Patent Application: US-7702899-A

Abstract:
a chimeric protein comprising a pseudomonas aeruginosa exotoxin moiety linked to a myelin basic protein moiety is disclosed . the mbp moiety is selected from the group comprising : mbp ; amino acids 69 - 88 of guinea - pig myelin basic protein or an antigenic portion thereof ; amino acids 84 - 102 of human myelin basic protein or an antigenic portion thereof ; amino acids 143 - 168 of human myelin basic protein or an antigenic portion thereof ; and an amino acid sequence in which one or more amino acids have been deleted , added , substituted or mutated in the amino acid sequences of , , or , the modified sequence of retaining at least 75 % homology with the amino acid sequences of , , or , respectively . each of the mbp moieties of , and are linked to the pe moiety by a pentapeptide linker repeated 1 - 3 times . the chimeric protein is useful in treating autoimmune diseases , and especially multiple sclerosis .

Description:
five peptide - toxin chimeric proteins were constructed . three contained the bpp sequence coding for 19 amino acids ( amino acids 69 - 88 ) of guinea pig - myelin basic protein ( 29a ). three oligonucleotides each containing this guinea pig sequence were synthesized ( oligos 1 , 1 ′ and 2 , table 1 ) and were each fused to the 5 ′ end of a dna fragment encoding a truncated form of pe ( pe40 ) or a mutated full length pe sequence ( pe66 4glu ). the bpp encoding sequences were preceded by a sequence encoding 2 amino acids met - val , in the cases of bpp1 - pe66 4glu and bpp1 ′- pe40 , and by a sequence encoding met - ala in the case of bpp2 - pe66 4glu . the bpp / mbp encoding sequences were fused either directly to the mutated pe sequence ( chimeric protein bpp2 - pe66 4glu ) or via a linker sequence of 5 amino acids ( gly - gly - gly - gly - ser ) that was inserted between the bpp sequence and pe66 4glu ( table 1 , oligo 1 chimeric protein bpp1 - pe66 4glu ) or pe40 ( table 1 oligo 1 ′, chimeric protein bpp1 ′- pe40 ). in the fourth plasmid , pis - 3 , a rat mbp sequence ( 30 ) was directly ligated to the pe66 4glu sequence ( fig1 ) whereas in the fifth psm - 8466 the sequence encoding for amino acids 84 - 102 of human mbp was ligated directly to the pe66 4glu sequence . 1 . bpp1 - pe66 4glu ( pis - 1 )— 69 - 88 guinea pig mbp & amp ; mutated pe , with linker 2 . bpp1 ′- pe40 ( phl15 )— 69 - 88 guinea pig mbp & amp ; truncated pe , with linker 3 . bpp2 - pe66 4glu ( pis - 2 )— 69 - 88 guinea pig mbp & amp ; mutated pe , no linker 4 . mbp - pe66 4glu -( pis 3 )— rat mbp & amp ; mutated pe , no linker 5 . bpp84 - pe66 4glu ( psm - 8466 )— 84 - 102 human mbp & amp ; mutated pe , no linker . plasmids for the expression of the chimeric proteins under the bacteriophage t7 promoter were constructed as shown in fig1 and 2 . plasmids pls - 1 , pls - 2 and psm - 8466 encoding bpp1 - pe66 4glu , bpp2 - pe66 4glu and bbp84pe66 4glu , respectively , were constructed from phl823 ( 28 ) cut with ndel and hindlll . the largest vector fragment ( 4 . 4 kb ) was ligated to either of three different synthetic oligonucleotides ( oligo 1 , 2 or 3 , table i and fig1 ). the resulting plasmids were examined for size and for the expression of the encoded chimeric proteins in bl 21 ( λde3 ) cells , see section g . plasmid phl15 was constructed from phl906 ( 29b ) cut with ndel . the largest vector fragment ( 4 kb ) was ligated to a synthetic oligonucleotide ( oligo 1 ′, table 1 and fig2 ). plasmid phl15 was examined as described above . plasmid pls - 3 , was constructed by fusing a 400 bp - pcr fragment corresponding to the whole rat mbp , to the larger fragment ( 4 . 4 kb ) of phl823 cut with ndel and hind lll ( fig1 ). the pcr fragment was synthesized using , is1 and is2 primers ( table 1 ) and plasmid pmbp - 1 , kindly provided by l . hood , california inst . of tech , usa . the pcr product was cut with ndel and hind lll and ligated to phl823 cut with the same enzymes . plasmid pmbp - 1 contains the full size cdna for rat mbp . the sequences of the chimeric genes constructed were confirmed by dna sequence analysis ( data not shown ). the plasmids encoding the fusion genes were transformed into e . coli strain bl - 21 de3 using the heat shock protocol ( 32 ). the transformed e . coli colonies were grown and selected from ampicillin ( 100 ( g / ml ) agar plates . selected colonies were further grown in slb medium containing ampicillin ( 50 ( g / ml ) until od650 reached 0 . 