EP0474764A1 - Method to treat rheumatoid arthritis - Google Patents

Method to treat rheumatoid arthritis

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Publication number
EP0474764A1
EP0474764A1 EP90909247A EP90909247A EP0474764A1 EP 0474764 A1 EP0474764 A1 EP 0474764A1 EP 90909247 A EP90909247 A EP 90909247A EP 90909247 A EP90909247 A EP 90909247A EP 0474764 A1 EP0474764 A1 EP 0474764A1
Authority
EP
European Patent Office
Prior art keywords
peptide
cells
sequence
raa
class
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Withdrawn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
EP90909247A
Other languages
German (de)
French (fr)
Other versions
EP0474764A4 (en
Inventor
Dennis A. Carson
Jean Roudier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Original Assignee
Scripps Clinic and Research Foundation
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Publication date
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Publication of EP0474764A1 publication Critical patent/EP0474764A1/en
Publication of EP0474764A4 publication Critical patent/EP0474764A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof

Definitions

  • the invention relates to regulation of the im ⁇ mune system to prevent and treat autoimmune diseases . More specifically, it concerns procedures to prevent and treat the immune response which is characteristic of rheumatoid arthritis in humans.
  • the B-lymphocytes and acrophages from more than 90% of patients with seropositive rheumatoid arthritis express the Class II HLA protein DR4 or DRl, as defined serologically. It has been shown that DR4 specificity is present on several different haplotypes, three of which, Dw4 , Dwl4 and Dwl5, are associated with the B-lymphocytes and macrophages from rheumatoid arthritis patients, and two of which, DwlO and Dwl3, are not.
  • the B-lympho- cytes and macrophages of subjects with rheumatoid arthritis express the Class II haplotypes Dw4, Dwl4, Dwl5 or DRl.
  • RA rheumatoid arthritis
  • the sequence of the region is either QKRAA (Dw4) or QRRAA (Dwl4, Dwl5 and DRl).
  • R arginine
  • K lysine
  • the sequence Q(K/R)RAA may be important in the development and symptoms of rheumatoid arthritis.
  • MHC major histocompatibility complex
  • the Class II protein contains a site capable of binding the foreign antigen (or a fragment thereof) and another site which recognizes a T-cell receptor. Structural studies by others have indicated that the position on the Class II MHC protein on which the Q(K/R)RAA sequence is located corresponds to the T-cell receptor recognition site.
  • Rheumatoid arthritis is thought to be an autoimmune disease wherein certain protein components of the synovial fluid are recognized as foreign by the immune system. Presumably they are presented to the T- lymphocytes in the context of an MHC Class II protein, which MHC Class II carrier is recognized as self by the T- lymphocyte. Based on the foregoing paragraphs, it may be supposed that this self-recognition site includes Q(K/ R)RAA. It has now been found that by administering a suitable vaccine, the response of T-lymphocytes to the synovial fluid-associated antigen can be either diverted from the helper T-cell class, or prevented. The vaccines of the invention thus prevent T-helper proliferation in response to the synovial antigen conjugated to the Class II recognition site Q(K/R)RAA.
  • the invention provides vaccines for the preven- tion and treatment of the autoimmune response to synovial protein which manifests itself in the symptoms of rheumatoid arthritis.
  • the vaccines of the invention ef ⁇ fectively prevent the T-helper cell response to these proteins presented in the context of the MHC Class II histocompatibility protein having the sequence Q(K/R)RAA at its recognition site.
  • the invention is directed to a method to immunize a subject to prevent or ameliorate rheumatoid arthritis
  • the vaccination protocol comprises administration of an effective amount of the peptide Q(K/R)RAA in immunogenic form in conjunction with an effective amount of one or more immunostimulatory or immunosuppresant substances which preferentially enhance the proliferation of suppressor and/or cytotoxic T-cells, and/or suppress the expansion of helper T-cells without effect on suppressor/cytotoxic expansion.
  • im unoaffecting substances include interleukin-6 (IL-6), cyclosporin A, antibodies against CD4 antigen, transforming growth factor-beta (TGF-beta), and combinations thereof.
  • the invention is also directed to vaccines useful in this method.
  • the invention is directed to a vaccine useful in preventing rheumatoid arthritis, which vaccine comprises the Q(K/R)RAA a ino acid sequence coupled to an invariant portion of a Class I histocompatibility sequence, and to methods of rheumatoid arthritis prevention and amelioration using this vaccine.
  • Figure 1 shows the stimulation (or not) of T- cells derived from EBV-exposed and EBV-nonexposed subjects by gpllO and by Dw4 peptide.
  • Figure 2 shows antibody titers measured against gpllO peptide or protein of a rabbit immunized with gpllO peptide.
  • Figure 3 shows immunoblots of HLA Class II Dw4 and Dw2 proteins with rabbit anti-D 4 antisera.
  • Figure 4 shows the comparative amino acid sequences of Dw4, Dwl4 and DwlO beta peptides.
  • the vaccines of the invention utilize as an es ⁇ sential component the amino acid sequence Q(K/R)RAA in an immunogenic form or an immunogenic form of the peptide sequence which forms that portion of a T-cell receptor binding to this epitope.
  • the specific peptide hapte ⁇ sequence may be part of a larger polypeptide or protein and/or may be conjugated, option ⁇ ally through a linker, to a carrier.
  • peptides are included within a larger protein
  • convenient larger proteins include additional amino acid residues found in the Class II DR-beta-1 chain or additional amino acid residues adjacent this peptide in glycoprotein gpllO as ⁇ sociated with the Epstein-Barr virus; for peptides representing the Q(K/R)RAA sequence in the context of the Class I MHC protein constant regions, extensions of these regions may also be employed.
