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EP0871660A1 - Proteine de 12 kda derivee de m. tuberculosis et efficace pour le traitement de maladies auto-immunes - Google Patents

Proteine de 12 kda derivee de m. tuberculosis et efficace pour le traitement de maladies auto-immunes

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Publication number
EP0871660A1
EP0871660A1 EP95943854A EP95943854A EP0871660A1 EP 0871660 A1 EP0871660 A1 EP 0871660A1 EP 95943854 A EP95943854 A EP 95943854A EP 95943854 A EP95943854 A EP 95943854A EP 0871660 A1 EP0871660 A1 EP 0871660A1
Authority
EP
European Patent Office
Prior art keywords
protein
ppd
kda
fractions
eae
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.)
Withdrawn
Application number
EP95943854A
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German (de)
English (en)
Other versions
EP0871660A4 (fr
Inventor
Avraham Ben-Nun
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Filing date
Publication date
Application filed by Yeda Research and Development Co Ltd filed Critical Yeda Research and Development Co Ltd
Publication of EP0871660A1 publication Critical patent/EP0871660A1/fr
Publication of EP0871660A4 publication Critical patent/EP0871660A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to agents that may be used for the treatment of autoimmune diseases, and more particularly to a protein having a molecular weight of about 12-kDa isolated and purified from the purified protein derivative (PPD), the major fraction of Mycobacterium tuberculosis (Mt) that protects mice against the induction of experimental autoimmune encephalomyelitis (EAE), and to salts, functional derivatives, analogs and active fractions thereof.
  • PPD purified protein derivative
  • Mt Mycobacterium tuberculosis
  • EAE experimental autoimmune encephalomyelitis
  • Ag antigen
  • EAE experimental autoimmune encephalomyelitis
  • PPD purified protein derivative
  • MBP myelin basic protein
  • Mt Mycobacterium tuberculosis
  • PLP proteolipid protein
  • RPLC reverse phase high-pressure liquid chromatography
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis
  • hsp heat shock protein.
  • Viruses have been most often implicated in the etiology of autoimmune diseases, although bacteria may also be involved : streptococcal infection may lead to rheumatic fever and myocarditis, Mycopiasma arthriditis or its toxins can cause arthritis in mice or rats, and arthritis has also been associated with reactivity to mycobacterial antigens, both in humans and rats.
  • streptococcal infection may lead to rheumatic fever and myocarditis
  • Mycopiasma arthriditis or its toxins can cause arthritis in mice or rats
  • arthritis has also been associated with reactivity to mycobacterial antigens, both in humans and rats.
  • viruses and bacteria may also enhance the natural propensity of mice to become resistant to an autoimmune disease.
  • non-obese diabetic mice infected with lymphocytic choriomeningitis virus become resistant to the development of insulin- dependent diabetes mellitus, and mice infected with lactic dehydrogenase virus are refractory to the development of experimental autoimmune encephalomyelitis (EAE).
  • EAE experimental autoimmune encephalomyelitis
  • bacteria may also be involved in conferring resistance to autoimmune diseases, as demonstrated in a previous study by the present inventor (Lehman and Ben- Nun, 1992).
  • EAE is an autoimmune disease of the central nervous system (CNS) that is caused by CD4 + T lymphocytes specific for myelin antigens, such as MBP or PLP (Ben-Nun and Lando, 1983).
  • CNS central nervous system
  • MBP myelin antigen
  • PLP myelin antigens
  • Mt can also protect Lewis rats against the induction of an autoimmune disease (Lisak and Kies, 1968; MacPhee and Mason, 1990; Hempel et al., 1985).
  • the inventor recently analyzed the effect of Mt and other bacteria on EAE in SJL/J mice, and observed that mice pre-exposed to Mt acquired long-lasting resistance to EAE, even when the bacteria were administered as an aqueous preparation, rather than as an emulsion in IFA. This study clearly demonstrated that Mt can not only promote the development of EAE, but can also protect against the disease, depending on the time of exposure to the bacteria (Lehman and Ben-Nun, 1992).
