WO1999027957A1 - Vaccination and methods against multiple sclerosis using specific tcr vbeta peptides - Google Patents

Abstract

The present invention provides vaccines and a means of vaccinating a mammal so as to prevent or control specific T cell mediated pathologies or to treat the unregulated replication of T cells. The vaccine is composed of a T cell receptor (TCR) or a fragment thereof corresponding to a TCR present on the surface of T cells mediating the pathology. The vaccine fragment can be a peptide corresponding to sequences of TCRs characteristic of the T cells mediating said pathology. The vaccine is administered to the mammal in a manner that induces an immunologically effective response so as to affect the course of the disease. The invention additionally provides specific β-chain variable regions of the T cell receptor, designated Vβ6.2/3, Vβ6.5, Vβ6.7, Vβ2, Vβ5.1, Vβ13 and Vβ7, which are central to the pathogenesis of multiple sclerosis (MS).

Description

VACCINATION AND METHODS AGAINST MULTIPLE SCLEROSIS RESULTING FROM PATHOGENIC RESPONSES BY SPECIFIC T CELL POPULATIONS

This invention is a continuation-in-part of U.S. Serial No. 08/055,006 filed on April 29, 1993, which is a continuation-in-part of U.S. Serial No. 08/010,483 filed on January 28, 1993, which is a continuation of U.S. Serial No. 07/530,229 filed on May 30, 1990, which is a continuation-in part of U.S. Serial No. 07/326,314 filed March 21, 1989, 07/382,085 filed July 18, 1989 and 07/382,086 filed July 18, 1989, the contents of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to the immune system and, more specifically, to methods of modifying pathological immune responses.

Higher organisms are characterized by an immune system which protects them against invasion by potentially deleterious substances or microorganisms. When a substance, termed an antigen, enters the body, and is recognized as foreign, the immune system mounts both an antibody-mediated response and a cell-mediated response. Cells of the immune system termed B lymphocytes, or B cells, produce antibodies which specifically recognize and bind to the foreign substance. Other lymphocytes termed T lymphocytes, or T cells, both effect and regulate the cell-mediated response resulting eventually in the elimination of the antigen.

A variety of T cells are involved in the cell- mediated response. Some induce particular B cell clones to proliferate and produce antibodies specific for the antigen. Others recognize and destroy cells presenting foreign antigens on their surfaces. Certain T cells regulate the response by either stimulating or suppressing other cells.

While the normal immune system is closely regulated, aberrations in immune response are not uncommon. In some instances, the immune system functions inappropriately and reacts to a component of the host as if it were, in fact, foreign. Such a response results in an autoimmune disease, in which the host's immune system attacks the host's own tissue. T cells, as the primary regulators of the immune system, directly or indirectly effect such autoimmune pathologies.

Numerous diseases are believed to result from autoimmune mechanisms . Prominent among these are rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Type I diabetes, myasthenia gravis and pemphigus vulgaris. Autoimmune diseases affect millions of individuals world-wide and the cost of these diseases, in terms of actual treatment expenditures and lost productivity, is measured in billions of dollars annually. At present, there are no known effective treatments for such autoimmune pathologies. Usually, only the symptoms can be treated, while the disease continues to progress, often resulting in severe debilitation or death.

In other instances, lymphocytes replicate inappropriately and without control. Such replication results in a cancerous condition known as a ly phoma. Where the unregulated lymphocytes are of the T cell type, the tumors are termed T cell lymphomas . As with other malignancies, T cell lymphomas are difficult to treat effectively. Thus there exists a long-felt need for an effective means of curing or ameliorating T cell mediated pathologies . Such a treatment should ideally control the inappropriate T cell response, rather than merely reducing the symptoms. The present invention satisfies this need and provides related advantages as well.

Summary of the Invention

The present invention provides vaccines and a means of vaccinating a mammal so as to prevent or control specific T cell mediated pathologies or to treat the unregulated clonal replication of T cells. The vaccine is composed of a T cell receptor (TCR) or a fragment thereof corresponding to a TCR present on the surface of T cells mediating the pathology. The vaccine fragment can be a peptide corresponding to sequences of TCRs characteristic of the T cells mediating said pathology. The vaccine is administered to the mammal in a manner that induces an immunologically effective response so as to affect the course of the disease.

The invention additionally provides specific β-chain variable regions of the T cell receptor, designated Vβ6.2/3, Vβ6.5, Vβ6.7, Vβ2, Vβ5.1, Vβl3 and Vβ7, which are central to the pathogenesis of multiple sclerosis (MS) . Also provided are means to detect, prevent and treat MS.

Brief Description of the Figures

Figure 1 shows the frequency of the various TCR Vβ genes expressed in the cultures from CSF of MS patients. Figure 2 shows the sequences of the Vβ gene family that are most frequently expressed in the cultures from CSF of the MS patients.

Figure 3 shows a summary of the Vβ7 CDR3 sequences. The top 3 monoclonal sequences were derived from cultured samples while the bottom sequences were obtained directly after sorting the CSF for CD25 (IL-2 receptor) and either CD3 or CD4.

Detailed Description of the Invention

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 a specific and sustained treatment which avoids problems associated with other potential avenues of therapy.

As used herein, the term "T cell-mediated pathology" refers to any condition in which an inappropriate T cell response is a component of the pathology. The term is intended to include both diseases directly mediated by T cells and those, such as myasthenia gravis, which are characterized primarily by damage resulting from antibody binding, and also diseases in which an inappropriate T cell response contributes to the production of those antibodies. The term is intended to encompass both T cell mediated autoimmune diseases and unregulated clonal T cell replication.

As used herein, "substantially the a ino acid sequence," or "substantially the sequence" when referring to an amino acid sequence, means the described sequence or other sequences having any additions, deletions or substitutions which do not substantially effect the ability of the sequence to elicit an immune response against the desired T cell receptor sequence. Such sequences commonly have many other sequences adjacent to the described sequence. A portion of the described immunizing sequence can be used so long as it is sufficiently characteristic of the desired T cell receptor as to cause an effective immune response against desired T cell receptors but not against undesired T cell receptors . Such variations in the sequence can easily be made, e.g. by synthesizing an alternative sequence, and tested, e.g. by immunizing a mammal, to determine its effectiveness.

