CA2195665A1 - Pharmaceutical compositions comprising a chimaeric tnf binding protein - Google Patents
Pharmaceutical compositions comprising a chimaeric tnf binding proteinInfo
- Publication number
- CA2195665A1 CA2195665A1 CA002195665A CA2195665A CA2195665A1 CA 2195665 A1 CA2195665 A1 CA 2195665A1 CA 002195665 A CA002195665 A CA 002195665A CA 2195665 A CA2195665 A CA 2195665A CA 2195665 A1 CA2195665 A1 CA 2195665A1
- Authority
- CA
- Canada
- Prior art keywords
- tnf
- binding protein
- human
- tnf binding
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The present invention is directed to the use of a chimaeric TNF binding protein which comprises a soluble part of the human p55- or the p75-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human immunoglobulin or of a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the treatment of autoimmune diseases.
Description
2 1 ~ 5 6 ~ ~ .~.,~ . ~788 phArmurrllti(~Al comnositions co~ -F a rhimAloric TNF hinllinF
proteirl The present invention is directed to the use of a chimaeric TNF
binding protein which cnmprie~e a soluble part of the human p55- or the p7~-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human ;lllllllllln~;lnhulin or a phArmArPllt;"Ally Ar~-~pt~hlr salt thereof for the method of treatment A..~ .l.P diseases, which are frequently A~o~ AIPd with infl-~ l...y processes, e.g. ~h,- ,." .1....1 arthritis, juvenile onset type I diabetes mellitus, systemic lupus 0 eryth~mAtr-s le, thyroiditis, especially multiple sclerosis and for the preparation of a pharmArentirAl rnmroeitinn for the treatment thereof.
Multiple Sclerosis (MS) is an inflAmmAtl~ry disease of the central nervous system (CNS), thought to originate in an ~ ..;""..,."/~ disorder, with components of myelin as the target. Support for the 5~.,1l.,...~,...,.r origin of 5 MS is provided by the fact that myelin basic protein (MBP) reactive T
lymphocytes are found in man which are phenotypically similar to MBP-specific encephalitogenic T cells in experimental allergic rnrephAlnmyelitis [Sun, Acta. Neurol. Scand. Suppl. ~,1-~6 (lg93);
Voskuhl et al., A~ l; L y 15, 137-143 (1993)], and there is evidence 20 that the frequencies of MBP reactive T cell clones in MS patients is elevated [Allegretta et al., Science ~Z, 718-721(1990)]. In addition, various experimental n1~toimmllnP rnrerhAlrJmyelitis animal models have been developed which mi_ic pathophysiologic aspects of human MS (see below). However, there are also environmental factors controling MS
2s incidence as evidenced by the existence of geographically well defined endemic areas.
MS is characterised by focal ArcllmlllAtir)nc of inflAmmAt.nry cells, edema snd demyelination in CNS tissue. It is an old finding that at autopsy more . ~
WO 96/03141 ~ ~ ~ 5 6 focal lesions are found than would have been predicted from the clinical status.
Typically, the lesions start as a perivenular infiltrate of ' cells in white matter with ensuing edema. The infiltrating cells appear to mediate the selective deQ Ll u~.Lu~ of myelin sheaths leaving the a~ons intact. In the further ~I~U~ ICSb;(JU, astrocyte ,u.uliÇc-uLion leads to a gliotic scar, the so-caUed MS plaque, of 0.1 to several cm diameter. The clinical course of MS
may be ~cll~iLLi~l~Glapdillg with a wide range in the duration of the disease free interval, or chronically ~J~U~ ., QU~;GC~IIiUg that the ulldeLlying lo pathoph.~ r processes are under hcl,_.ugcl~cous control.
Clinical r~t~-l Oy studies have revealed the presence of TNF producing cells in MS lesions. The majority of the TNF positive cells were morphnl ~ lly classified as reactive fibrous astrocytes, and double immnnn-~;";"g C--,UC.illlC~s cnnfirrr~~ d that most reactive cells were astrocytes, the rest 15 being monocyte / macrophages [Hofman et al., J. Exp. Med. 170, 607-612 (1989)]. An ;..~ ..1 study uu~li....ed the positive immunu~ca~Livi~y for TNF in asLIu..yLcd and IIIOIIG~ ~LCS in MS plaques, and also I~I~G ' _ ' ~
Iyl~ h~ (LT) immunu.~. Li~iLy. T ~ , "ly, in additon to the expected Iy u~h~ es~ microglial ce~ls were found positive for LT [Selmaj et al., J.
20 Clin. Invest. ~Z 949-954 (199la)]. Ful Ll._.l..u. c, iu~ . _ ' ~ - of the cytokine mRNA C.~IG ~ in MS lesions by in situ hy~ri~;Q~t;~n show that high levels of TNF, IL,6 and IFN-r (and other cytokine) mRNAs are present in all infl~mmQtnry perivagcular lesions. The levels of TNF, IL-6 and IFN-y mRNAs were particularly high in those lesions which had strong ~5 dGlLlycli~Liuu [Woodroofe and Cuzner, Cytokine ~, 583-588 (1993)].
A direct cytotoxic effect of TNF, but not of IFN-r, IL 2, T-cell s ~ or s~ntigs~ . . bluDide antiserum, on nli~o~Pn~1rocytes in myelinated cultures of mouse spinal chord tissue has been reported [&lmaj and Raine, Ann. Neurol. 2~, 339-346 (1988)]. TNF treatment of these cultures caused 30 oligodendrocyte necrosis; some nerve fibers ~lu~lc~ded to dc~ycliuation.
TNF concentrations in semm and ~.~ .cb~uQ~ al fluid (CSF) of MS patients have been measured by a number of hlvc~Li~;uLuld. There is a cnn~PnQllQ that a Qig~nifir~nt p_.. G~l~,. number of MS patients has elevated TNF in CSF, and much less 80 in serum. The ~.u~ Ll~Lion of TNF does not directly 3s relate to CSF cell content sl~gr~;ng that CSF TNF content might reflect W096/03141 2 i ~ i665 r~ oo TNF production~in and arolmd MS lesions, eQpecially in view of the frequent F v-~- ..I..;r..lvr location of lesions.
AnimAi studieQ in P~ 1 PnrPFh~ l;Lia (EAE) have ~i, ..;r~ y shaped the views on the ....I,~ .- . -~.~P origin and the role of cytokines in 5 human MS. EAE is induced either by active ~ .. I ~ n with myelin antigens or by adoptive transfer of specific T cell clones mto a o.~.6~ e ~
sllQcPphhl~ host animal. The finding that the ~ ivl induction of an i_mune response to myelin antigens leads to diseases with Rimil_ritiPQ to human MS is an - po. l~L argnvment for an ~ origin of MS~ The lD role of TNF in the . ' '~ ' ' and the effectvr ~ ~ rpcpnn for the the cl~-~,L~IihLic I ~ l Oy of the disease is d~:~uu~ DL v~l~d by the protective efficacy of anti-TNF mnnnrlnnol antibody therapy [Ruddle et al., J.
E~cp. Med~ ~, 1193-1200 (1990); Selma~ et al., Ann. Neurol. ;~Q, 694-700 (1991b)]. The PliminAti-n of cells producing TNF (and other cytokines) 16 similarly D~ .e3 EAE [Huitinga et al., J. E~cp. Med. 1~, 1025-1033 (1990N. Further support for an LU '~lUI L~L role of TNF in EAE is provided by the finding that PnrPpolitngPnirity in adoptive transfer stron61y depends on the TNF and LT production capability of various T cell clones, all r~6~ g the game MBP peptide IPowell et al., Intern. Tmmllnnl 2, 539-2D 544 (1990)].
The use of soluble parts of the human p55- and p75-TNF-receptor for the he, L~ L of ~ l PnrPrh-lrmyelitis (EAE) in rats has already been descnbed in EP 512 528. However, so far no data l~6dl~1i~g the ~.uLe~.Livt:
effect of rhim_Prjr TNF binding proteins and rh...".C ~-.I;rA11y _rrpp~
25 salts thereof have been reported. Therefore the use of a rhim ~~ TNF
binding protein which ~ ;APc a soluble part of the human p55- or the p75-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human immllnrgloblllin or a l.h_." ~ :r_lly arCprtohl? salt thereof for the preparation of a rh~ . r- -I r_l rAmroCit;~A~n for the 30 LL eaL.. e.,t of a . . l ; . ~ f diseases, e.g. multiple sclerosis, especially, wherein the, l - - TNF binding protein . v l~ .' the soluble part of the human p55-TNF-receptvr is an object of the present inventiom A
furthermore preferred o.. bc ' ' of the present invention is such a use wherein the chimaeric TNF binding prvtein r~ -.l - ~F~ all domains e~ccept 35 the first domain of the constant region of the heavy chain of human i ... .n~ hnlin which means that the soluble part of the human p55-TNF-receptor is fused at its C-terminal end tv the hinge region of the 2 ~ ~5~
wo 96f~3141 - PCTfEP~5102788 ;... ,.. ~lAhnlin heavy chain. Such ;.. ~ lAh~llin can be either IgG, IgA, IgM or IgE, especially IgG, like IgG1 or IgG3. The term "a soluble part of the human p55- or the p75-TNF-receptor means in the conte~ct of the present invention either the complete P trf-~Pll~ r domain of one of these s receptors or a part thereof which still binds human TNF, e.g. in case of the p55-TNF-receptor the ~ r domain lacking the first 12 N-terminal amino acids.
