CZ298A3 - Preparations and methods of treating diseases mediated by t cells - Google Patents

Abstract

Metabolizable lipid emulsions, such as Intralipid and Lipofundin, are excellent vehicles for peptide therapy of autoimmune diseases and of other TH1 T cell mediated diseases or conditions, as it promotes a TH1 to TH2 cytokine shift. Such emulsions may be used in conjunction with an antigen recognized by inflammatory T cells associated with the pathogenesis of a T cell mediated disease or condition for the therapeutic treatment of such a condition.

Description

(57)

This invention relates to the treatment of T cell mediated diseases and, in particular, to therapeutic agents, including T cell antigens involved in the pathogenesis of the disease, and preventing a metabolizable lipid emulsion as a biologically active carrier. Autoimmune disorders, such as insulin-dependent diabetes mellitus (IDDM, grip of diabetes 1), multiple sclerosis, rheumatoid arthritis and thyroitis, are characterized by the reactivity of the immune system to endogenous antigen, with subsequent tissue damage. These immune responses to the self-antigen are maintained by the persistent activation of endogenously reactive T cells. Using the inventive compositions, autoimmune T-cell responses shift from a damaging inflammatory mode, called TH1, to a T-cell response of TH2 mode that produces harmless antibodies, thereby suppressing inflammation and tissue damage. In preferred embodiments of the invention, the biologically active carrier is a fat emulsion in the therapeutic composition, including triglycerides of vegetable or animal origin, phospholipids of vegetable or animal origin, an osmo-regulator and an antioxidant (e.g. commercially available fat emulsion Intralipid /. The antigen is preferably a peptide that is recognized by inflammatory T cells and is associated with a pathogenesis of the disease, for example, in an IDDM peptide derived from human heat shock protein 60 (hsp60). A method of treating a patient suffering from a T cell-mediated disease or condition (including allergies) comprises administering to the patient a composition of the invention (i.e., the appropriate peptide in a biologically active carrier consisting of a fat emulsion).

Means and methods of treating diseases mediated

T cells

Technical field

The present invention relates to vaccine therapy for T cell mediated diseases and, in particular, to therapeutic agents comprising T cell antigens involved in the pathogenesis of T cell mediated diseases such as autoimmune diseases and comprising a matabolizable lipid emulsion as a biologically active carrier.

BACKGROUND OF THE INVENTION Insulin-dependent type I diabetes, multiple sclerosis, arthritis and thyroitis are characterized by the immune system to endogenous tissue damage. These immune responses to k are maintained by persistent activation

T lymphocytes.

helper type is divided into two groups according to cytokines that secrete when they are and Coffman, 1989). TH1 cells secrete T cell proliferation, and cytokines such as tissue inflammation. In contrast, it assists T cells and suppresses the production of al., 1994). IL-10 antigen presentation

Autoimmune disorders such as mellitus (IDDM or diabetes by rheumatoid reactivity with subsequent antigen reactive antigens intrinsic endogenously (Mosmann induces mediates secreting IL-4 and

IL-10. IL-4

IgG isotypes et.

CD4 characteristic T cells are activated by IL-2, which IFN-1, which TH2 cells secrete antibodies to certain THI inflammatory cytokines (Banchereau indirectly inhibits THI activation by affecting and production of inflammatory cytokines by macrophages (Moore et al., 1993). THI cells are involved in the pathogenesis of organ specific autoimmune diseases and THI type responses also appear to be implicated in other T cell mediated diseases or conditions, such as contact dermatitis (Romagnani, 1994).

Peptides suitable for immunologically specific therapy of autoimmune diseases are peptides that are recognized by T cells involved in the pathogenesis of autoimmune diseases. Each autoimmune disease will have its ideal peptide for therapeutic use. A disease such as multiple sclerosis in which self-reactive T cells such as myelin basic protein (MBP) is involved (Allegreta et al., 1990) will require a myelin basic protein peptide, such as which is described by Ot et al., 1990.

The present inventors show that autoimmune diseases, such as type 1 diabetes mellitus, can be treated by administering a suitable peptide in an oily vehicle. NOD mice develop spontaneously type I diabetes caused by autoimmune T cells that attack islet-producing β cells of insulin. This autoimmune attack is associated with the reactivity of T cells to a number of endogenous antigens, including the 60 kDa heat shock protein (hsp 60) and glutamic acid decarboxylase (GAD) peptide. Thus, for example, diabetes spontaneously developing in NOD / Lt mice can be treated with a peptide designated p227, which corresponds to positions 437-460 of the human hsp 60 sequence (PCT Patent, Publication No. WO90 / 10449; D. Elias and IR Cohen, Peptide The Lancet 343: 704-06, 1994), variants of the p227 peptide in which one or both of the cysteine residues at positions 6 and 11 have been replaced by valine and / or the threonine residue at position 16 has been replaced by Lys (see PCT patent , Publication No. WO90 / 19236) and peptides designated p12 and p12, respectively. p32, which correspond to positions 166-185, respectively. 466-485 of the human hsp 60 sequence. See Israeli Patent Application No. 114,407 of the same applicants as the present application, filed June 30, 1995. See also PCT Application No. PCT / US96 .... filed July 1, 1996, claiming priority to said Israeli Application No. 114,407, the entire contents of which are incorporated herein by reference.

For IDDM peptide therapy using p12, p32, p277 or variants thereof, the present inventor has been found to be effective when peptides are administered subcutaneously (sc) in mice in an oil vehicle such as a mineral oil emulsion known as non-Freund's adjuvant (IFA) ). However: 1FA as well as complete Freund's adjuvant (CFA, a mineral oil preparation containing varying amounts of killed Mycobacteria microorganisms) are not permitted for human use because the mineral oil is not metabolizable and cannot be degraded in the body. Therefore, it is desirable to find an efficient vehicle that is metabolizable for peptide therapy.

For many years, several fat emulsions have been used for intravenous nutrition in human patients. Two of the available commercial fat emulsions, known / as Tntralipid (Intralipid is a registered trademark of Kabi Pharmacia, Sweden, fat emulsions for intravenous nutrition, described in US Patent No. 3,119,094) and Lipofundin (registered trademark 13. Braun Melsungen, Germany) content]! as fat soybean oil (100 or 200 g in 1000 ml distilled water: 10% resp.

