Patent Abstract:
novel chimeric fusion proteins comprising immunodominant epitopes of gad and insulin are provided . also provided are immunomodulatory methods for the use of such proteins for both the prevention and treatment of type 1 diabetes mellitus . the chimeric fusion proteins of the invention are useful in predicting risk of onset of type i diabetes , determining prognosis of type 1 diabetes patients early in disease progression , and in evaluating patients for suitability as recipients of transplants of pancreatic cells or tissues . the administration of the proteins of the invention in accordance with the immunomodulatory methods of the invention results in beneficial effects on disease development and severity in patients suffering from or predicted to be at risk of developing type 1 diabetes , as well as on the outcome of transplants of pancreatic cells or tissues in type i diabetes patients .

Detailed Description:
chimeric fusion proteins all polypeptides synthesized in biological systems are initially made with an n - terminal methionine residue . the extreme n - terminal and c - terminal amino acid residues are not considered essential to the functional use of the polypeptides of the invention , which , although not particularly preferred , may be produced without such residues . for example , the polypeptides may be chemically synthesized without n - terminal methionines . preferred chimeric proteins of the invention thus include ig1 ( comprising amino acid residues starting at about residue 2 and ending at about residue 153 of seq id no : 1 ), ig2 ( comprising amino acid residues starting at about residue 2 and ending at about residue 173 of seq id no : 2 ), ig3 ( comprising amino acid residues starting at about residue 2 and ending at about residue 137 of seq id no : 3 ), ig4 ( comprising amino acid residues starting at about residue 2 and ending at about residue 175 of seq id no : 4 ), ig5 ( comprising amino acid residues starting at about residue 2 and ending at about residue 226 of seq id no : 5 ), ig6 ( comprising amino acid residues starting at about residue 2 and ending at about residue 387 of seq id no : 6 ) and ig7 ( comprising amino acid residues starting at about residue 2 and ending at about residue 439 of seq id no : 7 ). preferably ig1 comprises amino acid residues 1 - 154 of seq id no : 1 , ig2 comprises amino acid residues 1 - 174 of seq id no : 2 , ig3 comprises amino acid residues 1 - 138 of seq id no : 3 , ig4 comprises amino acid residues 1 - 175 of seq id no : 4 , ig5 comprises amino acid residues 1 - 226 of seq id no : 5 , ig6 comprises amino acid residues 1 - 387 of seq id no : 6 , and ig7 comprises amino acid residues 1 - 438 of seq id no : 7 . in certain preferred embodiments these chimeric proteins further comprise a histidine tag ( i . e ., a stretch of at least five and preferably six contiguous histidine residues , which facilitates the purification of the chimeric fusion protein by metal chelation chromatography ). preferably the histidine tag is located at the extreme c - terminus of the chimeric fusion protein . histidine tags are discussed in greater detail in copending u . s . patent application ser . nos . 08 / 431 , 644 and 08 / 482 , 114 , which are incorporated herein by reference to more fully describe such histidine tag sequences and their uses . accordingly , in certain of these preferred embodiments ig1 comprises amino acid residues 1 - 160 of seq id no : 1 and has a predicted molecular weight of about 18 . 8 kda , ig2 comprises amino acid residues 1 - 180 of seq id no : 2 and has a predicted molecular weight of about 21 . 2 kda , ig3 comprises amino acid residues 1 - 144 of seq id no : 3 and has a predicted molecular weight of about 17 . 1 kda , ig4 comprises amino acid residues 1 - 181 of seq id no : 4 and has a predicted molecular weight of about 19 . 8 kda , ig5 comprises amino acid residues 1 - 232 of seq id no : 5 and has a predicted molecular weight of about 25 . 3 kda , ig6 comprises amino acid residues 1 - 393 of seq id no : 6 and has a predicted molecular weight of about 43 . 7 kda , and ig7 comprises amino acid residues 1 - 444 of seq id no : 7 and has a predicted molecular weight of about 49 . 