Classifications

• • 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
  • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • 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 in the field of immunology and medicine relates to peptides which mimic a natural peptide and compete with the heat shock protein, hsp 65, or peptides derived therefrom for recognition by and activation of T lymphocytes.
• the peptides of the invention inhibit T lymphocyte activation and proliferative responses, and can protect a subject from immune reactions and immune-related disease including autoimmunity and graft rejection.
• autoimmune diseases are both long and disturbing. It includes, for example, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, and systemic lupus erythematosus. In all of these diseases, the immune response is potent and specific. The problem is that this immune response is directed at some essential component of the body. Millions of people are afflicted with chronic forms of arthritis which are thought to involve autoimmunity to constituents of the joints of connecting tissues of the body. These conditions include rheumatoid arthritis, ankylo ⁇ ing ⁇ pondyliti ⁇ , Reiter' ⁇ syndrome, and other forms of reactive arthritis.
• Adjuvant arthritis is an experimental model of human arthritis inducible by immunizing susceptible strains of rats to Mvcobacteria.
• the disease which develops about twelve days after immunization has many of the features of rheumatoid arthritis, and, indeed, adjuvant arthritis has been considered to be a model for rheumatoid arthritis.
• Rats immunized with adjuvant containing mycobacterial antigens to produce arthritis contain T lymphocytes specific for mycobacterial antigens. Indeed, long term T cell lines and clones have been isolated from rats so immunized; these T cell were able to induce and modulate the arthritic disease in vivo (Holoshitz, Science, 1983, 219;56; Holoshitz, J. et al.. J. Clin. Invest. ,
• T cell clone designated A2b
• the minimal antigenic structure, or epitope, responsible for this mimicry between a cartilage-as ⁇ ociated self structure and mycobacteria, recognized by both of the above T cell clone ⁇ , is the amino acid ⁇ equence from residue 180 to 188 of the mycobacterial 65 kD heat-shock protein (HSP) (Van Eden, W. et al. , Nature 1988, 331:171) .
• HSP heat-shock protein
• HSPs Heat shock proteins
• HSPs serve as important antigens of infectiou ⁇ agents, and possibly,of transformed cells. During infection, HSP ⁇ are induced in both microorganisms and host phagocytes. The dominant antigens of intracellular pathogenic microorganisms are HSP ⁇ known as hsp 65 (the human representative of the hsp 60 family of molecules) and hsp 70. Due to their extreme conservation, it is not surprising that HSPs bear a relationship with autoimmune diseases. Cloned "autoreactive" T cells have been obtained from normal donors that are reactive to human h ⁇ p 65 and h ⁇ p 70. D. Immunity to Heat Shock Proteins in Arthritis
• Adjuvant arthriti ⁇ can be induced u ⁇ ing M ⁇ . tuberculosis in oil in Lewis rats and, to a les ⁇ er extent, in Fisher and BN rats.
• the expression of the mammalian homolog HSP is selectively enhanced in affected joint tissue of rats with adjuvant arthritis and patients with rheumatoid arthritis (Karlsson-Parra, A.K. et al. , Scand. J. Immunol. 3.1:283-288 (1990) ) .
• the inventors and their colleagues reported that the minimal stimulatory sequence for two arthritis-specific T lymphocyte clones, A2b and A2c, was defined as the 180-187 amino acids sequence of the mycobacterial hsp 65 (Van der Zee, R. et al. , Eur. J. Immunol. :43 (1989)). Peptides having any amino acid replacement at po ⁇ ition ⁇ 187 and 188 fully stimulated both T cell clones, indicating that these positions did not affect the antigenic nature of the 180-186 heptapeptide. It was thought that mimicry occurs with an hsp 65 epitope of a proteoglycan-associated HSP or an HSP expressed in synovial ti ⁇ sue ⁇ .
• a refractory state can be "artificially" induced in the Lewi ⁇ rat adjuvant arthriti ⁇ model by prior treatment wit soluble mycobacterial h ⁇ p 65, which al ⁇ o down-regulates responses to the nonapeptide; this treatment also prevents SCW-induced arthritis and partially inhibits collagen-induced arthriti ⁇ .
• T cell re ⁇ pon ⁇ e ⁇ to mycobacterial hsp 65 are elevated in the synovial fluid, compared with blood; T cells from a few JRA patients with elevated antibody respon ⁇ e ⁇ to hsp 65 responded to the 180-188 nonapeptide (Lydard, P.M. et al. , Immunol. Today 11:228 (1990)).
• Stanford et al. discloses aqueous acetone soluble and insoluble fractions of certain mycobacteria, such a ⁇ Mvcobacterium H-37, M. kansa ⁇ ii and M. vaccae.
• the soluble fraction of H-37 was found to provoke an immune re ⁇ ponse leading to resistance to adjuvant arthritis wherea ⁇ the in ⁇ oluble fraction seemed to be responsible for the induction of the disease.
• M. vaccae was shown to be substantially free of adjuvant arthritis-inducing components.
• One T cell line, de ⁇ ignated A2 was found to induce arthritis upon intravenous (IV) injection into irradiated rat ⁇ .
• the A2 cell ⁇ were effective in vaccinating unirradiated rat ⁇ again ⁇ t ⁇ ubsequent induction of arthritis by active immunization with mycobacteria.
• Cell line A2 was cloned, yielding two distinct clone ⁇ , designated A2b and A2c.
• A2b cells induce arthritis but do not vaccinate against arthritis.
• Clone A2c cell ⁇ do not cause arthritis, but rather vaccinate against it, and additionally can be used to treat existing adjuvant arthritis. Moreover, clones A2b and A2c are used to identify antigens associated with arthritogenicity or with suppression of arthritogenicity. Both clones respond in vitro to whole mycobacteria as well as to cartilage proteoglycan protein.