%). Egg yolk phospholipids (12 g / l distilled water) and Lipofundin egg yolk lecithin (12 g / l distilled water) are used as emulsifiers in Intralipid. The isotonicity in both Intralipid and Lipofundin comes from the addition of glycerol (25 g / l). These fat emulsions are fairly stable and are used for intravenous nutrition of patients suffering from gastrointestinal or neurological disorders that prevent them from receiving the oral calories required to receive nutrition and sustain life. Ordinary who follow daily allowance

US et. al.

up to 1 liter per day.

No. al., describe parenteral patents No. 4,073,943 and Re. 32,393 issued

168 308 of the published carrier system in particular

Wretlind

US No. 4 issued February 14, 1978, May 1990 as

September 18th. 1979 Wretlind et.

29.

new edition.

for use in boosting intravenous administration

- a pharmacologically active oil-soluble agent comprising a stable oil-in-water emulsion comprising a pharmacologically inert lipoid as the hydrophobic phase dispersed in the hydrophilic phase, said lipid being dispersed in the emulsion as finely formed particles having a mean particle size of less than 1 micron to achieve a rapid start-up of an acceptable therapeutic effect, said carrier system being used with an effective dose of said pharmacologically active, oil-soluble agent dissolved predominantly in said lipoid according to its fractionation in the hydrophobic phase, and wherein said therapeutic effect of the agent. This administration of a water-soluble oil-soluble predominantly dissolves an effective dose of the active is said to be suitable for water-soluble, such anesthetics being attributed to said carrier system of the insoluble or pharmacologically active agent in the lipoid pharmacologically active agents phase. Examples are depressants, analgesics, stimulants, spasmolytics, muscle relaxants, vasodepressants, and diagnostics, eg, X-ray contrast agents. This carrier system is said to enhance the diagnostic or therapeutic effect of a given agent, with a quick start accompanied by a reduced incidence of body tissue damage.

Intralipid has been designed as a non-irritating vehicle for several adjuvants for use in vaccines such as e.g.

6-O- (2-tetradecylhexadecanoyl) - and 6-O- (3-hydroxy-2-docosylhexacosanoyl) -N-acetylmuramyl-L-alanyl-D-isoglutamine (Tsujimoto et al., 1986 and 1989), avridine (Woodard and Jasman, 1985), N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine (CP-20 961) (German Patent Application No. 29Ε 2945788; Anderson and Reynolds, 1979; Niblack et al. , 1979). Kristiansen and Sparrman, 1983, reported that the immunogenicity of hemagglutinin and neuraminidase in mice is greatly enhanced after adsorption to the lipid particles underlying Intralipid.

None of the publications mentioned above disclose the use of Intralipid as a vehicle for peptides in the treatment of autoimmune diseases.

Nor does it disclose that Intralipid could mediate a shift in the immune response from a TH1 type response to a TH2 type response.

SUMMARY OF THE INVENTION

It has now been found, in accordance with the present invention, that metabolizable lipid emulsions such as Intralipid and Lipofundin can function as vehicles for peptide therapy of autoimmune diseases and other TH1 T cell mediated diseases or conditions. It was further found that this activity is associated with a shift of TH1 to TH2 cytokines.

Accordingly, the present invention relates to a therapeutic composition for the treatment of an autoimmune disease or other T cell mediated disease or condition, the composition comprising a peptide or other antigen and a biologically active carrier, which peptide or other antigen is recognized by inflammatory T cells associated with the pathogenesis of said disease or condition, and the biologically active lipid carrier is a fat emulsion comprising 10-20% triglycerides of vegetable or animal origin, 1.2-2.4% phospholipids of vegetable or animal origin, 2.25-4.5% osmo-regulator, 0- 0.05% antioxidant and sterile water up to 100%.

The triglycerides and phospholipids of vegetable or animal origin may be derived from any suitable vegetable oil of cottonseed, coconut or egg yolk, or triglycerides derived from are

Preferably, a bovine serum oil ratio, such as soybean oil, ole] or olive oil, is derived from soybeans. Ξ advantage of soybean oil and phospholipids or egg yolk, triglycerides / phospholipids about 8: 1.

The fat emulsion may contain an osmo-regulator, preferably glycerol. The fat emulsion may optionally contain 0.05% tocopherol.

any suitable addition of xylitol or sorbitol.

an antioxidant, for example

In one embodiment of the invention, the fat emulsion as defined above is treated by centrifugation, e.g. at 10,000 g or more, forming a small triglyceride-rich layer (above about 1: 1 triglycerides / phospholipids) over an aqueous dispersion enriched in phospholipids (e.g. about 90% triglycerides), and said aqueous dispersion is used as a lipid vehicle in the compositions of the invention.

In one preferred embodiment of the invention, the composition is for the treatment of insulin-dependent diabetes mellitus (IDDM) and comprises a peptide derived from human heat shock protein 60 (hsp60) which is recognized by inflammatory T cells associated with IDDM pathogenesis, said peptide being selected from groups of peptides shown in Table 1:

TABLE 1

Peptides

SEQ ID NO. C.

Amino acid sequence (one letter code)

P3 1 (31-50) plO 1 (136-155) pil 1 (151-170) pl2 1 (166-185) pl4 1 (195-214) pl8 1 (255-274) p20 1 (286-305) p24 1 (346-365) p29 1 (421-440) p30 1 (436-455) p32 1 (466-485) p35 1 (511-530) p39 1 (343-366) p277 1 (437-460) p277 (Val ⁴ ) * 2 p277 (Val ¹¹ ) * * 3 p277 (Val ^{4 -} Val ' ⁵ ) * * * 4

KFGADARALMLQGVDLLADA NPVEIRRGVMLAVDAVIAEL VIAELKKQSKPVTTPEEIAQ EEIAQVATISANGDKEIGNI RKG VITVKDGKTLNDELEII QSIVPALEIANAHRKPLVIIA LVLNRLKVGLQWAVKAPGF GEVIVTKDDAMLLKGKGDKA VTDALNATRAAVEEGIVLGG IVLGGGCALLRCIPALDSLT EIIKRTLKIPAMTIAKNAGV VNMVEKGIIDPTKWRTALL GKVGEVIVTKDDAM VLGGGCALLRCIPALDSLTPANED VLGGGVALLRCIPALDSLTPANED VLGGGCAL.LRVIPALDSLTPANED

VLGGGVALLRVIPALDSLTPANED

to 437-460 that SEQ. id. C. 1 with C-442 changed on IN * * 437-460 that SEQ. id. C. 1 with C-447 changed on IN -k -k k 437-460 that SEQ. id. C. 1 with C-442 changed on IN and C- 447 altered on IN

The invention further relates to a method of treating a subject suffering from an autoimmune disease, or other disease or condition mediated ΤΗΊ _f which comprises administering an effective amount of a therapeutic composition of the invention to said subject.