As used herein, the term "fragment" is intended to cover such fragments in conjunction with or combined with additional sequences or moieties, as for example where the peptide is coupled to other amino acid sequences or to a carrier. The terms "fragment" and

"peptide" can, therefore, be used interchangeably since a peptide will be the most common fragment of the T cell receptor. Each fragment of the invention can have an altered sequence, as described above for the term "substantially the sequence."

As used herein, the term "vaccine" means compositions which, when administered into an individual, affect the course of the disease by causing an effect on the T cells mediating the pathology. This effect can include, for example, induction of cell mediated immunity or alteration of the response of the T cell to its antigen. Reference herein to a "fragment or portion of the T cell receptor" does not mean that the composition must be derived from intact T cell receptors. Such "fragments or portions" can be produced by various means well-known to those skilled in the art, such as for example manual or automatic peptide synthesis or methods of cloning.

As used herein when referring to the relationship between peptide fragments of the invention and sequences of TCRs, "corresponding to" means that the peptide fragment has an amino acid sequence which is sufficiently homologous to the TCR sequence to stimulate an effective regulatory response in the individual. The sequence need not be identical to the TCR sequence, however, as shown in Examples II and III.

By "substantially pure" it is meant that the TCR or fragment thereof is substantially free of other biochemical moieties with which it is normally associated in nature. For example, the TCR is normally found with moieties derived from the same species of origin. Such moieties may act as undesirable contaminants when the TCR is used, for example, as a vaccine.

By "immunologically effective" is meant an amount of the T cell receptor or fragment thereof which, is effective to elicit a change in the immune response to prevent or treat a T cell mediated pathology or an unregulated T cell clonal replication in the individual. Obviously, such amounts will vary between species and individuals depending on many factors. For example, higher doses will generally be required for an effective immune response in a human compared with a mouse. As used herein, "binding partner" means a compound which is reactive with a TCR. Generally, this compound will be a Major Histocompatibility Antigen (MHC) but can be any compound so long as when the TCR is bound in the normal course, T cell activation or proliferation occurs .

As used herein, "ligand" means any molecule that reacts to form a complex with another molecule.

As used herein, "selectively binds" means that a molecule binds to one type of molecule but not substantially to other types of molecules . In relation to Vβl7 "selective binding" indicates binding to Vβl7 containing TCRs but not substantially to other TCRs which lack Vβl7.

The immune system is the primary biological defense of the host (self) against potentially pernicious agents (non-self) . These pernicious agents may be pathogens, such as bacteria or viruses, as well as modified self cells, including virus-infected cells, tumor cells or other abnormal cells of the host.

Collectively, these targets of the immune system are referred to as antigens. The recognition of antigen by the immune system rapidly mobilizes immune mechanisms to destroy that antigen, thus preserving the sanctity of the host environment.

The principal manifestations of an antigen- specific immune response are humoral immunity (antibody mediated) and cellular immunity (cell mediated) . Each of these immunological mechanisms are initiated through the activation of helper (CD4+) T Cells. These CD4+ T cells in turn stimulate B cells, primed for antibody synthesis by antigen binding, to proliferate and secrete antibody. This secreted antibody binds to the antigen and facilitates its destruction by other immune mechanisms. Similarly, CD4+ T cells provide stimulatory signals to cytotoxic (CD8+) T cells which recognize and destroy cellular targets (for example, virus infected cells of the host) . Thus, the activation of CD4+ T cells is the proximal event in the stimulation of an immune response. Therefore, elaboration of the mechanisms underlying antigen specific activation of CD4+ T cells is crucial in any attempt to selectively modify immunological function.

T cells owe their antigen specificity to the T cell receptor (TCR) which is expressed on the cell surface. The TCR is a heterodimeric glycoprotein, composed of two polypeptide chains, each with a molecular weight of approximately 45 kD. Two forms of the TCR have been identified. One is composed of an alpha chain and a beta chain, while the second consists of a gamma chain and a delta chain. Each of these four TCR polypeptide chains is encoded by a distinct genetic locus containing multiple discontinuous gene segments. These include variable (V) region gene segments, junction (J) region gene segments and constant (C) region gene segments. Beta and delta chains contain an additional element termed the diversity (D) gene segment. (Since D segments and elements are found in only some of the TCR genetic loci, and polypeptides, further references herein to D segments and elements will be in parentheses to indicate the inclusion of these regions only in the appropriate TCR chains. Thus, V(D)J refers either to VDJ sequences of chains which have a D region or refers to VJ sequences of chains lacking D regions.) During lymphocyte maturation, single V, (D) and J gene segments are rearranged to form a functional gene that determines the amino acid sequence of the TCR expressed by that cell. Since the pool of V, (D) and J genes which may be rearranged is multi-membered and since individual members of these pools may be rearranged in virtually any combination, the complete TCR repertoire is highly diverse and capable of specifically recognizing and binding the vast array of binding partners to which an organism may be exposed. However, a particular T cell will have only one TCR molecule and that TCR molecule, to a large degree if not singly, determines the specificity of that T cell for its binding partner.