• Van Eden et al. European Patent Publication EP 262710 (4/6/88)
• a protein having an apparent molecular weight of about 64 kDa (the preparation of which is described in Thole, J.E.R. et al. , Infec. Immun. 50:800-806 (1985)) is useful as a vaccine for inducing resistance again ⁇ t autoimmune arthritis and similar autoimmune diseases.
• This publication also disclose ⁇ that this protein, also known a ⁇ antigen A, cross-reacted serologically with antigen ⁇ pre ⁇ ent in other bacterial ⁇ pecies, such as Mycobacteria.
• These polypeptides could be used as immunogens in pharmaceutical compositions, particularly a ⁇ vaccines, for the alleviation and the treatment of autoimmune disea ⁇ e ⁇ , a ⁇ well a ⁇ in diagnostic composition ⁇ for the diagnosis of these disease ⁇ .
• Thi ⁇ reference provided no guidance a ⁇ to which re ⁇ idue ⁇ could be ⁇ ub ⁇ tituted, and what amino acid ⁇ could serve a ⁇ appropriate ⁇ ubstitutes, in order to obtain a useful polypeptide for preventing or treating autoimmune disease.
• a peptide of at least seven amino acid residue ⁇ capable of inhibiting the proliferative response of a T lymphocyte to a specific antigen comprise ⁇ an amino acid sequence which corre ⁇ pond ⁇ to positions 180-186 of the Mycobacterium tuberculosis protein h ⁇ p 65 having the formula TFGLQLE but differing therefrom by one to three amino acid substitution ⁇ in the po ⁇ ition ⁇ 181-183, competes with hsp 65 for recognition by antigen reactive lymphocyte clones, such as clones A2b and A2c in experimental arthritis.
• These substitutions can be of any form wherein the modified hsp 65 180-186 ⁇ equence is recognized by antigen- reactive T lymphocytes, such as T lymphocytes reactive to M.
• tuberculosi ⁇ or any epitope thereof preferably to h ⁇ p 65, T lymphocyte ⁇ reactive to myelin ba ⁇ ic protein (MBP) , and the like, and the proliferative re ⁇ ponse of the T cell ⁇ is inhibited.
• MBP myelin ba ⁇ ic protein
• the present invention provides a peptide of at lea ⁇ t ⁇ even amino acid re ⁇ idue ⁇ capable of inhibiting the proliferative re ⁇ pon ⁇ e of a T lymphocyte to a specific antigen, said peptide comprising an amino acid sequence which corresponds to positions 180-186 of the Mycobacterium tuber ⁇ culosis protein hsp 65 having the formula:
• the peptide of the present invention has up to 20 amino acid residues, most preferably between 7 and 9 amino acid re ⁇ idue ⁇ .
• Preferred peptide of the pre ⁇ ent invention has the formula TFGAQLE, TFGAQLELT, TAGLQLE or TAGLQLELT.
• the above peptides are capable of inhibiting the proliferative response to hsp 65 of a T lymphocyte reactive thereto, a proliferative response to myelin basic protein of a T lymphocyte reactive thereto, or a proliferative response to Mycobacterium tuberculo ⁇ is of a T lymphocyte reactive thereto.
• the present invention i ⁇ further directed to a method for treating a ⁇ ubject having an autoimmune disease comprising administering to the subject an effective amount of a peptide of the present invention.
• the autoimmune di ⁇ ea ⁇ e i ⁇ arthritis In a preferred embodiment, the autoimmune di ⁇ ea ⁇ e i ⁇ arthritis.
• the present invention also provides a method for suppressing or preventing the rejection of a transplanted organ or tissue in a subject comprising administering to the subject an effective amount of a peptide according to the present invention, prior to and/or immediately after receipt of the transplanted organ or tis ⁇ ue.
• the pre ⁇ ent invention i ⁇ further directed to a T lymphocyte line or clone compri ⁇ ing T lymphocyte ⁇ ⁇ pecifically reactive to any of the peptide ⁇ of the pre ⁇ ent invention, and to the u ⁇ e of ⁇ uch cell ⁇ in the prevention or suppression of an immune-mediated disease, preferably arthritis.
• Figure 1 is a diagram showing two amino acid sub ⁇ titution networks for the nonapeptide comprising positions 180-188 of the mycobacterial 65 kD protein, hsp 65.
• Figure la pre ⁇ ents the "single substitution network," wherein every residue of the 180-188 sequence was replaced by each of 19 different amino acids.
• Figure lb presents the "combination network,” wherein substitution ⁇ at positions 180, 181, and 186 were combined.
• Figure 2 is a graph showing proliferative respon ⁇ es of the T cell clone, A2c, to the ⁇ timulatory h ⁇ p 65 180-188 peptide, and their inhibition by alanine-substituted peptides, where the competitive peptides were added prior to stimulation.
• Figure 3 is a graph showing rever ⁇ al of A183 inhibi ⁇ tion of T cell proliferation by increa ⁇ ing concentration ⁇ of peptide 180-188.
• Figure 4 i ⁇ a graph showing proliferative re ⁇ pon ⁇ es of the T cell clone, A2b, to the ⁇ timulatory hsp 65 180-188 peptide, and their inhibition by alanine-substituted peptides, where the competitive peptides were added at the time of stimulation.
• Figure 5 includes graphs showing results of experiments which demonstrate the dose dependency of the inhibitory activities of A183.
• Figure 6 is a graph showing that responses of an encephalitogenic T cell clone, Zla, specific for myelin basic protein, are inhibited by the A183 peptide.
• Figure 7 is a graph showing proliferative respon ⁇ e ⁇ to various doses of A183 of lymph node cells obtained from animals either immunized or not immunized with A183.
• Figure 8 is a graph showing that co-administration of A183 and an arthritogenic dose of Mycobacterium tuberculosi ⁇ reduces the severity of arthritis.
• Figure 9 is a graph showing the in vitro proliferative re ⁇ pon ⁇ es of clone A2b T cell ⁇ to single amino acid sub ⁇ titution variants of peptide 180-188.