· ·

Brief description of the pictures

Giant. 1 shows the production of ant.i-p277 under the action of the peptide p277 (Val? -Val ¹¹

2) phosphate buffered saline in Example 2.

induced in shows TH2-dependent

NOD mice acting

Intralipid, * antibodies in NOD mice (1) Intralipid or (PBS) as described against the substance isotypes of the peptide (Val) as described in Example 3.

Giant. 3 shows that p277 (Val? -Val ¹¹ ) / Intralipid therapy induces v

NOD mice specific shift in cytokine profile, to p277 (Val ⁶ -Val ^1] ) produced peptides as

Giant. 3A shows that after treatment

Reactive T cells is described in Example 4. p277 (Val ^ -Val ¹¹⁾ peptide in Intralipid and incubation of mouse spleen cells with p277 (Val -Val ⁸ *) peptide occurs reduc tions TH1 (IL-2, IFN-τ ) and an increase in TH2 (IL-4, IL-10) cytokines; Giant. 3B shows that there is no change in cytokines after treatment of the p277 (Val @ 1 -Val ¹¹ ) peptide with mice and incubation of spleen cells with Con A.

Giant. 4 shows that spontaneous T-cell proliferative responses to p277 (Val? -Val ¹¹ ) decrease after treatment with the p277 (Val ⁸ -Val ¹¹ ) peptide in Intralipid as described in Example 5.

Giant. 5 shows that treatment of rats with [beta] 1-90 myelin basic protein in Intralipid reduces the severity of experimental autoimmune encephalomyelitis (EAE) as described in Example 6.

Giant. 6 shows that treatment of rats with p71-90 myelin basic protein peptide in IFA reduces the severity of experimental autoimmune encephalomyelitis (EAE) as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention it was found that the effect of p277 (Val ⁶ -Val1 ¹⁾ the peptide in an appropriate carrier down-regulated the spontaneous proliferative T cell responses to epitopes of hsp60 and GAD and eliminate the production of autoantibodies to hsp60, to GAD and to insulin. Disease arrest was associated not with T-cell or anergy tolerance, but with a shift in cytokines produced by p277-reactive autoimmune T-cells (Val ⁶ -Val ³¹ ) from a TH1-like profile (IL-2, IFN-τ) to a similar profile TH2 (IL-4, IL-10). This modulation was immunologically specific: The spontaneous response of T cells of mice treated with bacterial hsp60 peptide remained in the TIII mode. Thus, the diabetic process, characterized by autoimmunity to several self antigens, can be treated using one of these antigens, eg, the peptide p277 (Val ⁶ -Val ³¹ ).

Connection p277 (Val -Val ^G ^ ³⁾ treatment with a shift in reactivity to p277 (Val ⁶ -Val ³¹⁾ from T-cell proliferation to antibodies indicates that the therapeutic effect results from a shift in the predominant cytokines produced by the autoimmune T cells in treated mice. TH1 cells secrete IL-2, which induces T-cell proliferation, and cytokines such as IFN-β, which mediate tissue inflammation, thereby contributing to disease pathogenesis, while TH2 cells secrete IL-4 and IL-10. IL-4 helps B cells secrete antibodies of certain IgG isotypes and suppresses the production of TIII inflammatory cytokines. IL-10 indirectly inhibits ΊΉ1 activation by influencing antigen presentation and inflammatory cytokine production by macrophages. Thus: TH2 cells suppress TH1 activity (see Liblau et al., 1995). This shift from TH1-like to TH2-like behavior was supported by isotype analysis of antibodies produced before and after p277 (Va1-Val ¹¹ ) therapy.

The fact that the mechanism of therapeutic effect of treatment of a given peptide in a lipid vehicle shows the involvement of a TH1 -> TH2 cytokine shift provides the possibility of using a TH1 -> TH2 cytokine shift as evidence that treatment is successful and elicits a beneficial response. In other words: The shift TH1 -> TH2 can serve

as a surrogate marker of response to treatment. For example: Missing shift may indicate the need for a second treatment. See Israeli Patent Application No. 114,459 filed July 5, 1995 and the corresponding PCT application filed on the same date, the entire contents of which are incorporated herein by reference.

The lipid emulsions of the invention, when used as a vaccine adjuvant with an antigenic substance to which active T cells are involved in the disease or condition being treated, mediate a shift from the TH1 T cell response before treatment to the TH2 T cell response after treatment. This finding determines that such lipid emulsions are tolerogenic biologically active carriers that can be used in vaccines to treat any THI cell-mediated disease or condition. In such vaccines, the antigen provides immunological specificity for a therapeutic effect, whereas the biologically active carrier of the invention provides a biological result, i.e. a TH1 -> TH2 shift. Due to a given shift mediated by said biologically active carrier of the invention, diseases with a certain spectrum of autoreactivities can be switched off by combining a single antigen and a carrier capable of inducing a T cell cytokine shift.

A preferred use of the invention is in the treatment of organ specific autoimmune diseases that are mediated by THI cells. Such diseases include, but are not limited to, autoimmune diseases such as IDDM, rheumatoid arthritis, multiple sclerosis, and thyroitis. The peptide used in such treatment is an autoantigenic peptide. Thus, for example for IDDM the peptide above-mentioned p277 peptide or the valine substituted analog p277 (Val ⁶ -Va1 ^31); for multiple sclerosis, such a peptide is derived from a myelin basic protein; for thyroitis, a thyroglobulin-derived peptide is intended, and for rheumatoid arthritis, the autoantigen may be derived from Mycobacterium, e.g., Mycobacterium tuberculosis organisms.

It is not critical that the antigen is a peptide. Thus, for example, THI-mediated allergic responses leading to skin irritation and inflammation, such as contact dermatitis, can be treated with a vaccine comprising an irritant antigen and a biologically active carrier of the invention that causes a shift in the cytokine response from TH1 type to TH2 type. Thus, while elevated antibody levels against a given antigen will persist in a patient, the inflammatory T-cell response causing skin irritation will be suppressed.

Accordingly, the tolerogenic biologically active carrier of the invention can be used at any time when it is desirable to create tolerance for a given antigen that attacks T cells, i.e. at any time when a vaccine is used to limit a T cell mediated condition, especially TH1-mediated condition. When determining which antigen activates the response when implant rejection or implant versus host disease, then administration of such antigen with the carrier of the invention can be expected to shift from unwanted inflammatory THJ responses to rather desirable TH2 responses, regardless of the overall complexity of the higher number of antigens, to which T cells are reactive in such a state.

