BG63183B2 - Antiidiotype vaccine against diseases caused by pathogenic responses of specific t-cell populations - Google Patents

Abstract

The invention relates to vaccines and means for vaccination of a host with the intention of preventing or controlling specific T-cell mediated pathologies. The vaccine is made of antiidiotype antibodies which are internal reflections of a segment of the T-cell receptor (TCR), found on the surface of the pathogenic T-cells. The vaccine is applied on the host in a manner which includes an immune reply, directed against the TCR of the pathogenic cells. This immune response regulates or eliminates the pathogenic T-cells, and in this way eliminates the pathogenesis. The invention relates to a mean for the determination of a suitable amino acid sequence of such a vaccine. 17 claims

Description

The invention relates to the immune system, 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 and regulate a cell-mediated 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 long-term need for effective agents for treating or ameliorating T-cell-mediated pathologies. Such treatment should ideally control a non-peculiar T-cell response rather than reduce symptoms. The invention satisfies this need by providing related advantages.

SUMMARY OF THE INVENTION

The invention relates to vaccines and mammalian vaccine agents so as 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 T-cell receptor (TCR) sequence sequences of the Cells that mediate such pathology or anti-idiotypic antibodies that are internal reflections of the peptides.

In addition, the invention provides vaccines for the treatment of unregulated T-cell propagation. The vaccine is composed of TCRs or fragments thereof corresponding to a 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 to prevent or improve 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 treatment approaches.

As used herein, the term "T-cell pathology" refers to any condition in which a non-specific 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 contributes to the production of these antibodies.

As used herein when referring to the association 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. In general, these immune system targets are referred to as antigens. The recognition of an antigen by the immune system quickly mobilizes the immune mechanisms to destroy 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 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. These include the variable (V) regions of the genius segments, the binding region (J) of the genius segments, and the constant (C) region of the genius segments. The B- and δ-chains contain an additional element called the difference segment (D). Since D segments and elements are located only in some TCR genetic loci 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 lymphocyte matures, the single V, (D) and J gene segments are rearranged to form a functional gene that determines the amino acid sequence of TCR expressed by that cell. Because the pooling of V, (D) and J genes that can be rearranged is polynomial, and since the individual members of these pools can be rearranged 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. Such an animal model, Experimental Allergic Encephalomyelitis (EAE), is an autoimmune disease of the central nervous system that can be induced in 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 are isolated from animals suffering from EAE and develop 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 also generally 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 analysis of 33 BMP-reactive T cells, only two α-chains from the V region of the gene segment and a single AveR gene gene segment were used. Similar restriction on the use of the β-chain TCR gene has also been observed in this T-cell population. Only two segments 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 region of β-chain V-D-J binding. These findings support the notion of a common TCR structure among T cells with similar antigenic specificities and indicate that TCR is an effective target for immunotherapeutic strategies aimed at eliminating the pathogenesis of EAE.

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 νβ8-specific monoclonal antibody, the major β-chain of the V region of the gene used by 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 was shown in rats with EAE with monoclonal antibody reacting with unidentified idiotypic determinant of TCR on BPM specific T cells (Burns et al., J. Exp. Med. 169: 27-39, (1989). While passive antibody therapy appears to have some enhancing effect on sensitivity to

