Patent Application: US-193401-A

Abstract:
the present invention relates to polypeptide compositions which bind to cell surface epitopes and , in multivalent forms , cause or lead to the killing of cells including lymphoid tumor cells , and in the case of monovalent forms , cause immunosuppression or otherwise inhibit activation of lymphocytes . the invention further relates to nucleic acids encoding the polypeptides , methods for the production of the polypeptides , methods for killing cells , methods for immunosuppressing a patient , pharmaceutical , diagnostic and multivalent compositions and kits comprising the polypeptides and uses of the polypeptides .

Description:
all buffers , solutions or procedures without explicit reference can be found in standard textbooks , for example current protocols of imlmunology ( 1997 and 1999 ) or sambrook et al ., 1989 . where not given otherwise , all materials were purchased from sigma , deisenhofen , del ., or merck , darmstadt , del ., or sources are given in the literature cited . hybridoma cell lines lb3 . 1 and l243 were obtained from lgc reference materials , middlesex , uk ; data on antibody 8d1 were generously supplied by dr . matyas sandor , university of michigan , madison , wis ., usa . to demonstrate that we could identify cytotoxic antigen - binding domains of human composition , we first prepared a purified form of a human antigen , the human mhc class ii dr protein ( dra * 0101 / drb1 * 0401 ) from the dr - homozygous b - lymphoblastoid line priess cells ( gorga et al ., 1984 ; gorga et al ., 1986 ; gorga et al ., 1987 ; stem et al ., 1992 ) and the human - mouse chimeric molecule dr - i e from the transfectant m12 . c3 . 25 ( ito et al ., j . exp . med . 183 : 2635 - 2644 , 1996 ) by using standard methods of affinity purification ( gorga et al ., 1984 ) as follows . first , priess cells ( ecacc , salisbury uk ) were cultured in rpmi and 10 % fetal calf serum ( fcs ) using standard conditions , and 1010 cells were lysed in 200 ml phosphate buffered saline ( pbs ) ( ph 7 . 5 ) containing 1 % np - 40 ( bdh , poole , uk ), 25 mm iodoacetamide , 1 mm phenylmethylsulfonylfluoride ( pmsf ) and 10 mg / l each of the protease inhibitors chymostatin , antipain , pepstatin a , soybean trypsin inhibitor and leupeptin . the lysate was centrifuged at 10 , 000 × g ( 30 minutes , 4 ° c .) and the resulting supernatant was supplemented with 40 ml of an aqueous solution containing 5 % sodium deoxycholate , 5 mm iodoacetamide and 10 mg / l each of the above protease inhibitors and centrifuged at 100 , 000 × g for two hours ( 4 ° c .). to remove material that bound non - specifically and endogenous antibodies , the resulting supernatant was made 0 . 2 mm with pmsf and passed overnight ( 4 ° c .) through a rabbit serum affigel - 10 column ( 5 ml ; for preparation , rabbit serum ( charles river , wilmington , mass ., usa ) was incubated with affigel 10 ( biorad , munich , del .) at a volume ratio of 3 : 1 and washed following manufacturer &# 39 ; s directions ) followed by a protein g sepharose fast flow column ( 2 ml ; pharmacia ) using a flow rate of 0 . 2 ml / min . second , the pre - treated lysate was batch incubated with 5 ml protein g sepharose fast flow beads coupled to the murine anti - hla - dr antibody lb3 . 1 ( obtained by protein g - sepharose ff ( pharmacia ) affinity chromatography of a supernatant of hybridoma cell line lb3 . 1 ) ( stern et al ., 1993 ) overnight at 4 ° c . using gentle mixing , and then transferred into a small column which was then washed extensively with three solutions : ( 1 ) 100 ml of a solution consisting of 50 mm tris / hcl ( ph 8 . 0 ), 150 mm nacl , 0 . 5 % np - 40 , 0 . 5 % sodium deoxycholate , 10 % glycerol and 0 . 03 % sodium azide at a flow rate of 0 . 6 ml / min ). ( 2 ) 25 ml of a solution consisting of 50 mm tris / hcl ( ph 9 . 0 ), 0 . 5 m nacl , 0 . 5 % np - 40 , 0 . 5 % sodium deoxycholate , 10 % glycerol and 0 . 03 % sodium azide at a flow rate of 0 . 9 ml / min ; ( 3 ) 25 ml of a solution consisting of 2 mm tris / hcl ( ph 8 . 0 ), 1 % octyl - β - d - glucopyranoside , 10 % glycerol and 0 . 03 % sodium azide at a flow rate of 0 . 9 ml / min . third , mhc class ii dr protein ( dra * 0101 / drb1 * 0401 ) was eluted using 15 ml of a solution consisting of 50 mm diethylamine / hcl ( ph 11 . 5 ), 150 mm nacl , 1 mm edta , 1 mm egta , 1 % octyl - β - d - glucopyranoside ( alexis corp ., lausen , ch ), 10 % glycerol , 10 mm iodoacetamide and 0 . 03 % sodium azide at a flow rate of 0 . 4 ml / min . 800 μl fractions were immediately neutralised with 100 μl 1m tris / hcl ( ph 6 . 8 ), 150 mm nacl and 1 % octyl - β - d - glucopyranoside . the incubation of the lysate with lb3 . 1 - protein g sepharose fast flow beads was repeated until the lysate was exhausted of mhc protein . pure eluted fractions of the mhc class ii dr protein ( as analyzed by sds - page ) were pooled and concentrated to 1 . 0 - 1 . 3 g / l using vivaspin concentrators ( greiner , solingen , del .) with a 30 kda molecular weight cut - off . approximately 1 mg of the mhc class ii dr preparation was re - buffered with pbs containing 1 % octyl - β - d - glucopyranoside using the same vivaspin concentrator to enable direct coupling of the protein to biacore cm5 chips . since the important biological activities of anti - dr mabs , e . g ., inhibition of cd4 t cell — antigen presenting cell ( apc ) interaction and tumoricide activity are associated with specificity for the first , n - terminal domains of dr molecules ( vidovic &# 39 ;, d . et al ., 1995 , eur . j . immunol . 25 : 3349 - 3355 ), we used purified dr molecules as well as human - murine chimeric mhc - ii molecules ( dr first domains grafted onto a murine class ii molecule , see ito , k . et al ., 1996 , j . exp . med . 183 : 2635 - 2644 ) for screeining the human combinatorial antibody library ( hucal ®) by alternating whole cell panning with protein solid - phase - panning . we identified certain antigen binding antibody fragments ( in this case , scfvs ) of human composition ( ms - gpc - 1 / scfv - 17 , ms - gp - 6 / scfv - 8a , ms - gpc - 8 / scfv - b8 , ms - gpc - 10 / scfv - e6 , etc ., see fig1 and 2 ) against the human antigen ( dra * 0101 / drb1 * 0401 ) from a human antibody library based on a novel concept that has been recently developed ( knappik et al ., 2000 ). a consensus framework resulting in a total of 49 different frameworks here represents each of the vh — and vl - subfamilies frequently used in human immune responses . these master genes were designed to take into account and eliminate unfavorable residues promoting protein aggregation as well as to create unique restriction sites leading to modular composition of the genes . in hucal - scfv , both the vh - and vl - cdr3 encoding regions of the 49 master genes were randomized . the hucal - scfv ( knappik et al ., 2000 ) library , cloned into a phagemid - based phage display vector pmorph13_scfv ( see fig1 ), in e . coli tg - 1 was amplified in 2 × ty medium containing 34 μg / ml chlorarnphenicol and 1 % glucose ( 2 × ty - cg ). after helper phage infection ( vcsm13 ) at 37 ° c . at an od 600 of about 0 . 5 , centrifugation and resuspension in 2 × ty / 34 μg / ml chloramphenicol / 50 μg / ml kanamycin / 0 . 1 mm iptg , cells were grown overnight at 30 ° c . phage were peg - precipitated from the supernatant ( ausubel et al ., 1998 ), resuspended in pbs / 20 % glycerol and stored at − 80 ° c . phage amplification between two panning rounds was conducted as follows : mid - log phase tg1 - cells were infected with eluted phage and plated onto lb - agar supplemented with 1 % of glucose and 34 μg / ml of chloramphenicol . after overnight incubation at 30 ° c . colonies were scraped off , adjusted to an od 600 of 0 . 5 and helper phage added as described above . wells of maxisorp ™ microtiterplates ( nunc , roskilde , dk ) were coated with mhc - class ii dra * 0101 / drb1 * 0401 ( prepared as above ) dissolved in pbs ( 2 μg / well ). after blocking with 5 % non - fat dried milk in pbs , 1 - 5 × 10 12 hucal - scfv phage purified as above were added for 1 h at 20 ° c . after several washing steps , bound phages were eluted by ph - elution with 100 mm triethylamine and subsequent neutralization with 1 m tris - cl ph 7 . 0 . three rounds of panning were performed with phage amplification conducted between each round as described above . three rounds of panning and phage amplification were performed as described in 2 . 3 . and 2 . 2 . with the exception that in the second round between 1 × 10 7 and 5 × 10 7 priess cells in 1 ml pbs / 10 % fcs were used in 10 ml falcon tubes for whole cell panning . after incubation for 1 h at 20 ° c . with the phage preparation , the cell suspension was centrifuged ( 2 , 000 rpm for 3 min ) to remove non - binding phage , the cells were washed three times with 10 ml pbs , each time followed by centrifugation as described . phage that specifically bound to the cells were eluted off by ph - elution using 100 mm hcl . alternatively , binding phage could be amplified by directly adding e . coli to the suspension after triethlyamine treatment ( 100 mm ) and subsequent neutralization . clones obtained after three rounds of solid phase panning ( 2 . 3 ) or mixed solid phase / whole cell panning ( 2 . 4 ) were screened by facs analysis on priess cells for binding to hla - dr on the cell surface . for expression , the scfv fragments were cloned via xbai / ecori into pm × 7_fs as expression vector ( see fig1 ). expression conditions are shown below in example 3 . 2 aliquots of 10 6 priess cells were transferred at 4 ° c . into wells of a 96 - well microtiterplate . scfv in blocking buffer ( pbs / 5 % fcs ) were added for 60 min and detected using an anti - flag m2 antibody ( kodak ) ( 1 : 5000 dilution ) followed by a polyclonal goat anti - mouse igg antibody - r - phycoerythrin - conjugate ( jackson immunoresearch , west grove , pa ., usa , cat . no . 115 - 116 - 146 , f ( ab ′) 2 fragment ) ( 1 : 200 dilution ). cells were fixed in 4 % paraformaldehyde for storage at 4 ° c . 10 4 events were collected for each assay on the facs - calibur ( bd immunocytometry systems , san jose , calif ., usa ). only fifteen out of over 500 putative binders were identified which specifically bound to priess cells . twelve scfv - s also bound to the chimeric mhc - ii molecule , but showed no reactivity to either i - e d ( the murine part of chimeric mhc - ii27 ), or unrelated proteins , such as lysozyme , transferrin , bovine serum albumine and human gamma globuline ( fig1 ), indicating that they were specific for the first domains of dr molecules . some of these clones were further analysed for their immunomodulatory ability and for their killing activity as described below . table 1 contains the sequence characteristics of clones ms - gpc - 1 ( scfv - 17 ), ms - gpc - 6 ( scfv - 8a ), ms - gpc - 8 ( scfv - b8 ) and ms - gpc - 10 ( scfv - e6 ) identified thereby . the vh and vl families and the cdr3s listed refer to the hucal consensus - based antibody genes as described ( knappik et al ., 2000 ); the sequences of the vh and vl cdrs are shown in table 1 , and the full sequences of the vh and vl domains are shorn in fig1 . the fine specificity of scfv - s was tested on a panel of dr - homozygous typing cells , and mhc - ii transfectants . ten of 12 scfv - s reacted with all major allelic froms of dr represented in the cell panel ( dr1 through 14 ), and 4 of 12 recognized additional mhc - ii molecules ( drw52 and w53 , dp and dq molecules ; fig2 ). thus , these antibodies potentially could be used widely as therapeutic agents across human populations virtually irrespective of polymorphic differences in mhc - ii molecules . most importantly , four of the 12 hits exhibited strong tumor killing activity , when cross - linked with anti - tag antibody ( see fig2 in bold ). the monovalent fragments were not tumoricidal , corresponding to previous observations ( vidovic &# 39 ;, d . et al ., 1995 , eur . j . immunol . 