5 . induction was then performed for 90 minutes using iso - propyl -(- d - galactoside ( iptg ) ( 1 mm final concentration ). a pellet of expressed cells was suspended in te buffer ( 50 mm tris ph 8 . 0 , 1 mm edta ) sonicated ( six 30 - sec bursts , at 100 w ) and centrifuged at 10 , 000 × g for 15 min . the supernatant ( soluble fraction ) was removed and kept for analysis . the pellet was denatured in 4 vol ( v / w ) of extraction buffer ( 7m gu - hci , 0 . 1m tris ph 8 . 0 , 1 mm edta , 1 mm dtt ) and sonicated ( six 30 - sec bursts 100 w ). the suspension was stirred for 1 hr at 4 ° c . the suspension was cleared by centrifugation at 12 , 000 g for 15 min and the pellet discarded . the clear supernatant was then diluted 4 - fold with phosphate buffer saline ( pbs ) and dialyzed against pbs . the dialyzed material was centrifuged at 12 , 000 g for 10 min . the resulting supernatant ( insoluble fraction , guanidine hydrochloride treated ) was used as a source of the chimeric proteins for most experiments . the new expressed chimeric proteins were characterized by sds polyacrylamide gel electrophoresis ( page ). these proteins migrated with an apparent molecular mass of approximately 42 . 9 kd and 68 . 3 - 68 . 9 kd , corresponding to the expected molecular mass of bpp1 ′- pe40 , bpp1 - pe66 4glu / bpp2 - pe66 4glu / bpp84 - pe66 4glu , respectively ( fig3 a ). the molecular weight of mbp - pe66 4glu is 80 kd . immunoblots showed that the chimeric proteins react with the polyclonal anti - pe antiserum ( fig3 b ). extracts from the cells displayed adp - ribosylation activity ( 31 ) as shown in table ii below . to identify the subcellular localization of the expressed chimeric proteins subcellular fractionation was performed . each subfraction was tested for its adp ribosylation activity using wheat germ extracts enriched in elongation factor 2 . according to the enzymatic activity the three different chimeric proteins ( bpp1 - pe66 4glu , bpp2 - pe66 4glu and bpp1 ′- pe40 ) are expressed and distributed similarly in the different subfractions . about 60 - 70 % of the total activity was found associated within the soluble fraction and only 30 - 40 % within the insoluble fraction . however , the insoluble fraction was found to be the most enriched one for the proteins , showing 7 to 16 - fold higher values ( specific activity ) than those calculated for the soluble fraction ( table ii ). thus bpp - pe chimeric proteins were purified from the insoluble fraction ( see below ). it should be noted that only basal levels of bpp - pe proteins were found in the periplasm fraction or in the incubation media . the cytotoxic activity of the different bpp - pe chimeric proteins was tested on mbp - specific encephalitogenic t cell lines . the cell lines (( mbp — specific t cell lines : z1a , d9 , pas , k1a and control cell lines : a2b . ppd , available from the inventors ) were maintained in culture by alternating specific antigen stimulation for 3 days and propagation phases for another 5 - 7 days . stimulation of mbp specific encephalitogenic mbp - t line cells ( 3 × 105 cells / ml ) was done in the presence of 15 μg / ml guinea - pig mbp for two to three days with 15 × 10 6 cells / ml syngeneic irradiated thymocytes as antigen presenting cells ( apc ) in rpmi - 1640 supplemented with 1 % syngeneic rat serum , 2 mm - l glutamine , 1 mm sodium pyruvate , 5 × 10 − 5 m β - mercaptoethanol , 100 μg / ml penicillin and 100 μg / ml streptomycin . the activated lymphoblasts were separated from the thymocytes and debris by centrifugation over a ficoll paque gradient ( pharmacia ). the blasts are found in the interface . blasts were then expanded in the same medium minus antigen and rat serum , enriched with 10 % heat inactivated fetal calf serum ( fcs ) and 10 % cona activated spleenocyte supernatant ( conditioned medium which contains tgcf ) or 25 units / ml of human rll2 . assays were performed using the insoluble fraction of the different chimeric proteins , diluted in pbs containing 0 . 