BG63182B2 - Vaccination by specific t-cell populations against diseases caused by pathogenic responses - Google Patents

Abstract

The invention relates to vaccines and vaccination means of a host with the intention of prevention or controlling specific T-cell determined proliferation diseases. The vaccine is made of pharmaceutically acceptable medium and a segment of T-cell receptor (TCR) found on the surface of the T-cells, determining the disease. The vaccine is applied on the host in a manner which induces an immune response, directed to the TCR of pathogenic T-cells. This immune response regulates or deletes the pathogenic T-cells, and in this way stops of the pathogenesis of the disease. The invention also relates to means for the determination of suitable amino acid sequence for such a vaccine. 6 claims

Description

The invention relates, in particular, to methods for modifying pathogenic immune responses.

BACKGROUND OF THE INVENTION

Higher organisms are characterized by an immune system that protects them from the invasion of potentially harmful substances or microorganisms. When a substance called an antigen penetrates the body and is recognized as foreign, the immune system responds with an antibody-mediated response and a cell-mediated response. Immune system cells called B lymphocytes or B cells produce antibodies that specifically recognize and bind to a foreign substance. Other lymphocytes, called T-lymphocytes or T-cells, simultaneously exert a regulatory cellular response, possibly leading to the removal of the antigen.

Different T cells are involved in the cellular response. Some excite specific B-cell clones for the proliferation and production of antigen-specific antibodies. Others recognize and destroy cells with foreign antigens on their surface. Some T cells regulate the response either by stimulating or suppressing other cells.

Although the normal immune system is well regulated, errors in the immune response are not uncommon. In some cases, the immune system functions abnormally and responds to a host component as if it were actually foreign. The result of such a response is an autoimmune disease in which the host immune system attacks the host's own tissue. T cells as primary regulators of the immune system directly or indirectly cause such autoimmune pathologies.

Many diseases are thought to result from autoimmune mechanisms. Known among them are rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, myasthenia gravis and pemphigus vulgaris. Autoimmune diseases affect millions of people worldwide, and losses from these diseases, expressed as actual costs for treatment and disability, are measured in billions of dollars annually.

No effective treatment for such autoimmune pathologies is currently known. Usually, only the symptoms can be treated as the disease continues to progress, often causing severe exhaustion or death.

In other cases, the lymphocytes replicate inappropriately and without control. Such replication leads to a cancer known as lymphoma. When unregulated lymphocytes are of T-cell type, tumors are defined as T-cell lymphomas. As with other malignancies, T-cell lymphomas are difficult to treat effectively.

Therefore, there is a need for effective agents for treating or ameliorating T-cell-mediated pathologies. Such treatment should ideally control the non-peculiar T-cell response rather than reduce the symptoms. The invention satisfies this need by providing related advantages.

SUMMARY OF THE INVENTION

The invention relates to vaccines and mammalian vaccine agents to prevent or control specific T-cell-mediated pathologies. The vaccine is composed of T-cell receptors (TCRs) or fragments thereof corresponding to TCRs located on the surface of the Pathogen-mediated T cells. Alternatively, the vaccine may be a peptide corresponding to the sequence of T cell receptor (TCR) characteristics of the T cells mediating such pathology or anti-idiotypic antibodies that are intrinsic to the peptides.

In addition, the invention provides vaccines for the treatment of unregulated T-cell propagation. The vaccine is composed of TCRs or fragments of the same, corresponding to the TCR located on the surface of unregulated T cells. Alternatively, the vaccine may be a peptide corresponding to the TCR sequences characteristic of unregulated T cells.

Means are also provided to determine suitable amino acid sequences for such vaccines. The vaccine is administered to the mammal in such a way as to elicit an immune response directed against TCR of the T cells mediating the pathology. This immune response completely regulates or destroys pathogenic T cells, thereby eliminating the pathogenesis of the disease.

The invention relates to vaccines and their use for preventing or ameliorating T-cell-mediated pathologies such as autoimmune diseases and T-cell lymphomas. Vaccination provides specific and sustained treatment that avoids problems associated with other possible therapies.

As used herein, the term " T-cell pathology " refers to any condition in which an unusual T-cell response is a component of the pathology. The term includes both T-cell mediated diseases and those such as myasthenia gravis, which are characterized mainly by damage due to antibody binding but also reflected in a non-specific cellular response that promotes the production of these antibodies.

