HUT77806A - Peptide compositions capable of down regulating an antigen specific immune response - Google Patents

Abstract

Therapeutic compsns. as follows are claimed: compsn. 1 comprising at least one isolated peptide having a defined sequence of amino acid residues, the compsn. being capable of down-regulating an antigen specific immune response (ASIR) in a population of humans subject to the ASIR, when administered in non-immunogenic form; and compsn. 2 comprising at least one isolated and purified peptide having a defined length, a defined sequence of amino acid residues and comprising at least one T cell epitope of a protein antigen, the compsn. being capable of down-regulating an ASIR to the protein antigen in a population of humans subject to the ASIR when administered in non-immunogenic form.

Description

Antigen-specific down-regulation peptide formulations

Immulogic Pharmaceutical Corporation, WALTHAM, MA, US

Inventors: Malcolm L. GEFTER, LINCOLN, MA, US

Ze'ev SHAKED, BERKELEY, CA, US

Malcolm MORVILLE, SHREWSBURY, MA, US

Date of filing: 02/09/1994

International Application Number: PCT / US94 / 10262

International Publication Number: WO 96/07428

BACKGROUND OF THE INVENTION

Since the beginning of human history, allergies have been the curse of human existence. People with allergies suffer from perpetual or recurrent attacks of sinusitis, rhinitis, itchy teary eyes, nose and mouth, allergic asthma, which may be so serious that they require hospitalization. The most serious human allergic reaction is anaphylaxis, which causes death without medical intervention.

Pharmaceutical formulations have been developed to alleviate many of the less serious symptoms, such as oral administration of antihistamines, inhaled anti-asthma formulations, and portable injection kits containing adrenaline to suppress anaphylactic shock pending medical attention. However, these pharmaceuticals do nothing more than cover the symptoms through the release of histamine, the end result of the human immune response that begins when the attacking allergen enters the body. In addition, many of the more effective antihistamines have undesirable side effects such as excessive dryness of the mucous membrane or drowsiness.

Over the past decades, a therapy has been developed that includes desensitization (desensitization, desensitization) of humans. Desensitization therapy involves repeated injections of the crude allergen extract of the attacking allergen in increasing doses. Although, in the clinical field, treatment with allergenic extracts has been shown to be somewhat effective in reducing allergen-related symptoms and is used as the most commonly used general therapy in allergy clinics, the mechanism of desensitization is still unknown. In addition, desensitization therapy should be given special attention because side effects may be significant or even fatal (such as anaphylaxis). Thus, it is necessary for the patient to undergo multiple injections of a significantly higher dose of the crude extract and to remain under medical observation for one or more hours after each injection. In addition, it is often necessary for the allergic patient to receive treatment until the maintenance dose is reached, which often requires one injection per week for 6 to 12 months and then for 4 to 6 weeks. Thus, this treatment is very time consuming, inconvenient and has no side effects or harm to the patient.

In attempts to make desensitization immunotherapy more effective and safer, many researchers have attempted to develop modified allergens with the intent of influencing immunological events occurring during immunotherapy (i.e., increasing blockade of IgG antibodies or reducing the number of allergen-specific IgE antibodies). Such modifications include urea (cabamide) denatured allergens, Ishizaka et al., E.J. Immunol. 114: 100-115 (1975); allergens produced by treating allergens with formalin, Norman et al., J. Allergy Clin Immunol. 70: 248-260 (1982); allergens bound to tolerogens such as D-glutamic acid D-lysine, Katz, D., Immunology, 41: 1-24 (1980); or polyethylene glycol, Sehon et al., J. Allergy Clin Immunol. 64: 242-250 (1979); and glutaraldehyde polymerized allergens, Metzger et al., N. Engi. J. Med. 295: 1160-1164 (1976).

Michael et al., U.S. Patent No. 4,338,297, disclose a polypeptide active pollen immunosuppressant produced by proteolytic enzyme digestion of pollen antigen followed by purification with an antipollen antibody, potentially useful as a therapeutic in desensitization. Later, the same group published an experiment that revealed that it was possible to suppress the immune response in mice against antigens such as bovine serum and honeybee venom phospholipase A2 with fragments derived from enzymatic digestion (Michael et al., J. Clin Immunol. 75: 200 (abst.) (1985); (Ferguson et al., Cell Immunol. 78: 1-12 (1983)), and it was found that fragmentation of allergenic pollen allergen proteins by peptide digestion and Use of digestive products with a molecular weight of -15 kD before or after intraperitoneal immunization with ragweed composition resulted in suppression of the immune response (Michael et al., Clin Exp. Allergy, 20: 669-674, 1990). suppression of the immune response also includes T cells and that fragments of the digestive material of the peptide are capable of stimulation of T cells that regulate essence.

In addition, the same research team tested for clinical efficacy a formulation containing peptide fragments of ragweed in humans (Litwin et al., Clin Exp. Allergy 21: 457-465, 1991). In this study, pre-seasonal immunotherapy was started with three groups of ragweed patients. One group received a traditional ragweed preparation, called Pool 2, enriched with ragweed antigen (Amb a I). In Pool 2, the proteins contain about 26% Amb a 1. Pool 2 was subjected to enzymatic digestion and the fraction containing proteins of 10 kD or less was used as a peptide fragment preparation and designated fSRW. The third group received placebo. Although the results indicated that groups receiving Pool 2 or fSRW had a significant reduction in symptom management compared to placebo, the difference between fSRW and Pool 2 was not significant. Thus, the fSRW peptic digestive formulation has not fully achieved its goal of providing significant efficacy or comfort compared to a conventional immunotherapeutic formulation containing an allergen, which is sensitive to the individual's crude extract, and has therefore not been approved in the United States. , or clinical trials of such a peptic preparation are currently underway in the United States.

In addition, the use of a formulation containing a protein allergen such as the peptide fragments described in Litwin et al. Has serious drawbacks. For example, the protein extract (Pool 2) subjected to digestion to form the fSRW peptide formulation was enriched in the desired Amb in protein I by only 26% and any of the different proteins and protein probably present in Pool 2 impurities, were transferred to fSRW digestive material. According to Litwin et al., The Amb content of Amb in fSRW cannot be accurately determined because the components of fSRW do not remain in solution. Thus, fSRW may contain unwanted proteins that may adversely affect the treated patient and thus not favor the use of conventional crude extract, and may be characterized by disadvantages associated with classical immunotherapy. In addition, enzymatic digestion of a crude protein does not always result in the same peptide composition each time. Therefore, it is virtually impossible to create a stable, well-defined, high purity drug peptide formulation for human therapeutic use, and most regulatory agencies in the world have these requirements for the use of enzymatically digested protein formulations, such as the fSRW described by Litwin et al.

In addition, like allergies, autoimmune diseases such as Ies type diabetes, multiple sclerosis, and rheumatoid arthritis are generally considered to be the result of an antigen-specific T cell-mediated response to an antigen that is the body's own tissue in autoimmune disease. Therefore, it is believed that one approach to treating autoimmune disease, which is essentially similar to treating allergies, would be appropriate. For example, WO 88/10120, WO 91/08760, WO 92/06704, WO 93/21222 and WO 94/07520 describe the oral or enteral use of whole autoantigens or fragments thereof, such as myelin basic protein , (MBP is a putative autoantigen in multiple sclerosis), the use of insulin in the treatment of diabetes, or the use of collagen in the treatment of rheumatoid arthritis. For the reasons described above, the clinical utility of autoantigens for oral, enteral or aerosol administration is limited, for example, it is not possible to characterize an active component of a therapeutic composition once it has been introduced into the stomach due to enzymatic degradation in the stomach. Thus, the predictive and reproducible therapeutic effects are difficult to achieve by these methods, not to mention the adverse side effects resulting from further unpredictable processing of the body therapeutically.