  • EAE is the well established and widely accepted animal model for studying the effects of various agents which may be implicated in human autoimmune diseases in general , and particularly multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and graft- versus-host disease.
  • an agent found to be capable of eliciting a protective effect against EAE in animals such as mice or rats is also considered to be an agent capable of eliciting protection against autoimmune diseases in humans.
  • the present invention is based on the identification of the major PPD component that confers resistance against EAE.
  • This component the 12-kDa protein of the invention
  • This purified 12-kDa protein which has also been partially sequenced, is capable of protecting mice against the development of EAE.
  • the present invention provides a protein derived from the "Purified Protein Derivative" (PPD) of Mycobacterium tuberculosis (herein 12-kDa PPD protein) capable of protecting mice against EAE and to stimulate PPD-specific T lymphocytes, said protein having a molecular weight of about 12-kDa when analysed by SDS-PAGE under reducing conditions, and comprising the following amino acid sequence obtained by N-terminal analysis:
  • PPD Purified Protein Derivative
  • 12-kDa PPD protein Mycobacterium tuberculosis
  • the invention relates to the 12-kDa PPD protein in substantially purified form.
  • the present invention also provides pharmaceutical compositions comprising the 12-kDa PPD protein, salts, functional derivatives, analogs or active fractions thereof or mixtures of any of the foregoing, according to the invention, and a pharmaceutical acceptable carrier, diluent or excipient.
  • These pharmaceutical compositions may be used against autoimmune diseases in general, and in particular against diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin- dependent diabetes mellitus and graft-versus-host disease.
  • the use of the protein, salts, functional derivatives, analogs or active fractions thereof or mixtures of any of the foregoing, for the treatment of autoimmune diseases in general or those mentioned above, in particular, is also encompassed by the present invention.
  • Figs. 1A-B show PPD-induced protection against EAE.
  • A Mice pre- immunized with PPD are resistant to induction of EAE. SJL/J mice were injected with 50 ⁇ g of PPD in IFA 21 to 25 days before the encephalitogenic challenge as described in Materials and Methods. X denotes mortality in all mice in the group, (a) and (b) correspond to p values of 0.0003 and 0.0001, respectively, when compared with paralysis (P) or mortality (M) in the combined control groups (none + IFA)
  • B Silver-stained SDS-gel of the PPD preparation used throughout in the present application. Twenty ⁇ g PPD were run on the gel. Molecular weights of standards (Bio- Rad, Richmond, CA) are shown on the right of the gel.
  • Figs. 2A-B show RPLC fractionation of PPD.
  • A Chromatographic profile. Two mg PPD were injected per RPLC run and eluted with a 0 to 50% linear gradient of 1-propanol in 0.1% trifluoroacetic acid (TFA). Pooled fractions are indicated by Roman numerals.
  • B Silver-stained SDS-gel of pooled fractions II through VIII. Four ⁇ g of pooled fraction were loaded per lane.
  • Figs. 3A-B show that protection against EAE by PPD fractions is not associated with a stimulatory effect on EAE-mediating T-cells, but correlates with their ability to stimulate PPD-specific T lymphocytes.
  • A Protective effect of PPD fractions . SJL/J mice were injected with 1.5 ⁇ g of the indicated PPD fractions emulsified in LFA 22 days prior to the encephalitogenic challenge.
  • B Effect of PPD fractions on T-cell lines specific for MBP 89-101 (DSBP cells) or PPD (DSP cells). The proliferative response to the indicated PPD fractions (0.5 ⁇ g/well) was measured as detailed in Materials and Methods. Vertical bars represent standard deviation from the mean of triplicate cultures. It should be noted that none of the PPD fractions were stimulatory for PLP-specific T cells (data not shown).
  • Figs. 4A-C show further purification of the PPD component responsible for imparting protection against EAE.
  • A RPLC refractionation of PPD fraction V, highly protective against EAE: chromatographic profile .