  • the individual peptide may also be used. In both cases arbitrary, non- interfering chain extensions may also be employed.
  • Suitable carriers which enhance immun ⁇ genicity of the hapten, or confer immunogenicity upon it include the commonly employed keyhole limpet hemocyanin (KLH) protein, tetanus toxoid, or other antigenically neutral materials, such as human serum albumin. If a separate carrier such as tetanus toxoid, KLH or HSA, is used, the peptide may be conjugated to the carrier using means standard in the art, including the use of linkers, many of which are commercially available from Pierce Chemical Company, Rockf ⁇ rd, IL. Typical linkers include, for example, SMCC and SPDP. Methods of effecting such conjugation are well known.
  • the vaccines of the invention are administered to persons at risk for rheumatoid arthritis as shown by the haplotypes described above.
  • individuals with haplotypes corresponding to Dw4, Dwl4, Dwl5 or DRl are administered the vaccine.
  • the immunogenic form of Q(K/R)RAA is administered in conjunction with an immunostimulant and/or immunosuppressant which results in a preferential expan- sion if T-suppressor and/or cyctotoxic cells at the expense of T-helper cells.
  • an immunostimulant and/or immunosuppressant which results in a preferential expan- sion if T-suppressor and/or cyctotoxic cells at the expense of T-helper cells.
  • stimulants which expand suppressor/cytotoxic populations but which do not affect helper populations may be used; or substances which actively suppress helper populations, but do not affect suppressor/cytotoxic T-cells may be used, or combinations of these factors may be used.
  • Certain substances which effect this differential expansion are known in the art; however, the invention includes the use of any substance which can be shown to have this effect.
  • Interleukin-6 is an appropriate immunostimulant for suppressor/cytotoxic T- cells. It is often desirable to include IL-2 to enhance this stimulation as well. Cyclosporin A, TGF-beta, and anti-CD4 antibodies (see, e.g., Carteron, N.L., et al. , J Immunol (1988) 140:713-716) are appropriate helper cell inhibitors.
  • administered “in conjunction with” is meant that either administration is in the same vaccine formula ⁇ tion (containing both the immunogenic form of Q(K/R)RAA and the immunostimulant and/or immunosuppressant) or each of these components may be administered within a conjunc ⁇ tive period of time so that their cooperative effect is obtained. Suitable conjunctive time periods are of the order of 4 hours to 2 days, preferably 4 hours to 1 day.
  • the immunostimulant/immunosuppressant and the immunogenic peptide may be administered in any order, and protocols of repeated administrations of each or both can also be employed.
  • Suitable routes of administration are systemic, and include administration by injection such as intra ⁇ venous, intramuscular, peritoneal, or subcutaneous injec- tion. Subcutaneous or intramuscular administration are preferred.
  • the immunogenic peptide and immunostimulatory/ immunosuppressant substance are formulated for injection using standard formulations for the administration of peptides which include suitable excipients such as buff- ers, Ringer's solution, or Hank's solution, and may further include wetting agents, stabilizers, and the like. Suitable adjuvants may also be included.
  • Other forms of administration such as transmembrane or transdermal administration using formulations suitable for these routes may also be employed. A wide range of formulations is found, for example, in Remington's Pharmaceutical Sci ⁇ ences, latest edition, Mack Publishing Company, Easton, Pennsylvania.
  • Suitable amounts of the immunogenic peptide are about 100 ug-100 mg per patient, preferably 10-100 mg.
  • the immunostimulant/immunosuppressant or combination should be administered in an amount according to the potency of the substance chosen; for IL-6, for example, suitable dosage ranges are 1-100 ug/kg, preferably 20-50 ug/kg. It is understood, of course, that dosage level is highly dependent on the particular immunogenic form of the peptide used, the i ⁇ vmunostimulant/immunosuppressant selected, the route of administration selected, the nature of the formulation, and the individual response of the subject. Establishment of optimum dosage protocols and regimens for a subject, taking account of the foregoing parameters, is routinely done and is well within ordinary skill.
  • the peptide Q(K/ R)RAA or a larger peptide containing this sequence is coupled to an invariant portion of a Class I histocompatibility sequence to generate the active component of the vaccine.
  • the invariant portion of Class I histocompatibility sequences are known and the relevant data are deposited in Genbank and other equivalent data- bases generally known in the art. (See referenced art in Chimini, G., et al. , J Exp Med (1989) 169:297-302. )
  • EBV Epstein-Barr virus
  • Dwl4/Dwl5/DR1 KDLLEQRRAAVDTYC
  • gpllO QENQEQKRAAQRAAGC
  • DwlO KDILEDERAAVDTYC.
  • peptides were used as test stimulating peptides for T-cell proliferation or were conjugated to KLH using m-maleimidobenzoyl-n-hydroxysuccinimide as described by Green, N., Cell (1982) ⁇ :477-487; Liu, F.T., et al., Biochemistry (1979) __ :690-697, for use in im ⁇ munization.
  • Example 2 Sensitization of T-Cells to Q(K/R)RAA Stimulation by Prior Exposure to EBV
  • the peptides synthesized in Example 2 were used to determine the response of T-cells in vitro as follows: The method was a modification of that disclosed in Ford, D., Cell Immunol (1983) 7_9:334-344; Thorley-Lawson, D.A. , et al., Proc Natl Acad Sci USA (1987) ⁇ 4:5384-5388, both incorporated herein by reference.
  • peripheral blood mononuclear cells were obtained both from a donor with no history of EBV infection, as indicated by the absence of antibody to the viral capsid antigen (VCA) and from an otherwise normal subject with a high titer of anti-VCA antibodies, indicating prior exposure to EBV.
  • the mononuclear cells were isolated by isopycnic centrifugation through Ficoll-Hypaque, and were washed three times in RPMI-1640 medium.