The present invention overcomes the drawbacks described above and provides a method for treating allergies and other immune response to protein antigen in humans (e.g., autoimmune diseases) using well-defined, highly purified formulations containing at least one peptide having a defined amino acid residue sequence, comprising at least the T cell epitope of the protein antigen and used in a non-immunogenic form. Preferably, compositions and methods for treating humans have been clinically tested in accordance with the present invention and have proven successful. The diverse nature of these successful clinical trials seems to have forever changed the face of allergic immunotherapy as well as altering the course of treatment of autoimmune diseases and other immune-related conditions.

SUMMARY OF THE INVENTION

The present invention provides therapeutic compositions and methods for treating a disease associated with an antigen, such as a protein antigen, associated with a specific immune response (e.g., allergies and autoimmune diseases) in humans. The therapeutic compositions of the present invention are highly defined, high purity, reconstitutable compositions suitable for use in human therapy. Preferred compositions of the present invention comprise at least one isolated, purified peptide free of other polypeptides or contaminants, the peptide having a defined amino acid residue sequence comprising at least one T cell epitope of the antigen of interest. A therapeutic composition of the present invention is capable of down-regulating an antigen-specific immune response to the antigen of interest in a human population subject to the antigen-specific immune response such that the symptoms of the disease are reduced or eliminated and / or the onset or worsening of symptoms of the disease or slow motion.

The compositions and methods of the present invention are useful in the treatment of susceptibility to protein allergens, such as ragweed, grasses, trees, dust particles (dust), cats, dogs and other animals, and any other airborne or contact allergens in humans. The compositions and methods of the present invention are useful in the treatment of autoimmune diseases such as rheumatoid arthritis, diabetes, myasthenia gravis, Grave's disease, Good Pasture's syndrome, thyroiditis and multiple sclerosis.

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BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the exacerbation of cat test syndrome (nose, lung, and total allergy) at 1 and 6 weeks post-treatment relative to placebo (PBO) at 75 pg and 750 pg for total allergy symptoms, at week 1, depending on the dose response, week 1 and week 6 test trends are reported for total allergic symptoms (see starred data).

DETAILED DESCRIPTION OF THE INVENTION

The scientific literature and patents cited in the present invention provide the knowledge available to those skilled in the art in this field. Thus, the disclosed US patents, PCT applications, and other cited publications are incorporated herein by reference.

The present invention provides therapeutic compositions and methods for treating disease states associated with an immune response to an antigen in humans. The therapeutic compositions of the present invention comprise at least one isolated, purified peptide, free of all other proteins and impurities, and having a defined amino acid residue sequence comprising at least one T cell epitope of the antigen. As used herein, the term isolated refers to a peptide that is free of any other polypeptide, impurity, starting reagent, or other material that is not attached to other molecules. The composition or compositions of the present invention, when administered to a patient in a non-immunogenic form during therapeutic treatment, are capable of down-regulating the antigen-specific immune response in a human population exposed to the antigen-specific immune response.

The compositions and methods of the present invention can be used to treat diseases related to antigens such as protein antigens in humans, i.e. ragweed, grasses, trees, house dust particles (dust), cats, dogs and other animals and any airborne or contact allergens. allergies. The human clinical trials described in the Examples describe the successful use of the compositions and methods of the present invention in the treatment of human allergy to cats. This invention is also useful in the treatment of autoimmune diseases such as rheumatoid arthritis, diabetes, myasthenia gravis, Grave's disease, Good Pasture syndrome, psoriasis, thyroiditis and multiple sclerosis, when the antigen responsible for the disease is present. is a protein autoantigen.

In accordance with the present invention, a peptide refers to a sequence of defined amino acid residues which preferably contains no more than about 50 amino acid residues and includes at least about 7 amino acid residues and preferably at least 12-40 amino acid residues and more preferably at least 13-30 amino acid residues. which, when derived from a protein antigen, contains less than about 75% of the amino acid residues of the total protein antigen, and preferably less than about 75% of the amino acid residues of the whole protein antigen. The peptides used in accordance with the invention comprise an antigen of at least one T cell of an antigen. The T cell epitope is the basic element or the smallest unit of T cell receptor recognition, where the epitope contains amino acids essential for receptor recognition. It is believed that the T cell epitope contains one of those

in the onset and persistence of an immune response to an antigen responsible for the clinical symptoms of an allergy, such as a protein allergen. It is believed that these T cell epitopes elicit early immune response events at the level of T helper cells by attaching to the appropriate HLA molecule on the surface of the antigen presenting cell and stimulating the corresponding T cell subpopulation. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, accumulation of additional immune cells in the area, and activation of B cell flooding, which results in the production of antibodies. One of these antibodies, the IgE isotype, is essential for triggering allergic symptoms and their production is regulated early in the triggering events at the level of T helper cells through the nature of secreted lymphokines. In an autoimmune disease, the antibodies produced are autoantibodies raised against an autoantigen, which are autoantibodies against an autoimmune disease such as a basic protein of myelin, which is thought to be a multiple sclerosis autoantigen.

In accordance with the present invention, a therapeutic / prophylactic treatment regimen (resulting in preventing or delaying the onset of symptoms of an attack by an antigen or resulting in a reduction, progression or amelioration of the symptoms caused by the attacking antigen, i.e., down-regulating the antigen-specific immune response) in a form (e.g., without an adjuvant), a therapeutic composition of the present invention, which comprises containing at least one isolated peptide which may be derived from a protein antigen responsible for the disease to be treated (or a peptide derived from an immunologically cross-reactive protein antigen). While not

It is intended that the theory be limited to the use of the therapeutic composition of the present invention.

a) inducing T cell insensitivity in the appropriate T cell subpopulation such that they do not respond to the attacking antigen and are not involved in stimulating an immune response when exposed to the attacking protein antigen (i.e., anergy or apoptosis);

b) modifying the lymphokine secretion profile by exposure to naturally occurring attack antigen (e.g., resulting in a decrease in IL-4 and / or an increase in IL-2);

c) removing the T cell subpopulation that normally participates in the response to the attacking antigen from the site of normal exposure (e.g., nasal mucosa, skin or lung allergy) to the site of application (this redistribution of the T cell subpopulation reduces or improves the ability of the individual's immune system to stimulate a normal immune response at the site of normal exposure to the attacking antigen, resulting in a reduction in allergic symptoms); obsession

d) induces T cell suppressor cells.

While not intended to be limited to any theory, it is believed that down-regulation of an antigen-specific immune response is induced if a proper costimulation signal, often referred to as a second signal, is not provided. Briefly, it is believed that stimulation of T cells requires two types of signals (signaling), the first being recognition by T cells via the T cell receptors of the corresponding MHC-linked processed antigens on antigen presenting cells (APCs), the second being signal is referred to as costimulation signal (s) or as a second signal provided by certain competent APCs. When used in a composition of the invention without a non-immunogenic form (e.g., no booster), it is believed that competent APCs that are capable of generating a second signal or costimulation signal are not involved in stimulating the corresponding T cells, It results in T cell insensitivity or decreased T cell sensitivity. In addition, there are many antibodies or other reagents that are capable of costimulatory signals such as the second signal, including but not limited to B7 (including B7-1, B7-2, and BB-1), CD28 , CTLA4, CD40, CD40L, CD54 and CD11a / 18 (Jenkins and Johnson, Current Opinion in Immunology, 5: 361-367, 1993), Clark and Ledbetter, Natur 367: 425-428, 1994). blocking the transport. Thus, in accordance with the present invention, a composition can be used in a non-immunogenic form as described above, in combination with a reagent capable of blocking costimulation signals in such a way as to increase the level of T cell insensitivity.