Animal models have contributed significantly to our understanding of the immunological mechanisms of autoimmune disease. One such 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 myelin basic protein (MBP) . The disease is characterized clinically by paralysis and mild wasting and histologically by a perivascular mononuclear cell infiltration of the central nervous system parenchyma. The disease pathogenesis is mediated by T cells with specificity for MBP. Multiple clones of MBP-specific T cells have been isolated from animals suffering from EAE and have been propagated in continuous culture. After in vitro stimulation with MBP, these T cell clones rapidly induce EAE when adoptively transferred to healthy hosts. Importantly, these EAE- inducing T cells are specific, not only for the same antigen (MBP) , but also usually for a single epitope on that antigen. These observations indicate that discrete populations of autoaggressive T cells are responsible for the pathogenesis of EAE. Analysis of the TCRs of EAE-inducing T cells has revealed restricted heterogeneity in the- structure of these disease-associated receptors. In one analysis of 33 MBP-reactive T cells, only two alpha chain V region gene segments and a single alpha chain J region gene segment were utilized. Similar restriction of beta chain TCR gene usage was also observed in this T cell population. Only two beta chain V region segments and two J region gene segments were found. More importantly, approximately eighty percent of the T cell clones had identical amino acid sequences across the region of beta chain V-D-J joining. These findings confirm the notion of common TCR structure among T cells with similar antigen specificities and indicate that the TCR is an effective target for immunotherapeutic strategies aimed at eliminating the pathogenesis of EAE.

Various attempts have been made to exploit the antigen specificity of autoaggressive T cells in devising treatment strategies for EAE. For example, passive administration of monoclonal antibodies specific for TCRs present on EAE-inducing T cells has been employed. In the mouse model of EAE, infusion of a monoclonal antibody specific for V_β8, the major beta chain V region gene used by MBP-specific T cells, reduced the susceptibility of mice to subsequent EAE induction (Acha- Orbea et al . , Cell 54:263-273 (1988) and Urban et al., Cell 54:577-592 (1988)). Similar protection has been demonstrated in rat EAE with monoclonal antibody reactive with an unidentified idiotypic determinant of the TCR on MBP specific T cells (Burns et al . , J. Exp . Med. 169:27- 39 (1989) ) . While passive antibody therapy appears to have some ameliorative effect on EAE susceptibility, it is fraught with potential problems. The protection afforded is transient, thus requiring repeated administration of the antibody. Multiple infusions of antibody increases the chances that the host will mount an immune response to the administered antibody, particularly if it is raised in a xenogeneic animal. Further an antibody response to a pathogenic T cell clone represents only one element in the complete immune response and neglects the potential contributions of cellular immunity in resolving the autoreactivity .

The role of cellular immunity in reducing the activity of autoaggressive T cells in EAE has been examined and potential therapies suggested. In a manner similar to the passive antibody approach, regulatory T cells have been derived e_x. vivo and readministered for immunotherapy . For example, Sun et al . , Nature, 332:843- 845 (1988), have recently isolated a CD8+ T cell line from convalescing rats in whom EAE had been induced by adoptive transfer of an MBP-specific CD4+ T cell line. This CD8+ T cell line displayed cytolytic activity in vitro for the CD4+ T cell used to induce disease. Moreover, adoptive transfer of this CTL line reduced the susceptibility of recipient rats to subsequent challenge with MBP. Lider et al., Science, 239:181-183 (1988) have also isolated CD8+ T cells with suppressive activity for EAE-inducing T cells. These CD8+ cells were isolated from rats vaccinated with attenuated disease-inducing T cell clones and, though they showed no cytolytic activity in vitro, they could suppress MBP-driven proliferation of EAE-inducing T cells. Although these studies indicate that the CD8+ T cells could downregulate EAE, it is hard to reconcile a major role for these selected CD8+ CTLs in the long-term resistance of the recovered rats since Sedgwick, et al . , (Eur. J. Immunol., 18:495-502 (1988)) have clearly shown 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, the administration of extant derived regulatory T cells overcomes the major obstacle of passive antibody therapy; it permits a regulatory response in vivo of prolonged duration. However, it requires in vitro cultivation with attenuated disease- inducing T cells to develop clones of such regulatory T cells, a costly and labor intensive process. Further, in an outbred population such as humans, MHC non-identity among individuals makes this a highly individualized therapeutic strategy. Regulatory clones need to be derived for each individual patient and then re- administered only to that patient to avoid potential graft versus host reactions .

Direct vaccination with attenuated disease- inducing T cell clones also has been employed as a therapy for EAE. MBP-specific T cells, capable of transferring disease, have been attenuated by gamma irradiation or chemical fixation and used to vaccinate naive rats. In some cases, vaccinated animals exhibited resistance to subsequent attempts at EAE induction (Lider et al . , supra; see Cohen and Weiner, Immunol. Today 9:332-335 (1988) for review). The effectiveness of such vaccination, however, is inconsistent and the degree of protection is highly variable. T cells contain a multitude of different antigens which induce an immune response when the whole T cell is administered as a vaccine. This phenomenon has been demonstrated by Offner et al., (J. Neuroimmunol . , 21:13-22 (1989)), who showed that immunization with whole T cells increased the delayed type hypersensitivity (DTH) response, as measured by ear swelling, to those T cells in an incremental manner as the number of vaccinations increased. However, positive DTH responses were found in both protected and non-protected animals. Rats responded similarly to both the vaccinating encephalitogenic T cells and control T cells .

Conversely, vaccination with PPD-specific T cells from a PPD-specific T cell line induced DTH to the vaccinating cells as well as to an encephalitogenic clone even though no protection was observed. The similar response of vaccinated rats to both disease-inducing and control cells, as quantified by delayed-type hypersensitivity (a measure of cell-mediated immunity) , indicates that numerous antigens on these T cells are inducing immune responses. Thus, vaccination with attenuated disease-inducing T cells suffers from a lack of specificity for the protective antigen on the surface of that T cell, as well as, variable induction of immunity to that antigen. As a candidate for the treatment of human diseases, vaccination with attenuated T cells is plagued by the same labor intensiveness and need for individualized therapies as noted above for infusion of CD8+ cells.

The present invention provides an effective method of immunotherapy for T cell mediated pathologies, including autoimmune diseases such as multiple sclerosis, which avoids many of the problems associated with the previously suggested methods of treatment. By vaccinating, rather than passively administering heterologous antibodies, the host's own immune system is mobilized to suppress the autoaggressive T cells. Thus, the suppression is persistent and may involve any and all immunological mechanisms in effecting that suppression. This multi-faceted response is more effective than the uni-dimensional suppression achieved by passive administration of monoclonal antibodies or extant-derived regulatory T cell clones.