  • the cells were then suspended at a density of 10 cells/ml in the same medium supplemented with 10% pooled human AB serum, 1% L- glut-amine, 100 U/ml penicillin, and 100 ug/ml streptomycin in the presence of 1 ug/ml of a stimulatory peptide selected from the list above.
  • a sample of 10 cells were taken from the bulk culture to evaluate the primary proliferative response.
  • the remaining cells were counted and serially diluted in fresh medium sup- plemented with the same peptide to be tested, or without said peptide as a control.
  • the cells were then distributed in 100 ul aliquots containing 50,000, 25,000, 10,000, or 5,000 viable cells from the primary culture in 96-well round-bottom culture plates and the number of cells in each well adjusted to 10 by the addition of ir ⁇ radiated (3,000 rad) peripheral blood mononuclear cells from the same donor. For each cell density, 18 wells with peptide and 6 control wells without peptide were assayed. After 4 days of this secondary culture, 1 uCi of tritiated thymidine was added to each well 18 hours before cell harvesting on glass fiber filters. For each cell dilution, positive wells were defined as having cpm higher than (mean + 2 standard deviations) of the cpm obtained for 6 control wells. Frequency of precursor T-cells was evaluated by plotting the percentage of negative wells for each dilution against the number of cells per well, as described by Ford et al. (supra) ⁇
  • Panels a and c show the response (in cpm/well) at various dilutions of cells derived from a VCA-positive donor; panels b and d, from VCA-negative donors. Panels a and b were treated with the gpllO peptide; panels c and d, with the Dw4 peptide.
  • RA-Susceptible Haplotype Antibodies The peptides prepared in Example 2 and conjugated to KLH were used to raise antibodies in rab ⁇ bits. New Zealand white rabbits were injected subcutane- ously with 1 mg of the conjugate emulsified in complete Freund's adjuvant, and then boosted three times at 3 week intervals with 1 mg of conjugate in incomplete Freund's adjuvant. The rabbits were bled four days after the last injection and sera were stored at -20 C.
  • Antibody titers of the resulting sera were tested using an ELISA assay or by i munoblot.
  • the ELISA assay was by the procedure of Luka, J. , et al., J Immunol Methods (1984) 7_:145-156, incorporated herein by refer ⁇ ence. Briefly, the antigen peptide was used to coat ELISA plates, and various solutions of preimmune or immune rab- bit sera, diluted in isotonic borate-buffered saline (BBS) were added in 100-ul aliquots to the wells and incubated overnight at 4°C.
  • BBS isotonic borate-buffered saline
  • ELISA determinations were made when reactivity of antibodies was tested to EBV gpllO.
  • the gpllO was purified by affinity chromatography from P3HR1 lymphoblasts according to the method of Kishishita, M. , et al., Virology (1984) 133:393-395.
  • the epitope recognized on the gpllO protein was that of the peptide, since a 20-fold dilution of serum preincubated with 100 ug/ml of gpllO peptide was no longer capable of binding gpllO protein.
  • a membrane extract from 2 x 10 cells was loaded into each lane of a 10% polyacrylamide gel containing 0.1% sodium dodecylsiilfate and 1 mM 2-mercaptoethanol. After electrophoresis, the reduced and denatured membrane polypeptides were electrophoretically transferred to nitrocellulose sheets, as described by Towbin, H. , et al. , Proc Natl Acad Sci USA (1979) 7_6:4350-4354; Billings, P.B., et al., ibid (1983) ⁇ 0:7104-7108.
  • the filters were then preincubated for one hour in a solution containing 0.05 M borate, 0.15 M NaCl, pH 3, and 3% powdered milk, followed by overnight incubation at 4°C with the antisera diluted at the same dilution.
  • candidate peptides were added to the antibody solu- tions prior to their incubation with the nitrocellulose sheets.
  • borate buffer After extensive washing of the sheets with borate buffer, bound antibody was detected using 125I- protein A (1 mCi/ l, ICN, Irvine, CA) .
  • the sheets were incubated for 1 hour with the detecting reagent, washed with BBS, dried, and exposed to Kodak XAR film overnight at -70 C using an intensifier screen.
  • the ability of Dw4 peptide to raise specific antibodies was also shown by assay of the rabbit antiserum by immunoblot as described above. The results of this assay are shown in Figure 3.
  • the immunoblot in panel A is a control testing anti-DR-beta monoclonal antibody diluted 1:1000 with extracts from Molt 4 (M4) cells, HLA Dw4 homozygous lymphoblastoid cells (D4), or HLA Dw2 homozygous lymphoblastoid cells (D2). Both DR beta proteins react, M4 does not.
  • M4 Molt 4
  • D4 HLA Dw4 homozygous lymphoblastoid cells
  • D2 HLA Dw2 homozygous lymphoblastoid cells
  • Lane set 1 represents the results when the electrophoresis was run using the membrane extracts alone. Only the D4 extract is immunoreactive with the antibody, confirming the specificity of the antiserum.
  • Lane set 2 shows the results when the antiserum was preincubated with 10 ug/ml Dw4 peptide; the binding to the membrane-derived D4 peptide in the gel is completely eliminated.
  • Lane set 3 shows the results when the antiserum was preincubated with the 100 ug/ml of the control peptide DwlO. No effect on the binding of the antibody to the D4 extract was noted.
  • Lane set 4 shows that preincubation with the related peptide Dwl4 at 10 ug/ml was partially effective in eliminating binding to D4; the binding was almost eliminated when the concentration of Dwl4 was increased to 100 ug/ml (lane set 5).
  • gpllO protein The recognition of gpllO protein was inhibited by preincubation of a 100-fold dilution of the serum with either 100 ug/ml gpllO peptide or 100 ug/ml Dw4 peptide.