Fu.lL,lL..ule it is an object of the present invention to provide a method of treating an u~ "~P disease in a mammal c.. ~ ;.. g ~-1,.. ;,.;~ .i- g 10 to said mammal a IL~ lly effective amount of a ~ .r~ ~ ;r TNF
binding protein or a salt thereof as defined above which meliorates the effects of such UI.l..;...~....~P disease, ~rerifir~lly such a method wherein the .l~... ..~P disease is multiple sclerosis.
The preparation of the chimaeric TNF binding proteins to be used for the L~ purpose of the present invention is described in detail in European Patent Application p~lhlir~tirn No. (EP) 417 563 and Loetscher et al., J. Biol. Chem.
2~L 18324-18329 (1991). Fu~ ILt L IILUI~ TNF binding proteins or parts t_ereof as described in the following patent pnhlir~t;~no~ can be used for the preparation of such ~-LuLat . ;c TNF binding proteins: EP 308 378, ~o EP 422 339, GB 2 218 101, EP 393 438, WO 90/13575, EP 398 327, EP 412 486, WO 9V03553, EP 418 014, JP 127,800/1991, EP 433 900, U.S. Patent No.
proteirl The present invention is directed to the use of a chimaeric TNF
binding protein which cnmprie~e a soluble part of the human p55- or the p7~-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human ;lllllllllln~;lnhulin or a phArmArPllt;"Ally Ar~-~pt~hlr salt thereof for the method of treatment A..~ .l.P diseases, which are frequently A~o~ AIPd with infl-~ l...y processes, e.g. ~h,- ,." .1....1 arthritis, juvenile onset type I diabetes mellitus, systemic lupus 0 eryth~mAtr-s le, thyroiditis, especially multiple sclerosis and for the preparation of a pharmArentirAl rnmroeitinn for the treatment thereof.
Multiple Sclerosis (MS) is an inflAmmAtl~ry disease of the central nervous system (CNS), thought to originate in an ~ ..;""..,."/~ disorder, with components of myelin as the target. Support for the 5~.,1l.,...~,...,.r origin of 5 MS is provided by the fact that myelin basic protein (MBP) reactive T
lymphocytes are found in man which are phenotypically similar to MBP-specific encephalitogenic T cells in experimental allergic rnrephAlnmyelitis [Sun, Acta. Neurol. Scand. Suppl. ~,1-~6 (lg93);
Voskuhl et al., A~ l; L y 15, 137-143 (1993)], and there is evidence 20 that the frequencies of MBP reactive T cell clones in MS patients is elevated [Allegretta et al., Science ~Z, 718-721(1990)]. In addition, various experimental n1~toimmllnP rnrerhAlrJmyelitis animal models have been developed which mi_ic pathophysiologic aspects of human MS (see below). However, there are also environmental factors controling MS
2s incidence as evidenced by the existence of geographically well defined endemic areas.
MS is characterised by focal ArcllmlllAtir)nc of inflAmmAt.nry cells, edema snd demyelination in CNS tissue. It is an old finding that at autopsy more . ~
WO 96/03141 ~ ~ ~ 5 6 focal lesions are found than would have been predicted from the clinical status.
Typically, the lesions start as a perivenular infiltrate of ' cells in white matter with ensuing edema. The infiltrating cells appear to mediate the selective deQ Ll u~.Lu~ of myelin sheaths leaving the a~ons intact. In the further ~I~U~ ICSb;(JU, astrocyte ,u.uliÇc-uLion leads to a gliotic scar, the so-caUed MS plaque, of 0.1 to several cm diameter. The clinical course of MS
may be ~cll~iLLi~l~Glapdillg with a wide range in the duration of the disease free interval, or chronically ~J~U~ ., QU~;GC~IIiUg that the ulldeLlying lo pathoph.~ r processes are under hcl,_.ugcl~cous control.
Clinical r~t~-l Oy studies have revealed the presence of TNF producing cells in MS lesions. The majority of the TNF positive cells were morphnl ~ lly classified as reactive fibrous astrocytes, and double immnnn-~;";"g C--,UC.illlC~s cnnfirrr~~ d that most reactive cells were astrocytes, the rest 15 being monocyte / macrophages [Hofman et al., J. Exp. Med. 170, 607-612 (1989)]. An ;..~ ..1 study uu~li....ed the positive immunu~ca~Livi~y for TNF in asLIu..yLcd and IIIOIIG~ ~LCS in MS plaques, and also I~I~G ' _ ' ~
Iyl~ h~ (LT) immunu.~. Li~iLy. T ~ , "ly, in additon to the expected Iy u~h~ es~ microglial ce~ls were found positive for LT [Selmaj et al., J.
20 Clin. Invest. ~Z 949-954 (199la)]. Ful Ll._.l..u. c, iu~ . _ ' ~ - of the cytokine mRNA C.~IG ~ in MS lesions by in situ hy~ri~;Q~t;~n show that high levels of TNF, IL,6 and IFN-r (and other cytokine) mRNAs are present in all infl~mmQtnry perivagcular lesions. The levels of TNF, IL-6 and IFN-y mRNAs were particularly high in those lesions which had strong ~5 dGlLlycli~Liuu [Woodroofe and Cuzner, Cytokine ~, 583-588 (1993)].
A direct cytotoxic effect of TNF, but not of IFN-r, IL 2, T-cell s ~ or s~ntigs~ . . bluDide antiserum, on nli~o~Pn~1rocytes in myelinated cultures of mouse spinal chord tissue has been reported [&lmaj and Raine, Ann. Neurol. 2~, 339-346 (1988)]. TNF treatment of these cultures caused 30 oligodendrocyte necrosis; some nerve fibers ~lu~lc~ded to dc~ycliuation.
TNF concentrations in semm and ~.~ .cb~uQ~ al fluid (CSF) of MS patients have been measured by a number of hlvc~Li~;uLuld. There is a cnn~PnQllQ that a Qig~nifir~nt p_.. G~l~,. number of MS patients has elevated TNF in CSF, and much less 80 in serum. The ~.u~ Ll~Lion of TNF does not directly 3s relate to CSF cell content sl~gr~;ng that CSF TNF content might reflect W096/03141 2 i ~ i665 r~ oo TNF production~in and arolmd MS lesions, eQpecially in view of the frequent F v-~- ..I..;r..lvr location of lesions.
AnimAi studieQ in P~ 1 PnrPFh~ l;Lia (EAE) have ~i, ..;r~ y shaped the views on the ....I,~ .- . -~.~P origin and the role of cytokines in 5 human MS. EAE is induced either by active ~ .. I ~ n with myelin antigens or by adoptive transfer of specific T cell clones mto a o.~.6~ e ~
sllQcPphhl~ host animal. The finding that the ~ ivl induction of an i_mune response to myelin antigens leads to diseases with Rimil_ritiPQ to human MS is an - po. l~L argnvment for an ~ origin of MS~ The lD role of TNF in the . ' '~ ' ' and the effectvr ~ ~ rpcpnn for the the cl~-~,L~IihLic I ~ l Oy of the disease is d~:~uu~ DL v~l~d by the protective efficacy of anti-TNF mnnnrlnnol antibody therapy [Ruddle et al., J.
E~cp. Med~ ~, 1193-1200 (1990); Selma~ et al., Ann. Neurol. ;~Q, 694-700 (1991b)]. The PliminAti-n of cells producing TNF (and other cytokines) 16 similarly D~ .e3 EAE [Huitinga et al., J. E~cp. Med. 1~, 1025-1033 (1990N. Further support for an LU '~lUI L~L role of TNF in EAE is provided by the finding that PnrPpolitngPnirity in adoptive transfer stron61y depends on the TNF and LT production capability of various T cell clones, all r~6~ g the game MBP peptide IPowell et al., Intern. Tmmllnnl 2, 539-2D 544 (1990)].
The use of soluble parts of the human p55- and p75-TNF-receptor for the he, L~ L of ~ l PnrPrh-lrmyelitis (EAE) in rats has already been descnbed in EP 512 528. However, so far no data l~6dl~1i~g the ~.uLe~.Livt:
effect of rhim_Prjr TNF binding proteins and rh...".C ~-.I;rA11y _rrpp~
25 salts thereof have been reported. Therefore the use of a rhim ~~ TNF
binding protein which ~ ;APc a soluble part of the human p55- or the p75-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human immllnrgloblllin or a l.h_." ~ :r_lly arCprtohl? salt thereof for the preparation of a rh~ . r- -I r_l rAmroCit;~A~n for the 30 LL eaL.. e.,t of a . . l ; . ~ f diseases, e.g. multiple sclerosis, especially, wherein the, l - - TNF binding protein . v l~ .' the soluble part of the human p55-TNF-receptvr is an object of the present inventiom A
furthermore preferred o.. bc ' ' of the present invention is such a use wherein the chimaeric TNF binding prvtein r~ -.l - ~F~ all domains e~ccept 35 the first domain of the constant region of the heavy chain of human i ... .n~ hnlin which means that the soluble part of the human p55-TNF-receptor is fused at its C-terminal end tv the hinge region of the 2 ~ ~5~
wo 96f~3141 - PCTfEP~5102788 ;... ,.. ~lAhnlin heavy chain. Such ;.. ~ lAh~llin can be either IgG, IgA, IgM or IgE, especially IgG, like IgG1 or IgG3. The term "a soluble part of the human p55- or the p75-TNF-receptor means in the conte~ct of the present invention either the complete P trf-~Pll~ r domain of one of these s receptors or a part thereof which still binds human TNF, e.g. in case of the p55-TNF-receptor the ~ r domain lacking the first 12 N-terminal amino acids.