  • Fraction V 150 ⁇ g derived from RPLC fractionation of 12 mg PPD (6 chromatographic runs) was refractionated by RPLC which was developed with a 0 to 60% linear gradient of 1-propanol. Resulting fractions were pooled as indicated and are represented by capital letters.
  • B Silver- stained SDS-gel of Fr D, protective against EAE. Four ⁇ g of protein were loaded on the gel.
  • C Analysis of the protective effect against EAE by the major pooled fractions of rechromatographed PPD fraction V.
  • Fractions B, D and F were also tested in vitro for their ability to stimulate EAE-mediating T lymphocytes specific for MBP 89-101 (DSBP line cells) or PLP 139-151 (NSPLP line cells) or T lymphocytes specific for PPD (DSP line cells) .
  • the results showed that none of the fractions had a stimulatory effect on EAE-mediating T lymphocytes. Only the fraction which induced protection against EAE (Fraction D) stimulated PPD-specific cells (DSP line cells).
  • Figs 5A-C show the protective activity of the 12-kDa PPD protein purified by preparative gel electrophoresis
  • A Silver-stained SDS-gel of Fraction D and the 12- kDa protein eluted from the gel.
  • the 12-kDa eluted protein also stimulates PPD- reactive T lymphocytes, but not MBP- or PLP-specific T cells.
  • Fraction D was electrophoresed on two preparative gels and the gel band corresponding to the 12-kDa protein was sliced out and the protein was eluted out of the gels as described in Materials and Methods.
  • Lane 1 shows the electrophoretic separation of Fraction D components (4 ⁇ g/lane) and lane 2 shows the 12-kDa protein (approx.
  • Fraction D eluted from the gel.
  • Fraction D and its 12-kDa component were identical in their ability to induce the proliferation of T lymphocytes specific for PPD (DSP line cells), but had no effect on EAE-mediating T lymphocytes specific for MBP 89-101 (DSBP line cells) or PLP 139-151 (NSPLP line cells).
  • B Two-dimensional gel electrophoretic analysis of gel-eluted 12-kDa protein of Fraction D. Approximately 3-4 ⁇ g were run on the first-dimension gel. The silver- stained second-dimension gel is shown. The gel-eluted 12-kDa protein appears as several contiguous spots, most likely representing post-translationally modified isoforms.
  • Fig. 6 shows that the N-terminal amino acid sequence of the 12-kDa protein purified from PPD is identical to that of the 10-kDa protein of Mt./M. bovis.
  • the 12- kDa protein of Fraction D was transferred onto PVDF membrane either by electroblotting of the electrophoretogram or by elution from the gel band and spotting. The protein adsorbed to the membrane was then subjected to amino acid microsequencing. Sequences of the 10-kDa protein ofM tuberculosis, M. bovis zn ⁇ M. leprae were from Shinnick et al. 1989, Yamaguchi et al. 1988 and Mehra et al. 1992, respectively. The sequence of E. coli GroES was from Shinnick et al. 1989.
  • Figs. 7A-B show that recombinant 10-kDa BCG-a protein does not protect against EAE and does not stimulate PPD line T cells.
  • the protective activity of Mt against induction of EAE in SJL/J mice is attributed to a protein of approximately 12-kDa in size purified from PPD. Sequencing of the first 17 N-terminal amino acids of this 12- kDa PPD protein seemed to indicate that it was identical, or closely related, to the 10- kDa BCG-a antigen of Mt. However, differences in protective activity and T-cell epitope specificity between the two proteins appear to refute this possibility. Thus, in contrast to the 12-kDa PPD protein, the 10-kDa BCG-a protein did not protect against EAE and did not stimulate PPD-specific T lymphocytes. Reciprocal analysis of the proliferative response to the 12-kDa PPD protein by 10-kDa BCG-a protein-reactive T cells demonstrated low cross-reactivity, supporting the notion that the two proteins share sequence homology but are not identical
  • the above noted biologic characteristics of the 12-kDa PPD protein of the invention opens the way for using this 12-kDa PPD protein in the treatment of human autoimmune diseases. It is stimulatory for PPD-specific T lymphocytes, but appears to do so without stimulation of other potentially pathogenic T lymphocytes, indicating that this 12-kDa PPD protein may be used as a potentially safe therapeutic agent for human autoimmune diseases.