As used herein, when referring to the link between peptides of the invention and TCR sequences, "corresponding to" means that the peptide has an amino acid sequence that is sufficiently homologous to the TCR sequence to stimulate an effective regulatory response without the need for be identical to the TCR sequence as shown in Examples 2 and 3.

The immune system is the primary biological protection of the host (he himself) against potentially harmful agents (not himself). These harmful agents can be pathogens, such as bacteria or viruses, modified own cells, including virus-infected cells, tumor cells or other abnormal host cells. Generally, these immune system targets are referred to as antigens. The recognition of an antigen by the immune system quickly mobilizes the immune mechanisms for the destruction of that antigen, thus preserving the inviolability of the host environment.

The main manifestations of the antigen-specific immune response are humoral immunity (mediated by antibodies) and cellular immunity (mediated by cells). Each of these immunological mechanisms is initiated by the activation of helper (CD4 +) T cells. These CD4 + T cells in turn stimulate B cells responsible for the synthesis of antibodies by binding to the antigen, for the proliferation and secretion of these antibodies. These secreted antibodies bind to the antigen and facilitate its destruction by other immune mechanisms. Similarly, CD4 + T cells provide stimulating signals for cytotoxic (CD8 +) T cells that recognize and destroy cellular targets, such as virus-infected host cells. Thus, activation of CD4 + T cells is the earliest event in stimulating the immune response. Therefore, activation of CD4 + T cells is crucial in any attempt to select modified immunological function.

T cells owe their antigen specificity to the T cell receptor (TCR), which is located on the cell surface, TCR is a heterodimeric glycoprotein composed of two polypeptide chains, each with a molecular weight of approximately 45 kD. Two forms of TCR have been identified. One is composed of α-chain and β-chain, while the second consists of γ-chain and δ-chain. Each of these four TCR polypeptide chains is encoded by a specific genetic locus containing multiple continuous gene segments. They include the variable (V) regions of the genomic segments, the binding region (J) of the genomic segments, and the constant (C) region of the genomic segments. The β and δ chains contain an additional element called the difference segment (D). Because D segments and elements are only found in some TCR genetic scales and polypeptides, further references here to D segments and elements will be in parentheses to indicate inclusion of these regions only in the appropriate TCR chains. Thus, V (D) J refers to either VDJ sequences of chains that have a D region or refer to VJ sequences of chains that lack D regions.

As the lymphocytes mature, the single V, (D) and J gene segments are rearranged to form a functional gene, determining the amino acid sequence of TCR expressed by that cell. Because the association of V, (D), and J genes that can be reordered is polynomial, and since the individual members of these associations can be reordered in virtually any combination, the complete TCR repertoire is very diverse and able to specifically recognize and to bind a large number of antigens to which an organism may be exposed. However, a single T cell has only one TCR molecule, and this TCR molecule largely, if not solely, determines the antigenic specificity of that T cell.

T-cell pathologies with autoimmune etiology.

Animal models have contributed significantly to the understanding of the immunological mechanisms of autoimmune diseases. One such animal model, Experimental Allergic Encephalomyelitis (EAE), is an autoimmune disease of the central nervous system that can induce mice and rats by immunization with the basic myelin protein (BPM). The disease is characterized clinically by paralysis and moderate attenuation and histologically by perivascular mononuclear cellular infiltration of the parenchyma of the central nervous system. The pathogenesis is mediated by T cells with specificity for BPM. Multiple clones of BPM-specific T-cells were isolated from EAE-bearing animals and reproduced in continuous culture. After in vitro stimulation with BPM, these T cell clones strongly induce EAE when transferred to healthy hosts. Importantly, these EAE-inducing T cells are specific not only to the same antigen (BPM) but usually also to a single epitope of that antigen. These observations indicate that rare autoaggressive T-cell populations are responsible for the pathogenesis of EAE.

TCR analysis of EAE-inducing T cells showed reduced heterogeneity of the structure of these disease-related receptors. In one assay of 33 BMP-reactive T-cells, only two strands from the V region of the gene segment and a single α-stranded J gene segment were used. Similar restriction on the use of the α-chain TCR gene has also been observed in this T-cell population. Only two segments a of the β-chain V region and two segments of the J region were identified. More importantly, approximately 80% of the T-cell clones have identical amino acid sequences in the domain of β-chain V-D-J binding. These findings support the notion of a common TCR structure between T cells with similar antigenic specificities and indicate that TCR is an effective target for immunotherapeutic strategies aimed at eliminating EAE pathogenesis.