To determine cytokine secretion by T cells after peptide activation, lymphocytes from the peripheral blood of patients are tested in an in vitro activation assay. Peripheral blood lymphocytes are isolated from total haparized blood for ficol-hypaque, and cultured with test peptide (s) at concentrations

5-50 µg / inl. Supernatants from cultured T cells are harvested at different time points and tested for the activity of different cytokines by ELISA or bioassays.

Examples of fat emulsions that may be used in the compositions of the invention include, but are not limited to, commercially available Intralipid nutrition, and fat emulsions of U.S. Pat. Nos. 3,169,094, and Lipofundin for intravenous administration described in the above mentioned

Nos. 073,943 and 4,168,308, incorporated herein by reference in their entirety. The finding according to the invention that these metabolizable lipids, previously administered for intravenous nutrition, can be effectively used as vehicles for the treatment of T cell mediated diseases, but is completely

- 12 • · tet *

t t * t t t t t t t t t t t t t t t t t t t t t t t t

In Vt · tt · unexpected. Similar findings biologically

TH1 tolerogenic mediate the unexpected shift.

That these active

TH2 means carriers are also completely covered

The fat emulsions as freshly prepared are not open to storage of the invention, with or after atmospheric advantage air storage.

Intralipid, when exposed, causes a decrease in pH activity.

air, biological

In one embodiment, the biologically%

of the invention a fat emulsion containing egg yolk phospholipids 2.5 to supplement 100 ml (Intralipid 10 vehicle fat emulsion containing egg yolk phospholipids 2.5 to supplement 100 ml.

a container that has a longer atmospheric duration, for example, corresponding reduction in the active carrier according to soybean oil, 1.2% glycerol and sterile water%). In another embodiment, it is 20% soybean oil, 2.4% glycerol, and sterile water

In yet another embodiment of the invention, the vehicle-treated lipid emulsion is obtained from the original fat emulsion defined herein by centrifugation, e.g., at 10,000 g or more, over a phospholipid-enriched aqueous dispersion over and containing about. 1: 1 triglycerides / phospholipids, creates a small layer rich in triglycerides (around 90% triglycerides). The two phases are separated, and the phospholipid-rich aqueous dispersion is used as a vehicle.

The compositions of the invention may comprise one or more peptides. Thus, for example, for the treatment of IDDM, the composition may comprise one or more of the peptides p12, p32, p277, p277 (Val ⁶ ), p277 (Val ¹¹ ), p277 (Val ¹¹ -Val ¹¹ ) or any other peptides of Table 1. In one preferred embodiment, the composition for treatment of IDDM comprises peptide p277 or p277 (Val-Val ¹¹ ) and a fat emulsion comprising 10% soybean oil, 1.2% egg yolk phospholipids, 2.5% glycerol and sterile water to make up 100 ml ( Intralipid 10%).

The present invention further relates to the use of a fat emulsion as defined herein, or a processed, phospholipid-enriched aqueous solution.

Μ ·· • · «· · · · ··" · · · · · · ·· - Ί Ί - · »· · · #» ·· ^J · ······ "·· ···· In a dispersion, prepared by centrifugation thereof, for the preparation of a therapeutic composition comprising one or more peptides or other antigens and said fat emulsion or treated aqueous dispersion as a vehicle, in the treatment of autoimmune diseases or other diseases or conditions mediated by THI.

The invention will now be explained by the following non-limiting examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Peptide therapy of type I diabetes using p277 (Val ⁶ -Val ¹¹ ) in oils

The efficacy of various lipid formulations as vehicles for peptide therapy of NOD mice was tested. In this model, autoimmune destruction of insulin-producing β-cells in the pancreas is mediated by T-lymphocytes. Around the pancreatic islets, in 5-8 infiltrates, 14 β-cell destruction, and open diabetes, the weeks leading to the week of age creating inflammatory conditions begin to manifest insulin deficiency in nearly 100% of female NOD mice at 25-40 weeks of age.

Female NOD mice were treated with 100 µg s- Ί Ί p277 peptide (Val-Val) per mouse sc in 0.1 ml: 1) phosphate buffered saline (PBS), or 2) 10% lipid emulsion consisting of 10% soybean oils, 1.2% egg phospholipids and 2.25% glycerol (Intralipid, Kabi Pharnacia AB, Sweden).

The incidence of diabetes at 6 months of age and the production of anti-p277 (Val ^ -Val ^) antibodies were studied. Diabetes was diagnosed as persistent hyperglycemia, blood levels «« • · · · · ···· _ _ Ί Λ · · · · · ··· «« * · ·· ^χ «« ··· · «« • • · Glucose above 11 mmol / l measured at least twice weekly on a Beckman Glucose Analyzer II. Successful peptide therapy was determined by maintaining normal blood glucose concentration (less than 11 mmol / L), remission of islet islet inflammation of the pancreatic islets (insulitis), and induction of antibodies to the therapeutic peptide as an indicator of TH-2 type immune response. The results are shown in Table 2.

Table 2: Incidence of diabetes at 6 months

Action

Diabetes (%) Incidence of death (%) p277 (Val ⁶ -Val ¹¹ ) / PBS p277 (Val ⁶ -Val ¹¹ ) / Intralipid none

90 80 45 # 20 # 100 ALIGN! 90

H p <0.01 compared to NOD mice without treatment.

As can be seen from Table 2, peptide therapy administered in Jntralipid was effective in reducing the incidence of diabetes and death. On the other hand, the treatment given in PBS was ineffective.

Example 2

Production of anti-p277 (Val? -Val ¹¹ ) antibodies

Protection against diabetes in treatment with the p277 peptide (Val ⁶ -Val ⁴⁴ ) depends on TH2 immunological reactivity to this peptide. Therefore, antibody production in mice immunized with p277 (Val? -Val? ¹ ) was measured by ELISA.

- 15 • · ··· *

Maxisorp microtiter plates (Nunc) were coated with peptide p277 (Val, 10 µg / ml for 18 hours and non-specific binding was blocked with 7% milk powder for 2 hours. Mouse sera, diluted 1:50, were allowed to bind for 2 hours and specific binding was detected by the addition of alkaline phosphatase conjugate and anti-mouse IgG (Serotec) for 2 hours and p-nitrophenyl statate substrate (Sigma) for 30 minutes.The staining intensity was measured in an FT..ISA reader (Anthos) as OD 405 nm.