As they relate to autoimmune disease, the vaccines of the present invention comprise TCRs of T cells that mediate autoimmune diseases. The vaccines can be whole TCRs substantially purified from T cell clones, individual T cell receptor chains (for example, alpha, beta, etc.) or portions of such chains, either alone or in combination. The vaccine can be homogenous, for example, a single peptide, or can be composed of more than one type of peptide, each of which corresponds to a different portion of the TCR. Further, these peptides can be from distinct TCRs wherein both TCRs contribute to the T cell mediated pathology.

The Vβ6 TCR subunits were sequenced from 8 patients. From these 8 patients three-quarters (6 of 8) were identified as members of the Vβ6.2/3 and Vβ6.5 subfamily shown in Figure 2. These two subfamilies of the Vβ6 gene family show considerable homology in the CDR2 region between residues 39 and 58. It appears that these two particular members of the Vβ6 family are particularly associated with multiple sclerosis.

Vβ6.7 also appears to be particularly associated with multiple sclerosis. In an expanded study of MS patients, CSF cells were analyzed for Vβ expression. In 21 of 47 patients analyzed, elevated levels of Vβ6 expression were observed in CD4+ T-cells. The Vβ6 family members most frequently detected were

Vβ6.5 and Vβ6.7. As shown in Figure 2, the CDR2 region sequences, encompassing amino acids 39-58, are very similar between these two Vβ family members. .

In a further specific embodiment, T cell receptors, whole T cells or fragments of the TCR which contain the Vβ chains designated Vβ6.2/3, Vβ6.5, Vβ6.7, Vβ2, Vβ5.1, Vβl3, Vβ7 can be used to immunize an individual having or at risk of having multiple sclerosis to treat or prevent the disease. The immune response generated in the individual can neutralize or kill T cells having the particular Vβ subunit and, thus, prevent or treat the deleterious effects of the Vβ-bearing T cells. Moreover, to the extent that these Vβ subunits are common to T cell receptors on pathogenic T cells mediating autoimmune diseases in general, such vaccines can also be effective in ameliorating such other autoimmune diseases.

The vaccines comprise peptides of varying lengths corresponding to the TCR or portions thereof. The peptides can be produced synthetically or recombinantly, by means well known to those skilled in the art. Preferably, the peptide vaccines correspond to regions of the TCR which distinguish that TCR from other nonpathogenic TCRs. Such specific regions can be located within the various region (s) of the respective TCR polypeptide chains, or spanning the various regions such as a short sequence spanning the V(D)J junction, thus restricting the immune response solely to those T cells bearing this single determinant.

The vaccines are administered to a host exhibiting or at risk of exhibiting an autoimmune response. Definite clinical diagnosis of a particular autoimmune disease warrants the administration of the relevant disease-specific TCR vaccines. Prophylactic applications are warranted in diseases where. the autoimmune mechanisms precede the onset of overt clinical disease. Thus, individuals with familial history of disease and predicted to be at risk by reliable prognostic indicators could be treated prophylactically to interdict autoimmune mechanisms prior to their onset.

TCR vaccines can be administered in many possible formulations, in pharmacologically accepable mediums. In the case of a short peptide, the peptide can be conjugated to a carrier, such as KLH, in order to increase its immunogenicity . The vaccine can be administered in conjunction with an adjuvant, various of which are known to those skilled in the art. After initial immunization with the vaccine, a booster can be provided. The vaccines are administered by conventional methods, in dosages which are sufficient to elicit an immunological response, which can be easily determined by those skilled in the art.

Appropriate peptides to be used for immunization can be determined as follows. Disease- inducing T cell clones reactive with the target antigens are isolated from affected individuals. Such T cells are obtained preferably from the site of active autoaggressive activity such as a lesion in the case of pemphigus vulgaris, central nervous system (CNS) in the case of multiple sclerosis or synovial fluid or tissue in the case of rheumatoid arthritis, or alternatively from blood of affected individuals. The TCR genes from these autoaggressive T cells are then sequenced. Polypeptides corresponding to TCRs or portions thereof that are selectively represented among disease inducing T cells (relative to non-pathogenic T cells) can then be selected as vaccines and made and used as described above.

Alternatively, the vaccines can comprise anti- idiotypic antibodies which are internal images of the peptides described above. Methods of making, selecting and administering such anti-idiotype vaccines are well known in the art. See, for example, Eichmann, et al . , CRC Critical Reviews in Immunology 7:193-227 (1987), which is incorporated herein by reference.

Multiple Sclerosis

T cells causative of multiple sclerosis (MS) have not previously been identified, though MBP-reactive T cells have been proposed to play a role due to the clinical and histologic similarities between MS and EAE. In rat and mouse models of EAE, MBP-reactive, encephalogenic T cells show striking conservation of β- chain VDJ amino acid sequence, despite known differences in MHC restriction and MBP-peptide antigen specificity. This invention is premised on the observation that a human myelin basic protein (MBP) -reactive T cell line, derived from an MS patient, has a TCR β-chain with a VDJ amino acid sequence homologous with that of β-chains from MBP-reactive T cells mediating pathogenesis in experimental allergic encephalomyelitis (EAE) , an animal model of MS. This line is specific for another epitope of MBP. This finding demonstrates the involvement of MBP-reactive T cells in the pathogenesis of MS and demonstrates that TCR peptides similar to those described herein for the prevention of EAE can be appropriate in treating MS. For example, peptides derived from the TCR selected from Vβ6.2/3, Vβ6.5, Vβ6.7, Vβ5.1, Vβ7, Vβl3 or Vβ2 can be used as vaccines to prevent or reduce the severity of MS. Such peptides can be used alone or together in various combinations.