  • the recognition of gpllO peptide was inhibited by preincubation with 10 ug/ml of either Dw4 or Dw4' peptide (Example 2) .
  • peptides are conjugated using dicyclohexylcarbodiimide to the peptide of the sequence Q(K/R)RAA and formulated into vaccines.
  • the gene encoding the desired portion of the above peptides with extensions of the residues Q(K/R)RAA are constructed and inserted into standard recombinant expression systems for production of fusion proteins. The resulting fusions are then formulated into vaccines as described above.

Abstract

Des sujets peuvent être immunisés contre le rhumatisme articulaire en leur administrant le peptide Q(K/R)RAA sous une forme immunogénique dans un protocole aboutissant à une expansion préférentielle des cellules T suppresseurs et/ou cytotoxiques plutôt que des cellules T auxilaires. Cette expansion préférentielle peut s'effectuer en administrant l'interleukine-6 et/ou la cyclosporine A. Dans une variante, le peptide Q(K/R)RAA couplé à la région invariante d'une séquence d'histocompatibilité de la classe I peut être administré. Sont également décrites des compositions pharmaceutiques utiles dans les protocoles de vaccination.Subjects can be immunized against rheumatoid arthritis by administering the Q (K / R) RAA peptide in immunogenic form in a protocol leading to preferential expansion of suppressor and / or cytotoxic T cells rather than auxiliary T cells. This preferential expansion can be carried out by administering interleukin-6 and / or cyclosporin A. In a variant, the Q (K / R) RAA peptide coupled to the invariant region of a class I histocompatibility sequence can be administered. Also described are pharmaceutical compositions useful in vaccination protocols.

Description

ETHOD TO TREAT RHEUMATOID ARTHRITIS
Technical Field
The invention relates to regulation of the im¬ mune system to prevent and treat autoimmune diseases . More specifically, it concerns procedures to prevent and treat the immune response which is characteristic of rheumatoid arthritis in humans.
Background Art
The B-lymphocytes and acrophages from more than 90% of patients with seropositive rheumatoid arthritis express the Class II HLA protein DR4 or DRl, as defined serologically. It has been shown that DR4 specificity is present on several different haplotypes, three of which, Dw4 , Dwl4 and Dwl5, are associated with the B-lymphocytes and macrophages from rheumatoid arthritis patients, and two of which, DwlO and Dwl3, are not. Thus, the B-lympho- cytes and macrophages of subjects with rheumatoid arthritis express the Class II haplotypes Dw4, Dwl4, Dwl5 or DRl. It has previously been shown that the haplotypes associated with rheumatoid arthritis (RA) are distinguished from those not associated with RA by the sequence of amino acids 70-74 in the third hypervariable region of the DR-beta-1 chain (Gregersen, P. , et al. , Proc Natl Acad Sci USA (1986) 8_3:2642-2646 ? Gregersen, P., et al., Arch Rheum (1987) 3_0: 1205-1213; Tonnelle, C, et al . , Ann Inst Pasteur Immunol (1988) 139:41-53) . The sequence of the region is either QKRAA (Dw4) or QRRAA (Dwl4, Dwl5 and DRl). The substitution of R (arginine) for K (lysine) is conservative. Thus, the sequence Q(K/R)RAA may be important in the development and symptoms of rheumatoid arthritis. The current thinking with regard to the role of the proteins encoded by the major histocompatibility complex (MHC) in permitting the immune system to distinguish between self and nonself postulates that foreign antigens are "presented" to the various classes of T-cells by Class II proteins encoded by the MHC. The Class II protein contains a site capable of binding the foreign antigen (or a fragment thereof) and another site which recognizes a T-cell receptor. Structural studies by others have indicated that the position on the Class II MHC protein on which the Q(K/R)RAA sequence is located corresponds to the T-cell receptor recognition site.
Rheumatoid arthritis is thought to be an autoimmune disease wherein certain protein components of the synovial fluid are recognized as foreign by the immune system. Presumably they are presented to the T- lymphocytes in the context of an MHC Class II protein, which MHC Class II carrier is recognized as self by the T- lymphocyte. Based on the foregoing paragraphs, it may be supposed that this self-recognition site includes Q(K/ R)RAA. It has now been found that by administering a suitable vaccine, the response of T-lymphocytes to the synovial fluid-associated antigen can be either diverted from the helper T-cell class, or prevented. The vaccines of the invention thus prevent T-helper proliferation in response to the synovial antigen conjugated to the Class II recognition site Q(K/R)RAA.
Disclosure of the Invention
The invention provides vaccines for the preven- tion and treatment of the autoimmune response to synovial protein which manifests itself in the symptoms of rheumatoid arthritis. The vaccines of the invention ef¬ fectively prevent the T-helper cell response to these proteins presented in the context of the MHC Class II histocompatibility protein having the sequence Q(K/R)RAA at its recognition site.
Thus, in one aspect, the invention is directed to a method to immunize a subject to prevent or ameliorate rheumatoid arthritis wherein the vaccination protocol comprises administration of an effective amount of the peptide Q(K/R)RAA in immunogenic form in conjunction with an effective amount of one or more immunostimulatory or immunosuppresant substances which preferentially enhance the proliferation of suppressor and/or cytotoxic T-cells, and/or suppress the expansion of helper T-cells without effect on suppressor/cytotoxic expansion. Such im unoaffecting substances include interleukin-6 (IL-6), cyclosporin A, antibodies against CD4 antigen, transforming growth factor-beta (TGF-beta), and combinations thereof. The invention is also directed to vaccines useful in this method.
