - r ) of the egf - r cdna ( livneh et al ., j . biol . chem ., vol . 260 , 12490 ( 1986 ). these transfected cells express approximately 10 6 mutant egf - r molecules / cell . the choice of ch - 71 cells allows the selection in the first screening test of only hybridomas secreting antibodies against the extracellular domain of the egf - r and avoids the selection of antibodies directed against the human specific carbohydrates linked to the human egf - r molecule . the mice were immunized three times on day 0 , 13 , and 32 . the two best responding mice were each boosted by three intraperitioneal injections of ch 71 cells three consecutive days before the fusion . on day 65 , the spleen cells of the mice were then fused with ns1 myeloma cells ( ratio 5 / 1 ) according to the general procedure of kohler and milstein , using peg 4000 ( merck ) as the fusing agent . ( kohler and milstein , eur . j . immuno ., vol . 6 , 511 - 519 ( 1976 ). the fusion product was diluted in hypoxanthineazaserine ( ha ) selection medium ( g . buttin et al ., current topics in microbiology and immunology , vol . 81 , 27 - 36 , ( 1978 )) instead of the hypoxanthine - amminopterin - thymidine ( hat ) selection medium and distributed in 96 well plates . the presence of specific antibodies in the medium of the wells of the growing hybridoma cells was first assayed by radioimmunoassay . cells expressing or not expressing the egf receptor were plated in 96 well plates . at confluency , they were washed once with binding medium ( dmem , 20 mm hepes , 0 . 2 bsa ) and incubated for 90 minutes at room temperature with 100 μl of culture supernatant from the different growing hybridomas . cells were then washed 3 times with binding medium and incubated for a further 60 minutes at room temperature with 100 μl of a solution of iodinated goat antimouse immunoglobulins ( 250 , 000 cpm / 100 μl .). after 3 washes with pbs ( phosphate buffered saline , ph 7 . 5 ), the cells were scraped from the wells and the radioactivity which was associated with their surface was counted using a gamma counter . the ability of the antibodies to bind specifically to the surface of cells expressing the egf receptor ( a 431 , human fibroblasts or mouse 3t3 cells transfected with human egf - r dna constructs ) was measured in this way and compared to their ability to bind to cells that do not express the egf - r ( a particular clone of mouse 3t3 cells ). the positive hybridomas were cloned by limiting dilution and further tested by measuring their ability to immunoprecipitate 35 s methionine or 32 p labeled egf - r from lysates of cell lines of different species ( human , mouse , chicken ). for this , goat antimouse immunoglobulins were bound to protein a sepharose by incubation of goat antimouse antibody solution with protein a sepharose beads for 30 minutes at room temperature . this was followed by washing 3 times with hepes 20 mm , ph 7 . 4 . then the goat mouse igs coated protein a sepharose beads were further incubated for 30 minutes at room temperature with the culture supernatant of the hybridomas , washed 3 times with hntg buffer ( hepes 20 mm , 150 mm , nacl , 0 . 1 % triton × 100 , 10 % glycerol ) and incubated for 1 hour at 4 degrees c . with the different cell lysates obtained by lysing cell monolayers with solubilization buffer ( 1 % triton × 100 , 150 mm nacl , 20 mm hepes , 1 . 5 mm egta , 1 . 5 mm mgcl 2 , 10 % glycerol , aprotinin , leupeptin and pmsf as protease inhibitors ) and centrifugation of the lysate to discard the nuclear pellet . for 32 p labeling , the immunoprecipitates were washed with hntg 3 times and then incubated for 15 minutes with a 32 p atp solution ( hntg with 5 mm mncl 2 and 3 μci / sample of 32 p atp ). electrophoresis sample buffer was then added and the samples boiled for 10 min at 95 degrees c . prior to loading on a 7 . 5 % sds polyacrylamide gel . monoclonal antibodies 108 , 96 and 42 were all found to be specific for the human egf - r . these antibodies were also tested for their ability to inhibit the binding of iodinated egf to the surface of cells expressing egf - r . these 3 antibodies inhibit the binding of egf to its receptor , but the level of inhibition varied with 96 & gt ; 108 & gt ; 42 . the kb human tumor cell line derived from oral epidermoid carcinoma was obtained from the american type tissue culture collection . the cells were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % fetal calf serum depleted of complement activity by incubation at 56 ° c . for 30 minutes and grown in glutamine , penicillin , streptomycin and sodium pyruvate , at 37 ° c . in 5 % co 2 : 95 % air atmosphere . b . culturing of human mammary epithelial cells ( 184 cells ) and human breast cancer cells ( mda - 468 cells ) 184ain4 and 184ain4 - t human mammary epithelial cells were provided by martha stampfer , lawrence berkeley laboratory , berkeley , calif . 