2 kda . secondary structure considerations in designing ig4 ( seq id no : 4 ), ig5 ( seq . id no : 5 ), and ig6 ( seq id no : 6 ), particular attention was paid to the predicted secondary structures of the polypeptides of the invention . ig4 was initially designed by hypothetically joining peptide epitopes of interest to form a single hypothetical sequence , ig4nhb ( seq id no : 8 ). secondary structure prediction of ig4nhb ( seq id no : 8 ) according to chou and fasman , 1978 ; chou 1990 ; and garnier et al ., 1978 ; was performed using lasergene sequence analysis software ( dnastar , madison wis .). this algorithm predicts secondary structure of proteins from their amino acid sequences . other sequence analysis software may also be used for this purpose , including other commercially available software such as gcg or macvector . the entire sequence from amino acid 77 ( phe 77 ) to amino acid 134 ( thr 134 ) of ig4nhb ( seq id no : 8 ) was predicted to have helix forming characteristics . the propensity for actual long helix formation diminishes very rapidly for sequences higher than 20 amino acids , with the longest unbroken helices typically containing no more than 26 amino acids . the 57 amino acid helix - forming sequence from amino acid 77 to 134 of ig4nhb ( seq id no : 8 ) would thus be expected to break at unpredictable , if not random , points so as to form a variety of different structures . such structures are undesirable in the chimeric fusion proteins of the invention , as they would be likely to be prone to uncontrollable aggregation and would be expected to differ from the native secondary structures of the epitopes in the isolated peptides comprised by the chimeric fusion proteins of the invention and in the native proteins from which the peptides are derived ( e . g ., insulin , gad 65 , or ia - 2 ), which native secondary structures are preferred for the epitopes contained within the chimeric fusion proteins of the invention . therefore , in certain preferred embodiments of the invention , helix breakers ( see following paragraph ) are inserted between the epitopes to 1 ) block the formation of a very long helix and 2 ) to predictably separate the epitopes into distinct structural entities with distinct secondary structures . helix breakers are amino acids , or groups of sequential amino acids that act to block the propagation of helical secondary structures in nascent polypeptide chains . gly and pro are known to be strong helix breakers , asn and tyr are weak helix breakers . the insertion of any of these amino acids or combinations thereof in a polypeptide will tend to cause a nascent polypeptide secondary structure helix to terminate at the point of the insertion . to assure helix termination and the separation of the desired epitopes , a helix breaker that is at least two amino acids long ( wherein each of the amino acids is the same or different from the other and is selected from the group consisting of gly , pro , asn , and tyr ) is preferred , more preferably , such a helix breaker is at least three amino acids long . most preferably , the helix breaker is exactly three amino acids long . highly preferred helix breakers are pro - pro - pro ( seq id no : 9 ) and gly - gly - gly ( seq id no : 10 ), with the latter being the most preferred of these . dosage in accordance with the present invention , when used as therapeutic agents , the chimeric proteins of the invention are administered to patients in need of such treatment in amounts ranging from about 6 . 9 pm / kg / patient to about 8 . 6 μm / kg / patient . preferably the amounts range from about 34 . 5 pm / kg / patient to about 5 . 2 μm / kg / patient . more preferably the amounts range from about 170 pm / kg / patient to about 3 . 5 μm / kg / patient . most preferably the amounts range from about 0 . 5 μm / kg / patient to about 3 . 5 sm / kg / patient . in certain of its aspects , the present invention also provides for the repeated administration of doses containing lower amounts of the chimeric fusion proteins of the invention to a patient in need of such treatment . although less preferred , doses containing amounts of below about 6 . 9 pm / kg / patient , and as low as about 1 pm / kg / patient may be used in the practice of the present invention . preferably the chimeric proteins are administered without the concomitant administration of an adjuvant , however , when used to perform t cell assays ( e . g ., in transgenic mice such as described in wong et al . 