EAE, it creates a number of potential problems. The protection achieved is transient, requiring repeated administration of antibodies. Repeated infusion of antibodies increases the chances of the host to detect an immune response to the administered antibody, especially if it is induced in heterogeneous organisms. Further, the antibody response to the pathogenic T-cell clone represents only one element of the complete immune response and ignores the potential possibilities 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 passive immunity approach, regulatory T cells were obtained ex vivo and reapplied for immunotherapy. For example, Sun et al., Nature, 332: 843-845, (1988) isolated a CO8 + T-cell clone from healing rats in which EAE was induced by adoptive transfer from a BPM-specific CD4 + T-cell line. The CD8 + T-cell clone showed cytolytic activity in vitro for CD4 + T-cells, used to induce the disease. Moreover, the adaptive transfer of this CTL clone reduced the susceptibility of the recipient rats to subsequent exposure to BPM. Lider etal., (Science, 239: 181-183, (1988) have also isolated a CD8 + T-cell clone with suppressive activity for EAE-inducing T cells. This CD8 + clone is isolated from rats vaccinated with attenuated disease-causing Clone clones and, since they do not show cytolytic activity in vitro, they could suppress the BPM-driven proliferation of EAE-inducing T cells, although these studies indicate that CD8 + T cells can completely regulate EAE, it is difficult to confirm the major role of these selected CD8 + T-cell lines in long-term resistance n recovered rats since Sedgwick et al., (Eur. J. Immunol., 18 495 502 (1988) clearly 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. It allows for an in vivo regulatory response of prolonged duration. However, this requires in vitro cultivation with attenuated disease-causing 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 that are not identical across individuals, this makes a highly individualized therapeutic strategy. 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, vaccinated animals show resistance in subsequent attempts to induce EAE (Lider et al., Supra; see Cohen and Weiner, Immunol. Today 9: 332 - 335, (1988) for reviews). However, the efficacy of such vaccines is variable and the degree of protection varies greatly. T cells contain many different antigens that elicit an immune response when the whole cell is introduced as a vaccine. This phenomenon is demonstrated 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 ear for these T cells as the rate of growth and the number of vaccinations increase, however, positive DTH responses were found in both the protected and unprotected animals. The rats responded similarly to vaccination with encephalitogenic T cells and controls. T-cells In contrast, vaccination with PPD-specific T-cells is PPD-specific cell lines induce 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-causing and control cells, as quantified by delayed type hypersensitivity (cell-mediated measurement) immunity) indicates that multiple antigens on these T cells have induced immune responses. Vaccination with attenuated T cells lacks the specificity of the protective antigen on the T cell surface, as well as variable induction of immunity to this antigen. As an opportunity for treatment of human diseases, vaccination with attenuated T cells is accompanied by the same laboriousness and need for individualized therapy as noted for infusion with 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 these methods of treatment. 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 of 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, for example α, β, etc., or portions of such chains alone or in combination. The vaccines may be homogeneous, for example a single peptide, or may be composed of more than one peptide, each corresponding to a portion of the 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 well known to those skilled in the art. Preferably the peptide vaccines correspond to the regions of

TCRs 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. Preferably, 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.

Alternatively, the vaccines may contain anti-idiotype antibodies that are internal reflections of the peptides described above. Those skilled in the art will be aware of methods for the creation, selection and administration of such anti-idiotypic vaccines. See, for example, Bichmann, et al., CRC Critical Reviews in Immunology 7: 193-227 (1987), included in the accompanying reference.

The vaccines are administered to a host who is at or at risk of an autoimmune response. Clear clinically active autoaggressive activity is observed 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 in diseases including T cells (versus non-pathogenic T cells), can then be selected as vaccines, and prepared and administered as described above.

T-cell pathologies with malignant etiology.

In order to illustrate the utility of TCR vaccination, autoimmune diseases are discussed. However, T-cell lymphoma is another T-cell pathology that may be susceptible to this type of treatment. The application of this technology in the treatment of T-lymphoma is practically identical. In one important aspect, however, this technology is more readily applicable to T-cell proliferative diseases, while isolation of pathogenic T-cells is more feasible. Once the clones have been isolated, the technology is applied as described above. Specifically, TCR genes of T lymphomas are sequenced, the corresponding regions of those

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.) will be immunized in each hind paw with 50 pg guinea pig basic protein emulsified in complete Fruend 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 - loosened tail; 2 - weakening of the hind leg; 3 - paralysis of the hind leg. After 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, which is incorporated herein by reference. A nine amino acid peptide having the VDJ sequence of the mouse TCR β-strand is synthesized by methods known to those skilled in the art. The sequence of this peptide is: SGDAGGGYE. (Amino acids are represented by the conventional one-letter code). The equivalent sequence in rats is known to be SSD-SSNTE (Bums 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, at a ratio of 7.5 mg peptide per mg KLH. The resulting conjugate was dialyzed with phosphate buffered sodium chloride (PBS).