2 % bovine serum albumin ( bsa ). they were then added at various concentrations to the ( mbp - t cells in a 96 - well plate ( 2 - 4 × 10 4 cells / well ) in triplicate at the third day ( d3 ) of the propagation phase . the cells were incubated in 5 % co 2 , in air at 37 ° c . for 18 - 24 hr , with the addition of 2 μci of [ 3h ]- leucine or 1 μci of [ 3h ]- thymidine for an additional 12 - 16 hr . cells were then harvested and incorporated radioactivity was measured . when assays were performed on hut102 control cells , the radioactive material was added for only 4 hr . before harvesting the cells . percentage of protein synthesis inhibition was calculated from control cells not exposed to any chimeric proteins . as shown in fig4 - 6 specific αmbp t cell lines ( d9 , z1a and k1a respectively ) were sensitive to the cytotoxic activity of these bpp - pe chimeric proteins demonstrating a 60 - 70 % inhibition of protein synthesis at the highest concentration tested . protein synthesis inhibition was concentration dependent . chimeric protein bpp1 - pe66 4glu was not cytotoxic to d9 target cells in contrast to the other two chimeras ( bpp2 - pe66 4glu and bpp1 ′- pe40 ) ( fig4 ). when the cytotoxic effect of bpp - pe chimeric proteins was tested on dna synthesis , similar results were observed ( results not shown ) demonstrating that the chimeric proteins irreversibly inhibit protein synthesis thus causing cell death . as the insoluble fraction of expressed cells was used in most of these experiments , we used the insoluble fraction of cells expressing only the toxin moiety of the chimeric protein , ( pe40 ), as a control to test for non - specific activity associated with the insoluble fraction preparations . the insoluble fraction of pe40 had no cytotoxic effect on target ( fig4 ) or non target cells . the insoluble ( ins .) fraction of mbp - pe66 4glu was cytotoxic to z85 cells . at the highest concentration tested ( 2 , 000 ng ) an inhibition of 58 % of protein synthesis was observed ( fig7 ). de novo expression of interleukin 2 receptor molecules ( il2r ) appears during the activation process of a t cell line induced by an antigen , thus we tested the cytotoxic activity of il2 - pe chimeric proteins on the different antigen specific t cell lines as positive controls . highly purified il2 - pe66 4glu caused 40 - 75 % inhibition of protein synthesis in the different αmbp t cell lines tested ( fig4 - 6 ). after demonstrating that bpp - pe chimeric proteins had a cytotoxic effect on αmbp t cell lines , we assessed the specificity of the response to the chimeric proteins . as shown in fig4 the cytotoxic effect of bpp - pe chimeric proteins on the αmbp t cell line d9 was completely blocked by excess of polyclonal αpe , demonstrating the specificity of their action . the effect of bpp - pe proteins was also tested on the non target cell lines a2b or ppd . the a2b cell line which is specific to mycobacterial antigen , cross reactive with rat cartilage and it is involved in mediating adjuvant arthritis . the ppd cell line is specific to purified protein derivative of mycobacterium tuberculosis . both t cells were maintained in culture in the same conditions as that of αmbp t cells . as shown in fig8 and 9 , a2b or ppd control cell lines were unaffected by the action of the bpp - pe chimeric proteins , showing that the effect is a highly specific response . the activity of bpp - pe chimeric proteins was also tested on other non target cells - hut 102 cells , a human htlv - i t cell leukemia cell line which expresses the high and the low affinity forms of il2r . this cells were maintained in rpmi 1640 supplemented with 10 % fcs . hut 102 was a gift of t . a . waldmann ( national cancer institutes ). no cytotoxic effect was observed by the bpp - pe chimeric . as another approach to confirm internalization of bpp - pe chimeric proteins into target cells we followed the existence of the proteins following incubation , within the cells , by immunoblot analysis . internalization of chimeric proteins into target cells was assayed by incubating chimeric proteins bpp2 - pe66 4glu and bpp1 ′- pe40 ( 5 μg , total protein concentration of insoluble fraction , guanidine hydrochloride - treated ), with αmbp t cells ( pas cells ) or with a2b non target cells for 5 hr . at the end of the incubation , cells were washed four times with hanks balanced sodium saline ( hbss ) buffer , saving samples from each washing step . cells were then lysed with lysis buffer ( 20 mm tris - hci ph 7 . 