25 : 3349 - 3355 ). since the tumoricidal hits had modest affinities ( k d - s ranging from 346 nm to 81 μm in single chain fv ( scfv ) format ), they were subjected to “ in vitro affinity maturation ”. the parental scfv - s were first converted into fab format that is less prone to aggregation and hence should give more reliable k off values . the fab - fragment antigen binding polypeptides ms - gpc - 1 - fab / 17 - fab , ms - gp - 6 - fab / 8a - fab , ms - gpc - 8 - fab / b8 - fab and ms - gpc - 10 - fab / e6 - fab were generated from their corresponding scfv fragments as follows . both heavy and light chain variable domains of scfv fragments were cloned into pm × 9 fab ( fig1 ), the heavy chain variable domains as mfei / styi - fragments , the variable domains of the kappa light chains as ecorv / bsiwi - fragments . the lambda chains were first amplified from the corresponding pmorph13_scfv vector as template with pcr - primers crt5 ( 5 ′ primer ) and crt6 ( 3 ′ primer ), wherein crt6 introduces a unique draiii restriction endonuclease site . the pcr product is cut with ecorv / draiii and cloned into pm × 9_fab ( see fig1 ). the fab light chains could be detected with a polyclonal goat anti - human igg antibody - r - phycoerythrin - conjugate ( jackson immunoresearch , west grove , pa ., usa , cat . no . 109 - 116 - 088 , f ( ab ′) 2 fragment ) ( 1 : 200 dilution ). 3 . 2 . expression and purification of hucal - antibody fragments in e . coli expression in e . coli cells ( jm83 ) of scfv and fab fragments from pm × 7_fs or pm × 9_fab , respectively , were carried out in one liter of 2 × ty - medium supplemented with 34 μg / ml chloramphenicol . after induction with 0 . 5 mm iptg ( scfv ) or 0 . 1 mm iptg ( fab ), cells were grown at 22 ° c . for 12 hours . cell pellets were lysed in a french press ( thermo spectronic , rochester , n . y ., usa ) in 20 mm sodium phosphate , 0 . 5 m nacl , and 10 mm imidazole ( ph 7 . 4 ). cell debris was removed by centrifugation and the clear supernatant filtered through 0 . 2 μm pores before subjecting it to strep tag purification using a streptactin matrix and purification conditions according to the supplier ( iba gmbh , göttingen , germany ). purification by size exclusion chromatography ( sec ) was performed as described by rheinnecker et al . ( 1996 ). the apparent molecular weights were determined by sec with calibration standards and confirmed in some instances by coupled liquid chromatography - mass spectrometry ( toplab gmbh , martinsried , germany ). in order to optimize certain biological characteristics of the hla - dr binding antibody fragments , one of the fab fragments , ms - gpc - 8 - fab / b8 - fab , was used to construct a library of fab antibody fragments by replacing the parental vlλ1 chain by the pool of all lambda chains λ 1 - 3 randomized in cdr3 from the hucal library ( knappik et al ., 2000 ). in the first round of optimization , both h - cdr2 - and l - cdr3 - sequences of clones ms - gpc - 1 / scfv - 17 , ms - gpc - 6 / scfv - 8a , ms - gpc - 8 / scfv - b8 and ms - gpc - 10 / scfv - e6 were randomized by substituting the parental sequence with randomized trim ®- based oligonucleotide - cassettes ( virnekäs et al ., 1994 ) leading to four different libraries with 7 . 6 × 10 6 to 1 . 0 × 10 7 primary transformants . for generation of h - cdr2 and l - cdr1 - libraries : trinucleotide - containing oligonucleotides starting from o - methyl trinucleotide phosphoramidites ( virnekäs 1994 ) were synthesized as described ( knappik et al ., 2000 ). the vh2 - cdr2 - design comprised an olionucleotide encoding for 16 amino acids which was randomized with up to 19 different amino acids ( all except for cystein ) at the following positions ( from n — to c - terminus ; amino acid - diversity and ratios in % are given in parentheses ): position - 1 ( 19 ), - 2 ( 40 % v / 20 % d , f , n ), - 3 ( 40 % v / 20 % d , v , n ), - 4 ( 19 ), - 5 ( 19 ), - 6 ( d ), - 7 ( 19 ), - 8 ( k ), - 9 ( 19 ), - 10 ( y ), - 11 ( 70 % s / 30 % g ), - 12 ( 50 % p / 50 % t ), - 13 ( s ), - 14 ( l ), - 15 ( k ), - 16 ( s ). for the l - cdr1 of the lambda - 1 - framework two different oligonucleotides ( termed as a and b ) were designed to encode : a ) position - 1 ( s ), - 2 ( g ), - 3 ( s ), - 4 ( 19 ), - 5 ( s ), - 6 ( 80 % n / 10 % d , k ), - 7 ( i ), - 8 ( g ), - 9 ( 19 ), - 10 ( 19 ), - 11 ( 19 ), - 12 ( v ), - 13 ( 19 ); b ) position 1 ( 50 % s , t ), - 2 ( g ), - 3 ( s ), - 4 ( 80 % s / 20 % n ), - 5 ( s ), 6 ( n ), - 7 ( 1 ), - 8 ( g ), - 9 ( 19 ), - 10 ( 19 ), - 11 ( 19 ), - 12 ( 19 ), - 13 ( v ), - 14 ( 19 ). the oligonucleotide for the cdr1 of lambda - 2 framework was designed to encode : position - 1 ( 19 ), - 2 ( g ), - 3 ( s ), - 4 ( 89 % s / 20 % t ), - 5 ( s ), - 6 ( d ), - 7 ( 80 % v , 20 % 1 ), - 8 ( g ), ) - 9 ( 19 ), - 10 ( y ), - 11 ( 19 ), - 12 ( 19 ), - 13 ( v ), - 14 ( 19 ). for framwork lambda 3 the following cdr1 - design was made : position - 1 ( 33 % g , q , s ), - 2 ( g ), - 3 ( 50 % d , n ), - 4 ( 19 ), - 5 ( 50 % l , i ), - 6 ( 33 % g , p , r ), - 7 ( 19 ), - 8 ( 19 ), - 9 ( 19 ), - 10 ( 50 % a , v ), - 11 ( 19 ). all cassettes were introduced into a promoter - less derivative of pmorph4 ( pack et al ., in preparation ). for all subsequent affinity - maturations the respective h - cdr2 or l - cdr1 - cassettes were derived from those plasmids using the respective flanking restriction - nuclease sites as described ( knappik et al ., 2000 ). prior to cloning of different libraries for affinity maturation all parental scfv were converted into the fab - format following the standard conversion protocol ( krebs et al ., 2001 ) for the modular hucal - library . based on each of the 4 parental fabs 17 , 8a , b8 and e6 ( all h2 lambdal ) a sub - library was constructed exhibiting a repertoire of different l - cdr3 - and h - cdr2 - cassettes . first cloning step included the subsitution of the parental xbai / draiii - fragment of fabs 17 , b8 , and e6 by a mix of corresponding fragments of all 3 v lambda consensus - genes encoding a repertoire of 5 . 7 × 10 6 different l - cdr3 cassettes . library - sizes for all 3 parental clones were in the range of 5 . 1 m - 6 . 0 × 10 6 transformants . these libraries were then used to introduce different h - cdr2 - library cassettes via substitution of the xhoi / eagi - fragments . final library sizes resulted in up to 1 . 2 × 10 7 transformants including 78 % correct clones based on dna - sequence analyis . in case of 8a the lcdr3 optimization was performed by exchanging the parental xbai / bsiwi - fragment for the corresponding hucal - scfv kappa3 sublibrary fragments . as before , this library was then used to insert different hcdr2 - cassettes via the xho / bsshii - fragment . library sizes were in the range of 1 . 7 × 10 6 cfu after l - cdr3 - and 1 . 0 × 10 7 cfu after h - cdr2 - cassette insertion including at least 65 % correct clones according to dna - sequence analysis . a fifth library has been constructed based on a consensus - sequence within h - cdr3 of binders 17 , b8 and e6 . for this purpose parental fab b8 has been chosen to randomize several positions within h - cdr3 by insertion of a synthetic trim - oligonucleotide comprising the following h - cdr3 - design from n — to c - terminus : position 1 ( all = all exept c ), - 2 ( all ), - 3 ( all ), - 4 ( 25 % of y / w / f / h ), - 4 ( r ), - 5 ( g ), - 6 ( 50 % g / a ), - 7 ( 50 % f / l ), - 8 ( all ). final library size was in the range of 6 . 8 × 10 6 different transformants comprising 63 % correct clones after sequence analysis . l - cdr1 - libraries were generated based on a pool of 20 different fab - clones derived from the combined light - chain - and h - cdr2 - based - optimization . equimolar amounts of vector dna from each parental clone was mixed after removal of the ecorv / bpuai - insert and religated by insertion of the corresponding fragments encoding a repertoire of different l - cdr1 - cassettes . final library - sizes were in the range of 4 . 2 × 10 8 cfu . since clones 17 , b8 and e6 exhibited a consensus - motif in h - cdr3 , a fifth library was constructed based on the parental clone b8 , in which several h - cdr3 positions were randomized while keeping the consensus motif constant . the latter library termed b8m gave rise to 6 . 8 × 10 6 initial transformants . all libraries were subjected to either two rounds of standard solid - phase panning on purified dr , or a solid phase and a whole cell panning . several panning - parameters including decreasing amounts of antigen ( 500 ng and 250 ng / well purified protein , see schier et al ., 1996a and 1996b ), or increasing concentrations of nh 4 scn ( 50 mm , 250 mm , 500 mm in pbs ) ( hall and heckel 1988 ; macdonald 1988 ; goldblatt 1993 ; ferreira & amp ; katzin 1995 ), or increasing the numbers of wash - cycles ( chen 1999 ; low 1996 ) were applied in the second panning - round to enhance panning - stringency and hence the probability of selecting high affinity fabs . phage - antibodes derived from the first round of a manual solid - phase - panning on 250 and 500 ng / well purified hla - protein , respecitvely , were pooled and used for the second panning round on either 12 ng / well purified protein according to a standard protocol ( krebs et al ., 2001 ), or on 250 ng coated antigen in combination with an additional 30 min incubation - step of different amounts of ammonium - isothiocyanate ( 50 mm , 250 mm , 500 mm and in pbs ) inbetween the standard wash - protocol ( 5 × tbst short and 5 × tbst for 5 min at room temperature ) and the elution step ( 100 mm glycine - hcl / 500 mm nacl , ph 2 . 2 ). alternatively , the second panning round was performed on different amounts of priess - cells ranging from 10 1 - 10 5 cells / well according to a standard whole - cell - panning - protocol ( krebs et al ., 2001 ). fab - clones for k off rankings were selected only from those panning wells which prior to and after treatment show a significant drop in phage - titer and thus indicating a maximum in bound phages at the highest panning - stringency . for example , the fab fragment ms - gpc - 8 - fab / b8 - fab ( see 3 . 1 ) was cloned via xbai / ecori from pm × 9_fab_gpc - 8 into pmorph18_fab , a phagemid - based vector for phage display of fab fragments , to generate pmorph18_fab_gpc - 8 ( see fig1 ). a lambda chain pool comprising a unique draiii restriction endonuclease site ( knappik et al ., 2000 ) was cloned into pmorph18_fab gpc - 8 cut with nsii and draiii ( see vector map of pmorph18_fab_gpc - 8 in fig1 ). the resulting fab optimization library was screened by two rounds of panning against mhc - class ii dra * 0101 / drb1 * 0401 ( prepared as above ) as described in 2 . 3 with the exception that in the second round the antigen concentration for coating was decreased to 12 ng / well . facs identified optimized clones as described above in 2 . 5 . finally , 12 fabs with improved k off values were selected from the b8 , b8m and 8a libraries . the best clone identified ( ms - gpc - 8 - 17 / 7ba ) had a k d of about 58 nm , corresponding to a 5 - fold affinity improvement compared to the best unoptimized clone ms - gpc - 8 / b8 ( table 3e ). libraries 17 , e 6 and 8 a did not yield many clones with improved k off values . binders selected from the b8 library showed different l - cdr3 - sequences , but all maintained the parental h - cdr2 - sequence ( knappik et al ., 2000 ), suggesting that the latter is critical for antibody - antigen interaction . for further affinity - improvement , we focussed on binders from the b8 and b8m library . seven of these clones , ms - gpc - 8 - 1 , ms - gpc - 8 - 6 , ms - gpc - 8 - 9 , ms - gpc - 8 - 10 , ms - gpc - 8 - 17 / 7ba , ms - gpc - 8 - 18 and ms - gpc - 8 - 27 , were further characterized and showed cell killing activity as found for the starting fragment ms - gpc - 8 / b8 . table 1 contains the sequence characteristics of ms - gpc - 8 - 1 , ms - gpc - 8 - 6 , ms - gpc - 8 - 9 , ms - gpc - 8 - 10 , ms - gpc - 8 - 17 / 7ba , ms - gpc - 8 - 18 and ms - gpc - 8 - 27 . the vh and vl families and the cdr3s listed refer to the hucal consensus - based antibody genes as described ( knappik et al ., 2000 ). the full sequences of the vh and vl domains of ms - gpc - 8 - 6 , ms - gpc - 8 - 10 , ms - gpc - 8 - 17 / 7ba and ms - gpc - 8 - 27are shown in fig1 . the optimized fab forms of the anti - hla - dr antibody fragments ms - gpc - 8 - 6 and ms - gpc - 8 - 17 showed improved characteristics over the starting ms - gpc - 8 / b8 . for example , the ec 50 of the optimized antibodies was 15 - 20 and 5 - 20 nm ( compared to 20 - 40 nm for ms - gpc - 8 / b8 , where the concentration is given as the concentration of the bivalent cross - linked fab dimer ), and the maximum capacity to kill mhh - call 4 cells determined as 76 and 78 % for ms - gpc - 8 - 6 and ms - gpc - 8 - 17 ( compared to 65 % for ms - gpc - 8 ) respectively . in the second round , l - cdr1 - optimization is performed . the l - cdr1 library was generated from a pool of the 20 best fab clones , of which 16 ( including 7ba ) derived from the l - cdr3 optimization and 4 from the h - cdr3 optimzation . to force off - rate selection , prolonged wash cycles and competing antigen were applied to the pool - library . specifically , the vl cdr1 regions of a set of anti - hla - dr antibody fragments derived from ms - gpc - 8 / b8 ( including ms - gpc - 8 - 10 and ms - gpc - 8 - 27 ) were optimized by cassette mutagenesis using trinucleotide - directed mutagenesis ( virnekäs et al ., 1994 ). in brief , a vλ1 cdr1 library cassette was synthesized containing six randomized positions ( total variability : 7 . 