Specifically, the invention provides a method of diagnosing or predicting susceptibility to T cell mediated pathologies in an individual comprising detecting T cells having the β-chain variable regions designated Vβ6.2/3, Vβ6.5, Vβ6.7, Vβ5.1, Vβ7, Vβl3, or Vβ2 in a sample from the individual, the presence of abnormal levels of these Vβ-containing T cells indicating the pathology or susceptibility to the pathology. The Vβ containing T cell can be qualitatively or quantitatively compared to that of normal individuals. Such diagnosis can be performed for example by detecting a portion of the Vβl7 which does not occur on multiple sclerosis associated β-chain variable region T-cell receptors. The Vβl7 can be detected, for example, by contacting the Vβl7 with a detectable ligand capable of specifically binding to Vβl7. Many such detectable ligands are known in the art, e.g. an enzyme linked antibody. Alternatively, nucleotide probes complementary to the Vβ subunit- encoding nucleic acid sequences can be utilized to detect T cells containing the corresponding Vβ subunit, as taught in Examples VIII and IX.

The invention also provides a method of preventing or treating a T cell mediated pathology comprising preventing the attachment of the Vβ subunit containing T-cell receptor to its binding partner. In one embodiment attachment is prevented by binding a ligand to the Vβ subunit. In an alternative embodiment attachment is prevented by binding a ligand to the binding partner . Attachment can be prevented by known methods, e.g. binding an antibody to the subunit or the binding partner to physically block attachment. The invention also provides a method of preventing or treating a T cell mediated pathology in an individual comprising cytotoxicly or cytostaticly treating T-cells containing the particular Vβ subunit in the individual. In one embodiment, the Vβ containing T- cells are treated with a cytotoxic or cytostatic agent which selectively binds Vβl7. The agent can be an antibody attached to a radioactive or chemotherapeutic moiety. Such attachment and effective agents are well known in the art. See, for example, Harlow, E. and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference .

The invention also provides a method of preventing or treating multiple sclerosis in an individual comprising cytotoxicly or cytostaticly treating T cells containing substantially the SGDQGGNE sequence in the individual. In one embodiment, T-cells are treated with a cytotoxic or cytostatic agent which selectively binds the sequence. The agent can be an antibody attached to a radioactive or chemotherapeutic moiety.

Gene Therapy

The present invention further relates to an alternative method of treating or preventing a T cell mediated pathology by gene therapy. In this method, a nucleic acid encoding for a TCR, or an immunogenic fragment thereof, that corresponds to the amino acid sequence of a T cell receptor present on the surface of a T cell mediating a pathology is first inserted into an appropriate delivery system, for example a plasmid. The nucleic acid can be DNA or RNA encoding for TCRs, immunogenic fragments thereof or anti-idiotype antibodies that can be used as vaccines in the present invention. Such DNA or RNA can be isolated by standard methods known in the art. The isolated nucleic acid can then be inserted into a suitable vector by known methods . Such methods are described, for example, in Maniatis et al . , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory 1982) , which is incorporated herein by reference .

The vector is subsequently administered directly into a tissue of an individual. Preferably, the DNA or RNA-containing vector is injected into the skeletal muscle of the individual. For example, a 1.5 cm incision can be made to expose the quadricep muscles of the subject. A 0.1 ml solution containing from 10-100 μg of a DNA or .RNA plasmid and 5-20% sucrose is injected over 1 minute into the exposed quadricep muscles about 0.2 cm deep. The skin is thereafter closed. The amount of DNA or RNA plasmid can range from 10 to 100 μl of hypotonic, isotonic or hypertonic sucrose solutions or sucrose solutions containing 2 mM CaCl₃. The plasmid containing solutions can also be administered over a longer period of time, for example, 20 minutes, by infusion. The in vivo expression of the desired gene can be tested by determining an increased production of the encoded polypeptide by the subject according to methods known in the art or as described, for example, in Wolff et al., Science 247:1465-1468 (1990).

The treated cells will respond to the direct injection of DNA or .RNA by expressing the encoded polypeptide for at about 60 days. Thus, the desired TCR, immunogenic fragment or anti-idiotype antibody can be effectively expressed by the cells of the individual as an alternative to vaccinating with such polypeptides . TCRs which can be used for the treatment of multiple sclerosis include, for example, nucleic acids encoding TCR amino acid sequences corresponding to Vβ6.2/3, Vβ6.5, Vβ6.7, Nβ2 , Vβ5.1, Vβ7 and Vβl3 or any combination thereof .

The present invention also relates to vectors useful in the gene therapy methods and can be prepared by methods known in the art . Compositions containing such vectors and a pharmaceutically acceptable medium are also provided. The pharmaceutically acceptable medium should not contain elements that would degrade the desired nucleic acids.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE I RAT MODEL OF EAE

Female Lewis rats, (Charles River Laboratories, Raleigh-Durham, ΝC) were immunized in each hind foot pad with 50μg of guinea pig myelin basic protein emulsified in complete Freund's adjuvant. The first signs of disease were typically observed 9-11 days post- immunization. Disease severity is scored on a three point scale as follows: 1=1imp tail; 2=hind leg weakness; 3=hind leg paralysis. Following a disease course of approximately four to six days, most rats spontaneously recovered and were refractory to subsequent EAE induction.

EXAMPLE II SELECTION AND PREPARATION OF VACCINES

Vaccinations were conducted with a T cell receptor peptide whose sequence was deduced from the DNA sequence of a T cell receptor beta gene predominating among EAE-inducing T cells of BIO. PL mice. The DNA sequence was that reported by Urban, et al . , . supra, which is incorporated herein by reference. A nine amino acid peptide, having the sequence of the VDJ junction of the TCR beta chain of the mouse, was synthesized by methods known to those skilled in the art. The sequence of this peptide is: SGDAGGGYE. (Amino acids are represented by the conventional single letter codes.) The equivalent sequence in the rat has been reported to be: SSD-SSNTE (Burns et al., J. Exp . Med. 169:27-39 (1989)). The peptide was desalted by Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, NJ) column chromatography in 0.1 M acetic acid and the solvent was subsequently removed by two cycles of lyophilization . A portion of the peptide was conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde at a ratio of 7.5 mgs of peptide per mg of KLH. The resulting conjugate was dialyzed against phosphate buffered saline (PBS) .