The compositions and methods of the present invention are useful in the treatment of humans allergic to any number of protein allergens, such as the protein allergen of the genus Dermatophagoides; the Felis genus against a protein allergen; against the protein allergen of the genus Ambrosia; against a protein allergen of the genus Lolium; against a protein allergen of the genus Cryptomeria; against a protein allergen of the genus Alternaria; against a protein allergen of the genus Alder; against a protein allergen of the genus Betula; against a protein allergen of the genus Quercus; against the protein allergen of the genus Olea; a protein allergen of the genus Artemisia; against a plant allergen of the Plantago genus; against a protein allergen of the genus Parietaria; against a protein allergen of the genus Canine; against a protein allergen of the genus Blattella; against the protein allergen of the genus Apis; a protein allergen of the genus Cupressus; a protein allergen of the genus Juniperus; against a protein allergen of the genus Thuya; a protein allergen of the genus Chemaecyparis; a protein allergen of the genus Perilplaneta; against a protein allergen of the genus Agropyron; against a protein allergen of the Secale genus; against a protein allergen of the genus Triricum; against a protein allergen of the genus Dactylis; against a protein allergen of the genus Festuca; against a protein allergen of the Poa genus; against the protein allergen of the genus Avena; against a protein allergen of the genus Holcus; a protein allergen of the genus Anthoxanthum; against the protein allergen of the genus Arrhenatherum; against a protein allergen of the genus Agrostis; against a protein allergen of the genus Phleum; against a protein allergen of the genus Phalaris; against a protein allergen of the genus Paspalum; and the protein allergen of the genus Sorghum.

Examples of various known protein allergens from some of the genera identified above include, for example, Dermatophagoides (pteronyssinus or farina), Der p /; Dér p II ·, Derp III, Dér p VII, Dér fi; Dér fii, Dér flll, Dér FVII; a Felis (domesticus) Fel d I; Ambrosia (artemisfolia) Amb a 1.1; Amb a 1.2; Amb a 1.3; Amb a 1.4; Amb a II; Lollium (perenne) Lol p I; Lol p II; Lol p III; Lol p IV; Lol p IX (Lol p V or Lol p Ib); Cryptomeria (japonica) Cry j I; Cry j II; Can f I; Can f II; Juniperus (sabinoides or virginiana) Jun s I; Jun v I; Dactylis (glomerata) Dac g I; Poa (pretensis) Poa p I; Phl p / and Sorghum (halepensis) Sor h

I

Peptides with defined amino acid residues containing at least one T cell epitope, which induce T cell insensitivity or reduced T cell sensitivity, have been identified and isolated for many of the above-mentioned protein allergens and are useful in the compositions and methods of the present invention. For example, Dér p 1; Peptides containing T cell epitopes derived from hoary p11, hoary f1, and hoary f11 are described in USSN

U.S. Patent Nos. 08 / 227,772 and USSN 07 / 963,381, which are hereby incorporated by reference and are incorporated by reference in WO 93/08279. Peptides containing T cell epitopes derived from Fel D1 are described in USSN 07 / 662,276, 07 / 884,718 and 08 / 006,116, which are incorporated herein by reference. Amb from 1.1; Amb from 1.2; Amb from 1.3; peptides containing T cell epitopes derived from Amb a 1.4 and Amb a II have been described in USSN 07 / 866,679 and WO 93/21321, which are incorporated herein by reference. From Lol p l; Peptides containing T cell epitopes derived from Dac g l, Poa p l and Phl p 1 are disclosed in USSN 08 / 031,001 and T cells derived from Lol p IX (Lol ρ V or Lol p lb) peptides containing cellular epitopes are described in WO 94/04564 and 08 / 106,016, which are incorporated herein by reference. Peptides containing T cell epitopes derived from Cry III and Cry III and Jun s l and Jun v l are described in USSN 08 / 226,248, which is incorporated herein by reference in its entirety. invention. Peptides containing T cell epitopes derived from Can f1 and Can f11 are described in USSN 08 / 156,549, which is incorporated herein by reference. THE

Peptides containing a T cell epitope from a series of h1 are described in AU 93/00559.

Many antigens (such as autoantigens) have been shown to cause disease symptoms in autoimmune diseases (e.g., autoantigens such as insulin, myelin basic protein; rh factor; acetylcholine receptors; thyroid cell receptors; basement membrane proteins; thyroid proteins) ICA-69 (PM-1), glutamic acid decarboxylase (64K or 65K), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), collagen (type II), heat shock protein and carboxypeptidase H) in autoimmune diseases such as diabetes, rheumatoid arthritis and multiple sclerosis. For example, peptides containing T cell epitopes derived from myelin oligodendrocyte protein (MOG), one of the autoantigens involved in multiple sclerosis, have been described in USSN 08/116824, which is incorporated herein by reference. Peptides believed to be capable of down-regulating antigen-specific response to MBP (a myelin basic protein), a protein considered to be an autoantigen in multiple sclerosis, have been identified in WO 93/21222, EP 0 304 279, WO 91/15225 and et al. (Letters to Naturre 346: 183-187 (1990)) and Wucherpfennig et al. (J. Exp. Med. 170: 279-290 (1994)). Peptides believed to be able to down-regulate antigen specific response to soluble type II collagen, a protein considered to be an autoantigen of rheumatoid arthritis, have been identified in WO 94/07520 and WO 94/07520.

92/06704. WO 92/06704 describes a method for identifying peptides of insulin which is considered effective in the treatment and prevention of type 1 diabetes.

In addition, peptides having a defined amino acid composition as well as T cell epitopes can be identified for any protein antigen or autoantigen. One method involves dividing and synthesizing the protein antigen into non-overlapping or overlapping peptides of desired length, purifying and testing these peptides to determine if the peptides contain at least one T cell epitope. For this, any number of assays, such as T cell proliferation assay, lymphokine secretion assay, and T cell insensitivity assays, are performed. In another method, an algorithm is used to calculate peptides that are likely to contain T cell epitopes, and then the peptides calculated by the algorithm are synthesized, purified and tested in T cell assays to determine whether the calculated peptides cause T cell proliferation or lymphokine secretion or T cell insensitivity and are therefore likely to contain a T cell epitope. As discussed in many of the previously cited documents, human T cell stimulatory activity can be tested by culturing T cells from an individual susceptible to a predetermined protein antigen (e.g., an allergen or an autoantigen) with a peptide derived from the antigen and determining that T cell proliferation occurs. e in response to the peptide as measured, for example, by cellular uptake of tritiated thymidine. The stimulation indices of T cell responses to peptides can be calculated by dividing the maximum number of minutes per minute (CPM) by the response to one peptide by the control CPM. A T cell stimulation index (S.l.) greater than or equal to background level is considered positive. Positive results are used to calculate average stimulation indices for each peptide of the peptide group tested. Preferred peptides for use in accordance with the present invention comprise at least one T cell epitope, preferably at least two or more T cell epitopes, and the average T cell stimulation index is equal to or greater than 2.0. A peptide having a T cell stimulation index of 2.0 or greater is considered to be a useful therapeutic agent. The preferred mean T cell stimulation index is at least 2.5, more preferably at least 3.5, more preferably at least 4.0, and most preferably at least 5.0.

In addition, the Peptides of the Preferred Peptides have a Positivity Index (P1) of at least 100, more preferably at least 150, more preferably at least 200, and most preferably at least 250. The Positive Index of a peptide is determined by multiplying the average T cell stimulation index by antigen-susceptible group of tested individuals (e.g., at least 9 individuals, more preferably at least 16 individuals or more, and more preferably at least 20 individuals or more, or more preferably at least 30 individuals or more) having peptide responsive T cells. Thus, the positivity index also represents the strength of the T cell response to a peptide (S.l.) and the frequency of a T cell response to a peptide in the population of individuals tested for the peptide tested.

An algorithm for calculating successfully used peptides with T cell stimulation activity has been reported by Rothbard, 1 st Forum in Virology, Annals of the Pasteur Institute, pp. 518-526 (December 1986), Rothbard and Taylor, Embo 7: 93-100 (1988) and EP 0 304 279. They report the determination of a generic T cell model (algorithm), its statistical significance and its correlation with known epitopes, as well as the successes in identifying previously unidentified T cell epitopes of various protein antigens and autoantigens. A general model of a peptide known as a class II MHC-binding peptide, as described previously. 18 reported, it seems to have a linear model consisting of a charged amino acid residue or two glycines followed by a hydrophobic residue. After determining whether a peptide conforms to the general model, the peptide can be tested for T cell reactivity. Other algorithms used to estimate T cell epitopes of previously unspecified proteins include the algorithm described by Margalit et al. (J. Immunol. 138: 2213-2229, 1987), which is based on an amphipathic helix model.