Example 3. Vaccination against EAE

The vaccines used in these studies contain free VDJ peptide and also KLH 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, aged 8-12 weeks, in a final volume of 100 μΐ per animal (50 μΐ) in each of the hind paws). 5 pg per rat of unconjugated VDJ peptide were administered. The KLH-VDJ conjugate was administered at doses equivalent to 10 pg KLH per rat. 29 days later, in each rat, 50 pg guinea pig basic protein of guinea pig was introduced into the forepaws in complete Freund's adjuvant. Animals were monitored daily starting from day 9 for clinical signs of EAE and counted 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 are ill, the severity of the disease is reduced and / or onset. The degree of protection varies with the form of the vaccine, and those incorporating CFA as adjuvant show a greater degree of protection.

Table 1.

Days after introduction

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

Reporting:

- no symptoms

1) Tailing

2) weakening of the hind leg

3) paralysis of the hind leg

Although the invention has been described with reference to a preferred embodiment, various modifications may be made without departing from its nature. Accordingly, the invention is limited only by the claims. 45

Claims (17)

Claims A composition for reducing the potency of T cell-mediated pathology, characterized by the use of a restricted T-cell receptor gene comprising an anti-idiotypic 35 antibody, which is an intrinsic reflection of a T-cell receptor (TCR) amino acid sequence located on the surface of autoaggressive T-cells mediating the pathology and a pharmaceutically acceptable 40 medium, wherein said anti-idiotypic antibody induces an immune response to said autoaggressive T cells cells, reducing the strength of said pathology. The composition of claim 1, wherein said anti-idiotype antibody corresponds to a fragment of said amino acid sequence from said T-cell receptor. The composition of claim 2, wherein said fragment includes a V (D) J accession sequence. The composition of claim 1, further comprising an adjuvant. The composition of claim 1 or 2, wherein said composition comprises anti-idiotype antibodies that are internal reflections of the amino acid sequences of more than one T-cell receptor or fragment thereof, or which are internal reflections of amino acid sequences corresponding to one or more different fragments from the same T-cell receptor. A method for reducing T-cell-mediated pathology, characterized by the use of a restricted T-cell receptor gene from an individual exhibiting or at risk of developing T-cell-mediated pathology, comprising administering an immunologically effective amount of the composition of claim 1 or 2. The method of claim 6, wherein said composition is attached to a carrier. The method of claim 6, wherein said composition is administered more than once. The method of claim 6, wherein said composition is administered in a formulation comprising an adjuvant. 10. Composition for reducing the strength of T-cell proliferative disease mediated by unregulated T-cell clonal replication, characterized by the use of a restriction T-cell receptor gene comprising an anti-idiotype antibody, which is an intrinsic reflection of an amino acid receptor acid sequence TCR) presented on the surface of said replicating T-cell clones, which anti-idiotypic antibody induces an immune response 5 to said replicating T-cell clones that reduce the potency of n the disorder. The composition of claim 10, wherein said anti-idiotype antibody is The yo corresponds to a fragment of said amino acid sequence of said cellular receptor. The composition of claim 11, wherein said fragment comprises V (D) J at 15 coupling sequence. The composition of claim 10, further comprising an adjuvant. A method for reducing the strength of T-cell-mediated pathology characterized by the limited use of an individual's T-cell receptor gene displaying or at risk of developing T-cell-mediated pathology, comprising administering the composition of claim 10. The method of claim 14, wherein said composition is attached to a carrier. The method of claim 14, wherein said composition is administered more than once. The method of claim 14, wherein said composition is administered in a formulation comprising an adjuvant.