• FIGS. 10 and Figure 11 are graphs showing proliferative responses of T cell clone A2b (Fig. 10) and T cell line Zla (Fig. 11) to single alanine sub ⁇ tituted variant peptides of peptide 180-188 corresponding to hsp 65 (for A2b) and of peptide 1020 corresponding to MBP (for Zla) .
• the peptide 180-188 comprise ⁇ wa ⁇ te ⁇ ted at concentration ⁇ of approximately 2-3 ⁇ g/ml, and the peptide 1020 series at concentrations of approximately 50 ⁇ g/ml.
• SI a ⁇ stimulation index
• Figure 12 and Figure 13 are graphs showing competition for antigen presentation between non-stimulatory single alanine ⁇ ub ⁇ tituted peptide analogue ⁇ and the unmodified 180-188 peptide.
• Non-stimulatory alanine ⁇ ub ⁇ tituted peptide analogue ⁇ of peptide 180-188 (Fig. 12) and peptide 1020 (Fig. 13) were preincubated with antigen presenting cells and A2b T cell ⁇ 2 h before the addition of the stimulatory peptide 180-188.
• Competition wa ⁇ asses ⁇ ed by determining the reduction of proliferation in the pre ⁇ ence of varying concentration ⁇ of the competitor peptide, ⁇ timulated by a suboptimal concentration of the stimulatory peptide 180- 188 (0.5 ⁇ g/ml) .
• Unstimulated control values were approximately 200 cpm. Results shown are the mean cpm of triplicate cultures. Error bars represent the ⁇ tandard error of the mean.
• Figure 14 is a graph showing the reduction of arthritis severity after disease induction with M. tuberculosis (Mt) when co-injected with peptide A183.
• the re ⁇ ults shown are the mean arthriti ⁇ ⁇ core ⁇ of each group. Error bar ⁇ represent the standard error of the mean.
• the pre ⁇ ent invention i ⁇ ba ⁇ ed on the unexpected di ⁇ covery by the inventors that peptide ⁇ at lea ⁇ t ⁇ even amino acid ⁇ , corresponding to a portion of the Mycobacterium tuberculosis hsp 65 protein, which have substituted amino acids in certain po ⁇ itions, but not in others, are potent generalized inhibitors of T lymphocyte proliferative re ⁇ ponse ⁇ as measured in vitro, and of immune-related diseases such a ⁇ autoimmune disea ⁇ e ⁇ in vivo.
• the peptide ⁇ of the pre ⁇ ent invention are those peptides which have at lea ⁇ t seven amino acid re ⁇ idues comprising an amino acid sequence which corresponds to positions 180-186 of the Mycobacterium tuberculosis protein hsp 65 having the formula: 180 186
• the peptide of the present invention has up to 20 amino acids, most preferably, between 7 and 9 amino acids.
• these peptide ⁇ are recognized by the immune system (either by T cell ⁇ or antigen-presenting cells) and are capable of inhibiting the proliferative re ⁇ pon ⁇ e of a T lymphocyte to an antigen to which the T lymphocyte is specific. Because of this recognition and inhibitory action, the peptides of the present invention can be used as com ⁇ petitors for recognition by any antigen-reactive T lymphocyte, or by an antigen-presenting cell. The peptide ⁇ inhibit the normal stimulation of such T lymphocytes and thereby protect - a subject from an immune-related disease, such as an autoimmune disea ⁇ e.
• ⁇ ub ⁇ titution of positions 181 and 183, most preferably position 183, of hsp 65, especially with alanine result ⁇ in a peptide having the de ⁇ ired propertie ⁇ a ⁇ taught herein, and u ⁇ eful for protection of a ⁇ ubject from an immune-related disea ⁇ e.
• a peptide of at lea ⁇ t ⁇ even amino acid residues comprising an amino acid sequence which correspond ⁇ to po ⁇ itions 72-85 of guinea pig yelin basic protein (MBP)
• a preferred peptide corresponding to the MBP sequence has the formula QKSQRSQAENPV. Identification of ⁇ ub ⁇ tituted peptide ⁇ which are useful in the methods of the pre ⁇ ent invention can be ea ⁇ ily accomplished by testing their ability to inhibit proliferative re ⁇ ponses in vitro of T cell ⁇ , a ⁇ exemplified by the arthritis-related rat T cell clone ⁇ A2b and A2c.
• immune-related di ⁇ ea ⁇ e a ⁇ u ⁇ ed herein refers to a disease in which the immune system is involved in the pathogenesi ⁇ of the disease, or in which appropriate stimulation of the immune system can result in protection from the di ⁇ ea ⁇ e.
• a preferred example of an immune-related disease to which this invention is directed is an autoimmune di ⁇ ea ⁇ e.
• Non-limiting example ⁇ of such autoimmune disea ⁇ e ⁇ are rheumatoid arthriti ⁇ , reactive arthritis, myasthenia gravis, multiple ⁇ clerosis, systemic lupus erythematosus, autoimmune thyroiditi ⁇ (Ha ⁇ himoto' ⁇ thyroiditi ⁇ ) , Graves' disea ⁇ e, inflammatory bowel di ⁇ ea ⁇ e, autoimmune uveoretiniti ⁇ , and polymyo ⁇ iti ⁇ .
• peptides are provided which can compete efficiently with Mycobacterium tuberculosis, the native Mycobacterium tuberculosis hsp 65 protein, , or its native peptide 180-188 for recognition by T lymphocyte ⁇ which are as ⁇ ociated with an immune-related disease, such as the arthriti ⁇ - ⁇ pecific T lymphocyte clone ⁇ , A2b and A2c.
• the peptide ⁇ compete with MBP for recognition by the MBP- ⁇ pecific and EAE-inducing T cell line, Zla.
• the peptide ⁇ of the pre ⁇ ent invention are ⁇ ub ⁇ tituted h ⁇ p 65 peptide ⁇ which can compete efficiently with the native h ⁇ p 65 peptide 180-188 for recognition by A2b and A2c T cell clone ⁇ , mea ⁇ ured a ⁇ inhibition of native hsp 65-induced, or native peptide 180-188-induced proliferation by the clones.