1998 ), initial administration to experimental animals is preferably made with adjuvant . administration on therapeutic t cell modulatory schedules in accordance with the invention , a therapeutic t cell modulatory schedule involves administration of doses containing the chimeric proteins of the invention , preferably in a pharmaceutically effective carrier , repeatedly to the patient , at least two times at an interval of at least six , and preferably at least twelve hours , with an interval of not more than seven days , preferably not more than 72 hours , more preferably not more than 48 hours , and most preferably not more than 24 hours between doses . in accordance with the present invention , the chimeric fusion proteins of the invention are preferably administered parenterally without the concomitant administration of an adjuvant . administration by a parenteral route will typically be via injection such as intravascular injection ( e . g ., intravenous infusion ), subcutaneous injection , or intramuscular injection . other non - oral routes of administration , e . g ., mucosal , inhalation , transdermal ultrasound , and the like , may be used if desired and practicable for the particular polypeptide to be administered . although less preferred , the chimeric fusion proteins of the invention may also be administered orally , however dosages for oral administration will typically be one to two orders of magnitude higher that those discussed above under the sub - heading “ dosage .” formulations suitable for injection and other routes of administration are well known in the art and may be found , for example , in remington &# 39 ; s pharmaceutical sciences , mack publishing company , philadelphia , pa ., 17th ed . ( 1985 ). preferred formulations for parenteral administration of the proteins of the invention are those described for insulin in the usp 23 / nf 18 { 1995 ). parenteral formulations must be sterile and non - pyrogenic , and generally will include a pharmaceutically effective carrier , such as saline , buffered ( e . g ., phosphate buffered ) saline , hank &# 39 ; s solution , ringer &# 39 ; s solution , dextrose / saline , glucose solutions , and the like . formulations may also contain pharmaceutically acceptable auxiliary substances as required , such as , tonicity adjusting agents , wetting agents , bactericidal agents , preservatives , stabilizers , and the like . further discussions of dosage and administration of polypeptides using therapeutic t cell modulatory schedules may be found in copending u . s . patent application ser . nos . 08 / 431 , 644 and 08 / 482 , 114 ( discussed above ) and copending u . s . patent application ser . no . 08 / 565 , 769 , filed dec . 1 , 1995 in the name of yi wang , each of which is hereby incorporated herein by reference in its entirety to more fully describe the state of the art to which the present invention pertains . without intending to limit it in any manner , the present invention will be more fully described by the following examples . experimental methods non - obese diabetic ( nod ) mice are a strain of mice that are prone to the development of diabetes , and represent an accepted model system for the study of diabetes mellitus . these mice , designated nod / mrktacfbr , were purchased from taconic farms ( germantown , n . y .). nod scid mice , designated nod / scid , are available from the jackson laboratories , bar harbor , me . the incidence of diabetes by 200 days of age in these mice is 80 % in females and 50 % in males . at 110 days of age , fewer than 15 % of the male nod mice became diabetic . cytoxan induction experiments : cytoxan ( cyclophosphamide ) treatment can increase the incidence and accelerate the onset of diabetes in non - diabetic nod mice . randomly selected , non - diabetic male nod mice , which were 100 to 110 days of age , were injected ( ip ) with cyclophosphamide ( 250 mg / kg ) on day 0 . all the mice were subjected to urine glucose measurement 2 - 3 times per week for a period of 21 days after cyclophosphamide injection . the onset of diabetes was recorded as the first point at which two consecutive days of positive urine glucose results were obtained . blood glucose was also measured to confirm the urine glucose results . ( exactech , medisense inc ., cambridge , mass .) in all the mice tested with positive urine glucose , blood glucose levels were greater than 150 mg / dl . at day 21 , these mice were sacrificed and examined histologically . experiments in the nod / scid adoptive transfer model : spleen mononuclear cells were harvested from diabetic nod mice of recent disease onset ( diabetic not more than 3 weeks ) and about 35 × 10 6 of these spleen cells in 0 . 