Fu.lL,lL..ule it is an object of the present invention to provide a method of treating an u~ "~P disease in a mammal c.. ~ ;.. g ~-1,.. ;,.;~ .i- g 10 to said mammal a IL~ lly effective amount of a ~ .r~ ~ ;r TNF
binding protein or a salt thereof as defined above which meliorates the effects of such UI.l..;...~....~P disease, ~rerifir~lly such a method wherein the .l~... ..~P disease is multiple sclerosis.
The preparation of the chimaeric TNF binding proteins to be used for the L~ purpose of the present invention is described in detail in European Patent Application p~lhlir~tirn No. (EP) 417 563 and Loetscher et al., J. Biol. Chem.
2~L 18324-18329 (1991). Fu~ ILt L IILUI~ TNF binding proteins or parts t_ereof as described in the following patent pnhlir~t;~no~ can be used for the preparation of such ~-LuLat . ;c TNF binding proteins: EP 308 378, ~o EP 422 339, GB 2 218 101, EP 393 438, WO 90/13575, EP 398 327, EP 412 486, WO 9V03553, EP 418 014, JP 127,800/1991, EP 433 900, U.S. Patent No.
5,136,021, GB 2 246 569, EP 464 533, WO 92fO1002, WO 92fl3095, WO 92fl6221, EP 512 528, EP 526 905, WO 93/07863, EP 568 928, WO 93J21946, WO 93fl9777, EP 417 563 and WO 94/u6476.
~; The term chimslPrir TNF binding protein as used IL-uu~Luu~ the ~lJP. .~rs~ :An . uLul,l;ses also such proteins in which any amino acid of the ;..... ~-~lrhlllin part or the TNF binding part has been deleted or sllhet;t -tPd by one or more amino acids or one or more aino acids have been added as long as the TNF binding part still binds TNF and the 30 immlmAglAh~ n parts shows one or more of its characteristic properties. In case the immllnAglAhlllin part consists of all domains including the hinge region ercept the first domain of the constant region of the heavy chain such hinge region may lack the first five N-terminal a_ino acids. Such chimaeric TNF binding protein muteins can be prepared by methods known in the art 35 and described e.g. by Sambrook et al. in "MA1 l~r Cloning" (second edition 1989, Cold Spring Harbor Laboratory Press, New York) or in one or more of the above tiAnPd patent pllh~ slt;AAO
WO 96/03141 ;~ 7 9 ~ 6 6 5 P~. I~r. _.
~5-FLLLiL~, IU~ the .1.:,..r . :r TNF binding protein of the present invention can be also used in modified form, e.g., when coupled to chemical entities without altering its basic biological activity. A preferred and well known mn~ifirAAtinn is the coupling to water soluble polymers, e.g., 5 pol.~ hi~ glycols or pol.yl,Lu~lu.-e glycols, within a wide range of mn1 ' weight of, e.g., from 500 to 20'000 daltons. This leads to protected proteins, which could be ~ 'ally non~ Several modes of coupling the polymer to the protein via different linkers are available in the state of the art and de~rrihe~l~ e.g., in general in 'I'~ c_L~. in o R~ j."~_L~ Chemistry", edt. by C.F. Meares, AmPrirAn Chemical Society, Woc~ingtnn 1993, and Ope~firAlly, e.g., in U.S. Patent No. 4,179,337.
Furthermore the rhimAPr r TNF binding proteins of the present invention can be in the form of a pl. . ",A ~ 1 'AAlly ~ r ' -L1A salt. Salta of a carboyl group may be formed by means known in the art and include inorganic salts, for e~ample, sodium, calcium, A- .. ;.. l.. , ferric or zinc salts, and the like, and salts with organic bases as those formed, for e 2ample, with amine~, such as t.riPthAnn1AminP arginine or lysine, rirPriAinP procaine and the like. Acid addition salts include, for e~cample, salts with mineral acids such as, for e~ample, hydrochloric acid or sulfi~ic acid and salts with organic acids such as, for e~ample, acetic acid or o~alic acid.
The . ' ~ - - TNF binding protein to be used for the purpose of the present invention is preferably a~lminic' ~d pa~ LI,_.dlly by i~ection, although other effective L 1~ ;F l ~Liu~l forms, such as h.~,~. L.,ular 25 inJection, or tr~qnAApTnAsl iu..~hu~ ;~ s,re also possible. One preferred carrier is physioloLical saline solution, but it is ~ tPd that other rl.,.. rA.. It;AA11y _A~Aprtqh1_ carriers may also be used. The primary solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other ~ e 1~ 11y qrrppfOh1~
.rip: for ~..odifj.. g or mqintsinine the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of rtiAAr.111tinn1 or odor of the fnrmn1qtiAn Similarly, the carrier may contain still other ph~ - 1~Agirq11y ArrPptsh1P
P~rir ' for ~..ulir.y g or ~ ~h~l ~h~ E the stability, rate of dissolution, release, or absorption of the . h;l..r.l . ;r TNF binding protein. Such ~
A~5 are those s-lhA~qnrpA usually and ~ ~- ..,.. ily employed to formulate dosages for parental administration in either unit dose or multi-dose form.
wo 96/03141 2 1 9 5 6 6 ~ r~ L . ~oo Once the tht~ euLc rAmr~r;t;~n has been frrmnlPtP~1 it mPy be stored in sterile vials as a solution, sllrpPnC;~n~ gel, Pmlllc;~n, solid, or in deh~dLul~d form, e g. as Iyophilized powder. Such fflrmlllPtion~ may be stored either in a ready to use form or a form requiring le~ 1 I.--I.;r,n 5 ;"..,.P~ .ly prior to a~ ,L;~Lull. The preferred storage of such fnrm~ t;~nf. is at k U~ LI.u1~,3 at least as low as 4~C and preferably at -70~C. It is also preferred that such form~lPt;r~nA are stored and P.l...;..;~ ~d at or near physiological pH.
A possible dosage range for the lLt:~Lut:..l, of multiple sclerosis may be between about 0.001-5.0, preferably 0.1-5.0, more preferably 0.1-2.0 mg per kg of patient body weight per 1-4 weeks a-l ..h.;~ d in a single dose and most preferably between about 0.1-2.0 mg per kg of patient body per 1-4 weeks and rrlminir' ad in a single dose of a rhim~Prir TNF binding protein which C~ r~ a soluble part of the human p55-TNF-receptor and all or parts of 15 the constant domains of the heavy or light chain of human immnnrglrhulin or a rh~....~- c,.l .rr~lly ~rCPp~s~hl~ salt thereof, preferably wherein such rhimPPrir TNF binding protein r~mrriRPr~ all domains except the first domain of the constant region of the heavy chain of human .. ....~. Inhlllin such as Ig&, IgA, IgM or IgE and most ~ .bl~ wherein the human 20 imm..nfgloh.llin is IgG, especially IgGl or IgG3. The Le(lueu~ ~ of dosing and the optimal dose will depend on rh~ ;rfL;ll?t;C parameters of the rhimPPrir TNF binding protein in the frrmlllPtifn used.
Regardless of the manner of aduliLu~L~Lion, the specific dose is rJ~lrnl~tPd according to the ~ylv~. u~.I,e body weight of the patient. Further ~i rPfinPmPn~ of the rPlr~ tirnr. nec&.,~ to rl_l.~....;~.P appropriate dosage for ~Le~LI,Luelll involving each of the above mPntirnpd fr,rmnlPt-/mq is routinely made by the man skilled in the art.