As can be seen from FIG. 3, p277 (Val-Val ¹¹ ) in Intralipid

NOD mice immunized generated anti-p277 (Val ⁶ -Val ^{I: L} ) peptide-specific antibodies in PBS while mice immunized showed no antibody response at all.

Example 3

Anti-substance isotypes induced by p277 treatment (Val ^ -Val ¹¹ )

The association between treatment with p277 (Val ⁶ -Val ¹¹ ) and Intralipid and the antibodies to p277 (Val 1 -Val ¹¹ ) shown in Example 2 suggests that the therapeutic effect may be due to a shift in the predominant cytokines produced by autoimmune T-cells. Helper-type CD4 T cells are divided into two groups according to the characteristic cytokines they secrete when activated (Mosmann and Coffman, 1989): TH1 cells secrete

II-2, which induces T-cell proliferation, and cytokines such as IFN-T, which mediates tissue inflammation; TH2 cells, on the other hand, secrete IL-4, which helps B cells to produce certain isotypes of antibodies, and IL-10 and other cytokines that can suppress tissue inflammation. The possibility of a shift from TH1-like to TH2-like behavior was supported by isotype analysis of antibodies produced after p277 therapy (Val ⁶ -Val ¹¹ ).

Groups of NOD mice, 3 months old, were treated with p277 (Val? -Val ¹¹ ) or PBS in oil as described in Example 2. The sera of individual mice were tested for isotypes of their antibodies to p277 (Val ⁶ -Val per group). ). Isotypes of determination

ELISA with (12-15 mice using (Southerη after treatment detected with

Associates,

Giant. 2, wherein: with control ^X1 ) the antibodies were antibody

Biotechnology shown in NOD mouse rings, Columns of mice treated with each attempt seemingly specific reduction

Birmingham,

The counteracting agents are p277 (Val ⁶ -Val ^{1: L} ) are the result of the number of rings being

Analysis of antibody isotypes of anti-p277 antibodies generated after treatment showed that they belong exclusively to the IqG1 and IgG2b classes, dependent on TH2 T-cells producing JI-4 mice, and isotypes

AL). Results for p277 (Val ^6- Val ¹¹ ) are shown with empty solid circles, equal number due to overlap. Antibody isotype treatment showed that (Snapper et al., 1993a) and possibly TGFP (Snapper et al., 1993b). There were no IgG2a TH1-type antibodies induced by p277 therapy (Val ^ -Val ^xx ). Generation of antibodies to a specific peptide used in therapy is an indication that autoimmune T cell responses shift from a damaging inflammatory mode called TH1 to a TH2 T cell response that produces harmless antibodies and suppresses inflammation and tissue damage (Rabinovitch, 1994).

Example 4

P277 (Val ⁶ -Val ^x1 ) / Intertripeptide therapy induces specific switch in cytokine profile

To confirm the idea of cytokine switching, cytokines produced by p277-reactive T cells (Val? -Val ¹¹ ) in p277 (Val ⁶ -Val ¹¹ ) treated mice were determined /

Intralipid and control mice. Concavalin A (ConA), a T cell mitogen, was used as a control to activate total spleen T cells.

Groups of 10 NOD mice, 3 months old, were treated with p277 (Val ⁶ -Val ¹ · ¹ ) in Intralipid (solid bars) or PBS · 4 · · Intralipid (open bars, see Example 2). After five weeks, mouse spleens were removed and spleen cells were pooled. Spleen cells were incubated with ConA or p277 (Val 1 -Val ¹¹ ) for 24 hours (for IL-2 and IL-4 secretion) or for 48 hours (for IL-10 and IFNγ secretion). The presence of these cytokines in the culture supernatants was quantified by ELISA using Pharmingen paired antibodies according to the Pharmingen cytokine protocol. Pharmingen recombinant mouse cytokines were used as standards for calibration curves. Briefly, 96-well microtiter flat bottom plates were coated with rat anti-mouse cytokine mAbs for 18h at 4 ° C and the culture supernatants or recombinant mouse cytokines were added for 18h at 4 ^P C. Plates were washed, and biotinylated rat were added mAb against mouse cytokines for 45 minutes at room temperature, then the plates were washed extensively and avidin-alkaline phosphatase was added. Plates were washed, chromogenic substrate (p-nitrophenyl phosphate) was added and samples were read at 405 nm in an ELISA reader. The results are shown in FIG. 3. Cytokine concentrations are shown as OD readings. * P <0.01.

Giant. 3A shows that spleen cells of control mice secrete both JJ-2 and IFN? By incubation with p277 (Val ⁶ -Val ¹¹ ). In contrast, mice treated with p277 (Val? -Val ¹ ?) Produced significantly less (P <0.01) IL-2 and IFN? In response to incubation with the peptide p277 (Val? Val ¹¹ ). This reduction in TH1 cytokines was specific: Mice treated with p277 (Val ⁶ -Val ³¹ ) maintained their IL-2 and IFNt cytokine responses to ConA (Fig. 3B). Giant. 3A and 3B show the amount of IL-10 and IL-4 produced by mouse mouse cells. Control mice produced very little IL-4 or IL-10 in response to p277 (Val? -Val ¹¹ ) or ConA. In contrast, significant increases in IL-10 and IL-4 occurred only in response to p277 (Val ¹ -Val ³ · ¹ ) and only in mice treated with p277 (Val ⁶ -Val ¹³ ) / intalipid (P <0.01). Decreases in IL-2 and IFN? And increases in IL-10 and IL-4 confirm a shift from TH1-like behavior to ΊΉ2-like behavior. Such a shift may help to explain how the loss in T cell proliferation to p277 shown by the inventors.

A · A A A

18 earlier (Elias et al., 1991), antibodies to p277 (Val ⁶ -Val ¹¹ ) and appearance of IgG1 according to the invention.

and IgG2b

Example 5

Spontaneous to p277 (Val ⁶ -Val ¹¹ )

T-cell proliferative responses are reduced by p277 therapy (Val ^ -Val ¹¹ )

Groups of 5 mouse NOD / Lt strain strains were treated at 3 months of age with 100 µg of peptide p277 (Val? -Val ¹¹ ) in Intralipid or with PBS mixed with Intralipid, sc on the back. After five weeks, the mouse spleens were removed and T-cell pro-ferromagnetic. responses to T cell mitogen ConA (1.25 pg / rol) or to p277 (Val-Val ¹¹ ) (10 pg / ml) were determined in vitro using standard assays. The results are shown in Fig. 4, where they are: ConA - black striped blocks; p277 (Val ^ -Val ¹¹ ) - gray blocks. T-cell responses were detected by incorporating [ ³ H] thymidine added to the wells of quadruplicate cultures for the last 18 hours of the three-day culture. The stimulation index (S1) was calculated as the ratio of the mean cpm of test cultures to the mean cpm of antigen-containing wells to control wells cultured without antigen or ConA. The standard deviation from mean cpm was always less than 10%.