43 × 10 6 ), and was cloned into a vλ1 framework . the cdr1 library was digested with ecorv and bbsi , and the fragment comprising the cdr1 library ligated into the lambda light chains of the ms - gpc - 8 - derived fab antibody fragments in pmorph18_fab ( as described above ), digested with ecorv and bbsi . the resulting library was screened as described above . the pool - library was subjected to two rounds of standard manual solid - phase panning using decreasing amounts of antigen ( 250 ng and 7 . 5 ng / well purified protein ) or increasing concentrations of nh 4 scn ( 100 mm , 500 mm and 2500 mm ), using either 2 - fold serial dilutions of purified hla - protein between 250 ng and 7 . 5 ng / well , or alternatively , constant amounts of 250 ng / well of protein in combination with an additional 30 min incubation step of different amounts of ammonium - isothiocyanate ( 100 mm , 500 mm and 2500 mm ) between the standard wash - protocol and the elution step . in order to enforce off - rate - selection an additional manual solid - phase - panning of 3 selection rounds was performed with the pool - library using 250 ng / well of coated hla - protein in combination with longer washes ( starting from 6 × 30 min in the first up to 24 × 30 min in the 3 rd panning - round ) and including different amounts of competing antigen ( from 20 nm up to 500 nm ) in the wash - buffer . this strategy yielded fabs with affinities of ˜ 3 nm ( table 3e ). ten clones were identified as above by binding specifically to hla - dr ( ms - gpc - 8 - 6 - 2 , ms - gpc - 8 - 6 - 19 , ms - gpc - 8 - 6 - 27 , ms - gpc - 8 - 6 - 45 , ms - gpc - 8 - 6 - 13 / 305d3 , ms - gpc - 8 - 6 - 47 , ms - gpc - 8 - 10 - 57 / 1c7277 , ms - gpc - 8 - 27 - 7 , ms - gpc - 8 - 27 - 10 & amp ; ms - gpc - 8 - 27 - 41 / 1d09c3 ) and showed cell killing activity as found for the starting fragments ms - gpc - 8 , ms - gpc - 8 - 10 and ms - gpc - 8 - 27 . table 1 contains the sequence characteristics of ms - gpc - 8 - 6 - 2 , ms - gpc - 8 - 6 - 19 , ms - gpc - 8 - 6 - 27 , ms - gpc - 8 - 6 - 45 , ms - gpc - 8 - 6 - 13 , ms - gpc - 8 - 6 - 47 , ms - gpc - 8 - 10 - 57 , ms - gpc - 8 - 27 - 7 , ms - gpc - 8 - 27 - 10 & amp ; ms - gpc - 8 - 27 - 41 . the vh and vl families and the cdr3s listed refer to the hucal consensus - based antibody genes as described ( knappik et al ., 2000 ), the full sequences of the vh and vl domains of ms - gpc - 8 - 6 - 13 , ms - gpc - 8 - 10 - 57 and ms - gpc - 8 - 27 - 41 are shown in fig1 . from these 10 clones , four fab fragments were chosen ( ms - gpc - 8 - 6 - 2 , ms - gpc - 8 - 6 - 13 / 305d3 , ms - gpc - 8 - 10 - 57 , c7277 and ms - gpc - 8 - 27 - 41 / 1d09c3 ) as demonstrating significantly improved ec 50 of cell killing as described in example 10 . table 1 shows the sequences of clones optimised at the cdr1 region . optimisation procedures not only increased the biological efficacy of anti - hla - dr antibody fragments generated by the optimisation process , but a physical characteristic — affinity of the antibody fragment to hla - dr protein — was also substantially improved . for example , the affinity of fab forms of ms - gpc - 8 / b8 and its optimised descendents was measured using a surface plasmon resonance instrument ( biacore , upsala sweden ) according to example 7 . the affinity of the ms - gpc - 8 / b8 parental fab was improved over 100 fold from 346 nm to ˜ 60 nm after vl cdr3 optimisation and further improved to single digit nanomolar affinity ( range 3 - 9 nm ) after vl cdr3 + 1 optimisation ( table 2 ). three fabs ( 305d3 , 1d09c3 , and 1c7277 ) obtained above were converted into igg 4 format , expressed and purified for affinity determination ( see below ). all 3 igg 4 mabs exhibited sub - nanomolar affinities ( 0 . 3 - 0 . 6 nm ; table 3e ), and retained their specificity ( fig2 ). heavy chains were cloned as follows . the multiple cloning site of pcdna3 . 1 +( invitrogen ) was removed ( nhei / apai ), and a stuffer compatible with the restriction sites used for hucal - design was inserted for the ligation of the leader sequences ( nhei / ecori ), vh - domains ( ecori / blpi ) and the immunoglobulin constant regions ( blpi / apai ). the leader sequence ( embl m83133 ) was equipped with a kozak sequence ( kozak , 1987 ). the constant regions of human igg 1 ( pir j00228 ), igg 4 ( embl k01316 ) and serum iga 1 ( embl j00220 ) were dissected into overlapping oligonucleotides with lengths of about 70 bases . silent mutations were introduced to remove restriction sites non - compatible with the hucal - design . the oligonucleotides were spliced by overlap extension - pcr . light chains were cloned as follows . the multiple cloning site of pcdna3 . 1 / zeo +( invitrogen ) was replaced by two different stuffers . the κ - stuffer provided restriction sites for insertion of a κ - leader ( nhei / ecorv ), hucal - scfv vκ - domains ( ecorv / bsiwi ) and the κ - chain constant region ( bsiwi / apai ). the corresponding restriction sites in the κ - stuffer were nhei / ecorv ( λ - leader ), ecorv / hpai ( vλ - domains ) and hpai / apai ( λ - chain constant region ). the κ - leader ( embl z00022 ) as well as the λ - leader ( embl l27692 ) were both equipped with kozak sequences . the constant regions of the human κ -( embl j00241 ) and λ - chain ( embl m18645 ) were assembled by overlap extension - pcr as described above . all cells were maintained at 37 ° c . in a humidified atmosphere with 5 % co 2 in media recommended by the supplier . cho - k1 ( crl - 9618 ) were from atcc and were co - transfected with an equimolar mixture of igg heavy and light chain expression vectors . double - resistant transfectants were selected with 600 μg / ml g 418 and 300 μg / ml zeocin ( invitrogen ) followed by limiting dilution the supernatant of single clones was assessed for igg expression by capture - elisa . positive clones were expanded in rpmi - 1640 medium supplemented with 10 % ultra - low igg - fcs ( life technologies ). after adjusting the ph of the supernatant to 8 . 0 and sterile filtration , the solution was subjected to standard protein a column chromatography ( poros 20a , pe biosystems ). the igg forms of anti - hla - dr antigen binding domains show improved characteristics over the antibody fragments . these improved characteristics include affinity ( example 7 ) and killing efficiency ( examples 9 , 10 and 14 ). to demonstrate that antigen - binding domains selected from the hucal library bound specifically to a binding site on the n - terminal domain of human mhcii receptor largely conserved between alleles and hitherto unknown in the context of cell killing by receptor cross linking , we undertook an assessment of their binding specificity , and it was attempted to characterise the binding epitope . the fab antibody fragments ms - gpc - 8 - 27 - 7 , ms - gpc - 8 - 27 - 10 , ms - gpc - 8 - 6 - 13 , ms - gpc - 8 - 27 - 41 / 1d09c3 , ms - gpc - 8 - 6 - 47 , ms - gpc - 8 - 10 - 57 11c7277 , ms - gpc - 8 - 6 - 27 , ms - gpc - 8 / b8 and ms - gpc - 8 - 6 showed specificity of binding to hla - dr protein but not to non - hla - dr proteins . fab fragments selected from the hucal library were tested for reactivity with the following antigens : hla - dr protein ( dra * 0101 / drb1 * 0401 ; prepared as example 1 , and a set of unrelated non - hla - dr proteins consisting of bsa , testosterone - bsa , lysozyme and human apotransferrin . an empty well ( plastic ) was used as negative control . coating of the antigen mhcii was performed over night at 1 μg / well in pbs ( nunc - maxisorp tm ) whereas for the other antigens ( bsa , testosterone - bsa , lysozyme , apotransferrin ) 10 μg / well was used . next day wells were blocked in 5 % non - fat milk for 1 hr followed by incubation of the respective antibodies ( anti - mhcli - fabs and an unrelated fab ( macl - 8a )) at 100 ng / well for 1 hour . after washing in pbs the anti - human igg f ( ab ′) 2 - peroxidase - conjugate at a 1 : 10 , 000 dilution in tbs ( supplemented with 5 % w / v non - fat dry - milk / 0 . 05 % v / v tween 20 ) was added to each well for 1 h . final washes were carried out in pbs followed the addition the substrate pod ( roche ). color - development was read at 370 nm in an elisa - reader . all anti - hla - dr antibody fragments ms - gpc - 8 - 27 - 7 , ms - gpc - 8 - 27 - 10 , ms - gpc - 8 - 6 - 13 , ms - gpc - 8 - 27 - 41 , ms - gpc - 8 - 6 - 47 , ms - gpc - 8 - 10 - 57 , ms - gpc - 8 - 6 - 27 , ms - gpc - 8 and ms - gpc - 8 - 6 demonstrated high specificity for hla - dr , as evidenced by the much higher mean fluorescence intensity resulting from incubation of these antibody fragments with hla - dr derived antigens compared to controls ( fig1 a ). in a similar experiment , the fab fragments ms - gpc - 1 , ms - gpc - 6 , ms - gpc - 8 and ms - gpc - 10 were found to bind to both the dra * 0101 / drb1 * 0401 ( preparaed as above ) as well as to a chimeric dr - ie consisting of the n - terminal domains of dra * 0101 and drb1 * 0401 with the remaining molecule derived from a murine class ii homologue ied ( ito et al ., 1996 ) ( fig1 b ). to demonstrate the broad - dr reactivity of anti - hla - dr antibody fragments and iggs of the invention , the scfv forms of ms - gpc - 1 , 6 , 8 and 10 , and igg forms of ms - gpc - 8 , ms - gpc - 8 - 10 - 57 , ms - gpc - 8 - 27 - 51 & amp ; ms - gpc - 8 - 6 - 13 were tested for reactivity against a panel of epstein - barr virus transformed b cell lines obtained from ecacc ( salisbury uk ), each homozygous for one of the most frequent dr alleles in human populations ( list of cell lines and alleles shown in fig2 ). the antibody fragments were also tested for reactivity against a series of l cells transfected to express human class ii isotypes other than drb1 : l105 . 1 , l257 . 6 , l25 . 4 , l256 . 12 & amp ; l21 . 3 that express the molecules drb3 * 0101 , drb4 * 0101 , dpo103 / 0402 , dp 0202 / 0201 , and dq0201 / 0602 respectively ( klohe et al ., 1988 ). reactivity of an antigen - binding fragment to the panel of cell - lines expressing various mhc — class ii molecules was demonstrated using an immunofluorescence procedure as for example , described by otten et al ( 1997 ). staining was performed on 2 × 10 5 cells using an anti - flag m2 antibody as the second reagent against the m2 tag carried by each anti - hla - dr antibody fragment and a fluorescein labelled goat anti - mouse ig ( bd pharmingen , torrey pine , calif ., usa ) as a staining reagent . cells were incubated at 4 ° c . for 60 min with a concentration of 200 nm of the anti - hla - dr antibody fragment , followed by the second and third antibody at concentrations determined by the manufacturers . for the igg form , the second antibody was omitted and the igg detected using a fitc - labeled mouse anti - human igg 4 ( serotec , oxford , uk ). cells were washed between incubation steps . finally the cells were washed and subjected to analysis using a facs calibur ( bd immunocytometry systems , san jose , calif ., usa ). [ 0286 ] fig2 shows that the scfv - fragments ms - gpc - 1 , 6 , 8 and 10 , and igg forms of ms - gpc - 8 , ms - gpc - 8 - 10 - 57 , ms - gpc - 8 - 27 - 51 & amp ; ms - gpc - 8 - 6 - 13 react with all drb1 all o types tested . this observation taken together with the observation that all anti - hla - dr antibody fragments react with chimeric dr - ie , suggests that all selected anti - hla - dr antibody fragments recognize the extracellular first domain of the monomorphic drα chain or a monomorphic epitope on extracellular first domain of the drβ chain . we then attempted to localize the binding domains of ms - gpc - 8 - 10 - 57 and ms - gpc - 8 - 27 - 41 further by examining competitive binding with murine antibodies for which the binding domains on hla - dr are known . the murine antibodies l243 and lb3 . 