2 ml of pbs were injected intravenously into 6 - 8 week old nod / scid mice . onset of diabetes was monitored biweekly by urine glucose testing and confirmed by blood glucose testing on day 28 relative to the time of spleen cell transfer . at day 28 , these mice were sacrificed and examined histologically . reagents : cytoxan ( cyclophosphamide , mead johnson oncology products ) was dissolved in distilled water ( 25 mg / ml ). oxidized bovine insulin chain b was purchased from sigma ( catalog no . i - 6383 ). both insulin chain b and the bsa control protein ( miles inc ., # 81 - 001 ) were dissolved in pbs / 1m hcl , ph2 , before dialysis against . pbs , ph7 . 2 , sterile filtration , and storage of frozen aliquots . ig1 . a synthetic chimeric gene encoding chimeric protein ig1 ( seq id no : 1 ) was constructed in two rounds of overlapping pcr ( ho et al ., 1989 ). each gene subdomain was synthesized in a standard pcr 100 μl reaction using 10 pmole of each of the appropriate oligonucleotide primers , as described below . the 5 ′ gene segment was amplified using the overlapping primers : prig1 ( seq id no : 11 ), and prig2 ( seq id no : 12 ). the 3 ′ gene segment was amplified using the overlapping primers prig3 ( seq id no : 13 ) and prig4 ( seq id no : 14 ). the prig1 oligo - nucleotide ( seq id no : 11 ) was designed to allow creation of a unique ndei restriction site at the 5 ′ end . the prig4 primer ( seq id no : 14 ) included a unique bamhi site , stop codon ( taa ) and 18 nucleotides that encoded a histidine tag sequence for purification of the recombinant chimeric protein by metal affinity chromatography . the two subdomains were combined using flanking oligonucleotides prig5 ( seq id no : 15 ) and prig6 ( seq id no : 16 ) in a second pcr reaction to yield a 492 bp gene product . the pcr product was cloned into expression plasmid vector pcr2 . 1 as described by the supplier ( invitrogen , san diego , calif .). kanamycin - resistant dh10b transformants were selected and the correct clones and orientations determined by restriction and sequence analysis . restriction fragments from two clones (# 6 and # 18 ) were combined to remove undesired mutations . following nucleotide sequence analysis , an independent plasmid pcr2 . 1 - ig1 was digested with ndei and bamhi and the 492 bp fragment inserted into compatible sites of the mp4 expression plasmid pet22b - mp4 ( elliott et al ., 1996 ), yielding plasmid pig1 . the insert from pcr2 . 1 - ig1 was also subcloned into plasmid vector pbluscript ® psk + ( stratagene cloning systems , la jolla , calif .) to yield plasmid psk + ig1 . the synthetic ig1 gene ( seq id no : 17 ) encodes a chimeric fusion protein with a predicted mass of about 18 . 8 kda . ig2 . a synthetic chimeric gene encoding chimeric protein ig2 ( seq id no : 2 ) was constructed by pcr amplification of an internal ig1 gene fragment using pig1 as a template along with the sense primer prig7 ( 5 ′- agatct gatg aacattctgc tgcagtatgt tgttaaaagc ttcgataaca tgtatgccat gatg - 3 ′— seq id no : 18 ; bglii site underlined ) in combination with the anti - sense oligonucleotide primer prig8 ( 5 ′- tgtaca gata tccgccagt tccagacatt ttttcagaga aaaatggcta tgttcagagg taaaggcaat cagacgcg - 3 ′— seq id no : 19 ; bsrg1 site underlined ). the 197 bp bglii - bsrg1 pcr fragment was subcloned into pcr2 . 1 . sequence analysis revealed a c & gt ; g substitution at position 183 in the coding strand of the 197 bp bglii - bsrg1 pcr fragment for all subclones analyzed , and the plasmid was named pcr2 . 1 - ig7 / 8c183g . subsequent analysis revealed an error in the original prig8 primer sequence ( seq id no : 19 ). a repair primer , prig12 ( seq id no : 20 ), was synthesized to correct the c & gt ; g substitution in pcr2 . 1 - ig7 / 8c183g . the internal 197 bp bglii - bsrg1 dna segment was corrected by pcr using pcr2 . 1 - ig7 / 8c183g as a template along with primers prig7 ( seq id no : 18 ) and prig12 ( seq id no : 20 ). the pcr fragments were subcloned into pcr2 . 1 , their sequence was determined using standard dideoxy sequencing to confirm that the desired sequence had been obtained , and a single clone designated pcr2 . 1 - ig7 / 12 was isolated and amplified . the 137 bp bglii - bsrg1 restriction fragment in psk + ig1 was exchanged with the 197 bp bglii - bsrg1 fragment from pcr2 . 