Various attempts have been made to use the antigenic specificity of autoaggressive T cells to find a strategy for the treatment of EAE. For example, passive administration of TCR-specific monoclonal antibodies represented in EAE-inducing T cells was used. In a EAE model in mice, infusion of a monoclonal antibody specific for νβ ⁸ , the major β-chain of the V region of the gene used BMP-specific T cells, reduces the susceptibility of mice to subsequent induction of EAE. (Acha-Orbea et al., Cell 54: 263-273 (1988) and Urban et al., Cell 54: 577-592 (1988). Similar protection has been shown in rats with EAE with a monoclonal antibody reacting with an unidentified idiotype determinant of TCR on BMP-specific T cells (Burns et al., J.Exp.Med. 169: 27-39 (1989). While passive antibody therapy seems to have some enhancing effect on EAE-sensitive, it creates a number of potential The protection achieved is transient, requiring repeated administration of antibodies. light response to the administered antibody, in particular if induced in heterogeneous organisms Further, the antibody response to a pathogenic T cell clone represents only one element of a full immune response and ignores the potential of cellular immunity in resolving autoreactivity.

The role of cellular immunity in reducing the activity of autoaggressive T cells in EAE has been investigated and potential therapies have been given. In a similar to the passive immunity approach, regulatory T cells were obtained ex vivo and re-administered for immunotherapy. For example, Sun et al., Nature, 332: 843-845 (1988) isolated CD8 + T-cell clones from healing rats in which EAE was induced by adaptive transfer from the BMP-specific CD4 + T-cell line CD8 + T-cell clone has cytolytic activity in vitro for CD4 + T cells used to induce the disease. Moreover, the adaptive transfer of this CTL clone reduces the susceptibility of the recipient rats to subsequent exposure to BPM. Liber et al., (Science, 239: 181-183, 1988) have also isolated a CD8 + T-cell clone with superactive activity for EAE-inducing T cells. This C08 + clone is isolated from rats vaccinated with attenuated pathogenic T-cell clones and, since they do not show cytolytic activity in vitro, could suppress the BPM-driven proliferation of EAE-inducing T cells. Although these studies indicate that CD8 + T cells can be fully regulated by EAE, it is difficult to confirm the major role of these selected CD8 + T cell lines in the long-term resistance of healthy rats, as Sedgwick et al., (Eur. J. Immunol., 18: 495502, 1988) show that depletion of CD8 + cells with monoclonal antibodies does not affect the disease process or recovery.

In the experiments of Sun et al. and Lider et al., described above, administration of existing regulatory T cells overcomes the major obstacle to passive antibody therapy. This allows for a prolonged regulatory response in vivo, which, however, requires in vitro cultivation with attenuated pathogenic T cells to develop clones of such regulatory T cells, which is a costly and time-consuming process. In addition, in populations such as human MHCs (major histocompatibility complex), it is not identical across individuals, and makes the therapeutic strategy strictly individualized. Regulatory clones should be obtained for each patient individually and then reapplied to that patient to avoid potential transplant response to the host.

Direct vaccination with attenuated disease-causing T-cell clones has also been used to treat EAE. BPM-specific T cells capable of transmitting the disease are attenuated by γ-irradiation or chemical fixation and used for vaccination in rats. In some cases, the vaccinated animals showed resistance in subsequent attempts to induce EAE (Lider et al., Supra; Coheh ahd Weiner, immunol. Today 9: 332335, 1988). However, the efficacy of such vaccines is not constant and the degree of protection varies considerably. T cells contain many different antigens that elicit an immune response when the whole cell is introduced as a vaccine. This phenomenon is shown by Offner et al., (J. Neuroimmunol., 21: 13-22, 1989), which shows that immunization with whole T cells increases the delayed type hypersensitivity (DTH) response as measured by swelling of the the ear for these T cells is an incremental way with the increase in the number of vaccinations. However, positive DTH responses were found in both protected and unprotected animals. Rats respond similarly to vaccination with encephalitogenic T cells and control T cells. In contrast, vaccination with PPD-specific T-cells from a PPD-specific T-cell line induces DTH to the vaccinated cells as well as to the encephalitogenic clone, even when no protection is observed. A similar response in vaccinated rats to both disease and control cells, as quantified by delayed type hypersensitivity (measurement of cell-mediated immunity), indicates that multiple antigens on these cells have induced immune responses. A disadvantage of attenuated T cell vaccination is the lack of specificity for the protective antigen on the surface of the T cell, as well as the variable induction of immunity to this antigen. As an opportunity for the treatment of human diseases, vaccination with attenuated T cells is accompanied by the same laboriousness and need for individualized therapy as noted above for the infusion of CD8 + cells.