1 are known to bind to the al domain , 1 - 1c4 and 8d1 to the β1 domain and 10f12 to the o 2 domain ( vidovic et al . 1995b ). to this end , an assay was developed wherein a dr - expressing cell line ( lg - 2 ) was at first incubated with the igg 4 forms of ms - gpc - 8 - 10 - 57 or ms - gpc - 8 - 27 - 41 , the fab form of ms - gpc - 8 - 10 - 57 or the fab form of gpc 8 , and an unrelated control antibody . subsequently murine antibodies were added and the murine antibodies were detected . if the binding site of ms - gpc - 8 - 10 - 57 or ms - gpc - 8 - 27 - 41 overlaps with the binding of a murine antibody , then a reduced detection of the murine antibody is expected . binding of the igg 4 forms of gpc - 8 - 27 - 41 and ms - gpc - 8 - 10 - 57 and the fab form of ms - gpc - 8 - 10 - 57 substantially inhibited ( mean fluorescence intensity reduced by & gt ; 90 %) the binding of 1 - 1c4 and 8d1 , whereas l243 , lb3 . 1 and 10f12 and a control were only marginally affected . the fab form of ms - gpc - 8 reduced binding of 1 - 1c4 by ˜ 50 % ( mean fluorescence dropped from 244 to 118 ), abolished 8d1 binding and only marginally affected binding of l243 , lb3 . 1 and 10f12 or the control . an unrelated control antibody had no effect on either binding . thus , ms - gpc - 8 - 10 - 57 and ms - gpc - 8 - 27 - 41 seem to recognise a β1 domain epitope that is highly conserved among allelic hla - dr molecules . the whole staining procedure was performed on ice . 1 × 10 7 cells of the human b - lymphoblastoid cell line lg - 2 was preblocked for 20 min . in pbs containing 2 % fcs and 35 μg / ml guinea pig igg (“ facs - buffer ”). these cells were divided into 3 equal parts a , b , and c of approximately 3 . 3 × 10 6 cells each , and it was added to a ) 35 μg ms - gpc - 8 - 10 - 57 or ms - gpc - 8 - 27 - 41 igg 4 , to b ) 35 μg ms - gpc - 8 - 10 - 57 fab or ms - gpc - 8 fab , and to c ) 35 μg of an unrelated igg 4 antibody as negative control , respectively , and incubated for 90 min . subsequently a , b , c were divided in 6 equal parts each containing 5 . 5 × 10 5 cells , and 2 μg of the following murine antibodies were added each to one vial and incubated for 30 min : 1 ) purified migg ; 2 ) l243 ; 3 ) lb3 . 1 ; 4 ) 1 - 1 c4 ; 5 ) 8d1 ; 6 ) 10f12 . subsequently , 4 ml of pbs were added to each vial , the vials were centrifuged at 300 × g for 8 min , and the cell pellet resuspended in 50 μl facs buffer containing a 1 to 25 dilution of a goat - anti - murine ig - fitc conjugate at 20 μg / ml final concentration ( bd pharmingen , torrey pines , calif ., usa ). cells were incubated light - protected for 30 min . afterwards , cells were washed with 4 ml pbs , centrifuged as above and resuspended in 500 μl pbs for analysis in the flow cytometer ( facs calibur , bd immunocytometry systems , san jose , calif ., usa ). the pepspot technique ( u . s . pat . no . 6 , 040 , 423 ; heiskanen et al ., 1999 ) is used to further identify the binding epitope for ms - gpc 8 - 10 - 57 . briefly , an array of 73 overlapping 15 - mer peptides is synthesised on a cellulose membrane by a solid phase peptide synthesis spotting method ( wo 00 / 12575 ). these peptide sequences are derived from the sequence of the α1 and β1 domains of hla - dr4dw14 , hla - dra1 * 0101 ( residues 1 - 81 ) and hla - drb1 * 0401 ( residues 2 - 92 ), respectively , and overlap by two amino acids . second , such an array is soaked in 0 . 1 % tween - 20 / pbs ( pbs - t ), blocked with 5 % bsa in pbs - t for 3 hours at room temperature and subsequently washed three times with pbs - t . third , the prepared array is incubated for 90 minutes at room temperature with 50 ml of a 5 mg / l solution of the igg form of gpc - 8 - 10 - 57 in 1 % bsa / pbs - t . fourth , after binding , the membrane is washed three times with pbs - t and subsequently incubated for 1 hour at room temperature with a goat anti - human light chain antibody conjugated to horseradish peroxidase diluted { fraction ( 1 / 5 , 000 )} in 1 % bsa / pbs - t . finally , the membrane is washed three times with pbs - t and any binding determined using chemiluminescence detection on x - ray film . as a control for unspecific binding of the goat anti - human light chain antibody , the peptide array is stripped by the following separate washings each at room temperature for 30 min : pbs - t ( 2 times ), water , dmf , water , an aequeous solution containing 8 m urea , 1 % sds , 0 . 5 % dtt , a solution of 50 % ethanol , 10 % acetic acid in water ( 3 times each ) and , finally , methanol ( 2 times ). the membrane is again blocked , washed , incubated with goat anti - human 1 light chain antibody conjugated to horseradish peroxidase and developed as described above . in order to demonstrate the superior binding properties of anti - hla antibody fragments of the invention , we measured their binding affinities to the human mhc class ii dr protein ( dra * 0101 / drb1 * 0401 ) using standard equipment employing plasmon resonance principles . surprisingly , we achieved affinities in the sub - nanomolar range for igg forms of certain anti - hla - dr antibody fragments of the invention . for example , the affinity of the igg forms of ms - gpc - 8 - 27 - 41 , ms - gpc - 8 - 6 - 13 & amp ; ms - gpc - 8 - 10 - 57 was measured as 0 . 3 , 0 . 5 and 0 . 6 nm respectively ( table 3a ). also , we observed high affinities in the range of 2 - 8 nm for fab fragments affinity matured at the cdr1 and cdr3 light chain regions ( table 3b ). fab fragments affinity matured at only the cdr3 light chain region showed affinities in the range of 40 to 100 nm ( table 3c ), and even fab fragments of non - optimised hucal antigen binding domains showed affinities in the sub μm range ( table 3d ). only a moderate increase in k on ( 2 - fold ) was observed following cdr3 optimisation ( k on remained approximately constant throughout the antibody optimization process in the order of 1 × 10 5 m − 1 s − 1 ), whilst a significant decrease in k off was a surprising feature of the optimisation process — sub 100 s − 1 , sub 10 s − 1 , sub 1 s − 1 and sub 0 . 1 s − 1 for the unoptimised fabs , cdr3 optimised fabs , cdr3 / cdr1 optimised fabs and igg forms of anti - hla - dr antibody fragments of the invention . the affinities for anti - hla antibody fragments of the invention were measured as follows . all measurements were conducted in hbs buffer ( 20 mm hepes , 150 mm nacl , ph 7 . 4 ) at a flow rate of 20 μl / min at 25 ° c . on a biacore3000 instrument ( biacore ab , sweden ). mhc class ii dr protein ( prepared as example 1 ) was diluted in 100 mm sodium acetate ph 4 . 5 to a concentration of 50 - 100 mg / ml , and coupled to a cm5 chip ( biacore ab ) using standard edc - nhs coupling chemistry with subsequent ethanolamine treatment as manufacturers directions . the coating density of mhcii was adjusted to between 500 and 4000 ru . affinities were measured by injection of 5 different concentrations of the different antibodies and using the standard software of the biacore instrument . regeneration of the coupled surface was achieved using 10 mm glycine ph 2 . 3 and 7 . 5 mm naoh . 8 . multivalent killing activity of anti hla - dr antibodies and antibody fragments to demonstrate the effect of valency on cell killing , a cell killing assay was performed using monovalent , bivalent and multivalent compositions of anti - hla - dr antibody fragments of the invention against granta - 519 cells . anti - hla - dr antibody fragments from the hucal library showed much higher cytotoxic activity when cross - linked to form a bivalent composition ( 60 - 90 % killing at antibody fragment concentration of 200 nm ) by co - incubation with anti - flag m2 mab ( fig3 ) compared to the monovalent form ( 5 - 30 % killing at antibody fragment concentration of 200 nm ). incubation of cell lines alone or only in the presence of anti - flag m2 mab without co - incubation of anti - hla - dr antibody fragments did not lead to cytotoxicity as measured by cell viability . treatment of cells as above but using 50 nm of the igg 4 forms ( naturally bivalent ) of the antibody fragments ms - gpc - 8 , ms - gpc - 8 - 6 - 13 , ms - gpc - 8 - 10 - 57 and ms - gpc - 8 - 27 - 41 without addition of anti - flag m2 mab showed a killing efficiency after 4 hour incubation of 76 %, 78 %, 78 % and 73 % respectively . furthermore , we observed that higher order valences of the anti - hla - dr antibody fragments further decrease cell viability significantly . on addition of protein g to the incubation mix containing the igg form of the anti - hla - dr antibody fragments , the multivalent complexes thus formed further decrease cell viability compared to the bivalent composition formed from incubation of the anti - hla - dr antibody fragments with only the bivalent igg form . the killing efficiency of anti - hla - dr antibody fragments selected from the hucal library was tested on the hla - dr positive tumor cell line granta - 519 ( dsmz , germany ). 2 × 10 5 cells were incubated for 4 h at 37 ° c . under 6 % co 2 with 200 nm anti - hla - dr antibody fragments in rpmi 1640 ( paa , germany ) supplemented with 2 . 5 % heat inactivated fbs ( biowhittaker europe , be ), 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 0 . 1 mg / ml kanamycin . each anti - hla - dr antibody fragment was tested for its ability to kill activated tumor cells as a monovalent anti - hla - dr antibody fragment or as a bivalent composition by the addition of 100 nm of a bivalent cross - linking anti - flag m2 mab . after 4 h incubation at 37 ° c . under 6 % co 2 , cell viability was determined by trypan blue staining and subsequent counting of remaining viable cells ( current protocols in immunology , 1997 ). the above experiment was repeated using karpas - 422cells against a multivalent form of igg forms of ms - gpc - 8 - 10 - 57 and ms - gpc - 8 - 27 - 41 prepared by a pre - incubation with a dilution series of the bacterial protein protein g . protein g has a high affinity and two binding sites for igg antibodies , effectively cross - linking them to yield a total binding valency of 4 . in a control using igg alone without preincubation with protein g , approximately 55 % of cells were killed , while cell killing using igg pre - incubated with protein g gave a maximum of approximately 75 % at a molar ratio of igg antibody / protein g of ˜ 6 ( based on a molecular weight of protein g of 28 . 5 kd ). higher or lower molar ratios of igg antibody / protein g approached the cell killing efficiency of the pure igg antibodies . experiments to determine the killing efficiency of the anti - hla - dr cross - linked antibody fragments against other tumor cell lines that express hla - dr molecules were conducted analogous to example 8 . tumor cell lines that show greater than 50 % cell killing with the cross linked fab form of ms - gpc - 8 after 4 h incubation include mhh - call4 , mn 60 , bjab , bonna - 12 which represent the diseases b cell acute lymphoid leukemia , b cell acute lymphoid leukemia , burkitt lymphoma and hairy cell leukemia respectively . use of the cross - linked fab form of the anti - hla - dr antibody fragments ms - gpc - 1 , 6 and 10 also shows similar cytotoxic activity to the above tumor cell lines when formed as a bivalent agent using the cross - linking anti - flag m2 mab . the method described in example 8 was used to determine the maximum killing capacity for each of the cross - linked bivalent anti - hla - dr antibody fragments against priess cells . the maximum killing capacity observed for ms - gpc - 1 , ms - gpc - 6 , ms - gpc - 8 & amp ; ms - gpc - 10 was measured as 83 %, 88 %, 84 % and 88 % respectively . antibody fragments generated according to example 4 , when cross linked using anti - flag m2 mab as above , also showed improved killing ability against granta and priess cells ( table 4 ). the optimized igg 4 mabs were tested for induction of tumor cell death on a panel of 24 dr + and 4 dr − cell lines , representing a variety of lymphoma / leukemia types ( table 5 ). compared to corresponding murine antibodies ( vidovic et al , 1995b ; nagy & amp ; vidovic , 1996 ; vidovic & amp ; toral ; 1998 ), we were surprised to observe significantly improved killing efficiency of igg forms of certain anti - hla - dr antibody fragments of the invention ( table 5 ). the killing is dependent on hla - dr expression , but is hls - dr subtype independent . for the cell killing assay , cells at 2 × 10 6 / ml concentration were incubated in rpmi 1640 supplemented with 2 . 