184ain4 cells were maintained at 37 c . in 5 % co2 and imem supplemented with glutamine ( 0 . 6 mg / ml ), fetal calf serum ( 0 . 5 %), hydrocortisone ( 0 . 5 μg / ml ), insulin ( 5 μg / ml ) and egf ( 10 ng / ml ). 184ain4 - t were maintained at 37 c . in 5 % co2 in imem ( biofluids , rockville , md .) supplemented with glutamine ( 0 . 6 mg / ml ), gentamicin ( 40 mg / ml ) and 10 % fetal calf serum . mda - 468 cells were cultured under the same conditions and medium as 184 ain4 - t cells . c . culturing of 96 igm and 108 igg2a hybridoma cell lines the 108 igg2a hybridoma cell line was generated by immunizing mice with ch 71 cells expressing the egf receptor and cultured under the same conditions as the kb cell line . the 96 igm hybridoma cell line was generated by the same procedure as that described for the 108 igg2a hybridoma cell line . ascites from animals injected with the 108 igg2a hybridoma cells were clarified by centrifugation in an eppendorf centrifuge at 4 ° c . for 10 min . monoclonal antibodies were precipitated by slow addition of saturated ammonium sulfate at 4 ° c . to a final concentration of 45 % ( v / v ), ph 7 . 5 , for 24 hours . the precipitate was collected by centrifugation at 10 , 000 g for 15 minutes and washed twice with 50 % v / v ammonium sulfate , ph 7 . 5 . at 4 ° c . further purification was carried out by affinity chromatography on sepharose cl protein a ( pharmacia ) in 0 . 14m tris buffer , ph 8 . 0 and the 108 monoclonal antibody was eluted with 0 . 1m citrate buffer , ph 3 . 0 , followed by extensive dialysis against pbs . ascites from animals injected with the 96 igm hybridoma cells were clarified by centrifugation in a low speed centrifuge at 3000 rpm for 15 minutes , at 4 ° c . monoclonal antibodies were precipitated by slow addition of saturated ammonium sulfate at 4 ° c . to a final concentration of 45 % ( v / v ), ph 7 . 5 , for 24 hours . the precipitate was collected by centrifugation at 10 , 000 g for 15 minutes and washed twice with 50 % v / v ammonium sulfate , ph 7 . 5 at 4 ° c . the precipitate was then dissolved in and dialyzed extensively against 50 mm tris , ph 8 , 0 . 5 m nacl . this material was semi - purified by gel filtration using sephacryl s - 3000 equilibrated in 50 mm tris , ph 7 . 8 , nacl 0 . 5 m . the peak containing the mab96 antibody was pooled and dialyzed against pbs . purification , specific activity and immunoreactivity of f ( ab )′ 2 , and f ( ab )′ fragment of 108 monoclonal antibody 108 monoclonal antibody ( 5 mg / ml ) in 0 . 1m sodium - acetate buffer at ph 3 . 9 was digested in the presence of 4 % w / w pepsin ( worthington biochemical corporation , new jersey ) for 7 hours at 37 ° c . digestion was terminated by adjusting the ph to 8 . 0 with 2m tris , followed by dialysis against pbs at 4 ° c . remaining intact igg molecules were removed by protein a affinity chromatography . the fc portion and smaller fragments were removed by gel filtration on sepharose g - 100 . for the preparation of monovalent fab ′ fragment , the f ( ab )′ 2 ( 2 mg / ml ) was reduced by 10 mm dithiothreitol in 20 mm tris buffer , ph 8 . 2 , for 1 hour at 37 ° c . alkylation was performed in 40 mm iodoacetamide for 30 minutes at 37 ° c ., followed by extensive dialysis against pbs at 4 ° c . purity and complete digestion of the various fragments were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis ( sds - page ). 125 i - labeling of 108 monoclonal antibody was performed by the chloramine t method ( hunter and greenwood , preparation of 131 iodine labeled human growth hormone of high specific activity , nature , vol . 196 , 465 - 6 , ( 1962 )). specific activities of about 3 × 10 6 cpm / μg igg were usually obtained . the f ( ab )′ 2 and f ( ab ) fragments of 108 monoclonal antilbody were fully immunoreactive when compared to native intact 108 monoclonal antibody in their capacity to compete with the binding of 125 i labeled 108 to egf receptors exposed on kb cells . a . 