• a most preferred embodiment i ⁇ the 180-186 or
• A183 180-188 peptide of h ⁇ p 65 with alanine at po ⁇ ition 183 (A183) .
• A183 a particularly effective competitor for binding to the T cell, pre ⁇ umably to the T cell receptor (TCR) , the MHC molecule of the antigen-pre ⁇ enting cell, or both.
• TCR T cell receptor
• the A183 ⁇ ub ⁇ titution al ⁇ o creates a peptide particularly effective in inhibiting activation and proliferation of arthritis-specific T cells. Additionally, because of its immunogenicity (see Examples) , A183 can induce T cells specific for itself, which apparently counteract the deleterious autoimmune response in vivo.
• A183 is considered to be not merely a competitor peptide but a “competitor-modulator” peptide.
• arthriti ⁇ - ⁇ pecific T cell i ⁇ intended any T lymphocyte, T lymphocyte line or clone, which i ⁇ particularly associated with arthritis by virtue of the T cell's ability to induce arthriti ⁇ , vaccinate against arthriti ⁇ , or otherwi ⁇ e modulate the induction, maintenance, or suppre ⁇ sion of arthritis in a disease-specific manner.
• an arthritis ⁇ pecific T cell i ⁇ immunoreactive in vitro or in vivo with antigens such as M.
• tubercu1osis tubercu1osis, hsp 65, or any amino acid sequence in the hsp 65 protein which is a ⁇ ociated with arthriti ⁇ .
• Examples of sub ⁇ titution ⁇ re ⁇ ulting in T cell inhib ⁇ itory activity include tho ⁇ e in which alanine has been substi ⁇ tuted as follows:
• substitution ⁇ are preferably with amino acids similar to ala, that i ⁇ small aliphatic, nonpolar or slightly polar residue ⁇ : ⁇ er, thr, or gly. However, other amino acid ⁇ ub ⁇ titution ⁇ are not excluded.
• a “chemical derivative” of the peptide of the present invention contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of thi ⁇ invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residue ⁇ . Many such chemical derivatives and methods for making them are well-known in the art. See, for non-limiting examples, U.S.
• Salts of a carboxyl group may be formed by means known in the art and include inorganic ⁇ alt ⁇ , for example, sodium, calcium, ammonium, ferric or zinc salt ⁇ , and the like, and ⁇ alt ⁇ with organic ba ⁇ e ⁇ ⁇ uch a ⁇ tho ⁇ e formed for example, with a ine ⁇ , ⁇ uch a ⁇ triethanolamine, arginine, or ly ⁇ ine, piperidine, procaine, and the like.
• Acid addition ⁇ alts include, for example, salt ⁇ with mineral acid ⁇ ⁇ uch a ⁇ , for example, hydrochloric acid or ⁇ ulfuric acid, and ⁇ alts with organic acids ⁇ uch a ⁇ , for example, acetic acid or oxalic acid.
• Al ⁇ o included within the ⁇ cope of the pre ⁇ ent invention are the peptides described herein attached to various carriers or immobilized matrices, as is well-known in the art.
• enzymatic degradation of the peptide ⁇ of the pre ⁇ ent invention in vivo may cau ⁇ e the peptide ⁇ to be relatively short-lived.
• One method of preventing such degradation would be by making synthetic peptides containing a D-amino acid.
• peptide by moieties intended to affect solubility, e.g, by the addition of a hydrophilic residue, such a ⁇ ⁇ erine, or a charged residue, such as glutamic acid.
• the peptide could be extended for the purpose of stabilization and preservation of a desired conformation, such as by adding cysteine re ⁇ idues for the formation of di ⁇ ulfide bridges.
• Another reason to modify the peptides would be to permit their detection after administration. This can be done by radioiodination with a radioactive iodine isotope, directly, or by adding tyrosine for ⁇ ubsequent radioiodination, as di ⁇ cussed above.
• an inhibitory peptide in accordance with the pre ⁇ ent invention i ⁇ that it be a ligand for the TCR and/or an MHC molecule such that the peptide is recognized by the T cell or antigen-presenting cell of interest, such a ⁇ the A2b and A2c clones, with sufficient affinity to compete successfully for binding with a native or other stimulatory protein or peptide (such as native h ⁇ p 65 or a ⁇ timulatory peptide derived therefrom) .
• a native or other stimulatory protein or peptide such as native h ⁇ p 65 or a ⁇ timulatory peptide derived therefrom
• the pre ⁇ ent invention provide ⁇ T lymphocyte ⁇ ⁇ pecific for the ⁇ e competitor-modulator peptides.
• An example of such an A183- ⁇ pecific T cell line similar in the rat is the ATL line described in Example XIV.
• Such cells are produced by appropriate immunization in vivo and restimulation in vitro, a ⁇ di ⁇ cussed below.
• Included within the scope of the present invention are human T cells specific for the peptides of the invention, preferably derived from the individual who will be the subject of the prevention or treatment of immune-related disea ⁇ e, ⁇ uch a ⁇ arthriti ⁇ .
• T cell lines are treated in a way which will permit their preventative or therapeutic efficacy, while inactivating them for any potential pathogenetic activity.
• Methods for inactivating cells or otherwi ⁇ e rendering them fit for vaccination again ⁇ t autoimmunity are well-known in the art, many of them having been developed by some of the present inventors.
• Such methods include treatment with hydrostatic pressure, with chemical cross-linking agents ⁇ uch a ⁇ glutaraldehyde, with a cyto ⁇ keletal di ⁇ rupting agent ⁇ uch a ⁇ cytochala ⁇ in B, or with low do ⁇ es of cells (U.S. Patents 4,634,590 and 4,716,038; Cohen, I.R. et al.