5 % fetal calf serum ( biowhittaker europe , belgium ) and different concentrations ( 50 nm in most experiments ) of human anti - dr mab at 37 ° c . for 4 hrs ( and 24 h in some experiments ). control cultures were without mab or with a murine anti - dr mab 10f12 that fails to induce cell death . cell cultures were set up in duplicate in flat bottom 96 well plates . since dead cells disintegrate very fast ( within 30 min ),% killing was determined based on viable cell recovery as follows : ( viable untreated — viable treated / viable untreated )× 100 . viable and dead cells were distinguished by trypan blue staining for light microscopy , fluorescein diacetate ( fda ; 100 μg / ml final concentration ; live cells ) and propidium iodide ( pi , 40 μg / ml final concentration ; dead cells ) for fluorescent microscopy , and pi for facs analysis . to obtain absolute cell counts by facs analysis , each culture was supplemented with equal amounts of facs “ truecount ” calibrating beads . cell counts were determined by the formula : viable cells x total beads / counted beads . the three different methods of cell counting ( light and fluorescent microscopy and facs ) yielded comparable results . following the method described in examples 8 and 9 and above but at 50 nm , repeated measurements ( 3 to 5 replica experiments where cell number was counted in duplicate for each experiment ) were made of the killing efficiency of the igg forms of certain antibody fragments of the invention . the mabs induced death in a wide range ( 23 of the 25 ) dr + lymphoid tumor lines . when applied at a final concentration of only 50 nm , iggs of the antibody fragments ms - gpc - 8 / b8 , ms - gpc - 8 - 6 - 13 / 305d3 , ms - gpc - 8 - 10 - 57 / 1c7277 & amp ; ms - gpc - 8 - 27 - 41 / 1d09c3 killed more than 50 % of cells from 17 , 20 , 19 and 22 respectively of a panel of 25 human tumor cell lines that express hla - dr antigen at a level greater than 10 fluorescent units as determined by example 11 . for comparison , two murine anti - dr mabs , l243 ( vidovic et al , 1995b ) and 8d1 ( vidovic & amp ; toral ; 1998 ) known to induce cell death 7 , 10 were tested on the same panel at 4 fold higher concentration ( 200 nm ) than the human mabs . the murine mabs usually killed less cells than human mabs , or failed to induce death in some dr + lines . over all , they reduced cell viability to a level below 50 % viable cells in only 13 and 12 of the 25 hla - dr expressing cells lines , respectively . in direct comparisons , the human mabs achieved 50 % killing efficiency at 20 to 30 fold lower concentrations than the murine mabs ( see below ). statistical analysis of the data in table 5 revealed a non - linear correlation between killing efficiency and the level of dr expression , with a significantly greater killing efficiency and better correlation for the human mabs because of the failure of the murine mabs to kill a number of dr + lines . indeed , even at the significantly increased concentration , the two murine antibodies treated at 200 nm showed significantly less efficient killing compared to the igg forms of anti - hla - dr antibody fragments of the invention . not only do igg forms of the human anti - hla - dr antibody fragments of the invention show an overall increase in cell killing at lower concentrations compared to the murine antibodies , but they show less variance in killing efficiency across different cell lines . the coefficient of variance in killing for the human antibodies in this example is 32 % ( mean % killing = 68 +/− 22 % ( sd )), compared to over 62 % ( mean % killing = 49 +/− 31 % ( sd )) for the mouse antibodies . statistically controlling for the effect on killing efficiency due to hla expression by fitting logistic regression models to mean percentage killing against log ( mean hla - dr expression ) supports this observation ( fig4 ). not only is the fitted curve for the murine antibodies consitently lower than that for the human , but a larger variance in residuals from the murine antibody data ( sd = 28 %) is seen compared to the variance in residuals from the human antibody data ( 16 %). the superior performance of human mabs could be explained , at least in part , by their higher affinity ( k d - s 0 . 3 - 0 . 6 nm , see table 3e , compared to l243 10 nm , and 8d1 & gt ; 30 nm ( z . a . nagy , unpublished )). the cell line mhh — preb - 1 was singled out and not accounted as part of the panel of 25 cell lines despite its expression of hla - dr antigen at a level greater than 10 fluorescent units due to the inability of any of the above antibodies to induce any significant reduction of cell viability . this is further explained in example 12 . 11 . killing selectivity of antigen - binding domains against a human antigen for activated versus non - activated cells since mhc - ii molecules are constitutively expressed on b lymphocytes , the most obvious potential side effect of anti - dr mab treatment would be the killing of normal b cells . human peripheral b cells were therefore used to demonstrate that human anti - hla - dr mab - mediated cell killing is dependent on cell - activation . 50 ml of heparinised venous blood was taken from an hla - dr typed healthy donor and fresh peripheral blood mononuclear cells ( pbmc ) were isolated by ficoll - hypaque gradient centrifugation ( histopaque - 1077 ; sigma ) as described in current protocols in immunology ( john wiley & amp ; sons , inc . ; 1999 ). purified b cells (˜ 5 % of peripheral blood leukocytes ) were obtained from around 5 × 10 7 pbmc using the b - cell isolation kit and macs ls + / vs + columns ( miltenyi biotec , germany ) according to manufacturers guidelines . successful depletion of non - b cells was verified by facs analysis of an aliquot of isolated b cells ( hla - dr positive and cd19 positive ). double staining and analysis is done with commercially available antibodies ( bd immunocytometry systems , san jose , calif ., usa ) using standard procedures as for example described in current protocols in immunology ( john wiley & amp ; sons , inc . ; 1999 ). an aliquot of the isolated b cells was tested for the ability of the cells to be activated by stimulation with pokeweed mitogen ( pwm ) ( gibco brl , cat . no . 15360 - 019 ) diluted 1 : 25 in rpmi 1640 ( paa , germany ) supplemented with 10 % fcs ( biowhittaker europe , be ), 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 0 . 1 mg / ml kanamycin by incubation at 37 ° c . under 6 % co 2 for three days . successful activation was verified by facs analysis of hla - dr expression on the cell surface ( current protocols in immunology , john wiley & amp ; sons , inc . ; 1999 ). the selectivity for killing of activated cells versus non - activated cells was demonstrated by incubating 1 × 10 6 / ml b cells activated as above compared to non - activated cells , respectively with 50 nm of the igg forms of ms - gpc - 8 - 10 - 57 , ms - gpc - 8 - 27 - 41 or the murine igg 10f12 ( vidovic et al , 1995b ) in the medium described above but supplemented with 2 . 5 % heat inactivated fcs instead of 10 %, or with medium alone . after incubation at 37 ° c . under 6 % co 2 for 1 or 4 h , cell viability was determined by fluorescein diacetate staining ( fda ) of viable and propidium iodide staining ( pi ) of dead cells and subsequent counting of the green ( fda ) and red ( pi ) fluorescent cells using a fluorescence microscope ( leica , germany ) using standard procedures ( current protocols in immunology , 1997 ). b cell activation was shown to be necessary for cell killing . in non - activated cells after 1 hr of incubation with the anti - hla - dr antibodies , the number of viable cells in the media corresponded to 81 %, 117 % 126 % and 96 % of the pre - incubation cell density for ms - gpc - 8 - 10 - 57 ( igg ), ms - gpc - 8 - 27 - 41 ( igg ), 10f12 and medium alone , respectively . in contrast , the number of viable activated b cells after 1 h incubation corresponded to 23 %, 42 % 83 % and 66 % of the pre - incubation cell density for ms - gpc - 8 - 10 - 57 ( igg ), ms - gpc - 8 - 27 - 41 ( igg ), 10f12 and medium alone , respectively . after 4 hr of incubation , 78 %, 83 % 95 % and 97 % of the pre - incubation cell density for ms - gpc - 8 - 10 - 57 ( igg ), ms - gpc - 8 - 27 - 41 ( igg ), 10f12 and medium alone were found viable in non - activated cells , whereas the cell density had dropped to 23 %, 24 % 53 % and 67 % of the pre - incubation cell density for ms - gpc - 8 - 10 - 57 ( igg ), ms - gpc - 8 - 27 - 41 ( igg ), 10f12 and medium alone , respectively , in activated cells . in conclusion , as shown in fig8 c , the viability of purified resting b cells was not significantly altered by human anti - dr mabs . in contrast , pokeweed mitogen - activated b cells from the same donor were killed by these mabs . no death of either unactivated or activated b cells was induced by the control antibody 10f12 . similar results were obtained with resting and lipopolysaccharide - stimulated spleenic b cells from dr - transgenic mice ( ito , k . et al . j . exp . med . 183 : 2635 - 2644 , 1996 ) ( data not shown ). thus , it appears that the mabs can kill activated but not resting mhc - ii positive normal cells in addition to tumor cells , suggesting a dual requirement of both mhc - ii expression and cell activation for mab - induced death . since the majority ( up to 99 %) of peripheral b cells is resting , the potential side effect due to killing of normally activated b cells in a leukaemia patient is negligible . 12 . killing activity of anti - hla antibody fragments against the cell line mhh preb 1 as evidenced in table 5 , we observed that our cross - linked anti - hla - dr antibody fragments or iggs did not readily kill a particular tumor cell line expressing hla - dr at significant levels ( mhh — preb - 1 ). we hypothesized that although established as a stable cell line , cells in this culture were not sufficiently activated . we therefore stimulated these cells with interferon - gamma , and lipopoysaccharide . activation was evidenced by an increase in the cell surface expression of cd40 and hla - dr . non - adherently growing mhh preb1 cells were cultivated in rpmi medium containing the following additives ( all from gibco brl and bio whittaker ): 10 % fcs , 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 1 × kanamycin . aliquots were activated to increase expression of hla - dr molecule by incubation for one day with lipopolysaccharide ( lps , 10 μg / ml ), interferon - gamma ( ifn - γ , roche , 40 ng / ml ) and phyto - hemagglutinin ( pha , 5 μg / ml ). the cell surface expression of hla - dr molecules was monitored by flow cytometry with the fitc - conjugated mab l243 ( bd immunocytometry systems , san jose , calif ., usa ). incubation of mhh preb1 for one day in the presence of lps , ifn - γ and pha resulted in a 2 - fold increase in hla - dr surface density ( mean fluorescence shift from 190 to 390 ). cell killing was performed for 4 hrs in the above medium but containing a reduced fcs concentration ( 2 . 5 %). a concentration series of the igg forms of ms - gpc - 8 - 27 - 41 / 1d09c3 & amp ; ms - gpc - 8 - 10 - 57 / 1c7277 was employed , consisting of final antibody concentrations of 3300 , 550 , 92 , 15 , 2 . 5 , 0 . 42 and 0 . 07 nm , on each of an aliquot of non - activated and activated cells . viable cells were identified microscopically by exclusion of trypan blue . whereas un - activated cell viability remains unaffected by the antibody up to the highest antibody concentration used , cell viability is dramatically reduced with increasing antibody concentration in activated mhh preb1 cells ( fig5 ). in addition , we found that cell proliferation was apparently not needed , since tumor cells in mitosis - arrest remained susceptible to mab - mediated killing ( data not shown ). in contracts to the mabs we describe here , two additional anti - hla - dr mabs with therapeutic potential , lym - 1 ( epstein et al ., cancer res . 