In addition, peptides containing cryptic epitopes can be determined and used in accordance with the methods of the present invention. In a protein antigen or protein autoantigen, cryptic epitopes are determinants that are not normally specific to the immune system due to processing and presentation of the native protein to the appropriate MHC moiety. However, a peptide containing a cryptic epitope is capable of rendering T cells non-susceptible, and when the subject is saturated with a peptide, T cells obtained from the subject proliferate in vitro to the peptide or protein antigen from which the peptide is derived. Peptides containing at least one cryptic epitope derived from a protein antigen or autoantigen are referred to as cryptic peptides. To confirm the presence of cryptic epitopes, a T cell proliferation assay may be performed as known in the art and described above. In this assay, antigen-saturated T cells are cultured in vitro in the presence of each peptide separately to generate peptide-reactive T cell lines. It is believed that a peptide contains at least one cryptic epitope if a T cell line can be created with a particular peptide and the T cells are able to proliferate when exposed to the peptide and the protein antigen from which the peptide is derived.

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It is also possible to modify the structure of any of the peptides described above for use in the present invention to increase solubility (particularly desirable when the composition is injected), increase therapeutic or prophylactic efficacy, or stability (e.g., ex vivo storage time and in resistance to carrier proteolytic degradation). A modified peptide may be generated in which the amino acid sequence is altered relative to the native protein sequence from which it is derived, either by substitution, deletion, or addition to modify the immunogenicity, or by adding a component for the same purpose. For example, WO 94/06828 discloses substituted peptides in which essentially each amino acid residue can be replaced by a conservative amino acid, an unnatural amino acid, or alanine, and yet the substituted peptide is still capable of down-regulating the antigen-specific immune response. . In addition, many of the references cited above describe various modifications of peptides of defined amino acid composition which have been shown to induce T cell insensitivity.

In addition, it is not necessary that a peptide used in accordance with the method of the present invention be derived from a known antigenic protein. Any peptide having a defined amino acid residue sequence, or a peptide capable of down-regulating an antigen-specific immune response to an antigen or an autoantigen, may be used in accordance with the method of the present invention. For example, peptides may be synthesized to contain a specific amino acid sequence based on a known protein antigen sequence and yet capable of down-regulating an antigen-specific immune response, e.g. or for other reasons, it is able to down-regulate the immune response, such as from a nearby antigen. Without being limited to any theory, it is believed that nearby antigens, which are also tissue-specific (but which are not targets of immune or autoimmune attack), have the property to affect suppressor T cells at the site of immune attack, which in turn results in down-regulation of the site of immune attack (for example, the affected self-tissue in autoimmune disease or nasal mucosa, skin and lung allergy). Nearby antigens include, but are not limited to, portions of the antigen which are not themselves the target of the immune attack and which have suppressive activity at the site of the immune attack.

In addition, any compound that mimics a downstream peptide capable of down-regulating an antigen-specific immune response to an antigen or autoantigen may be used in accordance with the invention. Such compounds may not be entirely subunits linked by peptide bonds but may be linked via other linkages (e.g. thiolester linkages), provided that the non-peptide bond mimics a peptide capable of down-regulating an antigen-specific immune response to the antigen of interest as indicated. , with effective therapeutic / prophylactic treatment of the symptoms.

In addition, the peptide compositions used in accordance with the present invention preferably contain an appropriate percentage of the T cell epitopes of the attacking protein antigen (i.e., at least about 10%, more preferably at least about 20%, more preferably at least 30%, more preferably at least 40% and more preferably at least 60% or more) and are present in the composition such that, in accordance with the present invention, a T-cell of an individual susceptible to a particular protein antigen will render the individual's T cells insensitive. protein antigen. An algorithm can be used to determine whether a peptide (labeled peptide) or a combination of labeled peptides is likely to contain an appropriate percentage of T cell epitopes of the protein antigen of interest in inducing T cell insensitivity in a significant percentage of protein antigen-sensitive individuals. In line with such an algorithm, an in vitro T cell proliferation assay (previously mentioned) for the peptide (s) in an in vitro T cell proliferation assay can be calculated for each individual tested in a population in which individuals are sensitive to a given protein antigen. In the in vitro T cell proliferation assay, the remaining peptides are overlapping peptides (about 5 to 15 amino acid residues overlapping) covering the remainder of the protein not labeled (peptide (s) remaining at least 12 amino acid residues in length). and preferably not longer than 30 amino acid residues, preferably no greater than 25 amino acid residues In a population of tested individuals, the T cell stimulation index of each of these remaining peptides is calculated and added to each other in a series of peptides generated by an in vitro T cell proliferation assay. For each individual, the human T cell stimulation index for the candidate peptide (s) is divided by the sum of the human T cell stimulation index of the remaining peptides in the peptide series tested to determine the percentage. and at least 20, preferably at least 30 individuals sensitive to rotein antigen, and averaging the percentage. About 10% on average, preferably about 20%, of the labeled peptide (s). preferably 30%, and more preferably about 50% or greater, with an average positivity of at least about 60%, preferably at least about 75%, more preferably at least about 90% (the indicated peptide or expressed as a percentage of positive T cell responses to a combination of labeled peptides (greater than or equal to Sl 2.0)) indicates that the candidate candidate peptide (s) selected are likely to contain a sufficient percentage of T cell epitopes to be sensitive to the antigen of the protein in question. induce T cell insensitivity in a significant percentage of the population.

For the treatment of allergies, it is preferred, in accordance with the methods of the invention, that the peptide used together does not bind to immunoglobulin Et (IgE) or Ig Et significantly less (i.e., at least 100-fold less, and more preferably at least 1000-fold). less binding) than the protein allergen binds from which the peptide is derived. The major complication of standard immunotherapy is IgE-mediated responses such as anaphylaxis. Immunoglobulin E is a mediator of anaphylactic reactions resulting from the binding or cross-linking of antigen to IgE on mast cells or basophils and the release of mediators (e.g., histamine, serotonin, eosinophil chemotatic factors) in patients with allergies (atopic). Thus, anaphylaxis in a significant percentage of the treated population of allergen-sensitive individuals can be avoided by employing a peptide or peptides that do not bind IgE to a significant percentage (i.e., at least 75%) of the population of allergen-sensitive individuals, or if the peptide binds to IgE, such binding will not result in the release of mediators from mast cells or basophils. The risk of anaphylaxis can be reduced by using a peptide or peptides with reduced IgE binding activity during immunotherapy. Furthermore, peptides with minimal IgE stimulating activity are desirable for therapeutic efficacy. Minimal IgE stimulatory activity refers to the production of IgE that is less than the amount of IgE production and / or IL-4 production stimulated by the native protein allergen (e.g., Der ρ I). If a peptide binds to IgE, it is preferred that such binding does not result in the release of mediators (e.g., histamine) from mast cells or basophils. To determine whether an IgE-binding peptide results in the release of mediators, a histamine release assay can be performed using, for example, standard reagents and protocol of Amac, Inc. (Westbrook, ME). Briefly, a buffered solution of the peptide to be tested is mixed with an equal volume of total heparinized blood of an allergic subject. After mixing and incubation, the cells are pelleted and the supernatant processed and analyzed by radio immunoassay to determine the amount of histamine released.

High purity peptides of any other peptide and impurity used in the therapeutic composition of the present invention having a defined amino acid residue sequence and containing at least one T cell epitope may also be synthesized using standard techniques. Various methods of chemical synthesis of peptides are known in the art, such as solid phase synthesis, which is fully or semi-automated on commercially available peptide synthesizers. Synthetically generated peptides can then be purified to homogeneity (i.e., at least 90%, more preferably at least 95% and more preferably at least 97%) such that they are free of all other polypeptides and contaminants, and there are many techniques known in the art for protein purification. at will.