  • EAE is the well-established and widely accepted animal model for ascertaining the effects of agents which may be involved in human autoimmune diseases.
  • the isolation and purification in accordance with the present invention of the 12-kDa PPD protein that affords protection against EAE in mice, is thus indicative that this protein is useful in the treatment of autoimmune diseases in human, in general, and, in particular, diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and graft- versus-host disease.
  • diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and graft- versus-host disease.
  • the present invention concerns the 12-kDa PPD protein characterized by its ability to elicit protection against EAE and to stimulate PPD-specific T lymphocytes, being thus useful for the treatment of autoimmune diseases, and comprising the following 17 amino acid N-terminal sequence : Ala-Lys-Val-Asn-Ile- Lys-Pro-Leu-Glu-Asp-Lys-Ile-Leu-Val-Gln-Ala-Asn.
  • salts, functional derivatives and analogs of the 12-kDa PPD protein and active fractions thereof which are also characterized by having the ability to elicit protection against EAE and to stimulate PPD-specific T lymphocytes.
  • the native 12-kDa PPD protein of the invention may be obtained from the PPD of Mt by any standard purification procedure, including affinity chromatography using antibodies specific for the protein.
  • the process for producing the substantially purified 12-kDa PPD protein comprises: (a) fractionating PPD derived from Mycobacterium tuberculosis (Mt) by RPLC; (b) selecting and isolating the biologically active fractions from (a) that provide highest protection of mice against EAE; (c) subjecting said 12-kDa PPD protein from the isolated fractions of (b) to RPLC and isolating the fractions having the highest protection against EAE; and (d) purifying the 12-kDa PPD protein from the isolated fractions of (c) by preparative gel electrophoresis.
  • Mt Mycobacterium tuberculosis
  • the 12-kDa PPD protein may be produced as a recombinant protein using standard recombinant DNA methods, and hence the present invention also encompasses all of the tools used in these procedures, i.e. the DNA molecules comprising the nucleotide sequence coding for the 12-kDa PPD protein or for a protein substantially homologous therewith, suitable recombinant expression vectors containing the DNA molecules, and suitable (eukaryotic or prokaryotic) host cells transformed by the expression vectors and capable of expressing the protein.
  • salts refers to both salts of carboxyl groups and to acid addition salts of amino groups of the protein molecule formed by means known in the art.
  • Salts of carboxyl group include inorganic salts, for example, sodium, calcium, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine.
  • Acid addition salts include, for example, salts with mineral acids and salts with organic acids.
  • “Functional derivatives” as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e.
  • These derivatives include aliphatic esters or amides of the carboxyl groups, N-acyl derivatives of free amino groups or O-acyl derivatives of dree hydroxyl groups formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) and derivatives with sugar or phosphate residues, e.g., those formed by post-translational modification.
  • acyl moieties e.g. alkanoyl or carbocyclic aroyl groups
  • sugar or phosphate residues e.g., those formed by post-translational modification.
  • Analogs are proteins having the activity of the 12-kDa PPD protein of the invention, but that represent modifications of the protein including deletions, substitutions or additions of one or more amino acid residues of the native 12-kDa PPD amino acid sequence; all such modifications being effected either by known chemical procedures (in the case of deletions and additions) or by standard recombinant DNA methods (in all cases). Suitable modifications, i.e. analogs are only those which retain the biological activity of the 12-kDa PPD protein.
  • active fractions of the substantially purified protein covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g. sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has the biological activity of the 12-kDa PPD protein.
  • the present invention also concerns pharmaceutical compositions comprising a pharmaceutically active carrier and the 12-kDa PPD protein, a salt, analog, functional derivative or an active fraction thereof.
  • These compositions may be used to elicit protection against autoimmune diseases, in general, and diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and graft-versus-host disease, in particular.