47 : 830 - 840 , 1987 ; denardo et al ., int . j . cancer 96 ( suppl . 3 ): 96 , 1988 ) and 1d10 ( gingrich et al ., blood 75 : 2375 - 2387 , 1990 ), achieve selectivity in a different way . these two mabs recognize what appear to be posttranslational modifications on dr molecules that occur preferentially in b - cell derived tumors , although some expression was noted also on normal b cells and monocytes ( epstein et al ., 1987 ; denardo et al ., 1988 ). neither of these mabs has inherent tumoricidal activity , and thus , lym - 1 is developed in a 131 i - labelled form ( oncolym ®, whereas the efficacy of 1d10 relies on intact immunological effector mechanisms of the patient , similarly to other mabs ( vose et al ., j . clin . oncol . 19 : 389 - 397 , 2001 ; dyer et al ., blood 73 : 1431 - 1439 , 1989 ) already available for the clinic . furthermore , lym - 1 is a murine mab with substantial immunogenicity for humans , and 1d10 is a humanized murine mab . our fully human mabs with strong inherent tumoricidal activity and selectivity for activated / tumor transformed cells demonstrate a substantially different profile and mechanism of action from these two mabs , and thus promise a novel therapeutic approach to lymphoma / leukemia . 13 . killing efficiency of anti - hla - dr igg antibodies of human composition against ex - vivo chronic lymphoid leukemia cells we investigated whether the human anti - dr mabs would also be active on freshly isolated leukemic cells , in addition to established cell lines . using purified malignant b cells obtained from the peripheral blood of 10 un - typed chronic lymphoid leukemia ( cll ) patients ( buhmann et al ., blood 93 : 1992 - 2002 , 1999 ), we demonstrated that igg forms of anti - hla - dr antibody fragments of the invention showed efficacy in killing of clinically relevant cells using an ex - vivo assay ( fig6 ). although the killing kinetics are slightly slower than those of in vitro experiments using established cell lines , significant killing is achieved over 24 hours of ab incubation , despite the low rate of cll cell proliferation . b - cells were isolated and purified from 10 unrelated patients suffering from cll ( samples kindly provided by prof hallek , ludwig maximillian university , munich ) according to standard procedures ( buhmann et al ., ( 1999 )). 2 × 10 5 cells were treated with 100 nm of igg forms of the anti - hla - dr antibody fragments ms - gpc - 8 , ms - gpc - 8 - 10 - 57 or ms - gpc - 8 - 27 - 41 and incubated for 4 or 24 hours analogous to examples 8 and 9 . a replica set of cell cultures was established and activated by incubation with hela - cells expressing cd40 ligand on their surface for three days before treatment with antibody ( buhmann et al ., 1999 ). as controls , the murine igg 10f12 ( vidovic et al ., 1995b ) or no antibody was used . cell viability for each experiment was determined as described in example 12 . surprisingly , igg forms of the anti - hla - dr antibody fragments of the invention showed highly efficient and uniform killing — even across this diverse set of patient material . after only 4 hours of treatment , all three human iggs gave a significant reduction in cell viability compared to the controls , and after 24 hours only 33 % of cells remained viability ( fig6 ). we found that on stimulating the ex - vivo cells further according to buhmann et al . ( 1999 ), the rate of killing was increased such that after only 4 hours culture with the human antibodies , only 24 % of cells remained viable on average for all patient samples and antibody fragments of the invention . the control murine anti - dr mab 10f12 , which has no inherent tumoricidal activity ( vidovic &# 39 ;, d . et al ., eur . j . immunol . 25 : 3349 - 3355 , 1995 ), had no effect on cll cells ( fig6 c ). we demonstrated superior effective concentration at 50 % effect ( ec 50 ) values in a cell - killing assay for certain forms of anti - hla - dr antibody fragments selected from the hucal library compared to cytotoxic murine anti - hla - dr antibodies ( table 6 ). the ec 50 for anti - hla - dr antibody fragments selected from the hucal library were estimated using the hla - dr positive cell line priess or lg2 ( ecacc , salisbury uk ). 2 × 10 5 cells were incubated for 4 h at 37 ° c . under 6 % co 2 in rpmi 1640 ( paa , germany ) supplemented with 2 . 5 % heat inactivated fbs ( biowhittaker europe , be ), 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 0 . 1 mg / ml kanamycin , together with dilution series of bivalent anti - hla - dr antibody fragments . for the dilution series of fab antibody fragments , an appropriate concentration of fab fragment and anti - flag m2 antibody were premixed to generate bivalent compositions of the anti - hla - dr antibody fragments . the concentrations stated refer to the concentration of bivalent composition such that the igg and fab ec 50 values can be compared . after 4 h incubation with bivalent antibody fragments at 37 ° c . under 6 % co 2 , cell viability was determined by fluorescein diacetate staining and subsequent counting of remaining viable cells ( current protocols in immunology , 1997 ). using standard statistical software , non - linear logistic regression curves were fitted to replica data points and the ec 50 estimated for each antibody fragment . when cross - linked using the anti - flag m2 antibody , the fab fragments ms - gpc - 1 , ms - gpc - 8 & amp ; ms - gpc - 10 selected from the hucal library ( example 4 ) showed an ec 50 of less than 120 nm as expressed in terms of the concentration of the monovalent fragments , which corresponds to a 60 nm ec 50 for the bivalent cross - linked ( fab ) dimer - anti - flag m2 conjugate . ( fig7 a ). when cross - linked using the anti - flag m2 antibody , anti - hla - dr antibody fragments optimised for affinity within the cdr3 region ( example 4 ) showed a further improved ec 50 of less than 50 nm , or 25 nm in terms of the bivalent cross - linked fragment ( fig7 b ), and those additionally optimised for affinity within the cdr1 region showed an ec 50 of less than 30 nm ( 15 nm for bivalent fragment ). in comparison , the ec 50 of the cytotoxic murine anti - hla - dr antibodies 8d1 ( vidovic & amp ; toral ; 1998 ) and l243 ( vidovic et al ; 1995b ) showed an ec 50 of over 30 and 40 nm , respectively , within the same assay ( fig7 c ). surprisingly , the igg form of certain antibody fragments of the invention showed approximately 1 . 5 orders of magnitude improvement in ec 50 compared to the murine antibodies ( fig7 d ). for example , the igg forms of ms - gpc - 8 - 10 - 57 & amp ; ms - gpc - 8 - 27 - 41 showed an ec 50 of 1 . 2 and 1 . 2 nm respectively . furthermore , despite being un - optimised for affinity , the igg form of ms - gpc - 8 showed an ec 50 of less than 10 nm . as has been shown in examples 11 and 12 , the efficiency of killing of un - activated cells ( normal peripheral b and mhh preb cells respectively ) is very low . after treatment with 50 nm of the igg forms of ms - gpc - 8 - 10 - 57 & amp ; ms - gpc - 8 - 27 - 41 , 78 % and 83 % of normal peripheral b cells , respectively , remain viable after 4 hours . furthermore , at only 50 nm concentration or either igg , virtually 100 % viability is seen for mhh preb1 cells . indeed , a decrease in the level of viability to below 50 % cannot be achieved with these unactivated cells using reasonable concentration ranges ( 0 . 1 to 300 nm ) of igg or bivalent cross - linked fab forms of the anti - hla - dr antibody fragments of the invention . therefore , the ec 50 for these un - activated cell types can be estimated to be at least 5 times higher than that shown for the non - optimised fab forms ( ec 50 ˜ 60 nm with respect to cross - linked bivalent fragment ), and at least 10 times and 100 times higher than ec 50 s shown for the vhcdr3 optimised fabs (˜ 25 nm with respect to cross - linked bivalent fragment ) and igg forms of ms - gpc - 8 - 10 - 57 (˜ 1 . 2 nm ) & amp ; ms - gpc - 8 - 27 - 41 (˜ 1 . 2 nm ) respectively . the examples described above show that cell death occurs — needing only certain multivalent anti - hla - dr antibody fragments to cause killing of activated cells . no further cytotoxic entities or immunological mechanisms were needed to cause cell death , therefore demonstrating that cell death is mediated through an innate pre - programmed mechanism of the activated cell . the mechanism of apoptosis is a widely understood process of pre - programmed cell death . we were surprised by certain characteristics of the cell killing we observed that suggested the mechanism of killing for activated cells when exposed to our human anti - hla - dr antibody fragments was not what is commonly understood in the art as “ apoptosis ”. for example , the observed rate of cell killing appeared to be significantly greater than the rate reported for apoptosis of immune cells ( about 10 - 15 hrs ; truman et al ., 1994 ). two experiments were conducted to demonstrate that the mechanism of cell killing proceeded by a non - apoptotic mechanism . first , we used annexin - v - fitc and propidium iodide ( pi ) staining techniques to distinguish between apoptotic and non - apoptotic cell death — cells undergoing apoptosis , “ apoptotic cells ”, ( annexin - v positive / pi negative ) can be distinguished from necrotic (“ dead ”) ( annexin - v positive / pi positive ) and fully functional cells ( annexin - v negative / pi negative ). using the procedures recommended by the manufacturers of the annexinv and pi assays , 1 × 10 6 / ml priess cells were incubated at 37 ° c . under 6 % co 2 with or without 200 nm anti - hla - dr antibody fragment ms - gpc - 8 together with 100 nm of the cross - linking anti - flag m2 mab in rpmi 1640 ( paa , de ) supplemented with 2 . 5 % heat inactivated fcs ( biowhittaker europe , be ), 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 0 . 1 mg / ml kanamycin . to provide an apoptotic cell culture as control , 1 × 10 6 / ml priess cells were induced to enter apoptosis by incubation in the above medium at 37 ° c . under 6 % co 2 with 50 μg / ml of the apoptosis - inducing anti - cd95 mab dx2 ( bd pharniingen , torrey pine , calif ., usa ) cross - linked with 10 μg / ml protein - g . at various incubation times ( 1 , 15 and 60 min ., 3 and 5 hrs ) 200 μl samples were taken , washed twice and stained with annexin - v - fitc ( bd pharmingen , torrey pine , calif ., usa ) and pi using annexin - v binding buffer following the manufacturer &# 39 ; s protocol . the amount of staining with annexin - v - fitc and pi for each group of cells is analysed with a facs calibur ( bd immunocytometry systems , san jose , calif ., usa ). cell death induced through the cross - linked anti - hla - dr antibody fragments shows a significantly different pattern of cell death than that of the anti - cd95 apoptosis inducing antibody or the cell culture incubated with anti - flag m2 mab alone . the percentage of dead cells ( as measured by annexin - v positive / pi positive staining ) for the anti - hla - dr antibody fragment / anti - flag m2 mab treated cells increases far more rapidly than that of the anti - cd95 or the control cells ( fig8 a ). in contrast , the percentage of apoptotic cells ( as measured by annexin - v positive / pi negative staining ) increases more rapidly for the anti - cd95 treated cells compared to the cross - linked anti - hla - dr antibody fragments or the control cells ( fig8 b ). second , we inhibited caspase activity using zdevd - fink , an irreversible caspase - 3 inhibitor , and zvad - fink , a broad spectrum caspase inhibitor ( both obtained from biorad , munich , de ). the mechanism of apoptosis is characterized by activity of caspases , and we hypothesized that if caspases were not necessary for anti hla - dr mediated cell death , we would observe no change in the viability of cells undergoing cell death in the presence of these caspase inhibitors compared to those without . 2 × 10 5 priess cells were preincubated for 3 h at 37 ° c . under 6 % co 2 with serial dilutions of the two caspase inhibitors ranging from 180 μm to 10 mm in rpmi 1640 ( paa , de ) supplemented with 2 . 5 % heat inactivated fcs ( biowhittaker europe , be ), 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 0 . 