4 , 150 mm nacl , 1 mm edta , 1 % np40 , 1 % deoxycholic acid , 0 . 1 % sds , 1 mm pmsf ) and incubated for 15 min . at rt . the cell lysate was centrifuged at 9000 g for 10 min . and the pellet ( dissolved in tris - hci ph 7 . 4 , 1 mm edta , te buffer ) and supernatant fractions of the lysed cells were kept for analysis . samples of the growing media , washing steps , and the lysed cells were applied for sds page analysis . electrophoresed samples were transferred from gels to nitrocellulose and immunoblotted using αpe antiserum . when internalization was assayed on a2b cells , immunobloting of the samples was performed by slot - blot analysis . after standard assay conditions , bpp2 - pe66 4glu and bpp1 ′- pe40 could be detected within lysates of target cells ( fig1 ). these chimeric proteins are cell associated as samples from the last washing step ( before lysing the cells , wash iv , fig1 ) didn &# 39 ; t reveal any immunoreactive material . no internalized chimeric proteins could be detected within the a2b non target cells under identical assay conditions ( fig1 ). as expected , chimeric proteins could be detected in this case only in the growing medium and in the first washing sample . mbp / bpp - pe chimeric proteins are expressed in prokaryotic systems . the expression in e . coli , as observed with many foreign eukaryotic proteins , results in the formation of insoluble inclusion bodies , which contain the recombinant protein in a non - native and non - active conformation . further ways for obtaining the fusion proteins differ and depend on nature of its moieties . the chimeric protein was expressed in e . coli strain bl21 ( λde3 ) cells . bacterial cells were grown in slb / amp ( 50 ( g / ml ) ( slb in il - 5 g nacl , 16 g tryptophan , 10 g yeast extract ) medium at 37 ° c ., until the od 600 reached 1 . 5 - 2 . 0 . induction with iptg ( 1 mm , 90 min . at 37 ° c .) resulted in the expression of the chimeric proteins . cultures were centrifuged at 2700 rpm for 15 min . pellets of cells were frozen at − 70 ° c . the frozen e . coli cells were thawed and suspended in lysis buffer ( 50 mm tris - hci ph 8 . 0 , 1 mm edta , 0 . 2 mg / ml lysozyme ) ( 10 ml / g cells ). the cell suspension was pulse sonicated 3 × 30 seconds . the disrupted cells were centrifuged at 35 , 000 × g for 30 min . at 4 ° c . to obtain the insoluble fraction , i . e . the inclusion bodies . the inclusion bodies were suspended in denaturing buffer ( 6m guhcl , 100 mm tris - hci ph 8 . 6 , 1 mm edta , 10 mm dtt , 50 mm nal ) ( 1 ml / g cells ). the mixture was collected by centrifugation ( 35 , 000 g for 15 min .). supernatant was diluted 100 - fold with refolding buffer ( 100 mm tris - hci , ph 8 . 0 , 1 mm edta , 0 . 25m arginine , 5 mm dtt , 0 . 25m naci ) and stirred for 48 hr . at 4 ° c . the diluted extract was clarified by centrifugation 35 , 000 g for 30 min . at 4 ° c . each gram of protein of the refolding solution was absorbed on to 0 . 7 - 0 . 8 ml of pre - treated ( with bsa ) hydroxyapatite . the fraction not bound to the absorbent was added with 0 . 25m nacl and 100 mm arginine and concentrated with a stirrer cell ( nucleopore , membrane cut 30 , 000 , sigma ) until final volume reached 1 - 2 ml . concentrated fraction was then loaded onto a sephacryl s - 200hr column ( 1 × 120 cm ) precalibrated with 50 mm k - phosphate buffer ph 7 . 0 and 0 . 25m naci . fractions of one ml were collected and tested for absorbance at 280 nm and on sds - page . peak fractions were collected and dialyzed against pbs and designed as purified bpp1 ′- pe40 . highly purified bpp1 ′- pe40 was kept at − 20 ° c . in aliquots . all purification steps were followed by sds - page electrophoresis , protein measurements , western blot analysis and adp - ribosylation activity of the samples . based on total activity ( as measured by adp - ribosylation activity ) a yield of 4 . 5 % from starting material ( table iii ) was obtained . according to specific activity of the chimeric protein , the degree of purification was 20 . 