In accordance with a process for the preparation of a highly purified homogeneous peptide composition, the peptide may be purified by preparative reverse-phase chromatography using synthetic chemical means (either solid phase synthesis on a polymeric support or conventional homogeneous chemical reactions). In this method, the synthetically generated crude peptide is dissolved in a suitable solvent (typically in aqueous buffer) and placed on a separation column (typically a reversed phase silica based medium, in addition to a polymer or carbon based medium). The peptide is eluted from the column with increasing concentrations of an organic component (typically acetonitrile or methanol) in an aqueous buffer (typically TFA, triethylamine phosphate, acetate, or the like). Fractions of the eluate were collected and analyzed by appropriate analytical techniques (typically reverse phase HPLC or CZE chromatography). Fractions of the desired homogeneity are pooled. The opposite ion present can be exchanged for further reverse phase chromatography with the salt of choice, or with ion exchange resins. The peptide can then be isolated in the form of acetate or other suitable salts. The peptide is then filtered and the water removed (typically by lyophilization) to form a homogeneous peptide composition containing at least 90%, more preferably at least 95% and more preferably at least 97% of the desired ingredient. Whenever possible, or in combination with reverse phase HPLC as described above, the purification process can be performed by affinity chromatography, ion exchange, size exclusion, countercurrent or normal phase separation system, or a combination of these methods.

The peptide may also be concentrated by ultrafiltration, rotary evaporation, precipitation, dialysis, or the like.

The highly purified homogeneous peptide composition may then be characterized by the following techniques or combinations of techniques:

(a) mass spectroscopy for molecular weight determination to verify peptide identity:

b) amino acid analysis to verify peptide identity through the amino acid composition

c) by amino acid sequencing (using automated protein sequencer or manually) to confirm the sequence of the specified amino acid residues

d) HPLC (multiple system if necessary) to verify peptide identity and purity (this identifies peptide impurities)

e) the water content to determine the water concentration of the peptide composition

(f) an ionic content in the peptide composition to determine the presence of salts; and

(g) by determining the residual organic matter for the presence of residual organic reagents, starting materials and / or organic impurities.

For synthetically produced peptides of the present invention, it is particularly desirable to have about 45 amino acid residues in length, most preferably about 30 amino acid residues in length, since increasing length causes difficulty in peptide synthesis. Peptides longer than this can be prepared by recombinant DNA techniques as described below.

The peptides useful in the present invention may also be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid sequence encoding such a peptide. When produced by recombinant techniques, host cells transfected with nucleic acid encoding the desired peptide are cultured in a culture medium suitable for culturing the cells and the isolated peptides are purified from the cell culture medium, the cells, or both using peptide and protein purification techniques known in the art. , ultrafiltration, electrophoresis, or immunopurification with antibodies specific for the desired peptide. Recombinantly produced peptides can be isolated and purified to homogeneity to be free of cellular material, other polypeptides, or the culture medium used, in accordance with the methods described above for synthetically generated peptides.

Under certain limited conditions, peptides can also be produced by chemical or enzymatic cleavage of a high purity full length native protein from which the chemical digestion or enzymatically cleaved region is predetermined and the resulting digestion region can be reproduced. High purity can be achieved for peptides having defined amino acid sequences and can be isolated from other polypeptides or impurities present in chemical or enzymatic digestion using the methods described above for high purity and isolated synthetically or recombinantly produced peptides.

High purity and isolated peptides prepared as described above may be formulated into therapeutic compositions of the present invention that are suitable for human therapy. When a therapeutic composition of the present invention is administered by injection (e.g., subcutaneous injection), it is preferred that the high purity peptide is dissolved in an aqueous solvent within a pharmaceutically acceptable pH range (e.g., pH 4-9) such that the composition is readily injectable. Preferably, the composition also comprises a pharmaceutically acceptable carrier. For the purposes of the present invention, a pharmaceutically acceptable carrier is any adjuvant, diluent, dispersion medium, coating, antibacterial and antifungal agent, toxicity, buffering agent, absorption delaying or promoting agent, surfactant and mycelial lipid, micelle? and liquid complexing agent, stabilizing agent and the like. The use of such media and substances as pharmaceutically active substances is known in the art. Exceptionally, if any conventional medium or substance is incompatible with the active substance, its use in therapeutic formulations should be considered. Additional active compounds may also be incorporated into the formulations.

Therapeutic compositions suitable for use in the injectable form of the present invention are preferably sterile aqueous solutions prepared by incorporating the active ingredient (e.g., the high purity and isolated peptide described above) in a suitable amount of a carrier with one of the ingredients listed below and above. or a combination thereof, as required, followed by filtration sterilization. Preferred pharmaceutically acceptable carriers include at least one adjuvant, such as sterile water, sodium phosphate, mannitol, sorbitol or sodium chloride, or any combination thereof. Other suitable pharmaceutically acceptable carriers include solvents or dispersion media containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and mixtures thereof. 0 · «0 0 0 0 0 and vegetable oils. Suitable fluidity may be maintained, for example, by coating materials such as lecithin, by maintaining the appropriate particle size in the case of dispersions and by the use of surfactants. Prevention of the activity of microorganisms can be achieved by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thyrerosol and the like. Prolonged absorption of the injectable preparation may be accomplished by the inclusion of a substance which retards absorption, such as aluminum monostearate and gelatin.

A therapeutic composition of the present invention must be sterile, stable during production, storage and distribution, and protected from contamination by microorganisms such as bacteria and filamentous fungi. A preferred means of maintaining the integrity of the composition (e.g., protection against contamination, prolonging shelf life, etc.) of a therapeutic composition of the present invention is to provide the peptide formulation and the pharmaceutically acceptable carrier in the form of a lyophilized powder. which can be reconstituted in a pharmaceutically acceptable carrier, such as sterile water, only by reconstitution prior to use. In the case of sterile powders for sterile injectable solutions, the preferred method of preparation is vacuum drying, freeze drying or centrifugation drying, which contains the active ingredient in powder form and other desired ingredients from the previously sterile filtered solution. Specific formulations of the therapeutic compositions of the present invention are described below in the Examples.

In many cases, the therapeutic compositions of the invention contain more than one isolated peptide. A therapeutic composition comprising multiple formulations suitable for pharmaceutical use in humans may be desirable for multiple active peptides. A multiple formulation comprises at least two or more isolated peptides having a defined amino acid sequence and capable of down-regulating an antigen-specific immune response. Aspects to consider when preparing a multiple formulation include maintaining the solubility and stability of the peptides in the formulation in an aqueous solution at a physiologically acceptable pH. This necessitates the selection of one or more pharmaceutically acceptable solvents and adjuvants that are compatible with all peptides in the multiple formulation. For example, suitable excipients include sterile water, sodium phosphate, mannitol, or sodium phosphate and mannitol. Another aspect of a multiple formulation is to prevent dimerization of the peptides, if necessary. Substances that prevent dimerization, such as EDTA, or any other substance or process known to prevent dimerization may also be included in the multiple formulation. The following is an example of a multiple formulation that is used as a therapeutic agent in cats in Phase II human clinical trials. The two active peptides, peptide X (SEQ ID NO: 1) and peptide Y (SEQ ID NO: 2), each have a defined amino acid residue sequence and are derived from the Fel d I protein allergen by chemical synthesis and purification as described above by homogenization. for use in a formula for the treatment of a human allergy to cats in accordance with the present invention. For this embodiment, peptide X (SEQ ID NO: 1) and Y peptide (SEQ ID NO: 2) are in lyophilized form which is redissolved in sterile water before use. Peptide X (SEQ ID NO: 1) and Y peptide (SEQ ID NO: 2) were mixed during preparation using known techniques to form a vial containing a sterile, pyrogen-free, lyophilized powder of the following composition:

Active: 0.75 mg of peptide X (SEQ ID NO: 1) and 0.75 mg of peptide Y (SEQ ID NO: 4)

Inactive: 0.05 M sodium phosphate, pH 6.2

5% w / v mannitol U.S.P.

Solvent: Sterile water for injection U.S.P. (initial reset)

0.9% sodium chloride for injection (initial reconstitution dilution)

The preparation of this multiple formulation requires reconstitution of the vials with sterile water for injection. In another embodiment, the multiple formulation may further comprise three (see Example 2), four or five additional peptides suitable for human therapy.