1 mg / ml kanamycin . hla - dr mediated cell death was induced by adding 200 nm of the human anti - hla - dr antibody fragment ms - gpc - 8 and 100 nm of the cross - linking anti - m2 mab . an anti - cd95 induced apoptotic cell culture served as a control for the activity of inhibitors ( drenou et al ., 1999 ). after further incubation at 37 ° c . and 6 % co 2 , cell viability after 4 and 24 h was determined by trypan blue staining and subsequent counting of non - stained cells . as we expected , cell viability of the anti - hla - dr treated cell culture was not significantly modified by the presence of the caspase inhibitors , while cell death induced through anti - cd95 treatment was significantly decreased for the cell culture pre - incubated with the caspase inhibitors . we therefore concluded that the cell death induced by the human anti - dr mabs does not occur via the classical apoptotic pathway that can be inhibited by zdevd - fin or zvad - fmk . 16 . in vivo therapy for cancer using an hla - dr specific antibody to test the in vivo efficacy , we inoculated immunocompromised ( such as scid , nude or rag - 1 knockout ) scid ( severe combined immunodeficient ) mice subcutaneously ( s . c .) or intraveneously ( i . v .) with the non - hodgkin b cell lymphoma line granta - 519 ( see in table 5 ), and monitored tumor development in mice treated with mab , in comparison to solvent - treated animals . in general , mice are treated i . v . or s . c with the igg form of the anti - hla - dr antibody fragments ms - gpc - 8 , ms - gpc - 8 - 10 - 57 , ms - gpc - 8 - 27 - 41 or others of the invention prepared as described above , using doses of 1 to 25 mg / kg over 5 days . survival of anti - hla - dr treated and control untreated mice is monitored for up to 8 weeks after cessation of treatment . tumor progression in the mice inoculated s . c . is additionally quantified by measuring tumor surface area . for example , eight weeks old female c . b .- 17 scid mice were injected with anti - asialogm1 antibody ( wako chemicals , neuss , germany ; 25 μl diluted 4 fold in pbs , i v .) to suppress natural killer ( nk ) cell activity , on days 0 , 1 , and 2 . on day 1 , 5 × 10 6 granta - 519 cells were injected s . c . into the right flank , or i . v . the endpoint in the s . c . model is a tumor surface area of & gt ; 5 cm 2 , skin ulceration above the tumor , or death , and in the i . v . model hind leg paralysis or death . mice were treated with 1 mg or 0 . 2 mg 1d09c3 mab s . c . or i . v . on days 5 , 7 and 9 . control mice received pbs . mice were monitored , and tumor length and width were measured by a slide - gauge twice a week . significant prolongation of survival of up to 80 % of anti - hla - dr treated mice is observed during the experiment , and up to 50 % mice survive at the end of the experiment . in the s . c . tumor experiment , at day 48 , 100 % of s . c . mab treated mice were alive and 80 % of i . v . mab treated mice were alive ( death is not related to mab treatment or tumor ), while all control mice died within the observation period ( fig1 a ). in s . c . inoculated and untreated mice , the tumor reaches a surface area of 2 - 3 cm 2 , while in anti - hla - dr treated animals the tumor surface area is significantly less . fig1 d shows representative tumor size in mice treated or untreated by mab of the instant invention . tumor growth was also significantly retarded in the treated animals ( fig1 b ). in the i . v . tumor experiment , a significant delay ( about 30 days ) in disease onset was observed in the mab treated groups ( fig1 c ). the 30 day survival rate for i . v . mab treated mice is 100 %, while the survival rate for control mice is 0 %. even at day 40 , the survival rate for i . v . mab treated mice is 50 %/ 20 % ( for high / low doses , respectively ). tumor - induced paralysis is also significantly reduced in the i . v . mab treated mice as compared to the control group mice which are all paralysized by day 40 . these experiments demonstrate that antigen - binding domains of human composition can successfully be used as a therapeutic for the treatment of cancer . the in vitro , ex vivo and in vivo efficacy data presented here are strong evidence that such mabs offer the potential to become useful and potent therapeutic agents for the treatment of different dr + lymphoma and leukemia . 17 . immunosuppression using anti - hla - dr antibody fragments measured by reduction in il - 2 secretion various diseases are caused by or associated with activated t - cells . for example , delayed - type hyper sensitivity ( dth ) is caused by t - cells activated by antigen - presenting cells ( apcs ) via mhc receptors . thus , inhibition of interaction between the mhc class ii molecule and the t - cell receptor ( tcr ) can inhibit certain undesirable immune responses . we were surprised to observe that certain anti - hla - dr antibody fragments of the invention also displayed substantial immunomodulatory properties within an assay measuring il - 2 secretion from immortalized t - cells ( t - cell hybridoma ). igg forms of the antibody fragments ms - gpc - 8 - 6 - 13 / 305d3 , ms - gpc - 8 - 10 - 57 / 1c7277 & amp ; ms - gpc - 8 - 27 - 41 / 1d09c3 showed very strong immunosuppressive properties in this assay with sub - nanomolar ic 50 values and virtually 100 % maximal inhibition ( fig9 a ). particularly surprising was our observation that certain monvalent compositions of the antibody fragments of the invention were able to strongly inhibit il - 2 secretion in the same assay . for example , fab forms of the vh cdr3 - selected and vl cdr3 / vl cdr1 optimised antibody fragments showed low single - digit nm ic 50 &# 39 ; s and also almost 100 % maximal inhibition ( fig9 b ). other monvalent anti - hla - dr antibody fragments of the invention showed significant immunosuppressive properties in the assay compared to control igg and fab fragments ( table 7 ). fig9 c also shows immunomodulatory properties of the mouse 1 - 2 c4 and l243 mab as well as the gpc 1 and 2 ab &# 39 ; s . the immunomodulatory properties of anti - hla - dr antibody fragments was investigated by measuring il - 2 secretion from the hybridoma cell line t - hyb1 stimulated using dr - transgenic antigen presenting cells ( apc ) under conditions of half - maximal antigen stimulation . il - 2 secretion was detected and measured using a standard elisa method provided by the optieia mouse il - 2 kit of pharmingen ( torrey pine , calif ., usa ). apcs were isolated from the spleen of unimmunized chimeric 0401 - ie transgenic mice ( ito et al . 1996 ) according to standard procedures . 1 . 5 × 10 5 apcs were added to 0 . 2 ml wells of 96 - well in rpmi medium containing the following additives ( all from gibco brl and paa ): 10 % fcs , 2 mm l - glutamine , 1 % non - essential amino acids , 1 mm sodium pyruvate and 0 . 1 g / l kanamycin . hen egg ovalbumin was added to a final concentration of 200 μg / ml in a final volume of 100 ul of the above medium , the cells incubated with this antigen for 30 min at 37 ° c . under 6 % co 2 . anti - hla - dr antibody fragments were added to each well at various concentrations ( typically in a range from 0 . 1 to 200 nm ), the plate incubated for 1 h at 37 ° c ./ 6 % co 2 and 2 × 10 5 t - hybl cells added to give a final volume of 200 μl in the above medium . after incubation for 24 h , 100 μl of supernatant was transferred to an elisa plate ( nunc - immuno plate maxisorp surface , nunc , roskilde , dk ) previously coated with il - 2 capture antibody ( bd pharmingen , torrey pine , calif ., usa ), the amount of il - 2 was quantified according to the manufacturer &# 39 ; s directions using the optieia mouse il - 2 kit and the plate read using a victor v reader ( wallac , finland ). secreted il - 2 in pg / ml was calibrated using the il - 2 standards provided in the kit . the t - cell hybridoma line t - hybl was established by fusion of a t - cell receptor negative variant of the thymoma line bw 5147 ( atcc ) and lymph node cells from chimeric 0401 - ie transgenic mice previously immunized with hen egg ovalbumin ( ito et al . 1996 ). the clone t - hyb1 was selected for the assay since it responded to antigen specific stimulation with high il - 2 secretion . 18 . immunosuppression using an hla - dr specific antibody measured by t cell proliferation immunomodulatory properties of the anti - hla - dr antibody fragments were also seen within an assay that measures t cell proliferation . the ic 50 value for inhibition of t cell proliferation of the igg form of ms - gpc - 8 - 10 - 57 / 1c7277 and ms - gpc - 8 - 27 - 41 / 1d09c3 were 11 and 20 nm respectively ( fig1 ). the anti - hla - dr antibody fragments were tested as follows to inhibit the proliferative t cell response of antigen - primed lymph node cells from mice carrying a chimeric mouse - human class ii transgene with an ra - associated peptide binding site , and lack murine class ii molecules ( muller et al ., 1990 ; woods et al ., 1994 ; current protocols in immunology , vol . 2 , 7 . 21 ; ito et al ., 1996 ). here , the immunization takes place in vivo , but the inhibition and readout are ex vivo . transgenic mice expressing mhc class ii molecules with binding sites of the ra associated molecule , drb * 0401 were commercially obtained . these mice lack murine mhc class ii , and thus , all th responses are channelled through a single human ra - associated mhc class ii molecule ( ito et al ., 1996 ). these transgenic mice represent a model for testing human class ii antagonists . the inhibitory effect of the anti - hla - dr antibody fragments and their igg forms were tested on t - cell proliferation measured using chimeric t - cells and antigen presenting cells isolated from the lymph nodes of chimeric 0401 - i e transgenic mice ( taconic , usa ) previously immunized with hen egg ovalbumin ( ito et al ., 1996 ) according to standard procedures . 1 . 5 × 10 5 cells are incubated in 0 . 2 ml wells of 96 - well tissue culture plates in the presence of ovalbumin ( 30 μg per well - half - maximal stimulatory concentration ) and a dilution series of the anti - hla - dr antibody fragment or igg form under test ( 0 . 1 nm - 200 nm ) in serum free hl - 1 medium containing 2 mm l - glutamine and 0 . 1 g / l kanamycin for three days . antigen specific proliferation is measured by 3 h - methyl - thymidin ( 1 μci / well ) incorporation during the last 16 hrs of culture ( falcioni et al ., 1999 ). cells are harvested , and 3 h incorporation measured using a scintillation counter ( topcount , wallac finland ). inhibition of t - cell proliferation on treatment with the anti - hla - dr antibody fragment and its igg form was observed by comparison to control wells containing antigen . fig9 d showed that the proliferation of the t - cell line ng - tcl ha - 10 was significantly inhibited by the two gpc antibodies ( ms - gpc - 8 - 10 - 57 / 1c7277 and ms - gpc - 8 - 27 - 41 / 1d09c3 ), at least to the same extent of the mouse 1 - 1 c4 positive control ab . [ 0352 ] fig9 e and 9 f showed that transgenic t - cell proliferation as measured by 3 h incorporation in two experiments were significantly inhibited by mab treatments , including ms - gpc - 8 - 10 - 57 / 1c7277 and ms - gpc - 8 - 27 - 41 / 1d09c3 human mab &# 39 ; s and mouse l243 , 11c4 and lb3 . 1 ab &# 39 ; s . in these experiments , t - cells are sensitized in vivo by specific antigens ( ovalbumin ( ova ) in one case , hen egg lysozyme ( hel ) in another case ), followed by re - stimulation ex vivo by these two antigens respectively for measuring immune stimulation in the form of antigen specific induction of t - cell proliferation . fig9 e and 9 f showed that more than 90 % inhibition of antigen specific induction of t - cell proliferation is achieved using the human mab &# 39 ; s of the instant invention . in order to select the most appropriate protein / peptide to enter further experiments and to assess its suitability for use in a therapeutic composition for the treatment of cancers , additional data are collected . such data for each igg form of the anti - hla antigen antibody fragments can include the binding affinity , in vitro killing efficiency as measured by ec 50 and cytotoxicity across a panel of tumor cell lines , the maximal percentage cell killing as estimated in vitro , and tumor reduction data and mouse survival data from in vivo animal models . the igg form of the anti - hla antigen antibody fragments that shows the highest affinity , the lowest ec 50 for killing , the highest maximal percentage cell killing and broadest across various tumor cell lines , the best tumor reduction data and / or the best mouse - survival data may be chosen to enter further experiments . such experiments may include , for example , therapeutic profiling and toxicology in animals and phase i clinical trials in humans . 