• the T cell ⁇ of the pre ⁇ ent invention are expected to induce regulatory anti-idiotypic or anti-clonotypic respon ⁇ e ⁇ directed again ⁇ t a ⁇ hared T cell receptor idiotope of the di ⁇ ease-inducing T cell ⁇ .
• a regulatory respon ⁇ e could prevent the development of the di ⁇ ea ⁇ e.
• Peptides according to the present invention are administered to patients having, or known to be susceptible to, an immune-related di ⁇ ea ⁇ e in amount ⁇ ⁇ ufficient to protect the patient from the disease by preventing the patient's immune system from activation leading to induction, maintenance or exacerbation of the disease state.
• the immune-related disea ⁇ es contemplated within the scope of the pre ⁇ ent invention are al ⁇ o di ⁇ ea ⁇ e ⁇ involving graft rejection or graft-ver ⁇ u ⁇ -ho ⁇ t di ⁇ ea ⁇ e.
• the peptides of the present invention are administered to organ transplant recipients- prior to receipt of the transplanted organ, immediately after receipt of the tran ⁇ planted organ, or both, in order to ⁇ uppre ⁇ s or prevent rejection of the tran ⁇ planted organ.
• the term "immediately after” as used herein i ⁇ intended to include administration beginning in the first few hours po ⁇ t-tran ⁇ plant. However the term i ⁇ not intended to limit the duration of treatment po ⁇ t-tran ⁇ plant, which can be readily determined by one of ⁇ kill in the art can without undue experimentation.
• T lymphocyte ⁇ which recognize foreign hi ⁇ tocompatibility antigens are transferred from donor to recipient and react against the recipient leading to a variety of inflammatory con ⁇ equences which may lead to significant morbidity and even mortality (See, for example, Roitt, I. et al. , Immunology, The C.V. Mosby Company, Gower Medical Publishing, London, 1985) .
• the peptides of the present invention are administered to an individual su ⁇ ceptible to a graft-ver ⁇ u ⁇ -ho ⁇ t reaction, ⁇ uch a ⁇ a bone marrow tran ⁇ plant recipient or a susceptible pregnant woman, to prevent the recognition of foreign cells i the "host" by the graft, thereby treating a graft-versu ⁇ -ho ⁇ t reaction.
• the dose ranges for the administration of the composition ⁇ of the pre ⁇ ent invention are those large enough to produce the desired effect, whereby, for example, an immune respon ⁇ e to a ⁇ timulatory peptide, as measured by T cell proliferation in vitro or a delayed hypersensitivity response in vivo, is sub ⁇ tantially prevented or inhibited, and further, where the immune-related disease is significantly treated.
• the dose ⁇ should not be so large as to cau ⁇ e adverse side effects, such as unwanted cros ⁇ reactions, generalized immunosuppre ⁇ ion, anaphylactic reaction ⁇ and the like.
• Effective do ⁇ e ⁇ of the peptides of this invention for use in treating an immune-related disea ⁇ e are in the range of about 1 ng to 100 mg/kg body weight.
• a preferred dose range i ⁇ between about 10 ng and 10 mg/kg.
• a more preferred dose range i ⁇ between about 100 ng and 1 mg/kg.
• the effective T lymphocyte do ⁇ e i ⁇ a function of the individual T cell line, the ⁇ ubject and hi ⁇ clinical ⁇ tatus, and can vary from about 10 6 to about 10 9 cell ⁇ /kg body weight.
• the dosage administered will be dependent upon the age, ⁇ ex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• the route of administration may include intravenous, ⁇ ubcutaneous, intraarticular, intramuscular, by inhalation, intraperitoneal, intranasal, intrathecal, intradermal, trans- dermal or other known routes, including the enteral route.
• composition ⁇ may contain ⁇ uitable pharmaceutically acceptable carrier ⁇ comprising excipients and auxiliaries which facilitate proces ⁇ ing of the active compound ⁇ into preparation ⁇ which can be u ⁇ ed pharmaceutically.
• the pharmaceutical compo ⁇ ition ⁇ of the invention may be admini ⁇ tered to any animal which may experience the beneficial effect ⁇ of the compo ⁇ ition ⁇ of the invention.
• Foremo ⁇ t among ⁇ uch animal ⁇ are human ⁇ , although the invention i ⁇ not intended to be ⁇ o limited.
• compositions of the present invention may be administered by any means that achieve their intended purpose, for example, by the routes described above. Alternatively, or concurrently, administration may be by the oral route.
• the peptides, T cell ⁇ and pharmaceutical compo ⁇ ition ⁇ can be administered parenterally by bolus injection or by gradual perfusion over time.
• the peptide ⁇ can be incorporated into lipo ⁇ omes using methods and compounds known in the art.
• Preparation ⁇ which can be administered orally in the form of tablets and capsules, preparations which can be ad ⁇ ministered rectally, such as suppositorie ⁇ , and preparation ⁇ in the form of solutions for injection or oral introduction, contain from about 0.001 to about 99 percent, preferably from about 0.01 to about 95 percent of active compound(s) , together with the excipient.
• Suitable excipients are, in particular, fillers such a ⁇ ⁇ accharides, for example lactose or ⁇ ucro ⁇ e, mannitol or sorbitol, cellulose preparations and/or calcium pho ⁇ phate ⁇ , for example tricalcium pho ⁇ phate or calcium hydrogen pho ⁇ - phate, a ⁇ well a ⁇ binder ⁇ ⁇ uch as starch paste, using, for example, maize ⁇ tarch, wheat ⁇ tarch, rice starch, potato ⁇ tarch, gelatin, tragacanth, methyl cellulose, hydroxy- propylmethylcellulo ⁇ e, ⁇ odium carboxymethylcellulo ⁇ e, and/or polyvinyl pyrrolidone.
• fillers such a ⁇ ⁇ accharides, for example lactose or ⁇ ucro ⁇ e, mannitol or sorbitol, cellulose preparations and/or calcium pho ⁇ phate ⁇ , for example tricalcium pho ⁇ phat
• compositions which can be used orally include push-fit capsules made of gelatin, a ⁇ well a ⁇ soft, sealed capsule ⁇ made of gelatin and a plasticizer such a ⁇ glycerol or sorbitol.