108 monoclonal antibody binding activity to cell surface egf receptors the antibody binding activity of 108 hybridoma supernatant was determined by an indirect immunofluorescence assay . kb cells ( 2 × 10 6 per sample ) were trypsinized 24 hours before the assay and placed in test tubes ( falcon , polystyrene round bottom tubes ). prior to assay , the kb cell suspensions were washed with cold pbs and incubated with 108 hybridoma supernatant for 45 min . at 4 ° c . after washing with pbs containing 1 % bovine serum albumin , the cells were incubated with fluorescein labeled rabbit anti - mouse igg for 45 min . at 4 ° c . cell samples were suspended in pbs and analyzed by a fluorescence cell sorter ( facs ii , bectin dickenson , mountainview , calif . u . s . a .). uniformity of receptor expression was shown by positive stain in at least 96 % of the cells compared with absence of staining observed with supernatant of hybridoma raised against human hepatitis b virus ( 7ho1 ). scatchard analysis of antibody binding parameters at 4 ° c . revealed an average of 2 × 10 5 binding sites per cell with kd of 1 . 8 × 10 − 9 m − 1 . b . a competitive radioimmunoassay of epidermal growth factor with 108 monoclonal antibody and its fragments kb cells ( 10 5 / well in 24 well plates ; nunc ) were grown for 24 hours , washed with pbs and incubated with different concentrations of either native antibody or its fragments in dmem containing 1 % bovine serum albumin for 1 hour at 4 ° c ., or at room temperature , in the presence of 125 i 108 monoclonal antibody ( about 1 × 10 6 cpm / ml .). the cells were then washed , solubilized in 0 . 5n naoh and their radioactivity was determined in a counter ( kontron , switzerland ). non - specific binding was determined by the addition of 100 - fold excess of unlabelled monoclonal antibody . results are presented as the percentage of radioactivity associated with the cells incubated with unlabelled antibody ( intact or fragmented ) vs . radioactivity associated with cells incubated without the addition of cold antibody . egf competes with the binding of the antibody to the receptor to a maximal level of about 70 %. kb cells ( 4 × 10 6 ) were inoculated subcutaneously on the back of nude mice ( 5 - 6 weeks old ). after 14 days , when the tumor reached a size of about 1 . 2 cm . diameter , 125 i 108 monoclonal antibody was injected intravenously or intraperitoneally ( 5 × 10 6 cpm ; 3 × 10 6 cpm / μg ). 7h01 125 i monoclonal antibody to human hepatitis b virus igg2a served as control . four days after the administration of antibodies , animals were killed and the radioactivity in the different tissues was determined . means of at least four animals per group are presented . both intravenous and intraperitioneal administration of the tagged 108 monoclonal antibody resulted in antibody concentration at the tumor mass . administration of control igg resulted in no concentration at the tumor mass when given intravenously , while a marginal concentration in the tumor was detected when the antibodies were administered intraperitoneally . the percentage of injected dose accumulated at the tumor mass 96 hours post intravenal injection were 7 . 8 ± 1 . 1 and 0 . 8 ± 0 . 1 for monoclonal antibody 108 and 7hoi monoclonal antibody ( control antibody ) respectively , and for the intraperitioneal injection 7 . 5 ± 0 . 4 and 1 . 8 ± 0 . 2 respectively . 96 monoclonal antibody binding properties : a competitive radioimmunoassay of epidermal growth factor with 96 monoclonal antibody washed , confluent mda - 468 cell monolayers in 24 - well culture plates were incubated at 4 c . for 2 . 5 hours with or without various concentrations of antibody or unlabeled egf in binding buffer ( imem , 0 . 1 % bsa , 50 mm hepes ) i [ 125 i ] egf ( s . a . 80 - 160 μci / μg , icn radiochemicals , ca ) was added for a final concentration of 1 nm . after incubation the monolayers were washed , solubilized with lysis buffer ( 10 mm tris , 1 mm edta , 0 . 5 % sds , ph 7 . 4 ) and radioactivity was determined using a gamma - counter ( lkb - pharmacia ). all four antibodies were able to inhibit the binding of labeled egf whereas nonspecific igg or igm were ineffective . the two antibodies most effective in inhibiting cell growth ( 125 igm and 225 igg ) were also the most effective in inhibiting [ 125 i ] egf binding . these antibodies were able to block [ 125 i ] egf binding to a greater extent than unlabeled egf . kb cells were seeded in petri dishes ( 50 × 15 mm 2 , nunc ) at a concentration of 2 × 10 2 cells per dish . after 16 to 24 hours medium was replaced with a fresh one containing different concentrations of either native or fragmented 108 monoclonal antibody with or without egf . on the sixth day cultures were fed with fresh medium containing the above ingredients . on the 15th day the cultures were washed with pbs , fixed with 4 % v / v formaldehyde in pbs for 15 min . and stained with hematoxylin . number of formed colonies ( 25 cells ) was then determined . exposure of kb cells to egf ( 160 nm ) resulted in an increase to 150 % in the number of colonies counted 15 days after seeding ( 14 days after the beginning of the treatment ) as compared to cells incubated in the absence of growth factor . in addition egf caused an increase in the size of kb cell colonies . when a similar experiment was performed in the presence of 108 monoclonal antibody ( 1 . 