F,~mnles F.Y~m~le I
30 ArntP EA~ mrrlPl (~/R r~tr) Female AEIH/R rats [Bloxham et al., J. Pharmacol. Exp. Ther. ~2, 1331-1340 (1990)] were inrclll~tPd with emulsion (0.05 ml) r----~ g g unea pig spinal cord and Freunds Complete Adjuvant (1:1) r....l:~.";"g Mycobacl~.i~
tuberculosis (Difco Laboratories, Detroit, MI, USA) (lOmg/ml) into the 3s planter surface of both hind paws. Animals were weighed and evaluated WO9G/03141 i~ ~ 95 ~ 65 PCT11~95102788 daily for n~ 1 sign~ a, L~g to a 4-point score system (clinical score): 0=normal; 1=flaccid tail; 2=parti~l hind limb paralysis; 3=full hind limb p~,.l~ ~, 4 L ' , ~ ~ ~ Groups of rats were dosed with a construct of the e- L~ ular dornain of the human p55-TNF-receptor (first 182 N-terminal amino acids of the receptor) fused to the hinge region of human IgG~1 heavy chain and e~ sJed in CHO-cells ("fusion construct") (i.p.) or with rh~ h-l~ buffered saline (PBS) vehicle only. The ~ ' ~ ~ ' ~Liul. of the PncPFhnlit~lEpnir r.lcrDn-;r.n in rats resulted in an onset of neurological ~yluluL4 .8 (day 10) which s lhseq~Pn~ly peaked on day 14. The neurological lû D.y~ JLulus were c~ u '~ ;Pd by a cignjfirnnt weight 1088. Treatment with the fusion construct (1, 5, and 10 mglkg, i.p., dosed daily, post-induction of EAE, days 8-14), cignifirnn~ly reduced the s.~...l Loll~b of EAE and the c_angesin body weights in a dose d~ manner [Figure~shows the mean clinical score values of 10 animals in each group from day 10 to 21 and Figure 2 shows the Wl l - ~ h ~g mean body weight from day 8 to 20; p-values were ~ d by the Mann-Wbitney-U-test (see s~otic~nl Methods in Biology, Bailey N.TJ., Hodder and .5~ngh~n, London)]. In a second series of ~ .L~ the fusion construct (10 mg/kg, i.p.) ~ ' ' ed post-induction Lluuu~sLuuL the disease "window" (days 8-20) ag~un cignifirnntly 2~ reduced t_e ~ uLullls of EAE and the changes in body weight up to day 14 [Figure~ shows the mean clinical score values of 10 animals from day 10 to 20 with p-values ~ , ...;..P~ by the Mamrl-Whitney-U-test, s.a. and Figure 4 shows the . ull.-~ l;..g mean body weight with p-values fi.~ ..Pd by the unpaired-T-test (see S1~h~~;~nl Methods in Biology, s.a.)]
rs3mnlP~ H
Chronir~ li',A~: m~8~1 (Bir77i mir~) Inbred Biozzi Selection I AB/H mice [Baker et al., J. Nt ul~ :.. 1 ) ~, 261-270 (1990)] were mnin~oinPd on standard pelleted diet and water ad libitum. Male mice were inr c~ tPd s~ . .PU " 1Y on the abdomen in two 30 sites with 0.3 ml (total 0.61) of an emulsion ~ ,.,i.~;";"g 6.6 ~/ml freeze-dried Biozzi spinal cord in ph.~crhnt.~-buffered saline (PBS) and 60 mg heat-killed IU,~- UbJ~ ~-. ;A [Mycobacterium tuberculosis H37Ra and M. butyricum (8:1); Difco, s.a.] in 0.15 ml of Freund's im , 1 adjuvant (Baker et al., s.a.). The inJections were repeated 7 days later. Animals were weighed and 35 evaluated daily for neu~ girnl signs according to a 5-point score system:
0=normal; l=limp tail; 2_ilul~il td righting refle~ (IRR); 3=partial hind limb paralysis, 4=crnnpl~~p hind limb paralysis; 5= death. Groups of mice were WO 96/03141 ;~ 1 9 ~! 6 6' 5 r~ oo -8- . --doaed v.~ith the fusion construct (see E~ample I) (i.p.) or l~hr~ buffered saline (PBS) vehicle. In the chronic EAE model, the Biozzi mice, the incidence of acute disease in the vehicle group was 90 %, witb n~ , ' 7 DyLu~l u~8 ~l~,u~ E on day 14 post-induction of EAE. Pl~lLe ' ' with the 5 fusion construct (7 mg/kg, i.p., dosed every 3 days IL~uu L~uL the P~l..-..1....l..l protocol), reduced the number of animala P~hihitinE paralysis (Nos. 1-4 in 5-point score system) during both the acute phase (3/9) and relapse phase (V7) of the disease as compared to the controls (9/10 and 6/8, e..Li~ . ly ,) [see Table 1 and Figure 5 which shovvs in Figure 5a the control "uell~ only with pure saline solution ("vehicle") and in Figure 5b the aituation with the fuaion conatruct wherein the bara give the clinical score values and the curve ia an indicator for the change in body weight (g)]. In a subsequent dose response study where the fusion construct (1-14 mg/kg, i.p.) was p h ; ~ . t d post-induction of the disease (days 10-30) beneficial effect was alsû d~ o~lD~L~.lad~ 7 mg/kg being the ~--;-.; .. . effective dose.
h~umrle ITT
GenPrPl mPthn~l~
Animals, reagents, and cell lines Inbred Lewis rats, 120 to 150 g in weight, were obtained from Charles RIVER WIGA (Sulzfeld, BRD). Guinea pig myelin basic protein (MBP) was isolated from whole brains [Eylar et al., J. Methods Enzymol. ~2~, 323 (1979)]. C1, the Pnrpphuli~ûgpnir peptide of MBP (amino acids 68-88) was 2~ purchaaed from Peptide Products (Porton Down, United Kingdom). Bovine S100~ protein waa obtained from Sigma Chemical Co. (St. Louia, MO, USA).
Protein purified d~Liv~lliv~: from M. tuberculosia (PPD) was obtained from Statens Ser--minPI;t~lt (Kf~pPnhu~Pn, De=rk). A mnnnrk~nul antibody against myelin r~ o~lPn~rûcyte ~ ,~hl [anti-MOG Mab 8-18C5, 30 S~hlii~PnPr et al., J. Immunol. ~, 4016 (1987)] was produced from hybridoma al~pprnutunto and purified on protein A ~IJL~ose beada (Sigma).
~ 9 Tmmnni~tinn Lewis rats were i~--- -...-;i~ into the hind footpads with either MBP
(100 ~rat), Cl (50 ~glrat), or S100,B protein (50 ~glrat) Pm~llP ~'iPd in Freund's adjuvant (Gibco BRL AG, Basle, CH) ~ with 4 mg/ml 5 M. tuberculosis (H37Ra, Difco, Detroit, IJSA).
Antigen-specific T cells T cell lines used in tbis study were specific for guinea pig MBP and/or Cl (F8, C1-C9, C1, LMBP) or bovine S100~ protein, a non-myelin, calcium binding protein (LS1). Two different protocols were used to estabhsh antigen-specific T cell lines. Ten day6 after immllnip~ a single cell sn~pPn~irn was prepared from the draining popliteal, inguinal, and p . _ lic lymph nodes. The primed cells were either cultured at a ~ 1"c.L.,.. of 107 cells/l (51, petri dish) and antigen (10 ~g/ml) to establish bulk T cell lines (LS1, C1, LMBP) or all~.L~Li~ly serially diluted to provide a large number of psnlrirlnnsll T line cellg (F8, C1-C9). In the latter case, lymph node cell g-.~pPn~irnD. were cultured in 96-well round-bottom l~ ..v~il~. plates at cell numbers ranging from 2 x 105 to 100 cells/well. The cells were snrpl~-nPntPd with antigen and i..~.di~l,ed (4,000 rad) Dy~ll~c thymus cells as source of antigen presenting cells (APC's) to give a final cell number of 2 x 105/well in 20 Eagle's medium OEA) enriched with L-as~ ;ill (36 g/ml), L-~1..1,..";,.~P (2mM), sodium pyruvate (1 mM), nrnP~Pnt ol ~minn~ri~l~ (1% v/v), penicillin (100 U/l), D~ 00 mg/ml; Gibco) and ~frlo~ P rat serum (l ~o).
After 72 hrs the medium was removed and the l~ ll~illg T cells expanded for further 7-10 days in media s~ppl~-n~ntpd with IL-2. The T cells were then ~5 ,~I;I,.l,l-led with antigen in the presence of 2 x 105 irradiated APC's. After visual inp~pect;~ of the cultures, those positive for growth were expanded in the presence of IL 2. Activated blastoid l~ Les b' ~ d in bulk cultures were collected from the interface of Lymphoprep-gradients (density 1.077 g/ml, Nycomed Pharma AS, Oslo, Norway). The resting T cells were 30 then re~iml~ P.1 in the presence of irradiated APC's (T cell to thymus cell ratio: 1:30). The cycle of prop~g~t;~m in the IL-2 .,....I..;..;..g medium and r~~fim--l~t;~.n with antigen plus APC's was then repeated until stable T cell lines were e-hlhli~hP-l WO96/03141 21~566~ r~ ,0O
-10- '--5p~ Iy assay To evaluate T cell ,~o~i.;i~,y a standard T cell proliferation test was used. T
cells (2 ~104/ml) were rc~;mlllAt~d in flat-bottom mi-,~u~ ~3 (200 ~lg/well) in the presence of APC's (6 ~c 1051ml) and the relevant antigens 5 MBP, C1 or S100~ (20 ~11), the non relevarlt antigen PPD (10 llgll) and ConA (2.5 ~g/l). After 72 hrs of culture the cells were L~ ~. ~ following a 16 hrs pulse with 1 ~LCilwell tritiated thy_idine (3H-dT; 2 Ci/mmol;
Amf~hAm-Buchler, Braunschweig, BRD). The rD~ oh~ d cells were collected on glassfiber filters and the radioactivity - - atPd with the ~D washed and dried filters was q ~ntifi~d with a Matri~c 96 counter (Canberra-Packard, Frankfurt, BRD).
Active EAE
Active EAE was induced in rats following ~ n with MBP: or C1.
The weight and clinical score were .ù~ d on a daily basis by clinical 15 ~ C~ l: . Clinical disease was evaluated as fo~lows: 0 = normal, 1 = tail limpness, 2 = pala~ei~;s with clumsy gait, 3 = hind limb paralysis, 4 = hind and fore limb paralysis, 5 = death. The study was done in a blinded fashion.