EXAMPLE III VACCINATION AGAINST EAE

Vaccines used in these studies consisted of free VDJ peptide and also of VDJ peptide conjugated to KLH. These were dissolved in PBS and were emulsified with equal volumes of either (1) incomplete Freund's adjuvant (IFA) or (2) complete Freund's adjuvant (CFA) made by suspending 10 mg/ml heat killed desiccated Mycobacterium tuberculosis H37ra (Difco Laboratories, Detroit, MI) in IFA. Emulsions were administered to 8-12 week old female Lewis rats in a final volume of 100 microliters per animal (50 μl in each of the hind footpads) . 5μg of unconjugated VDJ peptide were administered per rat. KLH-VDJ conjugate was administered at a dose equivalent to lOμg of KLH per rat. Twenty-nine days later each rat was challenged with 50 μg of guinea pig myelin basic protein in complete Freund's adjuvant in the front footpads . Animals were monitored daily beginning at day 9 for clinical signs of EAE and were scored as described above. The results are presented in Table I. As can be seen, not only was there a reduced incidence of the disease in the vaccinated individuals, but in those which did contract the disease, the severity of the disease was reduced and/or the onset was delayed. The extent of protection varied with the vaccine formulation, those including CFA as the adjuvant demonstrating the greatest degree of protection.

TABLE I

Animal Vaccination Days After Challenge No. (Adjuvant) 10 11 12 13 14 15 16 17 18

1 VDJ (IFA) - - 2 3 3 3 - - -

2 II - - 1 3 3 3 2 - -

3 II - - - 3 3 3 2 - -

4 VDJ (CFA) - - - - 1 1 1 - -

5 II

6 II - - - 1 3 3 3 2 -

7 KLH-VDJ (CFA) - - - 1 3 2 - - -

8 II - - - - 1 1 1 1 -

9 II

10 KLH-VDJ (IFA) - 1 3 3 2 2 1 - -

11 II - - 3 3 3 3 3 2 -

12 II - - 1 3 3 3 3 - -

13 NONE 1 3 3 3 3 1 - - -

14 II - 1 3 3 3 1 - - -

15 II 1 3 3 3 1 — — — —

Scoring : - no signs

1) limp tail

2) hind leg weakness

3) hind leg paralysis EXAMPLE IV Vaccination against EAE with Lewis Rat VDJ peptides

The VDJ peptide used in the previous examples was synthesized according to the sequence of TCR β chain molecules found on EAE-inducing T cells in BIO. PL mice. In addition, peptides were synthesized and tested which correspond to sequences found on encephalitogenic T cells in Lewis rats. These VDJ sequences are homologous with that of BIO. PL mice, but not identical. The rat peptides were synthesized according to the DNA sequences reported by Burns, et al . and Chluba, et al . , Eur . J. Immunol. 19:279-284 (1989) . The sequences of these peptides designated IRl, 2, 3 and 9b are shown below, aligned with the BIO. PL mouse sequence used in Examples I through III (VDJ) .

VDJ S G D A G G Y E

IRl C A S S D - S S N T E V F F G K

IR2 C A S S D - S G N T E V F F G K

IR3 C A S S D - S G N - V L Y F G E G S R IR9b A S S D - S S N T E

The preparation, administration and evaluation of these vaccines were conducted as described in Examples I through III with the following exceptions: 50 μg of the individual VDJ peptides were incorporated into vaccine formulations containing CFA; neither vaccinations in IFA nor vaccinations with peptides conjugated to KLH were conducted. Control animals were untreated prior to MBP challenge as in Example III or were vaccinated with emulsions of PBS and CFA to assess the protective effect of adjuvant alone. The results are shown in Table II below. TABLE II

Animal Vaccination Days After Challenge No. (Adjuvant) 10 11 12 13 14 15 16 17 18

1 None - 1 2 3 3 2 - - 2 II 1 3 3 3 2 - - - 3 11 - 2 3 3 3 1 - - 4 PBS -CFA 1 2 3 3 3 - - - 5 II 1 2 3 3 3 - - - 6 II - 2 3 3 3 - - - 7 IRl (50 μg) - - 2 1 - - - -

IT - - - - 1 3 - -

9 II - - - 1 1 1 1 - 10 IR2 (50μg) - - 1 3 3 3 - - 11 - - - - 2 2 3 3 12 1 13 IR3 (50μg) 1 3 3 3 2 - - - 14 fl — — 2 3 3 — - - 15 16 IR9b (50 μg) 17 18 19 Scoring: - no signs

1) limp tail

2) hind leg weakness

3) hind leg paralysis

As shown in Table II, disease in unvaccinated control animals was observed as early as day 10. Disease was characterized by severe paralysis and wasting, persisted for 4 to 6 days and spontaneously remitted. PBS-CFA vaccinated rats displayed disease courses virtually indistinguishable from those of unvaccinated controls. In contrast, delays in onset were observed in some of the IRl, 2 or 3 vaccinated animals and others showed both delayed onset as well as decreased severity and/or duration of disease. Overall, however, vaccinations with the rat VDJ peptides (IR1-3) were slightly less effective than those with the mouse VDJ peptide (Example III) . Vaccination with IR9b, however, afforded complete protection in all four animals in which it was tested. Importantly, no histologic lesions characteristic of disease were found in any of the four animals vaccinated with IR9b indicating that sub-clinical signs of disease were also abrogated.