Administration of a therapeutic composition as described above to an individual, in a non-immunogenic form, can be accomplished by known methods at a dose and for a period sufficient to down-regulate the antigen-specific immune response in the individual (e.g., reducing symptoms of antigen-specific immune response disease). necessary to achieve. Down-regulation of an antigen-specific immune response to an antigen associated with a disease state can be clinically determined in humans at any time. ··· · ··· ·································································································································································································································································································· ·

Effective amounts of the therapeutic compositions of the present invention will depend on factors such as the degree of susceptibility of the individual to the antigen, the age and sex of the patient, body weight, and the ability of the peptide to down-regulate the antigen-specific immune response. The therapeutic composition of the present invention may be administered in a non-immunogenic form by conventional means, for example, by injection (subcutaneous, intravenous, etc.), oral, sublingual, inhaled, transdermal, rectal or any other method known in the art for use in therapeutic agents. It may be desirable to provide the individual with a therapeutically effective amount of one or more of the therapeutic compositions of the present invention simultaneously or sequentially. Such compositions for simultaneous or sequential use may each contain only one peptide or may contain a multiple formulation as described above.

For subcutaneous injection of one or more therapeutic compositions of the invention, preferably from 1 pg to 3 mg, more preferably from 20 pg to 1.5 mg, and more preferably from about 50 pg to 750 pg, of the active component (peptide) per dosage unit. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, the term unit dosage form refers to physically discrete units suitable as unitary dosages for human patients to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect. with the desired pharmaceutical carrier. The specification of the dosage forms of the new unit of the present invention is dictated by, and dependent upon:

(a) the specific characteristics of the active compound and the specific therapeutic effect sought;

and

(b) the related scientific limitations in the formulation of the active compound for the treatment of human subjects.

The dosage regimen may be adjusted to provide an optimal therapeutic response. For example, multiple divided doses may be administered over days, weeks, months, years, or the dose may be increased or decreased proportionally with each successive injection, as dictated by the constraints of the therapeutic setting. In a preferred therapeutic schedule, subcutaneous injections of the therapeutic composition are administered once a week for 3-6 weeks. The dose may remain constant with each injection or may increase or decrease with successive injections. A booster injection may be given at intervals of about three to about one year from the initial treatment and may represent only one injection or a series of injections similar to the initial treatment.

Use of a composition of the invention other than parenteral administration (e.g., oral administration) may require wrapping or co-administration of the composition with a substance that prevents its inactivation or enhances its absorption and bioavailability. For example, a peptide formulation may be used in combination with an enzyme inhibitor or in a liposome. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP) and trasilol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., 1984, J. Neuroimmunol., 7:27). If you have a peptide

·· Properly protected can be administered orally, for example, with an inert diluent or an assimilable brain with an edible carrier. The peptide and other ingredients may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic use, the active compound may be incorporated into excipients and administered in the form of ingestible tablets, oral tablets, pills, capsules, elixirs, solutions, gels, suspensions, syrups, wafers, and the like. Such compositions and compositions must contain at least 1% by weight of active ingredient. The percentage of composition and composition may, of course, vary and may conveniently be from about 5 to about 80% by weight per unit. In such a therapeutically useful composition, the amount of active compound is such that a suitable dose is achieved. In addition, the active compound may be incorporated into sustained release or controlled release (sustained or pulsed) formulations and formulations.

The present invention is illustrated by the following non-limiting examples.

Example 1

Use of peptides in the treatment of cat allergy in human subjects

A. Composition of Phase 1 Formula

For Phase 1 clinical trials, the drug product comprises two freeze-dried peptide formulations, peptide X (SEQ ID NO: 1) and Y peptide (SEQ ID NO: 2). Peptide X (SEQ ID NO: 1) and Y peptide (SEQ ID NO: 2) are prepared separately and packaged in two different pyrogen vials.

Peptide X (SEQ ID NO: 1)

Active: 1.5mg / vial

Inactive: 0.1 M sodium phosphate, pH 5.7

Diluent: Sterile water for injection U.S.P.

Y peptide (SEQ ID NO: 2)

Active: 1.5mg / vial

Inactive: 0.1 M sodium phosphate, pH 5.7% w / v mannitol, U.S.P.

Diluent: Sterile water for injection U.S.P.

In this assay, the drug product is used in two different vials in the form of a sterile pyrogen-free, lyophilized powder containing sodium phosphate and mannitol as excipients, and the active compound is peptide X (SEQ ID NO: 1) and

Y peptide (SEQ ID NO: 2).

The drug product formulation in these assays requires reconstitution of the material in the vials with sterile water for injection. Dilution of reconstituted vials is required to provide lower doses at 7.5 or 75 pg / ml dose titration, also with sterile 0.9% sodium chloride for injection. If necessary, the vials can be prepared at only 7.5 or 75 pg per peptide without the need for dilution.

The lyophilized drug product is stored below 20 ° C and used immediately after reconstitution. Storage and use conditions are not limited.

· «• · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

B. Composition of Phase II Formula

For Phase II clinical trials, a single formulation containing a final concentration of 750 pg / ml of each peptide was prepared. Peptide X (SEQ ID NO: 1) and Y peptide (SEQ ID NO: 2) are mixed during the final filling process to form a vial containing a sterile, pyrogen-free, lyophilized powder.

Active: 0.75 mg of peptide X (SEQ ID NO: 1) and 0.75 mg of peptide Y (SEQ ID NO: 4)

Inactive: 0.05 M sodium phosphate, pH 6.2% w / v mannitol, U.S.P.

Diluent: Sterile water for injection U.S.P. (initial reset)

0.9% sodium chloride for injection (dilution beyond initial reconstitution)

The freeze-dried drug formulation is diluted at 750 pg / ml with sterile water for injection. For lower doses, dilution of reconstituted vials, for example to 7.5 or 75 pg / ml, is accomplished using sterile 0.9% sodium chloride. Subjects are administered the drug formulation according to the protocol of use provided in the clinical protocol.

The drug formulation is used immediately after reconstitution.

The lyophilized drug formulation is, but is not limited to, stored below -20 ° C.

··> * »* · ·· •« · · '4 «« · · · · · · · · · ············

D. Human Phase 1 and II Clinical Trials

Phase I and II studies of X and Y peptides (SEQ ID NOs: 1 and 2), respectively, are performed in feline allergic patients.

Protocol P92-01: Phase I Clinical Study of Safety and Activity of X and Y Peptides

This Phase I, open label, dose escalation safety study is conducted to determine the safety of using peptide X (SEQ ID NO: 1) and peptide Y (SEQ ID NO: 2). The antigenicity of the compounds is evaluated by the activity of the peptides in variable skin test sensitivity.

In this study, two centers - the Johns Hopkins Asthma and Allergy Center (Baltimore, Maryland) and the New England Medical Center (Boston, Massachusetts) - added a total of 19 cats with allergic patients. Peptide X (SEQ ID NO: 1) and Y peptide (SEQ ID NO: 2) were administered separately but simultaneously, by subcutaneous injection, at doses ranging from 7.5 pg to 1500 pg in increasing doses over a period of 5 weeks.

Safety is assessed by evaluating clinical laboratory parameters, physical observations, antibody tests, and noxious tests. Activity is assessed by skin test analysis. Puncture and intradermal skin testing was performed with affinity purified native Fel d 1 and peptides one week before and two and six weeks after the treatment period. Sixteen (16) patients were treated with X and Y peptides during the study. Peptides were well tolerated at doses of 1500 pg. No serious adverse effects were observed and the safety of the peptides was confirmed for further clinical development. Adverse events recorded during the experiment were typical of those reported in the open allergy studies. A * · · ·· ♦ · V »*« · · * «· · · · · · · · ·································••• all cats are allergic to this experiment and the tolerated dose is orders of magnitude higher than what can be achieved with conventional immunotherapy doses.

One patient discontinued the study due to a first asthma attack occurring after skin testing with cat-1 extract and peptides at Week 1 and a second asthma attack at weekly doses of 7.5 pg of each peptide. applied after. It is unclear whether there is a direct relationship between asthma and peptide treatment in this patient. It is worth noting that the study was conducted during the most severe pollen period of recent years, and this patient, like many of the other patients included in the study, is also seasonally allergic.