As shown in FIG. 4, the control mice tested with PBS / Intralipid showed T-cell responses proliterativní to both p277 (Val ⁸ -Va1 ¹¹⁾ and to the T-cell mitogen ConA. In contrast, mice treated with p277 (Va -Val ¹¹ ) in Intralipid showed a decrease in T-cell proliterative reactivity to p277 (Val ⁶ -Val ¹¹ ), but no decrease to ConA. Thus, the beneficial effect of p277 (Val 1 -Val ¹¹ ) peptide therapy is not caused by inactivating the autoimmune response, but by activating autoimmunity to a different cytokine mode of behavior (Cohen, 1995). Regulation of destructive autoimmunity is programmed within the immune system (Cohen, 1992); only need tt «« IV ·· φφ »·

19 to be activated by an appropriate signal requiring the peptide together with the lipid vehicle; neither the peptide itself nor the lipid without peptide are effective, as shown in Table 2. These results indicate that metabolizable lipid emulsions can be effectively used as vehicles in the treatment of autoimmune diseases. Each disease will require its own specific peptide, but metabolizable lipid emulsions can be used for various therapies.

Example 6

The administration of the peptide in Intralipid influences the development of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an experimental autoimmune disease of animals believed to model certain aspects of multiple sclerosis (Zamvil and Steinman, 1990). EAE can be induced in susceptible rat strains such as Lewis rat, immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA), a mineral oil emulsion containing killed mycobacteria. The disease develops about 12 days after immunization and is characterized by paralysis of various degrees, caused by inflammation of the central system. Paralysis can last up to 6 to 7 days and rats usually recover unless they die during the peak of acute paralysis. EAE is caused

T cells that define determinants on the MBP molecule.

the determinant is compounded in Lewis rats

Major MBPs from peptide sequence 71-90 (Zamvil and Steinman, 1990).

We have therefore attempted to test whether administration of the encephalitogenic MBP peptide p71-90 in IFA can also inhibit the development of EAE. Giant. 5 shows that administration of p71-90 in IFA days prior to EAE induction resulted in a significant decrease in the highest degree of paralysis compared to the control treatment with PBS emulsified in IFA, which had no effect on the degree of disease.

• 9

However, TFA cannot be administered to humans, as mentioned above, since it is not metabolizable in the body and causes inflammation. We therefore treated Lewis rats with p71-90 in Intralipid. Figure 6 shows the results. Rats receiving p71-90 in Intralipid developed significantly less paralysis than the control rats treated with PBS / Intralipid. It can therefore be concluded that a relevant peptide such as p71-92, administered in Intralipid, is capable of modulating EAE in rats. Thus, the effects of peptide / intralipid treatment are not limited to one peptide, one species or one autoimmune disease.

Example 7

Efficacy of new versus aged 10% Intralipid emulsion The% Intralipid emulsion was used to treat female NOD mice, 12 weeks old, with p277 (Val ⁶ -Val ¹¹ ). The emulsion was used either on the day the sealed bottle was opened or 4 months later after exposure to atmospheric air. The pH of the emulsion was tested at the time of preparation of the peptide + emulsion for treatment. Aging was characterized by a decrease in sawmills from 8.2 to 6.7. In each experiment, 10 mice were treated with peptide + emulsion, 10 mice received only emulsion, and 10 mice were untreated. The results are shown in Table 3.

- 21 - * «V * • · • · · • · · • · · • · · ··· · · • · · · • * · · ♦ · • · · · • «ti · · Table 3 Group Action Emulsion Diabetes Mortality PH (%) (%) 1 peptide + emulsion 8.2 20 * 10 * 2 emulsion _ II __ 90 70 3 peptide + emulsion 6.7 60 40 4 emulsion 80 60 5 without action - 90 80 * p <0.01

It is apparent that placebo-treated mice (emulsion only, groups 2 and 4) and untreated mice (group 5) developed a similar incidence of diabetes at 6 months of age, 80-90%. In contrast, the action of the peptide in the newly opened emulsion in mice protected% mice from diabetes. However, only 40% was protected when using the emulsion. Thus, the emulsion was chemically unstable upon exposure to air, as shown by a significant decrease in pH. This change is relevant to biological activity. Thus, intralipid is a biologically active carrier whose functional properties depend on pH and not just on the presence of an inert lipid.

Now that the invention has been fully described, those skilled in the art will understand that the same can be accomplished within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention by embodiments,

Although specific to amend.

and without undue experimentation, it has been described in connection with its understanding that the application is meant to include any variation, use or adaptation of the invention that will generally follow the principles of the invention and include such departures from the present disclosure as come within the scope of known conventional practice in the art to which the invention pertains, or which can be applied to its basic principles. the features of the appended claims.

All references herein, including journal articles and abstracts, published or unpublished US or foreign patent applications, issued US or foreign patents, or any other references are fully incorporated herein by reference, including all data, tables, figures and texts presented in the references cited therein. . Moreover, the full content of the references cited within the references cited herein is also fully incorporated by reference.

References to known method steps, conventional method steps, known methods or conventional methods do not in any way permit any aspect, description or embodiment of the invention to be disclosed, filed or proposed in the relevant art.

The foregoing description of particular embodiments of the invention will fully disclose the general nature of the invention that others, using the knowledge of the art (including the contents of references cited herein), can readily modify or adapt these particular embodiments for various applications, without undue experimentation, without diversion. from the general concept of this application. Such adaptations and such alterations are therefore to be understood as being within the meaning and scope of the equivalents of the disclosed embodiments, based on administration and guidance provided herein. It is understood that the phraseology of these specifications is to be interpreted by a person skilled in the art in light of the administration and guidance provided herein, in combination with the knowledge of a person ordinarily skilled in the art.