EXAMPLE V Vaccination with V region specific peptides

A peptide specific for the Vβ8 gene family was tested as a vaccine against EAE. Vβ8 is the most common β chain gene family used by encephalitogenic T cells in both rats and mice. A peptide was synthesized based on a unique DNA sequence found in the Vβ8 gene, and which is not found among other rat Vβ genes whose sequences were reported by Morris, et al . , Immunogenetics 27:174-179 (1988). The sequence of this Vβ8 peptide, designated IR7, is:

IR7 D M G H G L R L I H Y S Y D V N S T E K

The efficacy of this Vβ8 peptide was tested in the Lewis rat model of EAE (Example I) as described in Examples II and III. 50 μg of peptide were tested in CFA. Vaccinations in IFA or with peptide-KLH conjugates were not conducted. The results of these studies are shown in Table III. TABLE I I I

Animal Vaccination Days After Challenge

No. (Adjuvant) 10 11 12 13 14 15 16 17 1.

IR7 (50 μg) 3 3 1 1

Scoring: - no signs 1) limp tail

2) hind leg weakness

3) hind leg paralysis

The results of vaccinations conducted with the rat Vβ8 peptide are similar to those observed with the mouse and rat IRl, 2 and 3 peptides. Delayed onset as well as decreased severity and duration of disease was observed in one animal. One animal was completely protected.

EXAMPLE VI Vaccination with J region peptides

A peptide was synthesized which corresponds to the J gene segment, TA39, found among both rat and mouse encephalitogenic T cell receptors. The sequence of this peptide, designated IR5, is:

IR5 R F G A G T R L T V K

The efficacy of the JαTA39 peptide was tested in the Lewis rat model of EAE (Example I) as described in Examples II and III. 50 μg of peptide were tested in CFA. Vaccinations in IFA or with peptide-KLH conjugates were not conducted , The results of these studies are shown in Table IV .

TABLE IV

Animal Vaccination Days After Challenge No. (Adjuvant) 10 11 12 13 14 15 16 17 18 19 20

IR5 (50 μg) 1 -

2 3

Scoring: - no signs

1) limp tail

2) hind leg weakness

3) hind leg paralysis

The results of vaccinations conducted with the rat J o; TA39 peptide are more effective than those observed with the mouse VDJ peptide or the Vβ8 peptide. Two of three animals were totally protected and, in the third, disease onset was markedly delayed. Severity was also reduced in this animal though disease persisted for a normal course of 5 days. Importantly, the two animals which were completely protected showed no histologic evidence of T cell infiltration of the CNS . This result indicates that vaccinating with the J_αTA39 very efficiently induces a regulatory response directed at encephalitogenic T cells. Even sub-clinical signs of disease were abrogated.

EXAMPLE VII Vaccination with mixtures of TCR peptides

Vaccinations were conducted with a mixture of

TCR peptides. This mixture contained 50 μg of each of the peptides IRl, 2, 3 and 5 (the three rat VDJ peptides and the rat J TA39 peptide) .

The efficacy of this peptide mixture was tested in the Lewis rat model (Example I) as described in Examples II and III. Peptides were tested in CFA. Vaccinations in IFA or with peptide-KLH conjugates were not conducted. The results of these studies are shown in Table V.

TABLE V

Animal Vaccination Days After Challenge

No. (Adjuvant) 10 11 12 13 14 15 16 17

4 IRl, 2, 3, 5

5 (50 μg each)

Scoring: - no signs

1) limp tail

2) hind leg weakness

3) hind leg paralysis

The results of vaccinations conducted with the rat J TA39 and three VDJ peptides were as effective as those described for IR9b in Table II. All three animals were totally protected. In addition to the absence of any clinical signs of EAE, two of these three animals were completely free of histological evidence of T cell infiltration into the CNS while the third showed only two small foci of lymphocytic infiltration at the base of the spinal cord. EXAMPLE VIII Multiple Sclerosis Vaccine

CSF Cells: Cerebrospinal fluid (CSF) was obtained from 28 patients who were tapped at least once. Twelve were tapped twice and one tapped three times. In addition three pateient ' s CSF cells were cultured and assessed in duplicate. Fifty-150 thousand lymphocytes were recovered from 20 ml CSF; these cells were spun down and resuspended in 200 μl human T cell media (HTC) which consists of RPMI 1640 supplemented with human AB serum (15%), glutamine (2mM) , HEPES buffer (lOmM), 2- mercaptoethanol (0.05mM), and the antibiotics penicillin and streptomycin (each 5iu/ml) . A small aliquot (20 thousand cells) was set aside for flow cytometric analysis following staining with monoclonal antibodies Mabs specific for CD25 (IL-1R) , CD4 and CD8, and irrelevant (control antibodies) directly coupled to fluorescein isothiocyanate (FITC) and phycoerythrin (PE) .

The remaining cells (30-130 thousand) were exposed to washed DYNABEADS (Dynal Inc., Great Neck,

N.Y.) (ratio 1:20) coupled with Mab specific for human CD8 chains. CD8⁺ T cells coupled to these beads stick to the walls of the tube in a magnetic field and CD8^" T cells were recovered by pipetting off the fluid. Generally, this provides a yield of 70-80% (20-100 thousand cells) with greater than 95% depletion of cells bearing the CD8 marker .

The remaining cell population consists, at this point mostly of CD4⁺ T cells, half or more of which are activated and express the CD25 marker (IL-2R) . These activated T cells were expanded in cultures (20-50 thousand cells per well) of HTC medium supplemented with 20% Lymphocult-T-LF (Biotest Diagnostics Corp.; Denville, N.J.) and with recombinant human IL-2 and IL-4 (R & D Systems, Minneapolis, MN) (50 μ/ml) . Cultures were fed twice weekly; generally, after 1 week, cells began to overgrow their cultures. Each well was split into three, and four days later into six, wells. After 10 days to 2 weeks of culture, the initial inoculum generated more than 1 million cells. Flow cytometric analysis of the cultured cells indicated more than 95% were CD4⁺CD8^~CD3⁺TCRαβ⁺. Occasionally a majority of cells were CD4^"CD8^"CD3⁺, a population assumed to be rich in TCRγδ⁺ T cells.