The invention provides an effective method of immunotherapy for T-cell-mediated pathologies, including autoimmune diseases, which avoids many of the problems associated with the use of said therapies. By vaccination, unlike passive administration of heterologous antibodies, the host's own immune system is mobilized to suppress autoaggressive T cells. Thus, suppression is permanent and may involve any and all immunological mechanisms to effect this suppression. This multifaceted response is more effective than unilateral suppression achieved by passive administration of monoclonal antibodies or derivatives of available regulatory T-cell clones.

Insofar as they relate to autoimmune diseases, the vaccines according to the invention contain TCRs from T cells mediating autoimmune diseases. The vaccines can be fully TCR, substantially purified from T cell clones, individual T cell receptor chains, e.g., etc., or portions of such chains alone or in combination. Vaccines may be homogeneous, for example a single peptide, or may be composed of more than one peptide, each corresponding to a portion of a TCR. "Essentially pure" means that TCR is essentially free from other biochemical residues to which it is normally associated in nature. Alternatively, vaccines comprise peptides of different lengths corresponding to TCRs or portions thereof. The peptides can be produced synthetically or recombinantly with agents known to those skilled in the art. Preferably, the peptide vaccines correspond to regions of TCR that distinguish this TCR from other non-pathogenic TCRs. Such specific regions are preferably located in the V region (s) of the respective TCR polypeptide chains. It is preferred that the peptide corresponds to a short sequence having a V (D) J bond, thus limiting the immune response to only those T cells that carry this single determinant.

Vaccines are administered to a host who is or is at risk of an autoimmune response. The well-defined clinical diagnosis of a specific autoimmune disease justifies the administration of appropriate disease-specific TCR vaccines. Prophylactic applications are appropriate for diseases in which autoimmune mechanisms precede the onset of an open clinical picture of the disease, such as type I. Diabetes mellitus, thus individuals with a family history of the disease and with the assumption that they are at risk through reliable prognostic indicators can be treated prophylactically to prevent autoimmune mechanisms prior to their manifestation.

TCR vaccines can be administered in various formulations in pharmacologically acceptable media. In the case of a short peptide, the peptide can be conjugated to a carrier such as KLH to increase its immunogenicity. The vaccine can be administered with an adjuvant, many of which are known to those skilled in the art. After initial vaccination with the vaccine, a second injection can be made. Vaccines are administered by conventional methods at dosages sufficient to elicit an immunological response that can be readily determined by one of ordinary skill in the art.

Suitable peptides for use for immunization can be determined as follows. Disease-inducing T-cell clones that respond to target antigens are isolated from the affected individuals. Such T cells can be derived from the site of active autoaggressive activity such as a lesion in the case of pemphigus vulgaris, CNS in the case of multiple sclerosis, or myasthenia gravis or synovial fluid or tissue in the case of rheumatoid arthritis. The TCR genes from these autoaggressive T cells are then sequenced. Polypeptides corresponding to TCRs or portions thereof, which are selectively represented by pathogenic T cells corresponding to non-pathogenic T cells, can then be selected as vaccines and constructed and used as described above.

T-cell pathologies with etiology malignancies.

In order to illustrate the utility of TCR vaccination, autoimmune diseases are discussed. However, T-cell lymphoma is another T-cell pathology that could be susceptible to this type of treatment. The use of this technology in the treatment of 'Tlimphoma' is carried out in an identical manner. However, this technology is more easily applicable to T-cell proliferative diseases, while isolation of pathogenic T-cells is useful. Once the clones have been isolated, the technology is applied as described above. Specifically, TCRs of T lymphomas are sequenced, suitable regions of these TCRs are identified and used as vaccines. Vaccines may contain single or multiple peptides and may be administered in forms with or without adjuvants in known ways.