6 μm ) the number of cell colonies was reduced to 30 % of control values . moreover , a 100 fold excess of 108 monoclonal antibody added together with egf given at concentration which caused a 50 % increase in the colony number , reduced the number of colonies to 20 % of control values . under the same conditions , f ( ab )′ 2 fragments of 108 monoclonal antibody had no effect on the number of kb colonies . yet when added in 100 - fold excess to egf , the f ( ab )′ 2 fragments are able to abolish the effect of egf on the number of formed colonies ( from 150 % to 103 %). incubation with the same concentration of monoclonal antibody to dinitrophenyl ( dnp ) did not affect the number of formed colonies . kb cells ( 2 × 10 6 ) were injected subcutaneously into nude mice , followed by either one or several intravenal injections of the 108 monoclonal antibody , starting one day after tumor cell injection . tumor parameters were measured twice a week with a caliper and its volume was calculated according to the formula : tumor volume ( mm 3 )= length × width × height . in order to validate volume measurements , correlation between tumor volume and tumor weight at the day of animal killing was assessed . the antibody was assayed for its capacity to inhibit the growth of kb cells in nude mice . animals received 1 mg of either 108 monoclonal antibody or control monoclonal antibody to dinitrophenyl at days 1 , 5 , 12 and 18 after tumor inoculation . the fragments f ( ab )′ 2 and fab ′ were given at antibody equivalent doses . the 108 monoclonal antibody treated group significantly retarded tumor development and growth when compared to the group treated with control monoclonal antibody ( p & lt ; 0017 , student - t test ). the f ( ab )′ 2 , was found to affect tumor growth but less efficiently than the whole antibody ( p & lt ; 0 . 05 student - t test for days 12 , 17 , 22 , 25 ). fab ′ fragment did not affect the tumor growth . a single 2 mg dose of 108 native monoclonal antibody given one day after injection of tumor cells was found to be as efficient as four treatments of 1 mg given at days 1 , 5 , 12 and 18 after tumor inoculation . in another experiment , when animals were treated with a single dose of 0 . 66 mg f ( ab )′ 2 fragments , the antitumoral effect was slightly lower , yet a significant difference between the control and the treated group was found using the mann whitney analysis ( p & lt ; 0 . 03 for days 9 , 12 , 14 , 17 ) and student - t test ( p & lt ; 0 . 05 days 9 , 12 ). at the day of sacrifice , tumors were measured and then removed for weight determination . the correlation coefficient between the tumor volume and the tumor weight was 0 . 95 ( p & lt ; 0 . 0001 ). the injection of 3 × 10 6 kb cells intraperitoneally one week after mice ( nude in general background ) received x - irradiation ( 400 rads ), brought about the development of an ascitic growth . the intraperitioneal tumor - bearing mice died after 30 days . three intravenous injections of 108 monoclonal antibody ( 0 . 5 mg each ) prolonged the life span of animals with 30 % of animals not developing tumors at all . the metastatic form of the kb tumor could be obtained by the injection of the cells intravenously ( iv ). mice injected with , 1 . 5 × 10 6 kb cells developed tumor nodules in the lungs 4 - 6 weeks after their implantation . this tumor model mimics the situation in the clinic , where tumor cells infiltrate into internal organs . this is the major problem in the treatment of cancer . the kb cell injection was followed by 3 intravenous injections of 0 . 