That is, the observer was unaware of the protocol.
Passive EAE
Passive EAE was induced in byll~ ,;C rats foUowing i~ Ally iruection of freshly antigen-activated T cell blasts (5-7 x 106/rat). If S100~
specific T cells were used to induce EAE the rats received 107 cells followed by a single dose of an Mab 8-18C5 (3-5 mglrat) at day five after the cell transfer. The weight and clinical score was monitored daily as described ~5 above. At a~lu~;ald time points animals were sacrificed and used for conventional central and peripheral nerve histology.
Treatment of EAE
Activ or passively induced EAE was treated by repeated or single i--l,lAI~ oAl iIuections with 10 mg/kg of the fusion construct (see 30 E~ample I).
FACS-Analysis Mouse anti-rat T cell-specific mnn~-~lAn~l An~ho/h~ (MAb) pan-T (W 3.13), CD4 (W 3.25), CD8 (OX-8), a,~TcR [R73, Hunig et al., J. E~p. Med. lfia 73 21 '~66~
WO 96103141 r~ ,OO
(1989)] were purified from L.~ ;du~ r ~ on protein C ~~ u~,e beads (Sigma, Ta~il. Le.~, BRD) ûr ~u~.La32d from Camon (Wiesbaden, BRD). For TcR V~ ib~ y~ Je~iC MAbs B73 (V 8.5), G101 (V~ 10), and R78 (V~ 8.2) see Tûrres-Nagel, T.. n~ I. 305 (1993). Labelling of 6 specific MAbs was detected using a DTAF-labelled F (ab')2 fragment of goat anti-mûuse IgG (H+L chain). T.. --- - .n~ Arescence of viable cells e~luL~g iOdl~e (10 llg/ml, Sigma) was -- ~,d with a FACScan (Becton Dickinson, Heidelberg, BRD).
T~ rnnle m 1 T'ffPrt nf thP filPinn nnnP~uct on active ~ue~ uk,;l.lllll.. lr enrPnhqlnmvPlih~c (~,AP',) This model cûvers a relative broad spectrum of the pathogenesis including activation of ~uLu~~ ve T cells in vivo and variûus infl~..... l,l~.y stages of the disease within the central nervûus system. However the disease is normally limited orly to perivascular infiltrPt;~nc and not qooor;qt~d with primary large scale delll~eli~Liûn.
Groups of Lewis rats (n = 3) were ;.------~ d with C1 peptide which span the PnrDphqlitA~gPnir domain of guinea pig MBP for Lewis rats from ~3nnin ~ ' 68-88. All rats gained weight for the following 8 days (Fig. 6), followed then by fl~ttening of their weight curve bP~ e on day g.
Beginning day 9 the control, nûn-fusion construct treated group exhibited rapid weight lûss, developed tail limpness followed by distinct hind-limb weakness and rougbing of the fur. A partial lûss of the righting reflex and inrnntinPnrP wag also observed. These clinical signs of disease lasted until ~; day 16 when the rats began to recover and to regain weight. Those rats which received h~ e.;Lu.leal injec~ nP of fusion construct showed a fl qt~Pning of their weight curve starting on day 9 but the ~l ~..L.Il~.IL resulted in a dramatic reduction in the severity of clinical ~y~ u8. Overall they behaved clinically normal. Only a slight partial 1088 of tail tonicity was 30 observed which started at day 13 and lasted for 2-3 days with no further increase.
T~'Y~mnle ITT ~
T~'ffPct of thP filcinn ~nnctruct on T rPll mP~i~tPd tr~ncfpr EAT~' (tTb'AP') The basis of this model are T lymphocytes recovered from MBP or MBP-36 peptide ;.. - .. ;-- d rats. The cells are es~qhlichPd and .. ~h~ pd in vitro .. .
WO 96/03141 2 1 q ~ 6 ~ ~ P~.l/L~ . ~'A7788 as antigen specific linea They are ~ d a~ CD4+, CD8-, TcR V,B8.2+, MHC-class II .~ ' ' T cells which are able to predictively and ~lucibly induce EAE upon transfer in vivo into normal naive 1~4;~ _ .L~.
Similar as in the active model tEAE is not ~.~~.;~.~ d by dèmyelination 6 but focuses on the recirculation and inflA~ effector phase of EAE
including I thrE~nir interactions between the T cells and the blood brain barrier.
Groups of Lewis rats (n = 3) were passively l-~ . . e d with Cl-specific T
cells (C1-C9) iL1i-0~ ly~ The control group which was not treated with lo the fusion construct (Fig 7) developed typical severe signs of tEAE
a~ r~ d with severe'~veight 1088 bG~ ;..g at day 2 after cell transfer.
The onset of clinical signs began rapidly after day 2 and resemble those of active EAE. The rats recovered around day 6. Those rats which were treated with the fusion construct at three different time points showed only a slight ~5 weight 1088 starting at day 3. However, the clinical signs were greatly reduced to only partial 1088 of trail tonicity which was observed on day 3-6.
mpl P HT ?l F,ffr~rt of t.hP fuo;nn rnnctrnrt. nn trrncfPr ~W~ l.l.l.;l.. llP
h A l n m yel i ti c ( t F. A P ) 21) In thAis model S100~specific T cells derived from ;I l..l~ 1 rats mediate inflA.I..~lAL~l ~ lesions throughout the entire CNS, spinal cord, optic nerve, retina and uvea. Moreover, like in human multiple sclerosis (MS), the eyes are commonly affected showing retinal pPrirhlPhitiC Cellular infiltrates are observed perivascular but also in the surrounding pa~ y~ but, 2Ei generally, there are fewer ED1+ ,...qc~",hagl,d present as compared to MBP
induced EAE. Only mild clinical signs such as weight 1088 can be observed,.
A clinically more severe status appears after injection of Mab 8-18C6 [Linington et al., J. Immunol. 2;~, 1364 (1993)]. ~ictrl-'girAlly large confluent foci of demyelination arranged around the infiltrates are present bridging 30 this model to certain cased of human MS.
Groups of Lewis rats (n = 3) were passively trandferred with S100~ protein specific T cells (LS1). These cells induce a severe inflA1..l..Ai..ly response in the nervous system which, but only minimal n~ , r dysfunction in naive syngeneic ., , ' (Fig. B., #1-3). However, clinical signs became ouvert 36 when rat,s (Fig. 8, #~6) received anti-MOG rnAnnrltmAl AntihorliPo at day 6 after the cell transfer. All rats exhibited a complete 1088 of tail tonicity WO96/03141 '2 ~ 665 r-llr~r lasting 48 brs. In contrast, rats w~ich in addition to ~aab 8-18C5 al~o received the filsion construct did not show any clinical ~ ~ and behaved cr~r~ , normal (Fig 8, #7-9).
~; The term chimslPrir TNF binding protein as used IL-uu~Luu~ the ~lJP. .~rs~ :An . uLul,l;ses also such proteins in which any amino acid of the ;..... ~-~lrhlllin part or the TNF binding part has been deleted or sllhet;t -tPd by one or more amino acids or one or more aino acids have been added as long as the TNF binding part still binds TNF and the 30 immlmAglAh~ n parts shows one or more of its characteristic properties. In case the immllnAglAhlllin part consists of all domains including the hinge region ercept the first domain of the constant region of the heavy chain such hinge region may lack the first five N-terminal a_ino acids. Such chimaeric TNF binding protein muteins can be prepared by methods known in the art 35 and described e.g. by Sambrook et al. in "MA1 l~r Cloning" (second edition 1989, Cold Spring Harbor Laboratory Press, New York) or in one or more of the above tiAnPd patent pllh~ slt;AAO
WO 96/03141 ;~ 7 9 ~ 6 6 5 P~. I~r. _.
~5-FLLLiL~, IU~ the .1.:,..r . :r TNF binding protein of the present invention can be also used in modified form, e.g., when coupled to chemical entities without altering its basic biological activity. A preferred and well known mn~ifirAAtinn is the coupling to water soluble polymers, e.g., 5 pol.~ hi~ glycols or pol.yl,Lu~lu.-e glycols, within a wide range of mn1 ' weight of, e.g., from 500 to 20'000 daltons. This leads to protected proteins, which could be ~ 'ally non~ Several modes of coupling the polymer to the protein via different linkers are available in the state of the art and de~rrihe~l~ e.g., in general in 'I'~ c_L~. in o R~ j."~_L~ Chemistry", edt. by C.F. Meares, AmPrirAn Chemical Society, Woc~ingtnn 1993, and Ope~firAlly, e.g., in U.S. Patent No. 4,179,337.
Furthermore the rhimAPr r TNF binding proteins of the present invention can be in the form of a pl. . ",A ~ 1 'AAlly ~ r ' -L1A salt. Salta of a carboyl group may be formed by means known in the art and include inorganic salts, for e~ample, sodium, calcium, A- .. ;.. l.. , ferric or zinc salts, and the like, and salts with organic bases as those formed, for e 2ample, with amine~, such as t.riPthAnn1AminP arginine or lysine, rirPriAinP procaine and the like. Acid addition salts include, for e~cample, salts with mineral acids such as, for e~ample, hydrochloric acid or sulfi~ic acid and salts with organic acids such as, for e~ample, acetic acid or o~alic acid.