1 - ig7 / 12 to create psk +/ ig7 / 12 . the 552 bp nde1 - bamhi fragment from psk +/ ig7 / 12 was subcloned into the compatible sites of pig1 to create plasmid pig2 . the synthetic ig2 gene ( seq id no : 21 ) encodes a chimeric fusion protein with a predicted mass of about 21 . 2 kda . ig3 . a synthetic chimeric gene encoding chimeric protein ig3 ( seq id no : 3 ) was constructed by removing a 552 bp ndei - bamhi restriction fragment from plasmid pet22b - ig2 and substituting an ndei - bamhi digested 444 base pair pcr product therefor . this 444 base pair pcr product was made using primers prig5 ( seq id no : 15 ) and prig13 ( seq id no : 22 ) with the synthetic ig2 gene as a template . the synthetic ig3 gene ( seq id no : 23 ) encodes a chimeric fusion protein with a predicted mass of about 17 . 1 kda . ig4 . a gene sequence encoding chimeric protein ig4 ( seq id no : 4 ) is set forth below as seq id no : 24 , and encodes a chimeric fusion protein with a predicted mass of about 19 . 8 kda . ig5 . a synthetic chimeric gene encoding chimeric protein ig5 ( seq id no : 5 ) was constructed via ligation of pcr products using primers prig14 ( seq id no : 25 ), prig15 ( seq id no : 26 ), prig16 ( seq id no : 27 ), prig17 ( seq id no : 28 ), prig18 ( seq id no : 29 ), prig19 ( seq id no : 30 ), prig20 ( seq id no : 31 ), prig21 ( seq id no : 32 ), prig22 ( seq id no : 33 ), and prig23 ( seq id no : 34 ). the synthetic ig5 gene ( seq id no : 35 ) encodes a chimeric fusion protein with a predicted mass of about 25 . 3 kda . ig6 . a gene sequence encoding chimeric protein ig6 ( seq id no : 6 ) is set forth below as seq id no : 36 , and encodes a chimeric fusion protein with a predicted mass of about 43 . 7 kda . ig7 . a gene sequence encoding chimeric protein ig7 ( seq id no : 7 ) is set forth below as seq id no : 37 , and encodes a chimeric fusion protein with a predicted mass of about 49 . 2 kda . expression and purification of recombinant ig fusion proteins . for each ig fusion protein expression plasmid construction that was bacterially expressed , electrocompetent e . coli strain bl21 ( de3 ) was transformed with the expression plasmid and ampicillin - resistant colonies were selected for liquid culture . the 1de3 lysogen in strain bl21 ( de3 ) contains the gene for t7 polymerase behind the e . coli lacuv5 promoter for efficient expression of target genes under control of the strong bacteriophage t7 transcriptional and translation signals ( studier et al ., 1990 ). the recombinant chimeric fusion protein was purified from solubilized whole cell pellets by immobilized metal affinity chromatography and analyzed by sds - page / coomassie blue staining . four liters cultures were grown to od600 of 0 . 8 in terrific broth ( tb ) medium ( sambrook et al ., 1992 ). protein expression was induced for 5 hours with 1 mm isopropylthiogalactoside ( iptg ). induced cells were harvested by centrifugation and frozen overnight at − 20 ° c . cell pellets were thawed at room temperature and homogenized in 10 ml / g of buffer a ( 6 m guanidine - hcl / 10 % glycerol / 20 mm tris - cl ph 7 . 8 / 500 mm nacl / 200 mg sodium sulfite / 280 mg sodium tetrathionate ) using a tekmar homogenizer ( the tekmar co ., cincinnati , ohio ). cells were frozen at − 70 ° c . for 1 hour and thawed at room temperature to promote cell lysis . the cell suspension was gently mixed for 30 min at room temperature using a magnetic stirrer . the soluble fraction containing ig protein was collected as the supernatant following centrifugation of the cell lysate at 10 , 000 × g for 30 min at 4 ° c . in a beckman ja - 10 rotor . the supernatant was loaded on a ni - nta ( qiagen inc ., chadsworth , calif .) column previously equilibrated in buffer a at a flow rate of 8 ml / min . the column was washed to baseline absorbance using buffer a . the column was further washed with buffer b ( 6 m urea / 10 % glycerol / 20 mm tris - cl / 500 mm nacl ph 7 . 8 ) and contaminating e . coli proteins were removed with buffer c ( 6 m urea / 10 % glycerol / 20 mm tris - cl / 500 mm nacl ph 5 . 0 ). ig protein was eluted with buffer d ( 6 m urea / 10 % glycerol / 20 mm tris - cl / 500 mm nacl ph 4 . 0 ). all fractions were collected in batch and analyzed on a 4 - 20 % sds - polyacrlyamide gradient gel in the presence of reductant . the ig containing fractions ( buffer d wash ) were concentrated 10 - fold using an amicon stircell using a pm10 membrane . the sample was dialyzed against two changes of milli - q water at 4 ° c . the ig preparations were filter sterilized and the concentration determined spectrophotometrically using a conversion factor of 1 . 