  • the way of administration can be via any of the accepted modes of administration for similar agents and will depend on the condition to be treated, e.g. intravenously, intramuscularly, subcutaneously, by local injection, by topical application or per os, as the case may require.
  • compositions of the invention are prepared for administration by mixing the protein, a salt, analog, functional derivative or active fraction thereof with physiologically acceptable carriers, stabilizers and excipients, and prepared in dosage form, e.g. by lyophilization in dosage vials.
  • the amount of active compound to be administrated will depend on the route of administration, the disease to be treated and the condition of the patient. Local injection, for instance, will require a lower amount on a body weight basis than will e.g. intravenous injection.
  • the invention also encompasses a method for treatment of a patient afflicted with an autoimmune disease (i.e. multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and graft-versus-host disease) which comprises administering to said patient an effective amount of a 12-kDa PPD protein, a salt, functional derivative, analog or active fraction thereof or a mixture of any of the foregoing.
  • an autoimmune disease i.e. multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and graft-versus-host disease
  • mice Female SJL/J mice were purchased from Jackson Laboratories ( Bar Harbor, ME). All mice were 2-3 months old when used in the experiments.
  • Antigens and T-cell lines PPD was purchased from Statens Serum Institute (Copenhagen, Denmark). Recombinant 10-kDa BCG-a protein, batch MT10-1A, produced and purified by Dr. V. Mehra (see Mehra et al., 1992), and 65-kDa M. bovis protein were donated by Dr. J. van Embden of the National Institute of Public Health and Environmental Protection, Bilthoven, the Netherlands.
  • Peptides corresponding to amino acid residues 89-101 of MBP (MBP 89-101) and to amino acid residues 139-151 of proteolipid protein (PLP 139-151) were synthesized by the Weizmann Institute Synthesis Unit, using a solid phase technique on a peptide synthesizer (Applied Biosystems Inc., Foster City, CA).
  • T-cell lines encephalitogenic for SJL/J mice and specific for the 89-101 epitope of MBP or for the 139-151 epitope of PLP, were selected in vitro as previously described (Ben-Nun and Lando, 1983; Ben-Nun and Cohen, 1982), from SJL/J mice immunized with MBP 89-101 or PLP 139-151 peptides respectively.
  • T-cell line specific for PPD was selected in vitro from SJL/J mice immunized with complete Freund's adjuvant (CFA).
  • Fractionation of PPD was achieved by reverse phase liquid chromatography (RPLC) run on a wide-bore column (Aquapore RP-300, 7 ⁇ m, 200 x 6 x 4 mm), using a linear gradient of 1-propanol (0 to 50%) in 0.1% trifluoroacetic acid, with a flow rate of 0.75 or 1.0 ml/min (run time 50 min).Two mg of PPD were injected per run and the protein separation was performed with a Spectra Physics SP8750 HPLC system and a Waters Model 441 detector at 214 nm. Pooled fractions were freeze-dried and reconstituted in double-distilled H2O.
  • RPLC reverse phase liquid chromatography
  • PVDF polyvinyledene difluoride membrane
  • Two-dimensional gel electrophoresis Two -dimensional (2-D) gel electrophoresis of the 12-kDa PPD protein eluted from the gel band was performed according to the method of OTarrell (OTarrell, 1975), using Ampholine pH 3.5-10.0 from Sigma Chemical Co. (Cat. no. A-5174; St Louis, MO). The second-dimension electrophoresis was run on 15% gels and silver-stained. g) T-celi proliferative responses.
  • SJL/J T line cells (1.5 x 10 ⁇ ) specific for MBP 89-101 (DSBP line), PLP 139-151 (NSPLP line), or PPD (DSP line) were cultured in microtiter wells in the presence of irradiated (2500 rads) syngeneic spleen cells, as accessory cells, in 0.2 ml RPMI medium supplemented with L-glutamine, ⁇ - mercaptoethanol, antibiotics and 2.5% fetal calf serum as previously described (Ben- Nun and Lando, 1983). Popliteal lymph node cells (5 x 10 ⁇ ) isolated from SJL/J mice, injected s.c.