• the pu ⁇ h-fit cap ⁇ ule ⁇ can contain the active compound ⁇ in the form of granule ⁇ which may be mixed with filler ⁇ ⁇ uch a ⁇ lacto ⁇ e, binder ⁇ such a ⁇ starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds are preferably dis ⁇ olved or ⁇ u ⁇ pended in ⁇ uitable liquid ⁇ , ⁇ uch a ⁇ fatty oil ⁇ , or liquid paraffin.
• Po ⁇ ible pharmaceutical preparation ⁇ which can be u ⁇ ed rectally include, for example, ⁇ uppo ⁇ itories, which consist of a combination of one or more of the active compounds with a suppo ⁇ itory ba ⁇ e.
• Suitable ⁇ uppo ⁇ itory ba ⁇ es are, for example, natural or synthetic triglyceride ⁇ , or paraffin hydrocarbon ⁇ .
• Po ⁇ ible ba ⁇ e materials include, for example, liquid triglyceride ⁇ , polyethylene glycol ⁇ , or paraffin hydrocarbon ⁇ .
• Suitable formulation ⁇ for parenteral administration include aqueous solution ⁇ of the peptide ⁇ in water- ⁇ oluble form, for example, water-soluble salt ⁇ .
• ⁇ uspension ⁇ of the peptide ⁇ a ⁇ appropriate oily injection ⁇ uspen ⁇ ion ⁇ may be administered.
• Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglyceride ⁇ .
• Aqueou ⁇ injection ⁇ u ⁇ pensions may contain substances which increase the visco ⁇ ity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
• the ⁇ uspension may al ⁇ o contain stabilizers.
• the peptide ⁇ are formulated using conventional pharmaceutically acceptable parenteral vehicles for administration by injection. These vehicles are nontoxic and therapeutic, and a number of formulations are set forth in Remington's Pharmaceutical Sciences. (supra.. Nonlimiting examples of excipients are water, saline, Ringer' ⁇ ⁇ olution, dextrose solution and Hank's balanced salt solution.
• Formulation ⁇ according to the invention may also contain minor amounts of additives such as substance ⁇ that maintain isotonicity, physiological pH, and stability.
• the peptides of the invention are preferably formulated in purified form substantially free of aggregates and other protein materials, preferably at concentrations of about 1.0 ng/ml to 100 mg/ml.
• A2b and A2c helper T cell clones have been described previously (Holoshitz et al. , supra) . Briefly, a T cell line, A2, reactive to M. tuberculo ⁇ i ⁇ wa ⁇ first isolated from draining lymph nodes of Lewi ⁇ rat ⁇ immunized with ⁇ tuberculo ⁇ i ⁇ in incomplete Freund's adjuvant (IFA) . Subcloning of the A2 line revealed the presence of an arthritogenic T cell clone, A2b, and a protective T cell clone, A2c. Both clone ⁇ recognized the same epitope, contained in residues 180-188 of hsp 65.
• IFA incomplete Freund's adjuvant
• the T cell clone ⁇ were cyclically re ⁇ timulated for three day ⁇ with heat killed M. tuberculo ⁇ i ⁇ and propagated for one week in Iscove' ⁇ modification of Dulbecco' s Modified Eagle's Medium (Gibco) , supplemented with 10% fetal calf ⁇ erum (FCS) , 10% EL-4 ⁇ upernatant (containing variou ⁇ T cell growth factor ⁇ ) , 2 mM glutamine, antibiotics, and 1% nonessential amino acids.
• FCS fetal calf ⁇ erum
• EL-4 ⁇ upernatant containing variou ⁇ T cell growth factor ⁇
• MBP myelin basic protein
• helper T cell line ATL wa ⁇ i ⁇ olated from the popliteal lymph node ⁇ of a Lewis rat 10 days after hind footpad immunization with 100 ⁇ g/ml peptide A183 in CFA (containing 4 mg/ml of M. tuberculosi ⁇ (H37Ra) .
• T cell lines were cyclically restimulated in vitro for 3 or 4 day ⁇ with irradiated (3000 rad ⁇ ) thymocytes as APCs and 10 ⁇ g/ml heat-killed Mt in case of clone A2b, 10 ⁇ g/ml MBP in case of T cell line Zla or 10 ⁇ g/ml peptide A183 in case of T cell line ATL and propagated for 6 or 7 day ⁇ in I ⁇ cove's Modified Dulbecco ' ⁇ Medium (Gibco) , supplemented with 10% FCS, 10% EL-4 supernatant ⁇ (a ⁇ a source of IL-2) , 2 mM glutamine, 2-ME, antibiotics and 1% non-essential amino acids.
• the proliferative respon ⁇ e of T cell clones was assessed by measuring 3 H-thymidine incorporation into cells over the final 18-20 hours of culture.
• Cells (2 x 10 4 cells/well) were cultured for 4 days in flat-bottom 96-well microtiter plates in the presence of irradiated (1500 rads) syngeneic thymocyte ⁇ (1 x 10 6 cell ⁇ /well) a ⁇ antigen pre ⁇ enting cell ⁇ and variou ⁇ amount ⁇ of antigen.
• i ⁇ otope Following incorporation of the i ⁇ otope, cell ⁇ were harve ⁇ ted and counted using routine procedures.
• the competitor peptide wa ⁇ added to the culture containing the T cell ⁇ and irradiated thymocytes and responder cell ⁇ 1-2 hours before the addition of the ⁇ timulatory peptide 180-188.
• the single alanine sub ⁇ tituted peptide analog ⁇ derived from non peptide 180-188, the 180-188 amino acid ⁇ equence of the mycobacterial 65 kD protein, were synthesized with the PEPSCAN method (Geysen, H.M. et al. , Proc. Natl. Acad. Sci. USA, 1984, 11:3998; Proc. Natl.- Acad. Sci. USA 1985, £2 . :178)) and detached from their ⁇ olid ⁇ upports and used in T cell proliferation a ⁇ ays a ⁇ de ⁇ cribed previously (van der Zee et al. , supra) .