The following examples illustrate the invention without limitation.

Example 1. Model of EAE in rats

Lewis female rats (Charles River Laboratories, Raleigh-Durham, N.C) were immunized in each hind paw with 50 g guinea pig myelin basic protein emulsified in complete Eruend adjuvant. The first signs of the disease are usually seen 9-11 days after immunization. The severity of the disease is recorded on a three-point scale, as follows: 1-relaxed tail; 2-weakening of the hind leg; H-paralysis of the hind leg. After the course of the disease (approximately 4 to 6 days), most of the rats recover spontaneously and do not respond to subsequent EAE challenge.

Example 2. Selection and preparation of vaccines

Vaccination is performed with a T-cell receptor peptide whose sequence is derived from the DNA sequence of the T-cell receptor β-strand gene prevalent in EAE-inducing T cells of SJ / L mice. The DNA sequence is that indicated by Urban et al., Supra. A nine-amino acid peptide having the VDJ link sequence of the TCR mouse chain was synthesized by known methods. The sequence of this peptide is SGDAGGYE (amino acids are represented by the conventional one letter code). The equivalent sequence in rats is SSD-SSNTE (Burns et al., J. Exp. Med. 169-27-39, 1989). The peptide was salted by Sephadex G-25 column chromatography (Pharmacia Fine Chemicals, Piscataway, N.J.) in 0.1 M acetic acid, and the solvent was then removed in two lyophilization cycles. Part of the peptide was conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde in a ratio of 7.5 mg peptide per mg KLH.

The resulting conjugate was dialyzed with phosphate buffered sodium chloride (PBS).

Example 3. Vaccine against EAE

The vaccines used in these studies contain the free CDJ peptide as well as the KLD-conjugated VDJ peptide. They are dissolved in PBS and emulsified in equal volumes either by (1) incomplete Freund adjuvant (IFA) or (2) Freund complete adjuvant (CFA) prepared by suspending 10 mg / ml of heat-cured dry Mycobacterium tuberculosis H37ra (Difco Laboratories, Detroit, Mich.) At IFA. The emulsions were administered to female rats of the Lewis breed, 8-12 weeks old, in a final volume of 100 μΐ per animal (50 μΐ in each of the hind paws). 5 μg per rat of unconjugated VDJ peptide were administered. The KLH-VDJ conjugate was administered at doses equivalent to 10 g KLH per rat. For 29 days, 50 μg guinea pig base protein in guinea pig was introduced into the forepaws of each rat in complete Freund adjuvant.

Animals were monitored daily starting from day 9 for clinical signs of EAE and reported as described above. The results are presented in Table 1. As can be seen, vaccinated individuals not only have a reduction in the incidence of the disease, but in those who have taken the disease, the severity of the disease is reduced and / or onset and delayed. The degree of protection varies in the form of the vaccine and those incorporating CFA as adjuvant show a greater degree of protection.

Table ϊ

Days after introduction

Animal # Vaccination (adjuvant) 10 11 12 13 14 15 16 17 18 1 VDJ (IFA) - 2 ³ ΐ 3 3 - - - 2 U I - 1 ³ ί 3 3 2 - - 3 U - i - - 3 3 3 4 - - 4 VDJ (CFA) - - - -: 1 1 1 - - 5 and 6 U - - - 1 3 3 3 2 - 7 KLH-VDJ (CFA) - - - 1 3 2 - - - 8 U - - - - 1 1 1 1 - 9 m 10 KLH-VDJ (IFA) - 1 3 3 2 2 1 - - 11 m - - 3 3 3 3 3 2 - 12 And - - 1 3 3 3 3 - - 13 without 1 3 3 3 3 1 - - - 14 U - 1 3 3 3 1 - - - 15 and 1 3 3 3

Reporting:

- no tail release symptoms

- weakening of the hind leg

- Paralysis of the hind leg

Example 4. Vaccination against EAE with VDJ peptides from Lewis rats.