Antibody tests revealed that all patients under evaluation had IgG and IgE antibodies to Fel d I. Concentrations were unchanged for all patients except two patients during the study. Two patients showed IgE antibodies to peptide X (SEQ ID NO: 1) and / or Y peptide (SEQ ID NO: 2) at weeks 8 and 12, which did not occur at week 1. One of these patients showed a significant skin test reaction to peptide X (SEQ ID NO: 1) at week 8, which did not occur at week 12. 5 patients had endogenous IgG antibodies to peptide X (SEQ ID NO: 1) and 3 patients gave IgG antibodies to peptide Y (SEQ ID NO: 2) prior to exposure to the peptide. Four patients without prior anti-peptide IgG antibodies to peptide X (SEQ ID NO: 1) and / or Y peptide (SEQ ID NO: 2) developed such antibodies at increasing concentrations. Two patients had a positive immediate skin test response to a treatment peptide during the study, with no clinical correlation.

Another patient showed a delayed local reaction to skin test with peptide Y (SEQ ID NO: 2) at week 8, related to an intradermal dose of Y peptide (SEQ ID NO: 2).

Cat allergen skin test results show a statistically significant decrease in reactivity to feline extract containing fel dl at two or three dilutions in the puncture tests after 8 or 12 weeks, respectively. No statistically significant difference in reactivity was observed with the intradermal method. A statistically significant decrease in late phase reactivity to Fel dl (Σ | =) at 6 and 24 hours was observed at the Johns Hopkins Center when the 8-week results were compared to baseline (Week 1). The decrease was not significant when Week 12 was compared to baseline.

Although this study was conducted to evaluate safety, these skin test data suggest that peptides may cause desensitization to Fel dl.

P92-02 Protocol: Phase II Clinical Study of Safety and Activity of X and Y Peptides Using a Cat Room Trial Model

The Phase II safety and efficacy study was conducted with a double-blind, placebo-controlled study of peptides X and Y, administered subcutaneously at 7.5 pg, 75 pg or 750 pg every 4 weeks. Patients' sensitivity to natural exposure was determined by measuring symptom points and pulmonary function over a 60 minute period when the patient was placed in a small room filled with furniture and containing two cats. This cat room trial was performed prior to treatment w, and then after treatment 1 and 6 weeks. In the cat room, patients rated nasal, eye, and lung symptoms on a five-point scale every five minutes. Pulmonary function was tested every 15 minutes. Other parameters evaluated during the assay include skin test sensitivity to feline extract as well as X and Y peptides, specific IgE and IgG for Fel dl and component X and Y peptides, and T cellular response to feline antigen and selected peptides.

patients attended the trial at two centers - the Johns Hopkins Asthma and Allergy Center (Baltimore, Maryland) and New England Medical Center (Boston, Massachusetts). 91 patients completed the experiment. Four patients discontinued, two due to therapy-related temporary allergic symptoms and two due to scheduling conflicts.

Treatment was generally well tolerated. Analysis of the primary efficacy data from the study shows that there is a significant dose-response relationship, which is considered statistically significant (Figure 1) for the control of allergic symptoms (nasal, lung, and total allergy) induced by cat exposure after treatment. and 6 weeks. A statistically significant paired comparison with placebo was obtained at nasal and total allergic symptoms at 75 pg and 750 pg at week 6 (see Figure 1). The 7.5 pg dose could not be distinguished from placebo. Patients had a sting test negative during study treatment pre-treatment, but 13 patients had a positive sting and / or intradermal skin test for peptide treatment. Two patients gave a significant IgE titre against the treatment peptide and skin test positive after the study treatment. Only 5 patients with peptide-induced positive skin tests gave measurable IgE against the peptide. Some patients (placebo: 57.5%; 7.5 pg: 60.9%; 75 pg: 63.6%; 750 pg: 83.3%) developed cat allergic symptoms such as rhinorrhea, nasal congestion, pruritus , chest tightness, and / or asthma breathing during the test. The test treatment seems to produce mild transient symptoms, reminiscent of exposure to a natural cat. Cases are dose-dependent (placebo: 11.5-19.2%; 7.5 pg: 21.7-26.1%; 75 pg: 31.8-45.5%; 750 pg 50.0-62.5 %). These symptoms were generally mild self-limiting and did not require treatment or could be controlled with a beta agonist and / or antihistamine. One patient was treated with adrenaline and six patients showed asthma symptoms with droplets in the peak course. All of them were easily solved. The treatment does not alter antibody responses to induce allergies during the study period, nor does it affect the reactivity of the skin nipple (tea) relative to placebo, but is associated with an improvement in the overall allergic score in 80% of the study patients. Further studies are under way to further characterize the efficacy of the treatment.

Example 2

Use of peptides in humans to treat allergy to ragweed

A. Phase 1 formulation

For Phase I clinical trials, the drug product is a multiple formulation containing the three freeze-dried peptides of Amb a I, the residual amino acid sequence of each peptide comprising Amb a 1.1 (see WO 93/21321, incorporated herein by reference). is an epitope of at least one T-cell allergen of ragweed protein. Each peptide was purified to homogeneity (at least 97% purity) according to the methods described above. The multiple formula is prepared according to the procedures described herein. In the multiple formulation used in the Phase I clinical trial, each peptide is present as a freeze-dried powder cake in a vial. The formulation is redissolved in sterile water for injection just before use and normal saline (0.9%) is used for dilution in addition to the initial reconstitution.

B. Human Phase I Clinical Trials

Preliminary data indicate that all doses were tolerated and the multiple formulation appears to be safe. Further analysis and Phase II clinical trials are ongoing in the United States and Canada.

equivalents

One of ordinary skill in the art will recognize, or simply discover that there are a number of equivalents to the specific procedures described herein, using routine experimentation. Such suitable methods are within the scope of the present invention and are within the scope of the following claims.

SEQUENCE LIST (1) GENERAL INFORMATION:

(i) APPLICANT: IMMULOGIC PHARMACEUTICAL CORPORATION (ii) NAME OF THE INVENTION: PEPTID PREPARATIONS DERIVED FROM ANTIGEN SPECIFIC IMMUNE RESPONSE (iii) SEQ ID NO: 2 (iv)

ADDRESSE (A): IMMULOGIC PHARMACEUTICAL CORPORATION (B) STREET: 610 LINCOLN STREET (C) CITY: WALTHAM (D) STATE: Massachusetts (E) COUNTRY: USA (F) DIRECTORY: 02154 (v) COMPUTER:

(A) CARRIER: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: ASCII text (vi) APPLICATION DETAILS:

(A) APPLICATION NUMBER:

(B) DATE OF EDUCATION:

(C) CLASSIFICATION:

(vii) PRIORITY APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) DATE OF EDUCATION:

(C) CLASSIFICATION:

(viii) LAWYER / OFFICE INFORMATION:

(A) NAME: DARLENE A. VANSTONE (B) REGISTRATION NUMBER:

(C) REFERENCE / DOCUMENT NUMBER: 092.0US (ix) TELECOMMUNICATIONS DATA:

(A) TELEPHONE: (617) 466-6000 (B) TELEPHONE: (617) 466-6040 (2) SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (v) FRAGMENT TYPE: intimate (xi) SEQ ID NO: 1:

Lys Arg Asp Val Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val

10 15

Glu Gin Val Alá Gin Tyr Lys Alá Leu Pro Val

25

(2) DETAILS OF SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: peptide (v) FRAGMENT TYPE: internal (internal) (xi) DESCRIPTION OF SEQ ID NO: 2:

Lys Alá Leu Pro Val Val Leu Glu Asn Arg He Leu Lys Asn Cys

10 15

Val Asp Alá Lys Met Thr Glu Glu Asp Lys Glu

Claims (43)