In another aspect, the invention is directed to a vaccine useful in preventing rheumatoid arthritis, which vaccine comprises the Q(K/R)RAA a ino acid sequence coupled to an invariant portion of a Class I histocompatibility sequence, and to methods of rheumatoid arthritis prevention and amelioration using this vaccine.
Brief Description of the Drawings
Figure 1 shows the stimulation (or not) of T- cells derived from EBV-exposed and EBV-nonexposed subjects by gpllO and by Dw4 peptide.
Figure 2 shows antibody titers measured against gpllO peptide or protein of a rabbit immunized with gpllO peptide. Figure 3 shows immunoblots of HLA Class II Dw4 and Dw2 proteins with rabbit anti-D 4 antisera. Figure 4 shows the comparative amino acid sequences of Dw4, Dwl4 and DwlO beta peptides.
Modes of Carrying Out the Invention The vaccines of the invention utilize as an es¬ sential component the amino acid sequence Q(K/R)RAA in an immunogenic form or an immunogenic form of the peptide sequence which forms that portion of a T-cell receptor binding to this epitope. Accordingly, the specific peptide hapteπ sequence may be part of a larger polypeptide or protein and/or may be conjugated, option¬ ally through a linker, to a carrier. If the peptides are included within a larger protein, convenient larger proteins include additional amino acid residues found in the Class II DR-beta-1 chain or additional amino acid residues adjacent this peptide in glycoprotein gpllO as¬ sociated with the Epstein-Barr virus; for peptides representing the Q(K/R)RAA sequence in the context of the Class I MHC protein constant regions, extensions of these regions may also be employed. However, the individual peptide may also be used. In both cases arbitrary, non- interfering chain extensions may also be employed.
Suitable carriers which enhance immunσgenicity of the hapten, or confer immunogenicity upon it, include the commonly employed keyhole limpet hemocyanin (KLH) protein, tetanus toxoid, or other antigenically neutral materials, such as human serum albumin. If a separate carrier such as tetanus toxoid, KLH or HSA, is used, the peptide may be conjugated to the carrier using means standard in the art, including the use of linkers, many of which are commercially available from Pierce Chemical Company, Rockfσrd, IL. Typical linkers include, for example, SMCC and SPDP. Methods of effecting such conjugation are well known. The foregoing are exemplary of the art- recognized ways to confer immunogenicity on short peptide sequences such as the Q(K/R)RAA of the invention; any manner of conferring immunogenicity on this sequence may be employed, including synthesis of larger peptides, e.g., 10-100 amino acids, which include this sequence, prefer- ably repetitively.
The vaccines of the invention are administered to persons at risk for rheumatoid arthritis as shown by the haplotypes described above. Thus, individuals with haplotypes corresponding to Dw4, Dwl4, Dwl5 or DRl are administered the vaccine.
In one method of RA prevention or amelioration, the immunogenic form of Q(K/R)RAA is administered in conjunction with an immunostimulant and/or immunosuppressant which results in a preferential expan- sion if T-suppressor and/or cyctotoxic cells at the expense of T-helper cells. Thus, stimulants which expand suppressor/cytotoxic populations but which do not affect helper populations may be used; or substances which actively suppress helper populations, but do not affect suppressor/cytotoxic T-cells may be used, or combinations of these factors may be used. Certain substances which effect this differential expansion are known in the art; however, the invention includes the use of any substance which can be shown to have this effect. Interleukin-6 is an appropriate immunostimulant for suppressor/cytotoxic T- cells. It is often desirable to include IL-2 to enhance this stimulation as well. Cyclosporin A, TGF-beta, and anti-CD4 antibodies (see, e.g., Carteron, N.L., et al. , J Immunol (1988) 140:713-716) are appropriate helper cell inhibitors.
By administered "in conjunction with" is meant that either administration is in the same vaccine formula¬ tion (containing both the immunogenic form of Q(K/R)RAA and the immunostimulant and/or immunosuppressant) or each of these components may be administered within a conjunc¬ tive period of time so that their cooperative effect is obtained. Suitable conjunctive time periods are of the order of 4 hours to 2 days, preferably 4 hours to 1 day. The immunostimulant/immunosuppressant and the immunogenic peptide may be administered in any order, and protocols of repeated administrations of each or both can also be employed.
Suitable routes of administration are systemic, and include administration by injection such as intra¬ venous, intramuscular, peritoneal, or subcutaneous injec- tion. Subcutaneous or intramuscular administration are preferred. The immunogenic peptide and immunostimulatory/ immunosuppressant substance are formulated for injection using standard formulations for the administration of peptides which include suitable excipients such as buff- ers, Ringer's solution, or Hank's solution, and may further include wetting agents, stabilizers, and the like. Suitable adjuvants may also be included. Other forms of administration, such as transmembrane or transdermal administration using formulations suitable for these routes may also be employed. A wide range of formulations is found, for example, in Remington's Pharmaceutical Sci¬ ences, latest edition, Mack Publishing Company, Easton, Pennsylvania.
Suitable amounts of the immunogenic peptide are about 100 ug-100 mg per patient, preferably 10-100 mg. The immunostimulant/immunosuppressant or combination should be administered in an amount according to the potency of the substance chosen; for IL-6, for example, suitable dosage ranges are 1-100 ug/kg, preferably 20-50 ug/kg. It is understood, of course, that dosage level is highly dependent on the particular immunogenic form of the peptide used, the iπvmunostimulant/immunosuppressant selected, the route of administration selected, the nature of the formulation, and the individual response of the subject. Establishment of optimum dosage protocols and regimens for a subject, taking account of the foregoing parameters, is routinely done and is well within ordinary skill.