06 mg / ml / od 280 . five micrograms were analyzed under reducing and nonreducing conditions by sds - page so as to obtain an initial indication of purify and integrity of the protein preparations . chimeric fusion protein treatment — cytoxan model : groups of randomly selected nod mice were injected intravenously twice daily with either bsa as a control , or gad peptides , insulin chain b ( icb ) or various combinations of gad peptides and / or insulin chain b at the doses indicated in the figures on days 1 , 3 , and 5 following cytoxan treatment ( day 0 ). animals that received injections of bsa ( controls ) manifested a greater than 80 % incidence of diabetes by 21 days following cytoxan induction ( fig2 ). in contrast , animals treated with either icb or either of gad 65 250 - 273 or 520 - 555 peptides , experienced reductions to less than 50 % incidence of diabetes . the therapeutic effects of treatment with combinations of icb and the two gad peptides were then examined ( fig3 ). reduction in diabetes incidence to 25 % or less was achieved by the combination of gad 250 - 273 and icb at doses of 100 u g or 250 u g per injection . surprisingly , the addition of gad peptide 520 - 555 ( amino acid residues 139 - 173 of seq id no : 2 ) appeared to inhibit the therapeutic efficacy of the icb with gad 65 250 - 273 combination . evaluation in the cytoxan model of the ig1 and ig2 chimeric fusion proteins of the invention , showed that treatment with either of these polypeptides mediated a dose - dependent reduction in the incidence of diabetes compared to the bsa treated control animals ( fig4 ). in this experiment , 300 u g doses were more effective than 100 u g doses , and ig2 reduced the incidence of diabetes to a greater degree than ig1 . treatment with 300 ug doses of ig2 reduced disease incidence to less than 12 %, compared to greater than 80 % disease incidence in the bsa treated control animals . diabetogenic splenic mononuclear cells were harvested from newly diabetic nod mice ( onset less than 3 weeks previous ). to initiate the destruction of islet beta cells and the development of diabetes , 8 - 12 week old nod / scid mice were injected intravenously with 35 × 10 6 diabetogenic spleen cells . the incidence and onset of diabetes were monitored biweekly by urine glucose testing and confirmed by blood glucose testing at the end of the experiment . the average onset time of diabetes after disease induction was approximately 25 days . iv treatment with ig2 , ig3 , or islet beta cell antigens was initiated on day 3 . animals were treated with 300 ugs of antigen twice daily every other day for a period of six days ( from day 3 to day 9 , where the time of spleen cell transfer was day 0 ). the results of these experiments are set forth in fig5 . they surprisingly demonstrate that only the ig2 chimeric fusion protein prevented the onset of disease in the nod / scid recipients . in contrast , in this model only a slight delay in disease onset was observed in recipients of ig1 , insulin chain b ( icb ), or the combination of icb and gad peptide 250 - 273 . these profound differences in the effects of treatment with ig2 as compared to the other treatment regimens was unexpected . without wishing to be bound by any particular theory of operation , it is believed that this unexpected finding is a result of the in vivo processing of ig2 into unique antigenic peptides that are particularly effective at eliciting a state of immune tolerance protective against autoimmune diabetes , even in the demanding conditions resulting from challenge with heterogeneous t cell populations derived from the spleens of fully diabetic nod mice . although preferred and other embodiments of the invention have been described herein , further embodiments may be perceived by those skilled in the art without departing from the scope of the invention as defined by the following claims . abbas et al ., 1991 . cellular and molecular immunology w . b . saunders company , philadelphia . ausubel et al ., 1994 . current protocols in molecular bioloqy , john wiley & amp ; sons , new york . chou , 1990 . prediction of protein structure and the principles of protein conformation , plenum press 549 - 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