The VDJ peptide used in the previous 4θ examples was synthesized according to the sequence of TCR chain molecules found on EAE-inducing T cells in mouse B1O.PL. In addition, peptides corresponding to sequences found on encephalitogenic Tcells in Lewis rats were synthesized and tested. These VDJ sequences are homologous to those in B10-PL mice but not identical. Rat peptides were synthesized according to the DNA jq sequences reported by Burns, et al. and Chluba, et al., J. Immunol. 19: 279-284,

1989. The sequences of these three peptides, designated IR1, 2 and 3, are shown below, aligned with the B10.PL mouse sequences used in Examples 1 to 3 (VDJ).

VDJ SGDAGGYE

IR1 CASSD - SSNTEVFFGK

IR2 CASSD - SGNTEVFFGK IR3 CASSD - SGN - VLYFGEGSR

The preparation, administration and evaluation of these vaccines were performed as described in Examples 1 to 3 with the following exceptions: 50 pg of individual VDJ peptides included in the formulation of CFA-containing vaccines; neither vaccinations in IFA nor vaccinations with peptides conjugated to KLH have been performed. Control animals were not treated prior to administration of MBP as in Example 3, nor were vaccinated with PBS and CFA emulsions to achieve the protective effect of the adjuvant alone. These results are shown in Table 2 below.

Table 2

Days after introduction An animal N8 Vaccination (Adjuvant) 10 11 12 13 14 15 16 17 18 1 There is no - 1 2 3 3 2 - - 2 - 1 3 3 3 2 - - 3 - - 2 3 3 3 1 - 4 PBS-CFA 1 2 3 3 3 - - 5 1 2 3 3 3 - - 6 • 2 3 3 3 - - 7 IR1 (50 cent) - - 2 1 - - 8 * • - - 1 3 - 9 and - - 1 1 1 1 10 IR2 (50 µg) - 1 3 3 3 - 11 • - - - 2 2 3 3 12 • - - - 1 - - 13 IR3 (50 µg) 1 3 3 3 2 - - 14 • - 2 3 3 - - 15 and - - - - - -

Reporting:

(-) - no characters

- a loose tail

- weakening of the hind limb

- paralysis of the hind limb

As shown in Table 2, disease in non-vaccinated control animals was observed as early as 10 days. The disease is characterized by severe paralysis and helplessness, lasting 4 to 6 days and spontaneously resolving. PBS-CFA vaccinated rats show disease progression indistinguishable from unvaccinated rats. In contrast, in some of the IR1, 2, or 3 unvaccinated animals, disease was initially observed, and others showed a concurrent onset as well as a reduction in the severity and / or duration of the disease. In general, however, vaccines with rat VDJ peptides (IR1-3) were slightly less effective compared to those of the murine VDJ peptide (Example 3).

Example 5. Vaccination with specific peptides of V region V

A peptide specific for the νβ8 gene family is tested as a vaccine against EAE. νβ8 is a generally β-stranded gene family used by encephalitogenic T cells in both rats and mice. The peptide was synthesized based on a unique DNA sequence found in the νβ8 gene, which has not been identified among other rat V genes, whose sequences have been reported by Morris et al, immunogenetics 27: 174-179, 1988). The sequence of this V8 peptide, designated IR7, is:

IR7 DMGHGLRLIHYSYDVNBTEK

The efficacy of this νβ8 peptide was tested on an EAE model Lewis rat (Example 1) as described in Examples 2 and 3.50 μg of the peptide were tested in CFA. Vaccinations in IFA or with peptide KLN conjugates have not been performed. The results of these studies are shown in Table 3.

Table 3

Days after introduction Animal # Vaccination (Adjuvant) 10 11 12 13 14 15 16 17 18 1 IR7 (50 µg) - - 1 2 3 3 3 - - 2 and - - - - 1 1 - - - 3 - - - - - - - - -

Reporting:

(-) - no characters

- a loose tail

- weakening of the hind limb

- paralysis of the hind limb

The results of vaccinations performed with the rat νβ8 peptide are similar to those observed in mouse and rat VDJ peptides. Delay at onset as well as a reduction in the severity and duration of the disease were observed in one animal. One animal is completely protected.

Example 6. Vaccination with peptides with J region.

A peptide corresponding to the J gene segment, TA39, found in both rat and mouse encephalitogenic T cell receptors was synthesized.

The sequence of this peptide, designated IR5, is:

IR5 RFGAGTRLTVK

The efficacy of the JaTA39 peptide was tested on an EAE model Lewis rat (Example 1) as described in Examples 2 and 3.

pg of the peptide were tested in CFA. Vaccinations in IFA or with peptide-KLH conjugates have not been performed. The results of these studies are shown in Table 4.