• the SMPS ⁇ et-up wa ⁇ developed u ⁇ ing a ⁇ tandard auto ⁇ ampler (Gil ⁇ on model 221) .
• SMPS automated simultaneous multiple peptide synthe ⁇ i ⁇
• Peptides were obtained a ⁇ C-terminal amide ⁇ from 7.5 mg re ⁇ in/peptide (0.21 meq/g, PAL TM re ⁇ in, Milligen) .
• the activitie ⁇ of in vitro defined non- inhibitory and inhibitory peptide ⁇ ⁇ ynthesized by the PEPSCAN or the SMPS method were confirmed with the alanine ⁇ ub ⁇ tituted peptides, A184, 1029 and A183, 1028 prepared by conventional solid phase synthesi ⁇ .
• PEPTIDE A183 INHIBITS PROLIFERATIVE RESPONSES IN VITRO OF T CELLS OF CLONES TO NATIVE PEPTIDE 180-188
• Proliferative re ⁇ pon ⁇ e ⁇ of the T cell clone ⁇ and the capacity of ⁇ ynthetic peptide ⁇ to compete with the native ⁇ timulatory peptide 180-188 were a ⁇ e ⁇ sed as described above.
• Figure 2 show ⁇ response of A2c cells to a suboptimal concentration of the native peptide 180-188 in the ab ⁇ ence or presence of alanine- ⁇ ub ⁇ tituted peptides.
• A181 was also inhibitory, although to a les ⁇ er extent.
• a peptide with an alanine ⁇ ubstitution at position 182 showed only slight inhibition.
• Peptides with substitution ⁇ at positions 180. 184, 185, or 186 failed to inhibit response ⁇ to the ⁇ timulatory peptide.
• inhibitory peptide ⁇ were tested in the presence of increasing concentrations of stimulatory peptide 180-188.
• the stimulatory 180- 188 peptide overcame inhibition in a dose-dependent manner. Therefore, the inhibition by the substituted peptides followed the rules of dose-dependent competition.
• respon ⁇ e ⁇ to the T cell itogen, concanavalin A (Con A) were not affected by the presence of alanine-sub ⁇ tituted peptides in the concentrations which could inhibit the 180-188 respon ⁇ e.
• Figure 4 ⁇ how ⁇ that, when the competitor peptides were added at the ⁇ ame time a ⁇ the stimulatory peptide (here at 0.1 ⁇ g/ml), without preincubation, A183 competed for recognition by A2b cell ⁇ , but A181 did not.
• Figure 5 ⁇ how ⁇ a distinct set of experiments which demon ⁇ trate the do ⁇ e-dependency of the inhibitory activitie ⁇ of A183.
• A2b cells were incubated with APC and a fixed concentration of A183 (10 ⁇ g/ml) . After 1-2 hours of preincubation, increa ⁇ ing concentration ⁇ , from 0.1 - 10 ⁇ g/ml of stimulatory antigen ⁇ were added (Figure 5a: peptide 180-188; Figure 5b: hsp 65; Figure 5c: M. tuberculosi ⁇ ) .
• di ⁇ ea ⁇ e can be induced by immunization with the ba ⁇ ic protein of myelin (MBP) or by inoculation of MBP-reactive T cell line ⁇ , ⁇ uch a ⁇ Zla (Ben- Nun, A. et al. , J. Immunol. 129: 303 (1982)) .
• Figure 6 how ⁇ that proliferative re ⁇ ponses of Zla to MBP are inhibited by A183.
• the Zla cell line is known to recognize the amino acid sequence of re ⁇ idues 68-88 of MBP, which bears no ⁇ equence or structural similarity to residue ⁇ 180-188 Of hsp 65.
• A183 seems to inhibit respon ⁇ e ⁇ of both the arthriti ⁇ - ⁇ pecific T cell clone ⁇ and an encephaliti ⁇ - specific T cell line by competing for binding at the level MHC cla ⁇ II molecule ⁇ (Rt-1 B locu ⁇ product ⁇ ) involved in antigen presentation.
• Lewis rats were immunized ⁇ ubcutaneou ⁇ ly (SC) in their hind footpads with A183 (500 ⁇ g/rat) in IFA, or with IFA alone. Sixteen days later, rats were immunized intracutaneously at the ba ⁇ e of the tail with 1 mg of heat- killed M. tuberculosis in IFA. Eleven days later, the popliteal lymph nodes (LN) were removed and the T cells were tested in a ⁇ tandard proliferation a ⁇ say using 2 x 10 5 cells/well and variou ⁇ concentration ⁇ of A183, ranging from 1 - 50 ⁇ g/ml. 3 H-thymidine incorporation wa ⁇ measured after 4 day ⁇ of culture.
• SC ⁇ ubcutaneou ⁇ ly
• Figure 7 show ⁇ proliferative re ⁇ ponse ⁇ of immune or control LN cell ⁇ to variou ⁇ do ⁇ e ⁇ of A183, indicating that, under appropriate circum ⁇ tances, A183 can function as an immunogenic peptide.
• A183 REDUCES THE SEVERITY OF M. TUBERCULOSIS-INDUCED ARTHRITIS The re ⁇ pon ⁇ e to hsp 65 residue ⁇ 180-188 by the A2b T cell clone i ⁇ dominant in adjuvant arthriti ⁇ , and the re ⁇ pon ⁇ e to MBP by the T cell clone Zla i ⁇ dominant in experimental allergic encephalomyeliti ⁇ . Since A183 inhibited these clonal T cell reactivities in vitro, the in vivo effects of A183 admini ⁇ tration at the time of di ⁇ ease induction were investigated.
• Figure 8 shows that the concomitant administration of A183 with M. tuberculosis caused a significant reduction in the severity of arthritis (p ⁇ 0.02).