5 mg 108 monoclonal antibody at days 6 , 9 and 13 after the tumor cell injection . at the termination of the experiment , the lungs were removed , fixed in formaldehyde , and paraffin embedded . serial sections were cut 4 - 5 μm in thickness and stained with hematoxylin . the number of metastatic nodules of various depths through the lungs was obtained by light microscopy analysis . isolation of three metastatic cell clones from lungs of tumor bearing animals and their assay for receptor levels revealed persistence of receptor expression . treatment by the antibody reduced the number of lung tumor nodules to 15 % of those in the respective controls . ( p & lt ; 0 . 05 mann - whitney analysis ). 184ain4 and mda - 468 cells were passed ( 5 , 000 / well ) into triplicate wells of 24 - well plates and allowed to attach before antibody was added . 184ain4 growth media contained 1 ng / ml egf and differing amounts of egfr antibody which was added to the growth media simultaneously with the egf . mda 468 growth media contained no egf . growth media was changed after 48 hours and the cells were counted after 4 days . at the end of the experimental growth period cells were harvested with trypsin - edta and counted using a particle data cell counter ( particle data , inc ., elmhurst , ill .). data is % control cell numbers ( mean ± sd ). 96 igm (—), 42 igm (∘—∘), nonspecific igm ( δ — δ ), 225 igg (□—□), 108 igg (□—□), non - specific igg ( δ — δ ). ( see fig4 a - 4d ) 184ain4 - t cells were suspended in semisolid agar medium containing 0 . 4 % bacto - agar ( difco , detroit , mich . ), imem , 10 % fbs and treatments . cells were plated ( 10 , 000 / dish ) into triplicate 35 nm culture dishes containing 1 ml imem , 0 . 6 % agar and 10 % fbs . the dishes were incubated for 10 - 14 days at 37 c . in 5 % co in the presence of 20 nm aegfr or 20 nm nonspecific antibodies and increasing concentrations of egf . data are mean (± sd ) number of colonies greater than 60 μm . a ) igg : 225 igg (—), 108 igg (∘—∘), non specific igg ( δ — δ ). b ) igm : 96 igm (∘—∘), 42 igm (—), nonspecific igm ( δ — δ ). cell colonies larger than 60 um in diameter were counted using a bausch & amp ; lomb colony counter ( see fig5 a - 5 b ). mda - 468 cells were suspended in semisolid agar medium containing 0 . 4 % bacto - agar ( difco , detroit , mich .) imem , 10 % fbs and treatments . cells were plated ( 10 , 000 / dish ) into triplicate 35 mm culture dishes containing 1 ml imem , 0 . 6 % agar and 10 % fbs . the dishes were incubated for 10 - 14 days at 37 c . in 5 % c02 in the presence of 20 nm aegfr or 20 nm nonspecific antibodies and increasing concentrations of egf . data are mean (± sd ) number of colonies greater than 60 um . a ) igg : 225 igg (—) 108 igg ( δ — δ ) non - specific igg ( δ — δ ), egf alone (∘—∘). b ) igm : 96 igm ( δ — δ ), 42 igm (—) nonspecific igm ( δ — δ ) egf alone (∘—∘). cell colonies larger than 60 nm in diameter were counted using a bausch & amp ; lomb colony counter . ( see fig6 a - 6b ) monoclonal antibody 108 were injected to form a subcutaneous tumor . four doses of 0 . 45 mg of 108 monoclonal antibody and 37 . 5 μg of doxorubicin ( adriamycin ) were given 24 hours after the tumor injection and repeated 3 times at 3 - 4 day intervals . the volume of the tumor was compared to the controls : phosphate buffered saline antibody alone or drug alone . ( see fig1 .) a ) a single treatment comprising 1 . 8 mg 108 monoclonal antibody and 100 μg cisplatin was administered twenty four hours after the subcutaneous tumor inoculation with 2 × 10 6 kb cells . the results are presented in fig2 . b ) a single treatment comprising 1 . 9 mg 108 monoclonal antibody and 0 . 1 μg cisplatin were injected intravenously each in a separate needle 20 hours after the tumor transplantation . the combined treatment was significantly better than each of the treatments alone ( p & lt ; 0 . 02 by student - t - test , p & lt ; 0 . 007 by mann whitney analysis , fig3 ). expression and recombination of separate chain constructs of 96 and 108 v l and v h chains e . coli strain bl21 ( de3 ) and the plasmid expression vector pet8c were kindly provided by dr . f . w . studier of brookhaven national laboratories . this plasmid contains a fragment of t7 dna specifying the gene 10 promoter inserted into the bamhi site of pbr322 so as to direct transcription counterclockwise . this plasmid also provides a transcription terminator for t7 rna polymerase , a ribosome binding site and an atg for translation initiation , with the atg overlapping an ncol restriction site ( ccatgg ). the plasmid pet8c ( km r ) was also received from dr . studier and was constructed by removing the ampicillin resistance gene from pet - 8c [ 21 , 22 , 27 ] via excision of a bsphi - ecori fragment ( pbr322 bp 3195 - 4361 ) and replacing it with