  • the relevant antigen (MBP 89-101, PLP 139- 151, PPD or PPD-derived fraction or gel-eluted protein, or recombinant 10-kDa BCG-a protein) was added (2 ⁇ g/well for MBP and PLP peptides or PPD; 0.5 ⁇ g/well and 1.5 ⁇ g/well for PPD-derived fraction and gel-eluted protein, or recombinant 10-kDa BCG-a protein) in triplicate cultures. The cultures were incubated for 48 hrs at 37_C in humidified air containing 7.5% CO2.
  • [-1H]-thymidine (l ⁇ Ci/well) was added for the last 16 hrs of the incubation and the cultures were then harvested and counted using a Matrix 96 Direct beta counter (Packard Instr., Meriden, CT). The proliferative response is measured as the [ ⁇ HJ-thymidine incorporation expressed as mean counts per minute (cpm) of triplicate cultures.
  • h Induction of EAE and protection against the disease. EAE was induced in mice as previously described (Ben-Nun and Lando, 1983; Ben Nun et al., 1993).
  • mice were injected s.c.
  • mice were observed daily for clinical signs of EAE.
  • Example 1 RPLC fractionation of PPD yields discrete fractions with protective activity.
  • Fig. 1 A shows protection against the induction of EAE in SJL/J mice pre-treated with the PPD preparation used throughout the examples in the present application. The extent of protection was comparable to that previously observed (Ben-Nun et al., 1993).
  • SDS-PAGE analysis (Fig. IB) of this PPD preparation shows the presence of proteins of different sizes, with a band of an approximate molecular weight of 12-kDa being the most abundant.
  • PPD was fractionated by RPLC (Fig. 2A). Analysis of the resulting fractions revealed the presence of a 12-kDa protein(s), most predominantly in fractions III- VIII (Fig.2B). These fractions were tested in vivo for their protective activity (Fig. 3 A).
  • mice were injected s.c. with an equivalent of 1.5 ⁇ g PPD fraction emulsified in EFA, three weeks prior to the encephalitogenic challenge, and the effect of the various fractions on the development of EAE was followed.
  • Daily clinical scoring for EAE of mice pre-treated with some of the fractions (fractions III- VIII) is shown in Fig. 3A.
  • mice pre-treated with fractions V and VII were fully protected
  • Other fractions either had no protective effect or induced only partial protection against the induction of EAE (Fig.3A).
  • the partial protective effect of fractions IV and VI could be attributed to contamination of these fractions with fraction V, due to tailing of the fraction V peak.
  • Example 2 RPLC fractions which protect against EAE stimulate PPD cells, but not encephalitogenic T cells.
  • PPD fractions were assayed for their ability to stimulate encephalitogenic T lymphocytes in vitro. None of the fractions, including those which were highly protective against EAE, were stimulatory for T-lymphocytes specific for MBP (Fig.3B) or PLP (data not shown). Paradoxically, a correlation was observed between the protective effect of the fractions and their ability to stimulate PPD-specific T lymphocytes. Thus, fractions V and VII which conferred a complete protection against EAE also stimulated the PPD-specific T cells to the highest degree (Fig. 3, A and B).
  • Example 3 A 12-kDa protein purified bv RPLC refractionation and preparative gel electrophoresis imparts protection against EAE.
  • Fr. D that eluted at about 45% 1-propanol (Fig. 4A). This peak was highly protective against EAE (Fig.4C) and also had the ability to stimulate PPD-specific T-cells, but not MBP- or PLP-specific encephalitogenic T lymphocytes (Fig. 4A). SDS-PAGE analysis of Fr. D revealed a major protein of 12-kDa in size (Fig. 4B). Unexpectedly, two proteins of approximate molecular weight 16 kDa and 26 kDa were also detected (Fig. 4B). It is unclear whether these bands are a result of the enrichment of other proteins upon further purification, or whether they represent dimerization of the 12-kDa protein and/or aggregation of the 12-kDa protein with peptide fragments contained in the preparation.