In another form of the vaccine, the peptide Q(K/ R)RAA or a larger peptide containing this sequence is coupled to an invariant portion of a Class I histocompatibility sequence to generate the active component of the vaccine. The invariant portion of Class I histocompatibility sequences are known and the relevant data are deposited in Genbank and other equivalent data- bases generally known in the art. (See referenced art in Chimini, G., et al. , J Exp Med (1989) 169:297-302. )
In addition, the relevant sequences can be found in a publication entitled "Sequences of Proteins of Immunological Interest," 4th ed. (1987), published by the U.S. Dept. of Health and Human Services. On pages 337-358 of this publication are listed the residues occupying positions 1-360 of known Class I MHC antigens from human, urine and other animal sources . A review of these pages shows that the constant regions among the human MHC proteins are positions 1-8; 13-31; 46-53; 84-93; 117-136; 157-176; 200-238; 241-268. In general, peptides from these regions having 10-20 residues, preferably around 12- 15 residues, can be conjugated to the relevant Q(K/R)RAA sequence with or without additional extensions derived from those normally surrounding this sequence, as described above.
Methods to conjugate the Class I invariant sequence (which can be synthesized using standard techniques) and the antigen are known in the art, as described above. The conjugation of Class I portion and Q(K/R)RAA peptide can also be effected by recombinant production of the fusion protein. Routes of administration and formulations are also similar to those described above. The following examples are intended to il¬ lustrate, but not to limit, the invention.
Example 1
Ho ology of the Rheumatoid Arthritis Determinant and Epstein-Barr Virus gpllO As described by Roudier, J., et al. , Scand J Immunol (1988) 21_i 361-311 , and Roudier, J., et al. , Proc Natl Acad Sci USA (in press), incorporated herein by reference, the QKRAA/QRRAA sequence of the Class II MHC protein, which was known to be related to the incidence of rheumatoid arthritis, was matched through the National Biological Research Foundation database of protein sequences and the Genbank database of DNA sequences to segment 807-816 of the Epstein-Barr virus glycoprotein gpllO, which contains the 6-amino acid stretch EQKRAA, matching the HLA Dw4 sequence, followed by a nearly identical second copy, QRAA. Computer analysis of the secondary structure of residues 760-860 of gpllO by the method of Chou and Fassman (Adv Enthy ol (1978) 47:45-148) predicts that the relevant QKRAAQRAA stretch is part of an alpha-helix between two hydrophobic regions, representing a structure of a type postulated to constitute the epitope responsible for binding to T-cells. The result of this homology is the ability of this subsequence in Epstein- Barr virus glycoprotein to stimulate the proliferation of T-cells which mediate rheumatoid arthritis and which T- cells recognize the synovial protein antigen conjugated to the Class II MHC protein of susceptible haplotypes Dw4/ Dwl4/Dwl5/DR1.
The enhanced susceptibility of EBV-infected individuals to RA is known; however, this nexus has not been understood. The ability of Epstein-Barr virus (EBV) infection to stimulate the T-cell population to respond to both the gpllO peptide and the relevant Dw4 peptide is demonstrated in the following examples .
Example 2
Preparation of Peptides Four peptides representing the relevant portions of the Class II MHC protein haplotypes associated with RA, the gpllO peptide, and a control DwlO peptide were synthesized with the amino acid sequences:
Dw4 : KDLLEQKRAAVDTYC;
Dw4 ' : EQKRAAEQKRAA;
Dwl4/Dwl5/DR1: KDLLEQRRAAVDTYC; gpllO: QENQEQKRAAQRAAGC; DwlO: KDILEDERAAVDTYC.
These peptides were used as test stimulating peptides for T-cell proliferation or were conjugated to KLH using m-maleimidobenzoyl-n-hydroxysuccinimide as described by Green, N., Cell (1982) ^:477-487; Liu, F.T., et al., Biochemistry (1979) __ :690-697, for use in im¬ munization.
Example 3
Sensitization of T-Cells to Q(K/R)RAA Stimulation by Prior Exposure to EBV The peptides synthesized in Example 2 were used to determine the response of T-cells in vitro as follows: The method was a modification of that disclosed in Ford, D., Cell Immunol (1983) 7_9:334-344; Thorley-Lawson, D.A. , et al., Proc Natl Acad Sci USA (1987) ^4:5384-5388, both incorporated herein by reference. Briefly, peripheral blood mononuclear cells were obtained both from a donor with no history of EBV infection, as indicated by the absence of antibody to the viral capsid antigen (VCA) and from an otherwise normal subject with a high titer of anti-VCA antibodies, indicating prior exposure to EBV. The mononuclear cells were isolated by isopycnic centrifugation through Ficoll-Hypaque, and were washed three times in RPMI-1640 medium. The cells were then suspended at a density of 10 cells/ml in the same medium supplemented with 10% pooled human AB serum, 1% L- glut-amine, 100 U/ml penicillin, and 100 ug/ml streptomycin in the presence of 1 ug/ml of a stimulatory peptide selected from the list above. After 1 week culture at 37°C, a sample of 10 cells were taken from the bulk culture to evaluate the primary proliferative response. In a secondary culturing, the remaining cells were counted and serially diluted in fresh medium sup- plemented with the same peptide to be tested, or without said peptide as a control. The cells were then distributed in 100 ul aliquots containing 50,000, 25,000, 10,000, or 5,000 viable cells from the primary culture in 96-well round-bottom culture plates and the number of cells in each well adjusted to 10 by the addition of ir¬ radiated (3,000 rad) peripheral blood mononuclear cells from the same donor. For each cell density, 18 wells with peptide and 6 control wells without peptide were assayed. After 4 days of this secondary culture, 1 uCi of tritiated thymidine was added to each well 18 hours before cell harvesting on glass fiber filters. For each cell dilution, positive wells were defined as having cpm higher than (mean + 2 standard deviations) of the cpm obtained for 6 control wells. Frequency of precursor T-cells was evaluated by plotting the percentage of negative wells for each dilution against the number of cells per well, as described by Ford et al. (supra) ■
The results are shown in Figure 1. Panels a and c show the response (in cpm/well) at various dilutions of cells derived from a VCA-positive donor; panels b and d, from VCA-negative donors. Panels a and b were treated with the gpllO peptide; panels c and d, with the Dw4 peptide.