an 869 bp fragment encoding kanamycin resistance ( km r ), with the km r gene oriented clockwise in the vector . the km r gene derives from tn903 [ 28 ] and was obtained using the polymerase chain reaction with puc4kiss [ 29 ] as template . the fragment carrying the km r gene starts 50 nucleotides ahead of the km r initiation codon and ends exactly at the termination codon . a stratagene pbs plasmid dna ( bluescript ii sk +, stratagene ; la jolla , calif .) was used as a sub - cloning vector and transformed into commercially available e . coli host cell strains such as invitrogen dh - 1 competent cells ( invitrogen , san diego , calif .). oligonucleotides were synthesized on an applied biosystems model 380a synthesizer using the phosphoramidite method . all routine chemicals ( e . g . urea , tris buffer , dnp - lysine etc .) were purchased from standard suppliers such as sigma ( st . louis , mo .) and fisher ( pittsburgh , pa .). radioactive chemicals were purchased from new england nuclear ( boston , mass .). restriction and other dna - modifying enzymes ( e . g . t4 dna ligase , t4 polynucleotide kinase , calf intestinal phosphatase etc .) were purchased from standard biotechnology manufacturers such as new england biolabs ( beverly , mass .) and boehringer mannheim ( indianapolis , ind .). in order to obtain cdna clones for both 108 and 96 light and heavy chains , poly ( a )- containing rna was isolated from the respective hybridoma cell lines using standard methods [ 30 ]. the first strand cdna was synthesized using an oligo ( dt ) primer . the first strand cdna was then used as a template for second strand synthesis using the method of gubler and hoffman [ 31 ]. the double stranded cdna was then treated with ecori methylase and dna polymerase using reaction conditions described in maniatis [ 30 ]. the mixture was then cleaved with ecori and fractionated on an 8 % polyacrylamide gel . dna with a size greater than 600 bp was eluted from the gel and then collected by ethanol precipitation . the cdna was then inserted into ecori cleaved and phosphatase treated lambda gt11 dna using t4 dna ligase , to produce a library of approximately one million transformants . two separate libraries were constructed , one for identifying 108 sequences and the second for identifying 96 sequences . v h and v l cdna clones were identified by hybridization with an oligonucleotide probe specific for the constant region . insert dna from positive phage was subcloned into pbs vectors . the dna sequence for the v h and v l coding regions were verified for all v h and v l clones selected for further study . dna sequencing reactions were carried out as per manufacturers instructions ( sequenase , usb ; cleveland , ohio ). cdna clones encoding the variable regions of both monoclonal antibody 96 and 108 heavy and light chains were obtained from cdna libraries constructed from the respective hybridoma cell lines . the nucleotide sequence of all four variable regions is shown in fig9 - 12 . d . construction of expression vectors for v h and v l cdna in order to direct expression of the various v h and v l cdnas they were placed under the control of the bacteriophage t7 promoter [ 21 , 22 , 27 ]. in this system , the cdna is placed into a vector containing the promoter and translation initiation signals for the tø protein of bacteriophage t7 . t7 rna polymerase can then be delivered to the host cell by either induction or infection . in the present example the antibody expression vectors were placed into a cell that carries a prophage containing the gene for t7 rna polymerase under control of the lac uv5 promoter . addition of the lactose analog iptg to a growing culture of cells induces t7 rna polymerase , which in turn transcribes the target dna in the plasmid . transcription by t7 rna polymerase is so active that target rna can accumulate to amounts comparable to ribosomal rna and target proteins can constitute the majority of cellular protein . plasmids expressing the antibody v l or v h sequence and conferring resistance to kanamycin were constructed from pet - 8c ( km r ) and pcr products derived from the various cdnas . briefly , four oligonucleotides each capable of hybridizing to the 5 ′ of one of the various cdnas were designed . all four oligonucleotides incorporated an ncol restriction site . similarly , four oligonucleotides each capable of hybridizing to the 3 ′ of one of the various cdnas were also designed . in the latter case all four oligonucleotides incorporated an bamhi restriction site . four separate pcr reactions were carried out using the appropriate combination of template dna ( 108 v h or v l and 96 v h or v l ) and pcr primers . following 30 cycles of pcr the various reaction products were digested with ncol and bamhi and the insert fragment was then ligated to ncol / bamhi cleaved pet8c ( km r ). the resulting plasmid dna was then transformed into e . coli dh - 1 cells and a single isolate from each transformation was identified that released the appropriate size fragment by digestion with ncoi and bamhi . dna from a positive isolate for each of the four chains was then used to transform e . coli bl21 ( de3 ). a single isolate from each of these transformations was the used for expression of the various chains as described below . a schematic diagram of the expression vector constructs is indicated in fig8 . e . expression of v h , v l , and sfv genes in e . coli fresh overnight cultures were diluted 1 : 100 and grown to an o . d . 595 of ˜ 0 . 4 and then induced with 1 mm isopropyl β - d - thiogalactopyranoside ( iptg ). samples were removed at selected time points , centrifuged and the pellet resuspended in sample buffer ( 20 mm tris - hcl ph 6 . 8 , 3 . 0 % sds , 15 % glycerol , 0 . 1 - β - mercaptoethanol , 0 . 001 % bromophenol blue dye ) before analysis by sds gel electrophoresis [ 32 ]. expression vectors containing the various recombinant fv constructs under the control of the t7 promoter were introduced into bl21 ( de3 ) cells [ 21 , 22 , 27 ]. this cell line is an e . coli lysogen containing a single copy of the gene for t7 rna polymerase in the chromosome under the control of the iptg - inducible lac uv - 5 promoter . the addition of iptg to cell cultures elevates the expression levels of t7 rna polymerase and thus indirectly induces the expression of recombinant proteins under the control of t7 promoters . the first step in the purification of the individual v h , v l , or sfv proteins was their isolation in the form of bacterial inclusion bodies . e . coli cell pellets from 500 ml induced cultures ( 2 - 4 hours with 1 mm iptg ) were resuspended in 20 ml of 50 mm tris - hcl , ph 9 . 0 , 2 . 0 % glycerol and 0 . 1 mm edta . this suspension was sonicated 2 × 15 sec . on ice and then centrifuged at 15 , 000 g for 20 min . the precipitate ( containing essentially all of the v h , v l , or sfv proteins ) was resuspended in 8 m urea , 50 mm tris - hcl ph 8 . 0 , sonicated 2 × 15 sec . on ice , stored overnight at 4 ° c . and then clarified by centrifugation at 15 , 000 g for 20 min . supernatant samples in urea were adjusted to ˜ 1 mg / ml ( vh , vl ) or ˜ 0 . 1 mg / ml ( sfv ), as calculated from absorbance measurements using extinction coefficients e 280 nm 1 cm 0 . 1 % = 2 . 0 for v h , 1 . 0 for v l , or 1 . 5 for fv ( used also to estimate sfv ) [ 6 ] and stored overnight at 4 ° c . these samples were either used directly for analysis of refolding and recovery of active fv or processed for further purification . v h , v l , and sfv proteins purified from bacterial inclusion bodies were solubilized in 6 m guanidine hcl , 50 mm tris - hcl ph 8 . 0 , 5 mm edta and 1 mm β - mercaptoethanol . size exclusion chromatography was performed on a sephacryl s - 200 column ( 3 × 90 cm ). samples of s - 200 purified v h , v l , or sfv protein were further treated by ion - exchange chromatography following buffer exchange by dialysis to 8 m urea , 50 mm tris - hcl ph 8 . 0 , 20 mm nacl , 0 . 01 mm β - mercaptoethanol . samples were passed over a 5 ml q - sepharose anion exchange column and eluted with a 0 . 02 - 0 . 5 m nacl gradient in 8 m urea , 50 mm tris - hcl ph 8 . 0 . peptides from each of the separate chain constructs ( v h or v l ) and the sfv were found primarily in the form of insoluble inclusion bodies . this finding was consistent for proteins over - expressed in e . coli [ 34 ] and from a purification standpoint , this sequestration was useful since recombinant proteins were conveniently isolated in a highly enriched form . v h , v l , and sfv proteins exhibited minimal solubility in nondenaturing solvents and , therefore , were dissolved in either 8 m urea or 6 m guanidine hydrochloride ( guanidine hcl ). when these chaotropes were removed either slowly by dialysis or rapidly by dilution , v l remained soluble longer than v h . however , neither individual chain remained in solution in pbs except at low protein concentration ( less than 50 μg / ml ). significantly recombinant v h and v l chains did remain in solution in pbs at concentrations up to ˜ 1 mg / ml when later recovered as active fvs . further purification of recombinant v h , v l , or sfv proteins isolated in inclusion bodies and solubilized in guanidine hcl with reduction was performed by size exclusion chromatography . recoveries of s - 200 purified v h , v l , and sfv proteins following size exclusion chromatography varied with different inclusion body preparations and ranged from 100 - 200 mg / liter . the refolding of v h and v l peptides was carried out by the method of hochman et al . [ 18 , 33 ] equimolar amounts of v h and v l proteins were added together in 8 m urea , 50 mm tris - hcl , ph 8 . 