  • Fr. D was electrophoresed on SDS-polyacrylamide gel; the 12-kDa protein-containing band was sliced out and the protein was eluted. It migrated as a single, 12-kDa band on one- dimensional SDS-PAGE (Fig. 5A), was as highly protective against the induction of EAE as Fr. D (Fig. 5C) and strongly stimulated PPD-specific T cells, but could not induce a proliferative response by the encephalitogenic MBP- or PLP-specific T cells (Fig. 5A).
  • Two-dimensional gel analysis of the gel-eluted 12-kDa protein resulted in an electrophoretic pattern more consistent with post-translationally modified isoforms of a single protein than with a mixture of proteins (Fig. 5B), although the spread-out pattern of the charge isoforms makes it difficult to absolutely exclude the possibility that another protein is present in the gel-eluted band.
  • the likelihood that the two- dimensional gel pattern is caused by charge isoforms rather than by the presence of a mixture of proteins would be strengthened if an unambiguous N-terminal amino acid sequence could be obtained from the gel-eluted protein.
  • Example 4 The protective 12-kDa PPD protein shares identical N-terminal sequence with BCG-a protein. To gain some knowledge on the nature of the protective protein, Fr. D was electrophoresed and blotted onto PVDF-membrane and the 12-kDa protein band was cut out and subjected to N-terminal amino acid microsequencing. As shown in Fig. 6, the 17 N-terminal amino acids of the 12-kDa PPD protein were identified and found to be homologous to the 17 N-terminal amino acids of the 10-kDa BCG-a protein of M. tuberculosis.
  • Example 5 The 10-kDa BCG-a protein and the 12-kDa PPD protein differ in their ability to protect against EAE.
  • the 12-kDa PPD protein is identical to the 10- kDa BCG-a protein, as suggested by the N-terminal sequence data, the purified 12-kDa PPD protein and recombinant 10-kDa BCG-a protein were compared for their ability to protect against EAE.
  • the 10-kDa BCG-a protein was found to be ineffective in imparting protection against EAE (Fig. 7A). These two proteins were also compared for their ability to induce proliferative responses by PPD- specific T cells in vitro. As shown in Fig.
  • a major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein. J. Exp. Med 175:275.

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Abstract

L'invention concerne une protéine de 12 kDa de PPD isolée et purifiée à partir du dérivé de protéine purifiée (PPD), fraction principale de Mycobacterium tuberculosis protégeant les souris contre l'encéphalomyélite auto-immune expérimentale (EAE), ainsi que ses sels, dérivés fonctionnels, analogues et fractions actives et compositions pharmaceutiques les contenant afin de traiter des maladies auto-immunes.
EP95943854A 1994-12-04 1995-12-04 Proteine de 12 kda derivee de m. tuberculosis et efficace pour le traitement de maladies auto-immunes Withdrawn EP0871660A4 (fr)

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IL11186594A IL111865A (en) 1994-12-04 1994-12-04 Protein from mycobacterium tuberculosis and pharmaceutical compositions comprising it
IL11186594 1994-12-04
PCT/US1995/016444 WO1996017864A1 (fr) 1994-12-04 1995-12-04 Proteine de 12 kda derivee de m. tuberculosis et efficace pour le traitement de maladies auto-immunes

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EP0871660A1 true EP0871660A1 (fr) 1998-10-21
EP0871660A4 EP0871660A4 (fr) 2000-08-23

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JPH02169599A (ja) * 1988-12-21 1990-06-29 Ajinomoto Co Inc Ｂｃｇ菌由来ｍｐｂ５７蛋白の抗原決定領域を含むペプチド
IT1271074B (it) * 1994-11-21 1997-05-26 Italfarmaco Spa Peptidi ad attivita' antiinfiammatoria

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IL111865A0 (en) 1995-03-15
IL111865A (en) 2005-03-20
WO1996017864A1 (fr) 1996-06-13

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