The results in Figure 1 show that for T-cells derived from VCA-positive donors, either the gpllO peptide or Dw4 peptide was able to stimulate proliferation; for those derived from a VCA-negative donor, no stimulation occurred at any dilution. Corresponding results on a the primary stimulated cultures are shown below in Table 1.
Table 1
VCA+ VCA-
No stimulation 3976+1025 2691+141 gpllO 8720+1227 1976+162 Dw4 9604+1287 2448+578
The foregoing results clearly show that prior exposure to EBV sensitizes a population of T-cells to expansion upon subsequent contact with either the relevant portion of the gpllO peptide or the relevant portion of the Dw4 peptide.
These results further show that the precursor T- cell concentration in an individual with prior exposure to EBV was about 1 in 70,000 T-cells with respect both to gpllO and Dw4.
Example 4 Cross-Reactivity of gpllO and
RA-Susceptible Haplotype Antibodies The peptides prepared in Example 2 and conjugated to KLH were used to raise antibodies in rab¬ bits. New Zealand white rabbits were injected subcutane- ously with 1 mg of the conjugate emulsified in complete Freund's adjuvant, and then boosted three times at 3 week intervals with 1 mg of conjugate in incomplete Freund's adjuvant. The rabbits were bled four days after the last injection and sera were stored at -20 C.
Antibody titers of the resulting sera were tested using an ELISA assay or by i munoblot. The ELISA assay was by the procedure of Luka, J. , et al., J Immunol Methods (1984) 7_:145-156, incorporated herein by refer¬ ence. Briefly, the antigen peptide was used to coat ELISA plates, and various solutions of preimmune or immune rab- bit sera, diluted in isotonic borate-buffered saline (BBS) were added in 100-ul aliquots to the wells and incubated overnight at 4°C. After washing with BBS, 0.2% Tween-20, bound antibody was detected using alkaline phosphate- conjugated goat antirabbit IgG (Tago, Burlingame, CA) . The results are tabulated as absorbance ratios correspond¬ ing to O.D. at 405 nm for the immune serum divided by that of the preimmune serum. The antibody titer for each serum is defined as the highest dilution yielding an absorbance ratio of at least 2. In inhibition experiments, candidate inhibitor peptides were added to the antisera and allowed to react overnight at 4°C prior to performance of the ELISA.
ELISA determinations were made when reactivity of antibodies was tested to EBV gpllO. The gpllO was purified by affinity chromatography from P3HR1 lymphoblasts according to the method of Kishishita, M. , et al., Virology (1984) 133:393-395.
Using this assay, it was demonstrated that a rabbit immunized with the gpllO peptide produced antibod- ies which recognized both the gpllO peptide and the gpllO protein isolated as described. These results are shown in Figure 2.
Apparently, the epitope recognized on the gpllO protein was that of the peptide, since a 20-fold dilution of serum preincubated with 100 ug/ml of gpllO peptide was no longer capable of binding gpllO protein. These results indicate that the relevant sequence is an epitope associ¬ ated with B-cell response to the protein.
Reaction of antisera with Class II MHC proteins was tested by immunoblotting against membrane protein extract from Molt 4, T-cells (control) or from lymphoblastoid cells established from HLA Dw4 or HLA Dw2 homozygous donors (GM03161 and GM066821A, respectively, obtained from Human Genetic Mutant Cell Repository, Camden, NJ) . The proteins were extracted with Triton- X114, according to the method of Bordier, C, J Biol Chem (1981) 256:1604-1607.
A membrane extract from 2 x 10 cells was loaded into each lane of a 10% polyacrylamide gel containing 0.1% sodium dodecylsiilfate and 1 mM 2-mercaptoethanol. After electrophoresis, the reduced and denatured membrane polypeptides were electrophoretically transferred to nitrocellulose sheets, as described by Towbin, H. , et al. , Proc Natl Acad Sci USA (1979) 7_6:4350-4354; Billings, P.B., et al., ibid (1983) ^0:7104-7108. The filters were then preincubated for one hour in a solution containing 0.05 M borate, 0.15 M NaCl, pH 3, and 3% powdered milk, followed by overnight incubation at 4°C with the antisera diluted at the same dilution. (For inhibition determina¬ tion, candidate peptides were added to the antibody solu- tions prior to their incubation with the nitrocellulose sheets.) After extensive washing of the sheets with borate buffer, bound antibody was detected using 125I- protein A (1 mCi/ l, ICN, Irvine, CA) . The sheets were incubated for 1 hour with the detecting reagent, washed with BBS, dried, and exposed to Kodak XAR film overnight at -70 C using an intensifier screen.
The ability of Dw4 peptide to raise specific antibodies was also shown by assay of the rabbit antiserum by immunoblot as described above. The results of this assay are shown in Figure 3. The immunoblot in panel A is a control testing anti-DR-beta monoclonal antibody diluted 1:1000 with extracts from Molt 4 (M4) cells, HLA Dw4 homozygous lymphoblastoid cells (D4), or HLA Dw2 homozygous lymphoblastoid cells (D2). Both DR beta proteins react, M4 does not. In panel B, the gels were incubated with antisera from a rabbit immunized with the Dw4 peptide.
Lane set 1 represents the results when the electrophoresis was run using the membrane extracts alone. Only the D4 extract is immunoreactive with the antibody, confirming the specificity of the antiserum.