0 , to a final protein concentration of ˜ 1 mg / ml . refolding was initiated by the removal of chaotrope either by extensive dialysis in pbs or by rapid dilution 20 - fold into pbs . refolded material following rapid dilution ( final urea concentration equal to 0 . 4 m ) was maintained at room temperature for a minimum of 30 minutes . refolding of sfv protein was preceded by reduction of sfv in 8 m urea , 50 mm tris - hcl , ph 8 . 0 , at 37 ° c . for 1 hour with 0 . 1 m β - mercaptoethanol . reduction was carried out at protein concentrations of 1 mg / ml and then diluted with the same buffer to 50 - 100 μg / ml . the diluted sample was then dialyzed extensively , first against 8 m urea , 50 mm tris - hcl , ph 8 . 0 , and then to final equilibration in pbs . recombinant v h and v l and sfv peptides expressed to high levels in e . coli were found to be , as anticipated , sequestered in insoluble inclusion bodies . as a result , strong denaturants were required for protein solubilization . the recovery of active protein following this treatment was dependent upon the use of an effective in vitro refolding procedure . in general , protein refolding is initiated by the removal of the solubilizing chaotrope under conditions designed to promote the most effective outcome . fig1 illustrates this general scheme by outlining a simple model of the steps required for protein refolding of an antibody fv . in this model , oxidation of the individual v h and v l chains takes place separately , each in the presence of denaturant . intrachain disulfide bond formation within the relaxed chains is concentration dependent and the proper formation of these bonds presumably promotes the most effective subsequent refolding . refolding itself is initiated by the transfer of the combined v h and v l protein from denaturant into a physiological buffer ( e . g . pbs ). successfully refolded v h and v l chains can then associate together to form an active fv complex capable of specific ligand binding . a standard procedure for the refolding of recombinant 108 and 96 v l and v h was adopted based upon the conditions originally used by hochman et al . [ 18 , 33 ] to renature the native mopc315 v h and v l . solubilized recombinant 108 or 96 v h and v l chains ( either directly from inclusion bodies or after further purification ) were allowed to oxidize in air to greater than 90 %. the separate chains were combined in denaturant , diluted 1 : 20 in pbs , and allowed to refold at room temperature . the refolded chains were then used in the binding experiments described below . fig1 and 15 show that in a competition binding experiment , the 96 rfv competed for binding of mab 96 to a431 cells . similar results were observed for 108 rfv competing for mab 108 binding to a431 cells . the 96 rfv also inhibited the binding of radioiodinated egf to a431 cells , as shown in fig1 . antibody fragments may be produced by proteolytic degradation of entire immunoglobulin molecules , or by recombinant expression of dnas encoding antibody fragments . the antibody fragment 96 fv does not cause the receptor to dimerize , and does not activate the receptor . the antibody fragment induces internalization of the receptor without inducing its degradation . a toxin , radiochemical , or drug can be attached to the antibody fragment . the use of a variable region antibody fragment directed to a cellular receptor is useful in targeting drug delivery to cells expressing that receptor in order to affect cellular physiology and / or metabolism . 1 . roitt , i . m ., j . brosstoff , and d . k . male , immunology . 1985 , london : gower medical publishing . 2 . cabilly , s ., a . d . riggs , and h . pande et al ., generation of antibody activity from mmunoglobulin polypeptide chains produced in escherichia coli . proc . natl . acad . sci . usa ., 1984 . 81 : p . 3273 - 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