• ·

9 • · ·

Links

1. Allegretta, M., Nicklas, J. A., Sriram, S., and Albertini, R. J., Science 247: 718-721 (1990). 2. Banchereau, J. and Rybak, M. E., in: The Cytokine Handbook, 2nd Ed., Thompson Ed. Academic Press New York, pp. 99 (1994). 3. Cohen, I. R., Immunology Today 13: 490-494 (1992). 4 Cohen, I.R., in: Selective Immunosuppression: Basic Concepts and Clinical Applications (Eds. Adorini, L., Capra, D. J., Waldmann, H.) Chem. Immunol Karger, Basel, 60: 150-60 (1995). 5. Kristiansen, T., Sparrman, M., and Heller, L., J. Biosci 5 (suppl. 1): 149-155 (1983). 6. Liblau, R. S., Singer, S.M. and McDevitt, H.O., Immunology Today 16:34 38 (1995). 7. Moore, K.V., O'Garra, A., de Waal Malefyt, R., Vieira, P., and Mosmann, T.R., Ann. Roar. Immunol. 11.-165-190 (1993). 8. Mosmann, T.R. and Coffman R. L., Ann. Rev immunol. 7: 145,173 (1989). 9. Ota, K., Matsui, M., Milford, E. L., Mackin, G.A. , Weiner, H.I. and Hafler, D. A., Na ture 346: 103-187 (1990). 10. Rabinovitch, A., Sorensen, 0, Sua-Pinzon, W.K., Rajotta, R.V. and Bleakley, R.C., Diabetologia 37: 833-837 (1994). 11. Romagnani, S., Ann. Roar. Immunol. 12: 227-257 (1994). 12. Snapper, C.M. and Mond, J. J. Immunology Today 14: 15-17 (1993). 13 . Snapper, C.M., Waegell, W., Beernink, H., and Dasch, J.R., J. Immunol. 151: 4625-36 (1993). 14. Zarnvil, S.S. and Steinman, L., Ann. Roar. Immunol. 8: 579-621 (1990).

Sequence list

SEQ ID NO. no. 1

Met 1 Leu Ary Leu Pro Thr 5 Wall Phe Arg Gin Met 10 Arg For Wall Ser 15 Dec Arg Wall Leu Ala For 20 May His Leu Thr Arg Ala 25 Tyr Ala I.ys Asp Wall 30 Lys Phe Gly AI á Asp 35 Ala Aig Ala Leu Met 40 Leu Gin Gly Wall Asp 45 Leu Leu Ala Asp Ala 50 Wall Ala Wall Tlil Met 55 Gly For Lys Gly Arg 60 Thr Wall Ile Ile Glu os Gin Gly Trp Gly Ser 70 For Lys Wall Thr Lys 75 Asp Gly Wall Thr Wall B0 Ala Lys Ser Ile Asp 85 Leu Lys Asp Lys Tyr 90 Lys Asn Ile Gly Ala 95 Lys Leu Wall Gin Asp 100 ALIGN! Wall Ala Asn Asn Thr 105 Asn Glu Glu Ala Gly 110 Asp Gly Thr Thr Thr 115 Ala Thr Wall Leu Ala 120 Arg Ser Ile Ala Lys 125 Glu Gly Phe Glu Lys 130 Ile Ser Lys Gly Ala 135 Asn For Wall Glu Ile 140 Arg Arg Gly Wall Met 145 Leu Ala Wall Asp Ala 150 Wall Ile Ala Glu Leu 155 Lys Lys Gin Ser Lys 160 For Wall Thr Thr For 16S Glu Glu Ile Ala Gin 170 Wall Ala Thr Ile Ser 175 Ala Asn Gly Asp Lys 180 Glu Ile Gly Asn Ile 185 Ile Ser Asp A1 a Met 190 Lys Lys Wall Gly Arg 195 Lys Gly Wall Ile Thr 200 Wall Lys Asp Gly Lys 2 05 Thr Leu Asn Asp Glu 210 Leu Glu Ile Ile Glu 215 Gly Met Lys Phe Asp 220 Arg Gly Tyr Ile Ser 225 For Tyr Phe Ile Asn 230 Thr Ser Lys Gly Gin 235 Lys Cys Glu Phe Gin 240 Asp Ala Tyr Wall Leu 245 Leu Ser Glu Lys Lys 250 Ile Ser Ser Ile Gin 255 Ser Ile Wall For Ala 260 Leu Glu Ile Ala Asn 265 Ala His Arg Lys For 270 Leu Wall Ile Ile Ala 275 Glu Asp Wall Asp Gly 280 Glu Ala Leu Ser Thr 285 Leu Wall Leu Asn Arg 290 Leu Lys Wall Gly Leu 295 Gin Wall Wall Ala Wall 300 Lys Ala For Gly Phe 305 Gly Asp Asn Arg Lys 310 Asn Gin Leu Lys Asp 315 Met AI a Ile Ala Thr 320 Gly Gly Ala Wall Phe 32S Gly Glu Glu Gly Leu 330 Thr Leu Asn Leu Glu 335 Asp Wall Gin For His 340 Asp Leu Gly Lys Wall 345 Gly Glu Wall Ile Wall 3S0 Thr Lys Asp Asp Ala 355 Met Leu Leu Lys Gly 360 Lys Gly Asp Lys A1 a 365 Gin Ile Glu Lys Arg 370 Ile Gin Glu Ile Ile 375 Glu Gin Leu Asp wall 380 Thr Thr Ser Glu

Tyr Glu Lys. Glu Lys. Leu Asn Glu

385 390 395 400 Wall Ala Wall Leu Lys Wall Gly Gly Thr Ser Asp Wall Glu Wall Asn Glu 405 430 415 Lys Lys Asp Arg Wall Thr Asp Ala Leu Asn AI a Thr Arg Ala Ala Wall 420 425 430 Glu Glu Gly Ile Wall Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile 435 440 445 For Ala Leu Asp Ser Leu Thr For Ala Asn Glu Asp Gin Lys ile Gly 450 455 460 Ile Glu Ile ile Lys Arg Thr Leu Lys Ile For Ala Met Thr Ile Ala <65 470 475 480 Lys Asn Ala Gly Wall Glu Gly Ser Leu Ile Wall Glu Lys Ile Met Gin 485 490 495 Ser Ser Ser Glu Wall G1 y Tyr Asp Ala Met Ala Gly Asp Phe Wall Asn 500 505 510 MeL Wall Glu Lys Gly Ile Ile Asp For Thr Lys Wall Wall Arg Thr Ala 515 520 525 Leu Leu Asp Ala Ala Gly Wall Ala Ser Leu Leu Thr Thr Ala Glu Wall 530 535 540 Wall wall Thr Glu Ile For Lys Glu Glu Lys Asp For Gly Met Gly Ala 545 550 555 560 Met Gly Gly Hel Gly Gly Gly Met Gly Gly Gly Met Phe 565 570