Lane set 2 shows the results when the antiserum was preincubated with 10 ug/ml Dw4 peptide; the binding to the membrane-derived D4 peptide in the gel is completely eliminated. Lane set 3 shows the results when the antiserum was preincubated with the 100 ug/ml of the control peptide DwlO. No effect on the binding of the antibody to the D4 extract was noted.
Lane set 4 shows that preincubation with the related peptide Dwl4 at 10 ug/ml was partially effective in eliminating binding to D4; the binding was almost eliminated when the concentration of Dwl4 was increased to 100 ug/ml (lane set 5).
A comparison of the sequences of Dw4, Dw2, Dwl4, and DwlO (Figure 4) shows that the presence of residues 67, 70, and 71 are critical for recognition of the Dw4 antibody. The serum from this same rabbit, immunized with Dw4, was shown by ELISA to recognize Dw4 peptide, gpllO peptide, and gpllO protein, with the titers shown in Table 2. Table 2 Titers of Anti-Dw4 Serum
Titer
Dw4 104 gpllO peptide 103 gpllO protein 103
The recognition of gpllO protein was inhibited by preincubation of a 100-fold dilution of the serum with either 100 ug/ml gpllO peptide or 100 ug/ml Dw4 peptide. The recognition of gpllO peptide was inhibited by preincubation with 10 ug/ml of either Dw4 or Dw4' peptide (Example 2) .
The foregoing results show that the EQKRAA sequence shared by gpllO and HLA Dw4 is the basis for serological cross-reaction.
Example 5
Conjugation to Class I Constant Region The following peptides, representing constant regions of the human Class I MHC glycoprotein are constructed:
A-Y-D-G-K-D-Y-I-A-L-(K/N)-E-D-L-(R/S)-S-W-T-A-A-(D/N)- (M/T)-A-A-Q
representing positions 117-141 of the human HLA Class I protein; and
E-A-T-L-R-C-W-A-L-(G/S)-F-Y-P-A-E-I-T-L-T-W-Q-R-D-G-E-D-Q- T-Q-D-T-E-L-V-E-T-R-P-A-G-D-G-T-F
representing positions 198-241. Fragments of these peptides containing 12-15 residues from the sequences shown are also prepared.
These peptides are conjugated using dicyclohexylcarbodiimide to the peptide of the sequence Q(K/R)RAA and formulated into vaccines.
Alternatively, the gene encoding the desired portion of the above peptides with extensions of the residues Q(K/R)RAA (including constructions containing codons for multimers of the pentapeptide) are constructed and inserted into standard recombinant expression systems for production of fusion proteins. The resulting fusions are then formulated into vaccines as described above.

Claims

Claims
1. A method to immunize a subject against rheumatoid arthritis, which method comprises administering to said subject in need of such immunization, an amount of a peptide comprising the sequence Q(K/R)RAA in immunogenic form effective to stimulate the T-immune system, in conjunction with at least one immunostimulant and/or immunosuppressant capable of effecting the preferential expansion of suppressor and/or cytotoxic T- cells in comparison to helper T-cells.
2. The method of claim 1 wherein the immunostimulant is interleukin-6 (IL-6), with or without IL-2.
3. The method of claim 1 wherein the immunosuppressant is selected from cyclosporin A, TGF- beta, and antibodies immunoreactive with CD4 surface antigen.
4. A pharmaceutical composition which comprises, in admixture, a peptide containing the sequence Q(K/R)RAA in immunogenic form and an immunostimulant and/ or immunosuppressant capable of effecting the preferential expansion of suppressor and/or cytotoxic T-cells as compared to helper T-cells.
5. The composition of claim 4 wherein the immunostimulant is interleukin-6 (IL-6), with or without IL-2.
6. The composition of claim 4 wherein the immunosuppressant is selected from cyclosporin A, TGF- beta, and antibodies immunoreactive with CD4 surface antigen.
7. A method to immunize a subject against rheumatoid arthritis which method comprises administering to said subject in need of such immunization an amount of a peptide comprising the sequence Q(K/R)RAA conjugated to the invariant portion of the Class I histocompatibility sequence effective to stimulate the production of specific suppressor and/or cytotoxic T-cells.
8. A vaccine useful in immunizing human subjects against rheumatoid arthritis which contains as active ingredient a peptide comprising the amino acid sequence Q(K/R)RAA conjugated to the invariant portion of a Class I histocompatibility sequence.
9. A composition of matter which consists es¬ sentially of a peptide comprising the sequence Q(K/R)RAA conjugated to the invariant portion of a Class I histocompatibility sequence.
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US5194425A (en) * 1988-06-23 1993-03-16 Anergen, Inc. Mhc-mediated toxic conjugates useful in ameliorating autoimmunity
US5260422A (en) * 1988-06-23 1993-11-09 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
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US6682731B1 (en) * 1994-03-24 2004-01-27 Ludwig Institute For Cancer Research Isolated peptides derived from mage tumor rejection antigen precursors which complex with HLA-A2 molecules
US7094597B1 (en) 1994-05-20 2006-08-22 The Regents Of The University Of California Vaccine compositions and methods useful in inducing immune protection against arthritogenic peptides involved in the pathogenesis of rheumatoid arthritis
EP0762881B1 (en) * 1994-05-20 2004-03-17 The Regents of the University of California Use of active agents for the manufacture of medicaments for the treatment of rheumatoid arthritis
US5773570A (en) * 1994-05-20 1998-06-30 The Regents Of The University Of California Vaccine compositions and methods useful in inducing immune protection against arthritogenic peptides involved in the pathogenesis of rheumatoid arthritis
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US7074893B2 (en) 2002-06-03 2006-07-11 Regents Of The University Of Michigan Methods and compositions for the treatment of diseases associated with signal transduction aberrations
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