The . ' ~ - - TNF binding protein to be used for the purpose of the present invention is preferably a~lminic' ~d pa~ LI,_.dlly by i~ection, although other effective L 1~ ;F l ~Liu~l forms, such as h.~,~. L.,ular 25 inJection, or tr~qnAApTnAsl iu..~hu~ ;~ s,re also possible. One preferred carrier is physioloLical saline solution, but it is ~ tPd that other rl.,.. rA.. It;AA11y _A~Aprtqh1_ carriers may also be used. The primary solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other ~ e 1~ 11y qrrppfOh1~
.rip: for ~..odifj.. g or mqintsinine the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of rtiAAr.111tinn1 or odor of the fnrmn1qtiAn Similarly, the carrier may contain still other ph~ - 1~Agirq11y ArrPptsh1P
P~rir ' for ~..ulir.y g or ~ ~h~l ~h~ E the stability, rate of dissolution, release, or absorption of the . h;l..r.l . ;r TNF binding protein. Such ~
A~5 are those s-lhA~qnrpA usually and ~ ~- ..,.. ily employed to formulate dosages for parental administration in either unit dose or multi-dose form.
wo 96/03141 2 1 9 5 6 6 ~ r~ L . ~oo Once the tht~ euLc rAmr~r;t;~n has been frrmnlPtP~1 it mPy be stored in sterile vials as a solution, sllrpPnC;~n~ gel, Pmlllc;~n, solid, or in deh~dLul~d form, e g. as Iyophilized powder. Such fflrmlllPtion~ may be stored either in a ready to use form or a form requiring le~ 1 I.--I.;r,n 5 ;"..,.P~ .ly prior to a~ ,L;~Lull. The preferred storage of such fnrm~ t;~nf. is at k U~ LI.u1~,3 at least as low as 4~C and preferably at -70~C. It is also preferred that such form~lPt;r~nA are stored and P.l...;..;~ ~d at or near physiological pH.
A possible dosage range for the lLt:~Lut:..l, of multiple sclerosis may be between about 0.001-5.0, preferably 0.1-5.0, more preferably 0.1-2.0 mg per kg of patient body weight per 1-4 weeks a-l ..h.;~ d in a single dose and most preferably between about 0.1-2.0 mg per kg of patient body per 1-4 weeks and rrlminir' ad in a single dose of a rhim~Prir TNF binding protein which C~ r~ a soluble part of the human p55-TNF-receptor and all or parts of 15 the constant domains of the heavy or light chain of human immnnrglrhulin or a rh~....~- c,.l .rr~lly ~rCPp~s~hl~ salt thereof, preferably wherein such rhimPPrir TNF binding protein r~mrriRPr~ all domains except the first domain of the constant region of the heavy chain of human .. ....~. Inhlllin such as Ig&, IgA, IgM or IgE and most ~ .bl~ wherein the human 20 imm..nfgloh.llin is IgG, especially IgGl or IgG3. The Le(lueu~ ~ of dosing and the optimal dose will depend on rh~ ;rfL;ll?t;C parameters of the rhimPPrir TNF binding protein in the frrmlllPtifn used.
Regardless of the manner of aduliLu~L~Lion, the specific dose is rJ~lrnl~tPd according to the ~ylv~. u~.I,e body weight of the patient. Further ~i rPfinPmPn~ of the rPlr~ tirnr. nec&.,~ to rl_l.~....;~.P appropriate dosage for ~Le~LI,Luelll involving each of the above mPntirnpd fr,rmnlPt-/mq is routinely made by the man skilled in the art.
F,~mnles F.Y~m~le I
30 ArntP EA~ mrrlPl (~/R r~tr) Female AEIH/R rats [Bloxham et al., J. Pharmacol. Exp. Ther. ~2, 1331-1340 (1990)] were inrclll~tPd with emulsion (0.05 ml) r----~ g g unea pig spinal cord and Freunds Complete Adjuvant (1:1) r....l:~.";"g Mycobacl~.i~
tuberculosis (Difco Laboratories, Detroit, MI, USA) (lOmg/ml) into the 3s planter surface of both hind paws. Animals were weighed and evaluated WO9G/03141 i~ ~ 95 ~ 65 PCT11~95102788 daily for n~ 1 sign~ a, L~g to a 4-point score system (clinical score): 0=normal; 1=flaccid tail; 2=parti~l hind limb paralysis; 3=full hind limb p~,.l~ ~, 4 L ' , ~ ~ ~ Groups of rats were dosed with a construct of the e- L~ ular dornain of the human p55-TNF-receptor (first 182 N-terminal amino acids of the receptor) fused to the hinge region of human IgG~1 heavy chain and e~ sJed in CHO-cells ("fusion construct") (i.p.) or with rh~ h-l~ buffered saline (PBS) vehicle only. The ~ ' ~ ~ ' ~Liul. of the PncPFhnlit~lEpnir r.lcrDn-;r.n in rats resulted in an onset of neurological ~yluluL4 .8 (day 10) which s lhseq~Pn~ly peaked on day 14. The neurological lû D.y~ JLulus were c~ u '~ ;Pd by a cignjfirnnt weight 1088. Treatment with the fusion construct (1, 5, and 10 mglkg, i.p., dosed daily, post-induction of EAE, days 8-14), cignifirnn~ly reduced the s.~...l Loll~b of EAE and the c_angesin body weights in a dose d~ manner [Figure~shows the mean clinical score values of 10 animals in each group from day 10 to 21 and Figure 2 shows the Wl l - ~ h ~g mean body weight from day 8 to 20; p-values were ~ d by the Mann-Wbitney-U-test (see s~otic~nl Methods in Biology, Bailey N.TJ., Hodder and .5~ngh~n, London)]. In a second series of ~ .L~ the fusion construct (10 mg/kg, i.p.) ~ ' ' ed post-induction Lluuu~sLuuL the disease "window" (days 8-20) ag~un cignifirnntly 2~ reduced t_e ~ uLullls of EAE and the changes in body weight up to day 14 [Figure~ shows the mean clinical score values of 10 animals from day 10 to 20 with p-values ~ , ...;..P~ by the Mamrl-Whitney-U-test, s.a. and Figure 4 shows the . ull.-~ l;..g mean body weight with p-values fi.~ ..Pd by the unpaired-T-test (see S1~h~~;~nl Methods in Biology, s.a.)]
rs3mnlP~ H
Chronir~ li',A~: m~8~1 (Bir77i mir~) Inbred Biozzi Selection I AB/H mice [Baker et al., J. Nt ul~ :.. 1 ) ~, 261-270 (1990)] were mnin~oinPd on standard pelleted diet and water ad libitum. Male mice were inr c~ tPd s~ . .PU " 1Y on the abdomen in two 30 sites with 0.3 ml (total 0.61) of an emulsion ~ ,.,i.~;";"g 6.6 ~/ml freeze-dried Biozzi spinal cord in ph.~crhnt.~-buffered saline (PBS) and 60 mg heat-killed IU,~- UbJ~ ~-. ;A [Mycobacterium tuberculosis H37Ra and M. butyricum (8:1); Difco, s.a.] in 0.15 ml of Freund's im , 1 adjuvant (Baker et al., s.a.). The inJections were repeated 7 days later. Animals were weighed and 35 evaluated daily for neu~ girnl signs according to a 5-point score system:
0=normal; l=limp tail; 2_ilul~il td righting refle~ (IRR); 3=partial hind limb paralysis, 4=crnnpl~~p hind limb paralysis; 5= death. Groups of mice were WO 96/03141 ;~ 1 9 ~! 6 6' 5 r~ oo -8- . --doaed v.~ith the fusion construct (see E~ample I) (i.p.) or l~hr~ buffered saline (PBS) vehicle. In the chronic EAE model, the Biozzi mice, the incidence of acute disease in the vehicle group was 90 %, witb n~ , ' 7 DyLu~l u~8 ~l~,u~ E on day 14 post-induction of EAE. Pl~lLe ' ' with the 5 fusion construct (7 mg/kg, i.p., dosed every 3 days IL~uu L~uL the P~l..-..1....l..l protocol), reduced the number of animala P~hihitinE paralysis (Nos. 1-4 in 5-point score system) during both the acute phase (3/9) and relapse phase (V7) of the disease as compared to the controls (9/10 and 6/8, e..Li~ . ly ,) [see Table 1 and Figure 5 which shovvs in Figure 5a the control "uell~ only with pure saline solution ("vehicle") and in Figure 5b the aituation with the fuaion conatruct wherein the bara give the clinical score values and the curve ia an indicator for the change in body weight (g)]. In a subsequent dose response study where the fusion construct (1-14 mg/kg, i.p.) was p h ; ~ . t d post-induction of the disease (days 10-30) beneficial effect was alsû d~ o~lD~L~.lad~ 7 mg/kg being the ~--;-.; .. . effective dose.
h~umrle ITT
GenPrPl mPthn~l~
Animals, reagents, and cell lines Inbred Lewis rats, 120 to 150 g in weight, were obtained from Charles RIVER WIGA (Sulzfeld, BRD). Guinea pig myelin basic protein (MBP) was isolated from whole brains [Eylar et al., J. Methods Enzymol. ~2~, 323 (1979)]. C1, the Pnrpphuli~ûgpnir peptide of MBP (amino acids 68-88) was 2~ purchaaed from Peptide Products (Porton Down, United Kingdom). Bovine S100~ protein waa obtained from Sigma Chemical Co. (St. Louia, MO, USA).