8 - fold ( table iii ). the highly purified chimeric protein was tested for its nonspecific toxicity . this was done by injecting a single increasing dose of chimeric protein into mice ( strain sjl ). animals were observed for at least 2 weeks , although a toxic dose of pe caused death within 24 - 48 hours . none of the mice died at a dose of 50 μg , ⅔ of the mice died at a dose of 100 μg . use of an ion - exchange column ( in place of the stirrer cell ) after the hydroxyapatite step in the purification procedure described above , improves the purity of the end product . fractions found not to bind to the hydroxyapatite support were diluted ( 1 : 5 ) with te buffer ( te buffer : 10 mm tris hcl ph 8 . 0 , 1 mm edta ), added to q - sepharose ( 5 ml of wet column media to ˜ 20 mg protein of diluted bpp1 - pe40 solution ) and stirred for 30 min . at 4 ° c . the material was then packed into a column and washed with te buffer . elution was performed with 1 m nacl in te buffer . this step concentrated the protein samples that were then applied onto the sephacryl column . increasing amounts of purified bpp1 ′- pe40 chimeric protein were added to pas cells at day 1 ( d1 ) of the propagation phase . as shown in fig1 , the highly purified bpp1 ′- pe40 caused inhibition of protein synthesis in a dose dependent manner . nh 4 cl is known to be a lysosomotrophic agent which increases the lysosomal ph thus neutralizes the activity of its enzymes . different lysosomotrophic agents such as nh 4 ci , monensin , etc . caused enhancement of the immunotoxins &# 39 ; cytotoxicity . we examined the effect of nh 4 ci on bpp1 ′- pe40 cytotoxicity toward target cells . as can be seen in fig1 , 5 mm nh 4 ci dramatically increased the cytotoxic effect of bpp1 ′- pe40 on pas cells . while in the absence of nh 4 cl , 500 ng bpp1 ′- pe40 caused only 4 % inhibition of protein synthesis , adding nh 4 cl increased inhibition of protein synthesis to 98 % ( 5 mm of nh 4 cl caused in itself a 26 % decrease in protein synthesis ). peripheral was collected from ms patients or control healthy donors . lymphocytes were separated by application of 20 ml peripheral blood on ficoll - isopaque ( 1 . 077 ). lymphocytes were either activated with pha ( 5 ( g / ml ) for 72 hr . or used freshly within 24 hr . activated lymphocytes were transferred to rpmi 1640 supplemented with 10 % fcs and 10 units / ml rll2 . the cytotoxic assay ( 2 × 10 4 cells / well ) was done immediately after the cells transfer . fresh lymphocytes ( 10 5 cells / well ) were maintained in the same medium as described for the t cell lines supplemented with 20 % heat inactivated fcs and 10 units / ml rll2 . 1 ) fresh cells without any pre - activation period . calibration experiments were performed on control fresh blood samples to ensure good availability of the cells during the assay period . parameters such as density of cells seeded , addition of specific growth factors / cytokines , etc . were tested ; 2 ) cells following 2 - 3 days of activation pha . in parallel experiments , control blood samples taken from healthy individuals were tested in a similar manner , examining for specificity of the chimeric proteins used . the study was performed on ms patients mainly at relapses of the disease . eighteen ms patients were included in this study . fresh cells ( without any pre - activation ) from eleven patients were examined with bpp1 ′- pe40 at different stages of its purification ( following the progress in the purification protocol ). peripheral blood cells from six patients ( 54 %) were sensitive to bpp1 ′- pe40 mediated cytotoxicity with inhibition of protein synthesis ranging from 20 % to 92 %. fig1 demonstrates the effect of bpp1 ′- pe40 on a few ms patients , one that was very sensitive to the chimera ( msm - 18 ), one that did not show any response ( msm - 16 ) and two other patients that showed low to moderate sensitivity to bpp1 ′- pe40 mediated cytotoxicity ( msm - 13 , 14 ). samples from twelve patients ( part of the patients &# 39 ; samples were tested both as fresh cells and after activation ) were tested after 3 days of activation with pha . six out of the twelve samples ( 50 %) showed sensitivity to the chimera . cells taken from control donors showed no or very little response to the chimera . cells taken from a patient with another , unrelated neurological disease were also tested and found unresponsive to the cytotoxic effect of bpp1 ′- pe40 . 18 female sjl mice between 8 - 10 wk of age were immunized in 4 footpads with mice spinal cord homogenate ( 0 . 