Table 4

Days after introduction

Animal # Vaccination (Adjuvant) 10 11 12 13 14 15 16 17 18 19 20 1 IR5 (50 µg) - - - - 2 1 1 1 1 - 2 IR5 (50 rd) 3 IR5 (50 rd)

Reporting:

(-) - no characters

- a loose tail

- weakening of the hind limb paralysis of the hind limb

Vaccination results with the rat JaJA39 peptide were more effective than those observed with the mouse and rat VDJ peptides and the Υβ8 peptide. Two of the three animals are completely protected, and in the third the disease is initially noticeably delayed. The severity of the disease is also reduced in this animal and is thought to be a normal course of 5 days. Importantly, the two animals that are fully protected show no histological manifestation of CNS infectious cell infiltration. The To3h result shows that vaccination with JaTA39 very effectively induces a regulatory response targeting encephalitogenic T cells. Even subclinical signs of the disease have been eliminated.

Example 7. Vaccination with mixtures of TCR peptides.

Vaccinations with a mixture of TCR peptides were performed. This mixture contains 50 pg of each of the peptides IR1, 2, 3 and 5 (the three rats

VDJ peptide and rat JaTA39).

The efficacy of this peptide mixture was tested in a Lewis model rat (Example 1) as described in Examples 2 and 3. Peptides were CFA. Vaccinations in IFA or with peptide-KLH conjugates have not been performed. The results of these studies are shown in Table 5.

Table 5

Days after introduction An animal Ns Vaccination (Adjuvant) 10 11 12 13 14 15 16 17 18 4 IR1.2, 3.5 (50 µg each) 5 - - - - - - - - - 6 - - - - - - - - -

Reporting:

(-) - no characters

- a loose tail

- weakening of the hind limb

- paralysis of the hind limb

The results of vaccinations performed with rat JaA39 and three VFJ peptides were more effective than any of the examples described above. All three animals are fully protected. In addition to the absence of any clinical signs of EAE, two of these three animals are completely free of histological manifestation of T-cell infiltration in the CNS, while the third shows only a small focus on lymphocytic infiltration at the base of the spinal cord.

Although the preferred embodiments are described in the invention, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited to the following claims.

Claims (9)

A composition for reducing the potency of 30 T-cell proliferative disease caused by unregulated T-cell replication of T cells having limited T cell receptor heterogeneity in mammals, comprising: pharmaceutically acceptable 35 medium and substantially pure, immunogenic single-stranded peptide having the amino acid sequence of a non-permanent region of a TEC Receptor Polypeptide Chain located on the surface of said T cells, wherein the composition induces an immune response against said unregulated T cells, which reduces the severity of the disease. The composition of claim 1, wherein the said peptide has an amino acid sequence derived from a variable region of a T cell polypeptide chain sequence. The composition of claim 1, wherein the said peptide has an amino acid sequence derived from a V (D) J combination of a T cell receptor polypeptide chain. A composition according to claim 1 comprising more than one substantially pure immuno-5 gene single-stranded peptide derived from non-constant regions of different cellular receptor polypeptide chains. 5. A method for reducing the strength of a T cell proliferative disease caused by unregulated T cell clonal replication of T cells having limited T cell receptor heterogeneity in a mammal comprising administering claim 1. The method of claim 5, wherein said composition is bound to a carrier. The method of claim 5, wherein said composition is administered more than once. The method of claim 5, wherein 20 said composition is administered adjuvanted. 9. A method for selecting a composition for use in reducing the potency of T-cell proliferative disease caused by unregulated T-cell clonal replication of T-cells having limited T-cell receptor (TCR) heterogeneity in a mammal, comprising the steps of: a) Preparation of a clone of T cells causing said T cell proliferative disease; b) Determination of the amino acid sequence of non-constant TCR regions from the resulting T-cell clone. c) Selection of segments of said amino acid sequence of TCR from these T cells causing T cell proliferative disease, and d) Synthesis or selection of an amino acid sequence from said selected segments or their functional equivalents, which reduces the strength of said T cell proliferative disease. Publication of the Patent Office of the Republic of Bulgaria 1113 Sofia, 52-B Dr. GM Dimitrov Blvd.