Protein purified d~Liv~lliv~: from M. tuberculosia (PPD) was obtained from Statens Ser--minPI;t~lt (Kf~pPnhu~Pn, De=rk). A mnnnrk~nul antibody against myelin r~ o~lPn~rûcyte ~ ,~hl [anti-MOG Mab 8-18C5, 30 S~hlii~PnPr et al., J. Immunol. ~, 4016 (1987)] was produced from hybridoma al~pprnutunto and purified on protein A ~IJL~ose beada (Sigma).
~ 9 Tmmnni~tinn Lewis rats were i~--- -...-;i~ into the hind footpads with either MBP
(100 ~rat), Cl (50 ~glrat), or S100,B protein (50 ~glrat) Pm~llP ~'iPd in Freund's adjuvant (Gibco BRL AG, Basle, CH) ~ with 4 mg/ml 5 M. tuberculosis (H37Ra, Difco, Detroit, IJSA).
Antigen-specific T cells T cell lines used in tbis study were specific for guinea pig MBP and/or Cl (F8, C1-C9, C1, LMBP) or bovine S100~ protein, a non-myelin, calcium binding protein (LS1). Two different protocols were used to estabhsh antigen-specific T cell lines. Ten day6 after immllnip~ a single cell sn~pPn~irn was prepared from the draining popliteal, inguinal, and p . _ lic lymph nodes. The primed cells were either cultured at a ~ 1"c.L.,.. of 107 cells/l (51, petri dish) and antigen (10 ~g/ml) to establish bulk T cell lines (LS1, C1, LMBP) or all~.L~Li~ly serially diluted to provide a large number of psnlrirlnnsll T line cellg (F8, C1-C9). In the latter case, lymph node cell g-.~pPn~irnD. were cultured in 96-well round-bottom l~ ..v~il~. plates at cell numbers ranging from 2 x 105 to 100 cells/well. The cells were snrpl~-nPntPd with antigen and i..~.di~l,ed (4,000 rad) Dy~ll~c thymus cells as source of antigen presenting cells (APC's) to give a final cell number of 2 x 105/well in 20 Eagle's medium OEA) enriched with L-as~ ;ill (36 g/ml), L-~1..1,..";,.~P (2mM), sodium pyruvate (1 mM), nrnP~Pnt ol ~minn~ri~l~ (1% v/v), penicillin (100 U/l), D~ 00 mg/ml; Gibco) and ~frlo~ P rat serum (l ~o).
After 72 hrs the medium was removed and the l~ ll~illg T cells expanded for further 7-10 days in media s~ppl~-n~ntpd with IL-2. The T cells were then ~5 ,~I;I,.l,l-led with antigen in the presence of 2 x 105 irradiated APC's. After visual inp~pect;~ of the cultures, those positive for growth were expanded in the presence of IL 2. Activated blastoid l~ Les b' ~ d in bulk cultures were collected from the interface of Lymphoprep-gradients (density 1.077 g/ml, Nycomed Pharma AS, Oslo, Norway). The resting T cells were 30 then re~iml~ P.1 in the presence of irradiated APC's (T cell to thymus cell ratio: 1:30). The cycle of prop~g~t;~m in the IL-2 .,....I..;..;..g medium and r~~fim--l~t;~.n with antigen plus APC's was then repeated until stable T cell lines were e-hlhli~hP-l WO96/03141 21~566~ r~ ,0O
-10- '--5p~ Iy assay To evaluate T cell ,~o~i.;i~,y a standard T cell proliferation test was used. T
cells (2 ~104/ml) were rc~;mlllAt~d in flat-bottom mi-,~u~ ~3 (200 ~lg/well) in the presence of APC's (6 ~c 1051ml) and the relevant antigens 5 MBP, C1 or S100~ (20 ~11), the non relevarlt antigen PPD (10 llgll) and ConA (2.5 ~g/l). After 72 hrs of culture the cells were L~ ~. ~ following a 16 hrs pulse with 1 ~LCilwell tritiated thy_idine (3H-dT; 2 Ci/mmol;
Amf~hAm-Buchler, Braunschweig, BRD). The rD~ oh~ d cells were collected on glassfiber filters and the radioactivity - - atPd with the ~D washed and dried filters was q ~ntifi~d with a Matri~c 96 counter (Canberra-Packard, Frankfurt, BRD).
Active EAE
Active EAE was induced in rats following ~ n with MBP: or C1.
The weight and clinical score were .ù~ d on a daily basis by clinical 15 ~ C~ l: . Clinical disease was evaluated as fo~lows: 0 = normal, 1 = tail limpness, 2 = pala~ei~;s with clumsy gait, 3 = hind limb paralysis, 4 = hind and fore limb paralysis, 5 = death. The study was done in a blinded fashion.
That is, the observer was unaware of the protocol.
Passive EAE
Passive EAE was induced in byll~ ,;C rats foUowing i~ Ally iruection of freshly antigen-activated T cell blasts (5-7 x 106/rat). If S100~
specific T cells were used to induce EAE the rats received 107 cells followed by a single dose of an Mab 8-18C5 (3-5 mglrat) at day five after the cell transfer. The weight and clinical score was monitored daily as described ~5 above. At a~lu~;ald time points animals were sacrificed and used for conventional central and peripheral nerve histology.
Treatment of EAE
Activ or passively induced EAE was treated by repeated or single i--l,lAI~ oAl iIuections with 10 mg/kg of the fusion construct (see 30 E~ample I).
FACS-Analysis Mouse anti-rat T cell-specific mnn~-~lAn~l An~ho/h~ (MAb) pan-T (W 3.13), CD4 (W 3.25), CD8 (OX-8), a,~TcR [R73, Hunig et al., J. E~p. Med. lfia 73 21 '~66~
WO 96103141 r~ ,OO
(1989)] were purified from L.~ ;du~ r ~ on protein C ~~ u~,e beads (Sigma, Ta~il. Le.~, BRD) ûr ~u~.La32d from Camon (Wiesbaden, BRD). For TcR V~ ib~ y~ Je~iC MAbs B73 (V 8.5), G101 (V~ 10), and R78 (V~ 8.2) see Tûrres-Nagel, T.. n~ I. 305 (1993). Labelling of 6 specific MAbs was detected using a DTAF-labelled F (ab')2 fragment of goat anti-mûuse IgG (H+L chain). T.. --- - .n~ Arescence of viable cells e~luL~g iOdl~e (10 llg/ml, Sigma) was -- ~,d with a FACScan (Becton Dickinson, Heidelberg, BRD).
T~ rnnle m 1 T'ffPrt nf thP filPinn nnnP~uct on active ~ue~ uk,;l.lllll.. lr enrPnhqlnmvPlih~c (~,AP',) This model cûvers a relative broad spectrum of the pathogenesis including activation of ~uLu~~ ve T cells in vivo and variûus infl~..... l,l~.y stages of the disease within the central nervûus system. However the disease is normally limited orly to perivascular infiltrPt;~nc and not qooor;qt~d with primary large scale delll~eli~Liûn.
Groups of Lewis rats (n = 3) were ;.------~ d with C1 peptide which span the PnrDphqlitA~gPnir domain of guinea pig MBP for Lewis rats from ~3nnin ~ ' 68-88. All rats gained weight for the following 8 days (Fig. 6), followed then by fl~ttening of their weight curve bP~ e on day g.
Beginning day 9 the control, nûn-fusion construct treated group exhibited rapid weight lûss, developed tail limpness followed by distinct hind-limb weakness and rougbing of the fur. A partial lûss of the righting reflex and inrnntinPnrP wag also observed. These clinical signs of disease lasted until ~; day 16 when the rats began to recover and to regain weight. Those rats which received h~ e.;Lu.leal injec~ nP of fusion construct showed a fl qt~Pning of their weight curve starting on day 9 but the ~l ~..L.Il~.IL resulted in a dramatic reduction in the severity of clinical ~y~ u8. Overall they behaved clinically normal. Only a slight partial 1088 of tail tonicity was 30 observed which started at day 13 and lasted for 2-3 days with no further increase.
T~'Y~mnle ITT ~
T~'ffPct of thP filcinn ~nnctruct on T rPll mP~i~tPd tr~ncfpr EAT~' (tTb'AP') The basis of this model are T lymphocytes recovered from MBP or MBP-36 peptide ;.. - .. ;-- d rats. The cells are es~qhlichPd and .. ~h~ pd in vitro .. .
WO 96/03141 2 1 q ~ 6 ~ ~ P~.l/L~ . ~'A7788 as antigen specific linea They are ~ d a~ CD4+, CD8-, TcR V,B8.2+, MHC-class II .~ ' ' T cells which are able to predictively and ~lucibly induce EAE upon transfer in vivo into normal naive 1~4;~ _ .L~.
Similar as in the active model tEAE is not ~.~~.;~.~ d by dèmyelination 6 but focuses on the recirculation and inflA~ effector phase of EAE
including I thrE~nir interactions between the T cells and the blood brain barrier.