1 mg / mice ) solubilized in pbs , ph 7 . 4 , and emulsified 1 : 1 in cfa ( 5 mg / ml mycobacterium tuberculosum ) in a total volume of 100 μl / mice . in addition , pertusis toxin ( 200 ng / 200 μl / mice ) was i . v . injected . boosting was done in 48 h . later with another dose of pertusis toxin , i . v . injected ( 200 ng / 200 μl / mice ). treatment consisted of i . p . injection every 12 h . of bpp1 ′- pe40 at 5 ug / mice ( based on 2 . 5 μg / g animal ) in 0 . 5 ml pbs ph 7 . 4 or 0 . 5 ml pbs alone . the treatment was initiated 8 days after immunization and continued for 10 days . grading of the severity of eae was made every day during treatment and at least 1 month later . the scoring ranged from 1 to 6 as follows : 1 = mild tail weakness , 2 = tail paralysis , 3 = tail paralysis and hind leg paralysis , 4 = hind leg paralysis or mild fore - limb weakness , 5 = quadriplegia , 6 = death . bpp1 ′- pe40 treated mice did not develop eae at all ( mean severity = 0 ) and none of them died from the disease . in contrast , in the pbs control group , 6 out of 9 mice developed eae with the peak of disease on day 14 . the 6 affected mice displayed differences in severity and duration of the disease , the mean severity on day 14 being 3 . in addition , one of the 6 mice in the control group died from the disease . the treated group was followed for an additional 4 weeks and no sign of a late appearance or late relapse of the disease was observed . several additional chimeric proteins based on the human mbp pathogenic sequences were constructed . the targeting sequences in these chimeric proteins were based on peptides 84 - 102 and 143 - 168 of the human mbp ( table ivb ). the short peptide sequences were synthesized on a dna synthesizer and purified by conventional protocols . sequences of the oligo &# 39 ; s used for the constructs are shown in table iva . 5 ′ phe lys gly val asp ala gln gly thr leu ser lys ile phe lys leu gly gly arg asp 3 ′ ( seq id no : 16 ) the new chimeric proteins were constructed using gene fusion techniques . schematic representations of the plasmids encoding the new chimeric proteins are shown in fig1 a - d . the targeting sequences were cloned at the 5 ′ end of a full length modified pe ( pe66 4glu ) ( fig1 a , b ) or to a truncated form of pe - cdna ( pe40 ) ( fig1 c , d ). both forms of the pe - cdna had been used for constructing il2 - toxin chimeric proteins and were found to exhibit a differential activity towards human and murine freshly activated t cells . oligo &# 39 ; s 1 - 2 were used to construct bpp 143 - 168 - pe40 ; oligo &# 39 ; s 3 - 4 were used to construct bpp 84 - 102 - pe40 ; oligo &# 39 ; s 5 - 6 were used to construct bpp 84 - 102 - pe66 4glu ; and oligo &# 39 ; s 7 - 8 were used to construct bpp 143 - 168 - pe66 4glu . the new chimeric cytotoxins were expressed in e . coli expression systems , similar to the production of previous mbp - pe . expression was followed by sds - page of cell extracts ( fig1 a - c ), by western blot analysis using anti - pe antibodies and by measuring adp - ribosylation activity of the expressed proteins . all new chimeric proteins were highly expressed and primarily concentrated in the insoluble fraction of the expressing cells . these human chimeric proteins were tested on target cells such as peripheral blood from ms patients . bpp 84 - 102 - pe66 4glu was tested on the cells of two ms patients ( already tested before , msm 16 , 18 ) and found to be cytotoxic ( table v ). one of the patients ( msm 16 ) responding to the new chimeric proteins based on the human sequence did not respond before to bpp1 - pe40 ( guinea pig - based sequence ) mediated cytotoxicity . cells from an additional ms patient had no response to bpp 84 - 102 - pe66 4glu . effect of bpp 84 − 102 − pe66 4glu on pbl from ms patients 1 ) aharoni , r ., teitelbaum , d ., arnon , r . & amp ; puri , j . 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( 1971 ) j . biol . chem . 256 : 1496 - 1503 . it will be appreciated by persons skilled in the art that the present invention is not limited to what has been thus far described , but rather the scope of the present invention is limited only by the following claims : phe lys gly val asp ala gln gly thr leu ser lys ile phe lys leu