SEQ ID NO. No 2

Val Leu Gly Gly Val Ala Leu Leu Arg Cys Ile Pro Ala Leu Asp 1 5 í 15

Ser Leu Thr Pro Al Asn Glu Asp

- 26 Seq ID. No 3

Val Leu Gly Gly Gly Cys Ala

Ile Pro Ala Leu Asp

Thr

For Ala Asn Civ Asp

SEQ ID NO. No. 4

Val Leu Gly Gly Val Ala Val Leu Arg Val

10

Ile Pro Ala Leu Asp

Ser Leu Thr Pro Al Asn Glu Asp

Claims (26)

PATENTOVÉ Claims 1. Therapeutic means to treat illness or condition mediated T cells v y z learning comprising an antigen and a biologically active carrier, wherein the antigen is an antigen which is recognized by inflammatory T cells associated with the pathogenesis of said disease or condition and said carrier is a fat emulsion comprising 10-20% triglycerides of plant or animal origin, 1,2- 2.4% phospholipids of vegetable or animal origin, 2.25-4.5% osmo-regulator, 0-0.05% antioxidant and sterile water to make up 100 ml. 2. A composition according to claim 1 wherein the triglycerides are of vegetable origin. 3. A composition according to claim 2 wherein the triglycerides are derived from soybean oil. 4. The composition of claim 1 wherein the triglycerides are of animal origin. 5. A composition according to claim 4 wherein the triglycerides are derived from egg yolk or bovine serum. A composition according to any one of claims 1 to 5, characterized by a plant origin. The method of claim 1, wherein the phospholipids are The composition of claim 6 wherein the phospholipids are derived from soybeans. Composition according to any one of claims 1 to 5, characterized in that the phospholipids are of animal origin. * «« » 9. A composition according to claim 8 wherein the phospholipids are derived from egg yolk or bovine serum. 10. Means according to any of claims 1 to 5, mark and c í by that osmo-regulator ίθ chosen from you kupiny Consisting of glycerol, sorbitol and xylitol 11. Means according to any of claims 1 to 5, mark and c i by that contains 0.05 O o tocopherol as an antioxidant. The composition of claim 1, wherein the biologically active carrier is a fat emulsion comprising 10% soybean oil, 1.2% egg yolk phospholipids, 2.5% glycerol and sterile water to make up 100 ml. The composition of claim 1, wherein the biologically active carrier is a fat emulsion comprising 20% soybean oil, 2.4% egg yolk phospholipids, 2.5% glycerol and sterile water to make up 100 ml. The composition of claim 1, wherein the biologically active carrier is a fat emulsion comprising 5% soybean oil, 5% medium chain triglycerides, 1.2% egg yolk lecithin, 2.5% glycerol, and sterile water to replenish 100 ml. A composition according to any one of claims 1 to 5 or 12 to 14, which causes a shift in the T-cell cytokine response of an individual from TH1 to TH2. * «« A composition according to any one of claims 1 to 5 or 12 to 14 which causes a decrease in IL-2 or IFN-τ T cell cytokine response and an increase in IL-4 or IL-10 T cell cytokine response . A composition according to any one of claims 1 to 5 or 12 to 14 for the treatment of insulin-dependent diabetes mellitus (IDDM), characterized in that it comprises a peptide derived from human heat shock protein 60 (hsp60), which is recognized by inflammatory T cells, associated with the pathogenesis of IDDM, wherein said peptide is selected from the group of peptides listed in Table 1. The composition comprises a fat emulsion according to claim 10, wherein the phospholipids are water to replenish, comprising 10 egg yolk, 2.5 100 ml. 17 for the treatment of IDDM that comprises the soybean oil p277 peptide, 1.2% glycerol, and sterile The composition of claim 17 for the treatment of IDDM, comprising the peptide p277 (Val-Val- ¹¹ ) and a carrier consisting of a fat emulsion comprising 10% soybean oil, 1.2% egg yolk phospholipids, 2.5% glycerol, and sterile water to make up 100 ml. A composition according to any one of claims 1 to 5 or 12 to 14 for the treatment of multiple sclerosis, comprising a peptide derived from myelin basic protein which is recognized by T cells associated with the pathogenesis of multiple sclerosis. • «* * * Use of a fat emulsion containing 10-20% triglycerides of vegetable or animal origin, 1.2-2.4% phospholipids of vegetable or animal origin, 2.25-4.5% osmo-regulator, 0-0.05% antioxidant and sterile water for replenishment of 100 ml, characterized in that it is used for the manufacture of the therapeutic composition according to claim 1. Use of a fat emulsion comprising 10% soybean oil, 1.2% egg yolk phospholipids, 2.5% glycerol and sterile water to make up 100 ml, characterized in that it is used for the manufacture of the therapeutic composition according to claim 1 . 23. A method of treating a patient suffering from a T cell mediated disease or condition comprising administering to said patient a composition comprising an antigen recognized by inflammatory T cells associated with the pathogenesis of said disease or condition in a biologically active carrier, comprising a fat emulsion comprising 10-20% triglycerides of vegetable or animal origin, 1.2 - 2.4% phospholipids of vegetable or animal origin, 2.25 - 4.5% osmo-regulator, 0 - 0.05% antioxidant and sterile water to make up 100 ml. 24. Method according to claim 23, v y z n and no u se se se that carrier consists of fat emulsion, including 10 soybean oil 1, 2% phospholipids egg yolk, 2.5 glycerol and sterile water to replenish 100 ml. 25. The method of claim 23 or 24, wherein said T cell mediated disease is an autoimmune disease and said antigen is a peptide. 26. The method of claim 23 or 24, wherein said T cell mediated disease is TH1 mediated disease. Mt t Mtl 4 4 · » 27. The method of claim 26, wherein said autoimmune disease is an organ specific autoimmune disease. 28. A method of treating a patient suffering from IDDM comprising administering a composition comprising one or more of the peptides listed in Table 1 and a biologically active carrier consisting of a fat emulsion comprising 10% soybean oil, 1.2% egg phospholipids yolk, 2.5% glycerol and sterile water to make up 100 ml. The method of claim 28, wherein the composition comprises the p277 peptide. 30. The method of claim 28, wherein said composition comprises the peptide p277 (Val ⁶ -Va.l- ^1]). 31. The method of claim 23 or claim 24, wherein said T cell mediated disease is a T cell mediated allergic condition and said antigen is an allergen that triggers said condition.