PATENT CLAIMS A therapeutic composition comprising an isolated peptide having at least one defined amino acid residue sequence, said composition having the ability to down-regulate an antigen-specific immune response when used in a non-immunogenic form in a human subject subject to said antigen-specific immune response. The composition of claim 1, wherein at least one peptide is derived from a protein antigen and said antigen specific immune response is directed against said antigen. The composition of claim 1, wherein the at least one peptide is purified to at least 95% purity. A therapeutic composition comprising at least one isolated and purified peptide, a sequence of at least one said peptide of defined length and defined amino acid residues, and comprising at least one T cell epitope of a protein antigen when used in a non-immunogenic form. is capable of down-regulating an antigen-specific immune response in a human subject subject to said antigen-specific immune response. Composition according to claim 1 or 4, characterized in that the at least one peptide consists of residues of defined length and up to 50 amino acids. ··· · ··· · 6. The composition of claim 4, wherein the at least one peptide comprises at least 12 amino acid residues but not more than 40 amino acid residues. 7. The composition of claim 4, wherein said antigen is a protein allergen. The composition of claim 7, wherein said protein allergen is a protein allergen of the genus Drematophagoides; a protein allergen of the genus Felis; a protein allergen of the genus Ambrosia; a protein allergen of the genus Lolium; a protein allergen of the genus Cryptomería; a protein allergen of the Alternaria genus; a protein allergen of the genus Alder; a protein allergen of the genus Betula; a protein allergen of the genus Quercus; a protein allergen of the genus Olea; a protein allergen of the genus Artemisia; a protein allergen of the Plantago genus; a protein allergen of the genus Parietaria; a protein allergen of the Canine genus; a protein allergen of the genus Blattella; a protein allergen of the Apis genus; a protein allergen of the genus Cupressus; a protein allergen of the genus Juniperus; a protein allergen of the Thuya genus; a protein allergen of the genus Chamaecyparis; a protein allergen of the genus Periplaneta; an allergen of a protein of the genus Agropyron; a protein allergen of the Secale genus; a protein allergen of the genus Triticum; a protein allergen of the genus Dactylis; a protein allergen of the genus Festuca; a protein allergen of the Poa genus; a protein allergen of the Avena genus; a protein allergen of the genus Holcus; a protein allergen of the genus Anthoxanthum; a protein allergen of the genus Arrhenatherum; a protein allergen of the genus Agrostis; a protein allergen of the Phleum genus; a protein allergen of the genus Phalaris; selected from the group consisting of a protein allergen of the genus Paspalum and a protein allergen of the genus Sorghum halepensis. The composition of claim 8, wherein the protein allergen is Dérp 1; Derp II; Derp III; Derp VIII; Derf 1; Derf II; Dér fllll; Dér f Vll; Fel d 1; Amb 1.1; Amb. 1.2; Amb. 1,3; Amb a 1.4; Amb a ll her; Lol p 1; lol p ll; Lolp III; Lolp IV; Lol p IX (Lol ρ V or Lol p lb); Cry II; Cry j ll; Can f 1; Can f ll; Jun s 1; Jun v 1; Dac g 1; Poa p l; selected from the group consisting of Phl p l and Ser h l. 10. The composition of claim 4, wherein said protein antigen is an autoantigen. 11. The composition of claim 10, wherein the autoantigen is insulin, myelin basic protein, myelin oligodendrocyte protein, rh factor, acetylcholine receptors, thyroid cell receptors, basement membrane proteins, thyroid proteins; ICA-69 (PM-1); glutamic acid decarboxylase (64K and 65K); proteolipid protein (PLP), myelin-associated glycoprotein (MAG); collagen (type II); heat shock protein; and the carboxypeptidase H is selected from the group consisting of H. 12. The composition of claim 4, wherein at least one peptide is not derived from said protein antigen and said peptide is capable of mimicking a T cell epitope of said antigen. 13. The composition of claim 4, wherein the at least one peptide represents a cryptic peptide of said protein antigen. The composition of claim 4, wherein the at least one peptide has an average T cell stimulation index of at least 3.5 as determined by an in vitro T cell proliferation assay on T cells obtained from said allergen-sensitive human population. The composition of claim 14, wherein the at least one peptide has at least 150 positivity index as determined by an in vitro T cell proliferation assay on T cells obtained from said allergen-sensitive human population. • · • · • · · · · · · ....... · »··. ·.:. 16. The composition of claim 4, wherein said at least one peptide comprises an adequate percentage of a T cell epitope of said protein antigen such that application of the composition reduces the development and progression of symptoms of the antigen-induced disease. 17. The composition of claim 16, wherein said at least one peptide comprises at least 20% of allergenic T cell epitopes of said protein. 18. The composition of claim 7, wherein said at least one peptide does not bind to IgE or IgE substantially less than the naturally occurring allergen of said protein binds to IgE. 19. The composition of claim 4, wherein said at least one peptide is purified to homogeneity. 20. The composition of claim 19, wherein said at least one peptide is purified to at least 95% purity. The composition of claim 4, wherein said at least one peptide is produced in accordance with a method selected from chemical synthesis, recombinant DNA techniques, chemical cleavage of the purified whole protein, and enzymatic cleavage of the purified whole protein. • · • ί! · · · ........ · * *. ·. j "50 22. A therapeutic composition according to claim 4, characterized in that it comprises at least one pharmaceutically acceptable carrier. 23. The therapeutic composition of claim 22, wherein said pharmaceutically acceptable carrier is selected from the group consisting of sterile water, sodium phosphate, mannitol, sorbitol, sodium chloride, and combinations thereof. 24. The composition according to claim 1, 4, 16 or 22, characterized in that it is soluble in aqueous solution at physiologically acceptable pH. 25. The composition of claim 1, 4, 16, or 22, wherein said at least one peptide comprises an appropriate percentage of a T cell epitope of said protein antigen such that the antigen is the onset and progression of symptoms of the disease is eliminated. 26. The composition of claim 4, wherein said at least one peptide is present in an amount of about 1 pg to 3.0 mg peptide / dose per unit. 27. The composition of claim 26, wherein said at least one peptide is present in an amount of about 20 pg to 1.5 mg peptide / dose per unit. • ·. . . . . ··. . • ::::: ···· ··· ··. _β 28. The composition of claim 27, wherein said at least one peptide is present in an amount of about 50 pg to 750 pg peptide / dose per unit. 29. A composition according to claim 1, 4, 16 or 22, characterized in that it comprises at least two peptides. 30. A method of treating a human susceptible to antigen, comprising treating at least one therapeutic composition according to claim 4. 31. A method of treating a human susceptible to antigen, comprising the use of a therapeutic composition comprising at least two isolated and purified peptides, each having a defined amino acid residue sequence of defined length and containing at least one T cell epitope of a protein antigen, said composition being capable of down-regulating an antigen-specific immune response to said protein antigen in a human population subject to said antigen-specific immune response when the composition is administered in a non-immunogenic form and said composition is soluble in aqueous solution at physiologically acceptable pH and remains. 32. A method of treating a human susceptible to antigen, wherein the at least two compositions of claim 4 are administered simultaneously or sequentially. 33. A method of treating a human susceptible to antigen, wherein the at least two compositions of claim 31 are administered simultaneously or sequentially. 34. The method of claim 30, wherein said composition is administered orally. 35. The method of claim 30, wherein said composition is administered subcutaneously. 36. The method of claim 35, wherein said composition is for initial treatment, said initial treatment comprising subcutaneous injection of the composition for at least 3 weeks but less than 6 weeks. 37. The method of claim 36, wherein said vaccine is administered a booster dose of at least three months after said initial treatment. 38. The method of claim 36, wherein said initial treatment comprises administering each successive injection at increasing doses. 39. The method of claim 30, wherein said antigen is a protein allergen or an autoantigen. • · * ·· «» • · · · · · · · · · · · · · · · · · · 40. The method of claim 36, wherein said initial treatment comprises administering each successive injection at a decreasing dose. 41. The method of claim 30, wherein said use is sublingual application of said composition. 42. The method of claim 30, wherein said composition is administered intravenously. 43. A method of treating a human susceptible to antigen, comprising the use of at least one therapeutic composition according to claims 1, 16, 22 or 26.