20 . in vivo efficacy of immunosuppression using an hla - dr specific antibody in treating delayed - type - hypersensitivity ( dth ) in order to determine the in vivo efficacy of the immunosuppression activity of the mab &# 39 ; s of the instant invention , we conducted experiments using a mouse model for delayed - type - hypersensitivity ( dth ). in this system , mouse ear - swelling in response to treatments by haptens such as oxazalone ( oxa ) or dinitroflurobenzene ( dnfb ) were measured to determine the in vivo efficacy of the mab &# 39 ; s of the instant invention . specifically , 0 . 05 ml of 2 % oxa or dnfb were applied to the bellies of treatment group mice on day 1 and 2 . on day 5 , different doses of test mab &# 39 ; s 1d09c3 or control treatments were administered i . v . after waiting for 4 or 8 hours , mice were challenged with 0 . 02 ml of 0 . 5 % oxa or dnfb . ear thickness was measured on day 6 , 8 , 9 and 12 , and the results were presented in fig9 g , 9 h and 9 i . in fig9 g , dth to oxa as measured by ear - thickness was blocked by roughly 75 % if 1 mg or 0 . 75 mg of mab was administered i . v ., while 0 . 5 mg of mab or less has no significant effect . in fig9 h , the time course of inhibition , by human anti - dr mab , of dth to dnfb in dr - tg mice as measured by ear - thickness was presented . dth was almost completely blocked ( p & lt ; 0 . 005 ) at 7 th hour after treatment with the mab 1d09c3 , followed by a 60 % block ( p & lt ; 0 . 01 ) at 18th hr and no effect at 4 hr . fig9 i showed a positive correlation between the dose of mab ( 1d09c3 ) used at the 7 th hour and the effectiveness of the inhibition of dth in dr - tg mice . both 1 mg and 0 . 5 mg of 1d09c3 significantly ( p & lt ; 0 . 005 ) inhibited dth while lower doses have no effect . these experiments demonstrates that mab &# 39 ; s of the instant invention is capable of specifically inhibiting the very part of the immune system responsible for the unwanted immune reaction . it is an inhibition of immune reaction rather than suppression of existing immune reactios . since the mab &# 39 ; s of the instant invention are fully human antibodies , rather than murine mab or humanized murine antibodies , they are expected to have very low immunogenicity in the host and a much longer half life . in addition , most mab &# 39 ; s of the instant invention also have very high affinity in the pico molar range . these mab &# 39 ; s shall prove to be useful for a variety of immune diseases such as dth and graft v . host disease ( gvhd ). 21 . selection of useful polypeptide for the treatment of diseases of the immune system in order to select the most appropriate protein / peptide to enter further experiments and to assess its suitability for use in a therapeutic composition for the treatment of diseases of the immune system , additional data are collected . such data for each monovalent antibody fragment or igg form of the anti - hla antigen antibody fragments can include the affinity , reactivity , specificity , ic 50 - values , for inhibition of il - 2 secretion and of t - cell proliferation , or in vitro killing efficiency as measured by ec 50 and the maximal percentage cell killing as estimated in vitro , and dr - transgenic models of transplant rejection and graft vs . host disease . the antibody fragment or igg form of the anti - hla antigen antibody fragments that shows the lowest ec 50 , highest affinity , highest killing , best specificity and / or greatest inhibition of t - cell proliferation or il - 2 secretion , and high efficacy in inhibiting transplant rejection and / or graft vs . host disease in appropriate models , might be chosen to enter further experiments . such experiments may include , for example , therapeutic profiling and toxicology in animals and phase i clinical trials in humans . [ 0366 ] table 3a affinities of selected igg 4 monoclonal antibodies constructed from f ab &# 39 ; s . errors represent standard deviations binder ( igg 4 ) k on [ m − 1 s − 1 ] × 10 5 k off [ s − 1 ] × 10 − 5 k d [ nm ] ms - gpc - 8 - 27 - 41 1 . 1 ± 0 . 2 3 . 1 ± 0 . 4 0 . 31 ± 0 . 06 ms - gpc - 8 - 6 - 13 0 . 7 ± 0 . 1 3 . 0 ± 1 . 0 0 . 50 ± 0 . 20 ms - gpc - 8 - 10 - 57 0 . 7 ± 0 . 2 4 . 0 ± 1 . 0 0 . 60 ± 0 . 20 [ 0367 ] table 3b affinities of binders obtained out of affinity maturation of cdr1 light chain optimisation following cdr3 heavy chain optimisation . errors represent standard deviations binder ( f ab ) k on [ m − 1 s − 1 ] × 10 5 k off [ s − 1 ] × 10 − 3 k d [ nm ] ms - gpc - 8 - 6 - 2 1 . 20 ± 0 . 10 0 . 94 ± 0 . 07 7 . 6 ± 0 . 3 ms - gpc - 8 - 6 - 19 1 . 10 ± 0 . 10 1 . 00 ± 0 . 20 9 . 0 ± 1 . 0 ms - gpc - 8 - 6 - 27 1 . 80 ± 0 . 20 1 . 10 ± 0 . 20 6 . 3 ± 0 . 6 ms - gpc - 8 - 6 - 45 1 . 20 ± 0 . 07 1 . 03 ± 0 . 04 8 . 6 ± 0 . 6 ms - gpc - 8 - 6 - 13 1 . 90 ± 0 . 30 0 . 55 ± 0 . 05 3 . 0 ± 0 . 5 ms - gpc - 8 - 6 - 47 2 . 00 ± 0 . 30 0 . 62 ± 0 . 04 3 . 2 ± 0 . 3 ms - gpc - 8 - 10 - 57 1 . 70 ± 0 . 20 0 . 44 ± 0 . 06 2 . 7 ± 0 . 3 ms - gpc - 8 - 27 - 7 1 . 70 ± 0 . 20 0 . 57 ± 0 . 07 3 . 3 ± 0 . 3 ms - gpc - 8 - 27 - 10 1 . 80 ± 0 . 20 0 . 53 ± 0 . 05 3 . 0 ± 0 . 2 ms - gpc - 8 - 27 - 41 1 . 70 ± 0 . 20 0 . 49 ± 0 . 03 2 . 9 ± 0 . 3 [ 0368 ] table 3c binders obtained out of affinity maturation of gpc8 by cdr3 light chain optimisation binder ( f ab ) k on [ m − 1 s − 1 ] × 10 5 k off [ s − 1 ] × 10 − 3 k d [ nm ] ms - gpc 8 - 18 1 . 06 8 . 30 78 . 3 ms - gpc 8 - 9 1 . 85 16 . 60 90 . 1 ms - gpc 8 - 1 1 . 93 20 . 90 108 . 0 ms - gpc 8 - 17 1 . 00 5 . 48 54 . 7 ms - gpc - 8 - 6 a ) 1 . 20 ± 0 . 10 5 . 50 ± 0 . 70 8 . 0 ± 12 . 0 [ 0369 ] table 3d binders obtained out of hucal in scfv form and their converted fabs scf v f ab k on [ m − 1 s − 1 ] × k off [ s − 1 ] × k on [ m − 1 s − 1 ] × k off [ s − 1 ] × binder 10 5 10 − 3 k d [ nm ] 10 5 10 − 3 k d [ nm ] ms - gpc 1 0 . 413 61 1500 0 . 639 53 820 ms - gpc 6 0 . 435 200 4600 0 . 135 114 8470 ( 1 curve ) ms - gpc 8 0 . 114 76 560 0 . 99 29 . 0 346 a ) +/− 0 . 40 b ) +/− 8 . 4 +/− 141 ms - gpc 10 0 . 187 180 9625 0 . 22 63 2860 [ 0370 ] table 3e affinity improvements achieved by antibody optimization mab format optimization k on [ s − 1 m − 1 ] × 10 5 k off [ s − 1 ] × 10 − 3 k d [ nm ] a b8 fab parental 0 . 99 ± 0 . 4 b 29 . 0 ± 8 . 4 346 . 1 ± 140 . 5 7ba fab l - cdr3 0 . 96 ± 0 . 14 5 . 48 ± 0 . 73 58 . 6 ± 11 . 7 305d3 fab l - cdr3 + 1 1 . 90 ± 0 . 26 0 . 55 ± 0 . 05 2 . 96 ± 0 . 46 1c7277 fab l - cdr3 + 1 1 . 65 ± 0 . 21 0 . 44 ± 0 . 06 2 . 67 ± 0 . 25 1d09c3 fab l - cdr3 + 1 1 . 67 ± 0 . 16 0 . 49 ± 0 . 03 2 . 93 ± 0 . 27 305d3 igg 4 l - cdr3 + 1 0 . 71 ± 1 . 6 0 . 33 ± 1 . 0 0 . 5 ± 0 . 20 1c7277 igg 4 l - cdr3 + 1 0 . 11 ± 2 . 0 0 . 31 ± 0 . 4 0 . 3 ± 0 . 06 1d09c3 igg 4 l - cdr3 + 1 0 . 71 ± 1 . 2 0 . 41 ± 1 . 1 0 . 6 ± 0 . 20 [ 0371 ] table 4 killing efficiency after 4 hour incubation of cells with cross - linked anti - hla - dr antibody fragments , and maximum killing after 24 hour incubation cross - linked killing efficiency against maximum killing against fab fragment granta priess ms - gpc - 1 + + ms - gpc - 6 + + ms - gpc - 8 + + ms - gpc - 10 + + ms - gpc - 8 - 6 ++ ++ ms - gpc - 8 - 17 ++ ++ ms - gpc - 8 - 6 - 13 +++ +++ ms - gpc - 8 - 10 - 57 +++ +++ ms - gpc - 8 - 27 - 41 +++ +++ [ 0372 ] table 5 killing efficiency of anti - hla - dr igg antibodies of human composition compared to murine anti - hla - dr antibodies against a panel of lymphoid tumor cell lines . hla - dr expression a % killing by mabs cell lines mfl murine mabs human mabs name dr type tumor type l243 l243 8d1 b8 1d09c3 1c7277 305d3 lg - 2 1 , 1 b - lymphoblastoid 458 79 85 86 87 88 82 priess 4 , 4 b - lymphoblastoid 621 87 83 85 88 93 74 arh - 77 12 b - lymphoblastoid 301 88 73 84 85 88 87 granta - 519 2 , 11 b cell non - hodgkin 1465 83 56 76 78 78 73 karpas - 422 2 , 4 b cell non - hodgkin 211 25 32 51 66 68 71 karpas - 299 1 , 2 t cell non - hodgkin 798 78 25 81 82 79 76 dohh - 2 1 , 2 b cell lymphoma 444 29 23 58 59 60 53 sr - 786 1 , 2 t cell lymphoma 142 3 8 1 53 44 26 mhh - call - 4 1 , 2 b - all 348 35 41 43 63 46 43 mn - 60 10 , 13 b - all 1120 46 22 71 69 66 67 bjab 12 , 13 burkitt lymph . 338 53 59 49 71 67 64 raji 10 , 17 burkitt lymph . 617 69 64 81 84 86 83 l - 428 12 hodgkin &# 39 ; s lymph . 244 82 81 82 91 91 92 hdlm - 2 hodgkin &# 39 ; s lymph . 326 77 73 89 88 84 90 hd - my - z hodgkin &# 39 ; s lymph . 79 35 39 49 69 57 72 km - h2 hodgkin &# 39 ; s lymph . 619 81 56 75 86 88 87 l1236 hodgkin &# 39 ; s lymph . 41 52 62 44 63 66 66 bonna - 12 hairy cell leuk . 2431 92 91 91 92 91 86 hc - 1 hairy cell leuk . 372 88 89 89 93 86 93 nalm - 1 1 , 4 cml 1078 44 4 83 82 78 65 l - 363 plasma cell leu . 49 6 5 26 26 24 19 eol - 1 aml ( eosinophil ) 536 22 13 36 69 49 53 lp - 1 multiple myeloma 315 12 0 61 73 70 73 rpmi - 8226 multiple myeloma 19 6 0 14 29 26 19 mhh - preb - 1 b cell non - hodgkin 175 3 3 2 4 8 11 mhh - call - 2 b cell precursor leu . + 5 5 opm - 2 multiple myeloma 3 13 0 8 1 4 5 kasumi - 1 aml 5 0 0 8 10 10 6 hl - 60 aml 3 18 0 3 15 9 22 lama - 84 cml 7 7 9 5 11 5 7 [ 0373 ] table 6 ec 50 values for certain anti - hla - dr antibody fragments of the invention in a cell - killing assay against lymphoid tumor cells . all ec 50 refer to nanomolar concentrations of the bivalent agent ( igg or cross - linked fab ) such that values for cross - linked fab and igg forms can be compared . antibody ec 50 of cell killing ( nm ) +/− fragment form cell line tested se for bivalent agent ms - gpc - 1 fab priess 54 ± 14 ms - gpc - 8 fab priess 31 ± 9 ms - gpc - 10 fab priess 33 ± 5 ms - gpc - 8 - 17 fab priess 16 ± 4 ms - gpc - 8 - 6 - 2 fab priess 8 ± 2 ms - gpc - 8 - 10 - 57 fab lg2 7 . 2 ms - gpc - 8 - 27 - 41 fab lg2 7 . 2 ms - gpc - 8 - 27 - 41 fab priess 7 . 7 ms - gpc - 8 igg 4 priess 8 . 3 ms - gpc - 8 - 27 - 41 igg 4 priess 1 . 1 ± 0 . 1 ms - gpc - 8 - 10 - 57 igg 4 priess 1 . 1 ± 0 . 2 ms - gpc - 8 - 27 - 41 igg 4 lg2 1 . 23 ± 0 . 2 ms - gpc - 8 - 10 - 57 igg 4 lg2 1 . 0 ± 0 . 1 8d1 migg priess 33 l243 migg priess 47 [ 0374 ] table 7 ic 50 values for certain anti - hla - dr antibody fragments of the invention in an assay to determine il - 2 secretion after antigen - specific stimulation of t - hyb 1 cells . ic 50 for the igg forms ( bivalent ) are represented as molar concentrations , while in order to provide easy comparison , ic 50 s for the fab forms ( monovalent ) are expressed in terms of half the concentration of the fab to enable direct comparison to igg forms . ic 50 ( igg / nm ) anti - hla - dr (( fab )/ 2 / nm ) maximum antibody fragment form mean se inhibition (%) ms - gpc - 8 - 10 - 57 igg 0 . 31 0 . 01 100 ms - gpc - 8 - 27 - 41 igg 0 . 28 0 . 07 100 ms - gpc - 8 - 6 - 13 igg 0 . 42 0 . 06 100 ms - gpc - 8 - 6 - 2 igg 3 . 6 1 . 1 100 ms - gpc - 8 - 6 igg 6 . 7 2 . 0 100 ms - gpc - 8 igg 11 . 0 0 . 8 100 ms - gpc - 8 - 6 - 2 fab 4 . 7 1 . 9 100 ms - gpc - 8 - 6 - 13 fab 2 . 1 0 . 8 100 ms - gpc - 8 - 6 - 19 fab 5 . 3 0 . 2 100 ms - gpc - 8 - 10 - 57 fab 2 . 9 1 . 0 100 ms - gpc - 8 - 6 - 27 fab 3 . 0 1 . 2 100 ms - gpc - 8 - 6 - 47 fab 2 . 6 0 . 6 100 ms - gpc - 8 - 27 - 7 fab 5 . 9 2 . 2 100 ms - gpc - 8 - 27 - 10 fab 7 . 3 1 . 9 100 ms - gpc - 8 - 27 - 41 fab 3 . 6 0 . 7 100 ms - gpc - 8 - 6 fab 20 100 ms - gpc - 8 fab 110 100 [ 0375 ] table 8 antibody name conversion table ms - gpc - 8 b8 ms - gpc - 8 - 17 7ba ms - gpc - 8 - 6 - 13 305d3 ms - gpc - 8 - 10 - 57 1c7277 ms - gpc - 8 - 27 - 41 1d09c3 ms - gpc - 1 17 ms - gpc - 6 8a ms - gpc - 10 e6 the following is a partial list of references cited in the instant application . the contects of these references are hereby incorporated herein by reference . adorini l , mueller s , cardinaux f , lehmann p v , falcioni f , nagy z a , ( 1988 ), nature 334 : 623 . ausubel , f . m ., brent , r ., kingston , r . e ., moore , d . d ., seidman , j . g ., smith , j . a . and struhl , k . 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