Groups of Lewis rats (n = 3) were passively l-~ . . e d with Cl-specific T
cells (C1-C9) iL1i-0~ ly~ The control group which was not treated with lo the fusion construct (Fig 7) developed typical severe signs of tEAE
a~ r~ d with severe'~veight 1088 bG~ ;..g at day 2 after cell transfer.
The onset of clinical signs began rapidly after day 2 and resemble those of active EAE. The rats recovered around day 6. Those rats which were treated with the fusion construct at three different time points showed only a slight ~5 weight 1088 starting at day 3. However, the clinical signs were greatly reduced to only partial 1088 of trail tonicity which was observed on day 3-6.
mpl P HT ?l F,ffr~rt of t.hP fuo;nn rnnctrnrt. nn trrncfPr ~W~ l.l.l.;l.. llP
h A l n m yel i ti c ( t F. A P ) 21) In thAis model S100~specific T cells derived from ;I l..l~ 1 rats mediate inflA.I..~lAL~l ~ lesions throughout the entire CNS, spinal cord, optic nerve, retina and uvea. Moreover, like in human multiple sclerosis (MS), the eyes are commonly affected showing retinal pPrirhlPhitiC Cellular infiltrates are observed perivascular but also in the surrounding pa~ y~ but, 2Ei generally, there are fewer ED1+ ,...qc~",hagl,d present as compared to MBP
induced EAE. Only mild clinical signs such as weight 1088 can be observed,.
A clinically more severe status appears after injection of Mab 8-18C6 [Linington et al., J. Immunol. 2;~, 1364 (1993)]. ~ictrl-'girAlly large confluent foci of demyelination arranged around the infiltrates are present bridging 30 this model to certain cased of human MS.
Groups of Lewis rats (n = 3) were passively trandferred with S100~ protein specific T cells (LS1). These cells induce a severe inflA1..l..Ai..ly response in the nervous system which, but only minimal n~ , r dysfunction in naive syngeneic ., , ' (Fig. B., #1-3). However, clinical signs became ouvert 36 when rat,s (Fig. 8, #~6) received anti-MOG rnAnnrltmAl AntihorliPo at day 6 after the cell transfer. All rats exhibited a complete 1088 of tail tonicity WO96/03141 '2 ~ 665 r-llr~r lasting 48 brs. In contrast, rats w~ich in addition to ~aab 8-18C5 al~o received the filsion construct did not show any clinical ~ ~ and behaved cr~r~ , normal (Fig 8, #7-9).
Claims (7)
1. Use of a chimaeric TNF binding protein which comprises a soluble part of the human p55- or the p75-TNF-receptor and all or parts of the constant domains of the heavy or light chain of human immunoglobulin or of a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the treatment of autoimmune diseases.
2. Use according to claim 1, wherein the autoimmune disease is multiple sclerosis.
3. Use according to claim 1 or 2, wherein the chimaeric TNF binding protein comprises the soluble part of the human p55-TNF-receptor.
4. Use according to any one of claims 1 to 3, wherein the chimaeric TNF
binding protein comprises all domains except the first domain of the constant region of the heavy chain of human immunoglobulin such as IgG, IgA, IgM or IgE.
binding protein comprises all domains except the first domain of the constant region of the heavy chain of human immunoglobulin such as IgG, IgA, IgM or IgE.
5. Use according to claim 4, wherein the human immunoglobulin is IgG, especially IgG1 or IgG3.
6. A method of treating an autoimmune disease in a mammal comprising administering to said mammal a therapeutically effective amount of a chimaeric TNF binding protein or a salt thereof as defined in any one of claims 1 and 3 to 5 which ameliorates the effects of such autoimmune disease.
7. A method as claimed in claim 6 wherein the autoimmune disease is multiple sclerosis.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94111455.5 | 1994-07-22 | ||
| EP94111455 | 1994-07-22 |
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| EP (1) | EP0772449A1 (en) |
| JP (1) | JPH09508140A (en) |
| AU (1) | AU3111995A (en) |
| BR (1) | BR9508419A (en) |
| CA (1) | CA2195665A1 (en) |
| CZ (1) | CZ283219B6 (en) |
| FI (1) | FI970247A (en) |
| HU (1) | HUT76666A (en) |
| NO (1) | NO970264L (en) |
| PL (1) | PL318501A1 (en) |
| WO (1) | WO1996003141A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6350730B1 (en) | 1994-08-17 | 2002-02-26 | The Rockefeller University | OB polypeptides and modified forms as modulators of body weight |
| US6471956B1 (en) | 1994-08-17 | 2002-10-29 | The Rockefeller University | Ob polypeptides, modified forms and compositions thereto |
| US6001968A (en) | 1994-08-17 | 1999-12-14 | The Rockefeller University | OB polypeptides, modified forms and compositions |
| US6429290B1 (en) | 1994-08-17 | 2002-08-06 | The Rockefeller University | OB polypeptides, modified forms and derivatives |
| US20030040467A1 (en) | 1998-06-15 | 2003-02-27 | Mary Ann Pelleymounter | Ig/ob fusions and uses thereof. |
| US6936439B2 (en) | 1995-11-22 | 2005-08-30 | Amgen Inc. | OB fusion protein compositions and methods |
| CA2253557A1 (en) * | 1996-05-08 | 1997-11-13 | Louis Martin Renzetti | Treatment of asthma with tnfr-ig |
| TW555765B (en) * | 1996-07-09 | 2003-10-01 | Amgen Inc | Low molecular weight soluble tumor necrosis factor type-I and type-II proteins |
| US7070771B1 (en) | 1996-12-09 | 2006-07-04 | Regents Of The University Of California | Methods of expressing chimeric mouse and human CD40 ligand in human CD40+ cells |
| JP2001526632A (en) * | 1996-12-12 | 2001-12-18 | ジェネンテク，インコーポレイテッド | HVEM polypeptides and their uses |
| MXPA01005267A (en) | 1998-11-27 | 2002-04-24 | Synaptics Uk Ltd | Position sensor. |
| MXPA03007563A (en) * | 2001-02-23 | 2003-12-11 | Immunex Corp | Increased recovery of active proteins. |
| WO2003011323A1 (en) * | 2001-07-30 | 2003-02-13 | Genset S.A. | Agonists and antagonists of contabix for use in the treatment of metabolic disorders |
| US7786282B2 (en) | 2001-12-06 | 2010-08-31 | The Regents Of The University Of California | Nucleic acid molecules encoding TNF-α ligand polypeptides having a CD154 domain |
| DK1471974T3 (en) | 2002-02-06 | 2007-12-03 | Ares Trading Sa | Tumor necrosis factor combined with interferon in demyelinating diseases |
| NZ554520A (en) | 2004-10-22 | 2010-03-26 | Amgen Inc | Methods for refolding of recombinant antibodies |
| US11111284B2 (en) | 2014-08-21 | 2021-09-07 | The General Hospital Corporation | Tumor necrosis factor superfamily and TNF-like ligand muteins and methods of preparing |
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| IL98078A0 (en) * | 1991-05-07 | 1992-06-21 | Yeda Res & Dev | Pharmaceutical compositions comprising an anticytokyne |
| DE10399023I2 (en) * | 1989-09-12 | 2006-11-23 | Ahp Mfg B V | TFN-binding proteins |
| GB9015908D0 (en) * | 1990-07-19 | 1990-09-05 | Celltech Ltd | Multivalent immunoglobulin |
| EP0620739A4 (en) * | 1992-09-15 | 1997-01-15 | Immunex Corp | Method of treating tnf-dependent inflammation using tumor necrosis factor antagonists. |
| WO1994008609A1 (en) * | 1992-10-15 | 1994-04-28 | Dana-Farber Cancer Institute, Inc. | TREATMENT OF INSULIN RESISTANCE IN OBESITY LINKED TYPE II DIABETES USING ANTAGONISTS TO TNF-α FUNCTION |
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1995
- 1995-07-15 CZ CZ97193A patent/CZ283219B6/en not_active IP Right Cessation
- 1995-07-15 AU AU31119/95A patent/AU3111995A/en not_active Abandoned
- 1995-07-15 HU HU9700180A patent/HUT76666A/en unknown
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- 1995-07-15 WO PCT/EP1995/002788 patent/WO1996003141A1/en not_active Application Discontinuation
- 1995-07-15 EP EP95926902A patent/EP0772449A1/en not_active Withdrawn
- 1995-07-15 PL PL95318501A patent/PL318501A1/en unknown
- 1995-07-15 JP JP8505421A patent/JPH09508140A/en active Pending
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| HUT76666A (en) | 1997-10-28 |
| WO1996003141A1 (en) | 1996-02-08 |
| BR9508419A (en) | 1997-11-18 |
| EP0772449A1 (en) | 1997-05-14 |
| NO970264L (en) | 1997-03-24 |
| CZ283219B6 (en) | 1998-02-18 |
| PL318501A1 (en) | 1997-06-23 |
| FI970247A0 (en) | 1997-01-21 |
| FI970247A (en) | 1997-01-21 |
| AU3111995A (en) | 1996-02-22 |
| CZ19397A3 (en) | 1997-06-11 |
| JPH09508140A (en) | 1997-08-19 |
| NO970264D0 (en) | 1997-01-21 |
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