• the disease developing in A183-treated rats was very mild and did not cause irreversible joint damage (average maximum disease score of 4) .
• Animals which did not receive A183 developed much more severe disease with irreversibly ankylosed joints.
• EAE was induced in Lewi ⁇ rats by injecting 0.1 ml of a 1:1 emul ⁇ ion of MBP in ⁇ aline and complete Freund' ⁇ adjuvant (containing 4 mg/ml M. tuberculo ⁇ i ⁇ ) in both hind footpad ⁇ .
• Clinical ⁇ ign ⁇ of di ⁇ ea ⁇ e were monitored daily, and rated on a ⁇ cale of 0 to 4: 0, no ⁇ ign ⁇ ; 0.5, lethargy and weight lo ⁇ ; 1, limp tail; 2, hind leg weakness?
• the A183 peptide appears to be capable of interacting with different MHC products, both rat and human. A183 not only inhibits antigen specific T cell recognition in both specie ⁇ , but in addition, it effectively interferes with the m. vivo generation of autoimmune effector T cells, as shown in adjuvant arthriti ⁇ and allergic encephalomyeliti ⁇ experiment ⁇ , above.
• a ⁇ ingle ⁇ ub ⁇ titution network of peptide 180-188 wa ⁇ prepared. In thi ⁇ network every re ⁇ idue of the nonapeptide wa ⁇ replaced by all 19 other po ⁇ ible amino acid ⁇ (See Figure 1) .
• Sub ⁇ titution peptide ⁇ that induced high proliferative T cell re ⁇ pon ⁇ es were: position 180 T- ->S; position 181 F—>L; and position 186 E—>D or E—>Q.
• Peptides having the following substitution ⁇ were al ⁇ o stimulatory for both T cell clones: position 180 T—>D or — >K; position 182 F—>I; position 186 E—>H.
• the degree of proliferative responses found in the presence of the latter ⁇ ub ⁇ titutions peptides was clearly inferior to those obtained with native peptide 180-188.
• Sub ⁇ titution peptides with two or three replacements were found to be non-stimulatory. Based upon the result ⁇ obtained with the ⁇ ingle ⁇ ub ⁇ titution network, the better ⁇ timulatory sub ⁇ titution ⁇ , po ⁇ ition 180 T—>S, po ⁇ ition 181 F—>L, and po ⁇ ition 186 E—>D were selected for ⁇ ynthe ⁇ is of combined substitution peptides. Proliferative re ⁇ pon ⁇ es of the A2b and A2c T cell clones to the combined-substitution peptide ⁇ were determined.
• a ⁇ can be ⁇ een from the re ⁇ ult ⁇ for clone A2b, ⁇ hown in the lower half of Table 5, none of the peptide ⁇ with two or three combined ⁇ ub ⁇ titution ⁇ at po ⁇ ition ⁇ 180, 181, and 186 was stimulatory.
• T cell clone A2b responded to the native peptide 180-188 and to the alanine- ⁇ ub ⁇ tituted peptides A187 and A188.
• Figure 11 shows that T cell line Zla responded not only to the native MBP peptide 1020 but also to the alanine-sub ⁇ tituted peptide ⁇ 1021, 1024, 1026, 1030, and 1032, and a very modest response to peptide 1031 was noted.
• Peptide 1026 induced a larger proliferative re ⁇ pon ⁇ e than did peptide 1020.
• inhibitory peptide A183 or 1028 When a fixed dose of inhibitory peptide A183 or 1028 was tested in the pre ⁇ ence of increasing concentrations of stimulatory peptide 180-188, the inhibition could be overcome in a dose-dependent manner, arguing against any non-specific toxic effects of the inhibitory peptides on the T cells. Furthermore, both A183 and 1028 inhibited the proliferative response of Zla cells to either MBP or peptide 1020, whereas peptide ⁇ A184 and 1029 did not inhibit.
• peptide A183 and 1028 were selected for testing in the adjuvant arthriti ⁇ and EAE di ⁇ ease model ⁇ .
• peptide A184 and 1029 were used as control ⁇ .
• Rat ⁇ were ob ⁇ erved daily and graded on a four-point ⁇ cale. k Number with di ⁇ ea ⁇ e/number te ⁇ ted. c Average day of disease onset of those animals that develope disease (1SD) .
• di ⁇ ea ⁇ e can be induced by a well-defined MBP-related peptide
• the di ⁇ ea ⁇ e-inducing antigen for adjuvant arthritis is whole heat-killed
• rats were immunized at day 0 with Mt (250 ⁇ g) in IFA emul ⁇ ified with PBS or A183 (250 ⁇ g) or A184 (250 ⁇ g) in PBS.
• Rat ⁇ were immunized with 100 ⁇ g A183 in CFA in the hind footpads and, 10 days later, popliteal lymph node cell ⁇ were obtained and ⁇ timulated in vitro with A183 (10 ⁇ g/ml) for 4 day ⁇ .
• the cell ⁇ were then expanded for 1 week i IL2-containing medium and restimulated for 4 day ⁇ by A183 (10 ⁇ g/ml) in the pre ⁇ ence of ⁇ yngeneic irradiated thymocyte ⁇ a ⁇ antigen pre ⁇ enting cell ⁇ .
• Table 8 ⁇ how ⁇ the antigen ⁇ pecificity of a CD4 + T cell line, de ⁇ ignated ATL, obtained after 6 restimulation cycles in vitro.
• DDA dimethyl - dioctadecylammonium bromide
• a di ⁇ ea ⁇ e induced by a highly complex antigen, whole mycobacteria, disea ⁇ e inhibition by co-immunization with a competitor peptide apparently involve ⁇ the ⁇ ynergistic effect of competition for MHC binding between competitor and disea ⁇ e- eliciting epitope ⁇ , a ⁇ well a ⁇ the concomitant induction of a regulatory (protective) immune re ⁇ pon ⁇ e.

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