Total cellular RNA was isolated from T cell populations following lysis in guanidinium isothiocyanate and phenol extraction (Chomczynski, P., and N. Sacchi,

"Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction," Analy. Biochem. 162:156 (1987), which is incorporated herein by reference. The RNA (3-5 μg) was first denatured in methyl mercuric hydroxide (lOmM final concentration; Alfa Products, Ward Hill, MA) and then converted to cDNA in Taq ( Thermus aqua ticus) DNA polymerase buffer (Perkins Elmer Cetus; Norwalk, CT) (50mM KC1, lOmM Tris-HCl pH8.3, 2.5mM MgCl₂, and 0.01% gelatin) in the presence of RNasin (20 units, Promega, Madison, WI), β-mercaptoethanol (40mM), dNTPs (0.5mM, Pharmacia), Cβ specific oligonucleotide primer (Cβ-1; lμM) and AMV (Avian myeloblastosis virus) reverse transcriptase (8 units, U.S. Biochemical, Cleveland, OH) in a 25μl reaction for 90 minutes at 42°C. The Cβ oligonucleotide primer, (Cβ- 1), is complementary to a sequence found at the C terminal of human Cβl and Cβ2 mRNA. PCR amplification: cDNA was transferred to a tube containing the following: a Cβ-2 primer (0.6μM) corresponding to a sequence more internal to Cβ-1 used in cDNA synthesis, dNTPs (200μM) and Taq DNA polymerase (23u, Perkin Elmer Cetus, Norwalk, CT) , in Taq polymerase buffer as above except for the presence of 1.5mM MgCl₂. Fiftyμl of this mixture is added to each of 30 individual wells of a microtest III U bottom flexible assay plate (Falcon, 3911; Becton Dickinson and Co., Oxnard, CA) . Each well contained a different oligonucleotide Vβ primer specific for one of the 30 known human Vβ families shown in the accompanying table (0.6μM in lμl) or no Vβ primer as control. The wells were overlain with light mineral oil (Sigma; St. Louis, MO), heated to 94°C for 5 minutes to denature DNA/RNA duplexes and then subjected to 27 amplification cycles of 1 minute at 94°C for melting, 1.5 minutes at 55°C for annealing, and 2 minutes at 72°C for extension in a 96 well thermal cycler (MJ Research, Inc. ; Watertown,MA) .

Quantitation of Vβ expression: Following amplification, 15 μl of the PCR product was denatured for 20 minutes at room temperature by the addition of 15 μl IN NaOH. The samples were then neutralized by the addition of 15μl IN HC1 and 15μl 20X SSC. 15μl of the neutralized samples was spotted onto nitrocellulose filters (BA85, Schleicher & Schuell; Keene, NH) using a Bio-Dot microfiltration apparatus (Bio-Rad Laboratories; Richmond, CA) and then cross-linked to the filter using a UV Stratalinker 1800 according to manufacturer's recommendations (Stratagene; San Diego, CA) . The relative level of amplification in each well was assessed by probing with a gamma ³²P (DuPont; Boston, MA) end- labelled Cβ specific oligonucleotide, Cβ-3, which was further 5' to the Cβ oligonucleotide used in the PCR. Filters were pre-hybridized at 37°C for 1-2 hours in a mixture containing 6X SSC, IX Denhardt ' s solution, 0.5% SDS, 0.05% sodium pyrophosphate, and 100 μg/ml sonicated salmon sperm DNA (Salmon sperm DNA Cat. #1626; Sigma; St. Louis, MO) . The filters were then hybridized with the radiolabeled oligonucleotide Cβ primer overnight at 37°C in a mixture containing 6X SSC, IX Denhardt' s, 0.1% SDS, 0.05% sodium pyrophosphate and 20μg/ml wheat germ tRNA (Type V, Sigma; St. Louis, MO). Following hybridization, the filters were washed twice at 37°C for 30 minutes in 6X SSC containing 0.05% sodium pyrophosphate and one time more at 47°C for 10 minutes. The level of hybridization for each Vβ was measured using an AMBIS radioisotope detector (Ambis; San Diego, CA) . All values are corrected by subtracting counts incorporated into the water blank control well. Relative Vβ expression was calculated by summing all counts detected and dividing this value into the net counts for any given well.

A summary of results of TCR β chain usage among T cells in the CSF of MS patients is presented in Figure 1. As can be seen, Vβ2 was expressed on greater than 70% of the cultured CSF T cells from 1 patient, 50-59% of T cells from another, 20-29% from 4 others, and so forth.

Assuming 30-50 different Vβ members in the repertoire of human T cell receptors, each being represented randomly, the frequency of any one TCR Vβ member would be expected to be approximately 2-3% of the total in a given T cell population. In fact, one rarely finds expression of any particular Vβ chain exceeding 10% in the peripheral blood T cell pool of a normal individual. Figure 1 indicates the disproportionate usage (greater than 20%) of at least 4 different Vβ members among the T cells of CSF from MS patients. In order of most frequent usage (greater than 20%) Vβ6.2/3 or Vβ6.5 are used disproportionately in 13 samples, Vβ2 in 6 samples, Vβ5.1 and Vβl3 in 4 samples each. Thus, among 39 samples, 27 of them show a disproportionate usage of one or more of these 4 different TCR Vβ members.

The predicted amino acid sequences of these Vβ members is shown in Figure 2.

Based on preliminary data gathered over the past year, a peptide of the CDR2 region of Vβ6.5 encompassing amino acids 39-58 was made 39- LeuGlyGlnGlyProGluPheLeuThrTyrPheGlnAsnGluAlaGlnLeuGluLys Ser-58 (LGQGPEFLTYFQNEAQLEKS) . This region is nearly identical to the corresponding sequences found in Vβ6.2/3, Vβ6.8 and Vβ6.9, and differs only slightly from sequences found in Vβ6.4, Vβ6.7, and Vβ6wl. This peptide is effective in provoking an immune response against TCR of most of the Vβ6 family.

Of the 13 samples (from 12 patients) where Vβ6.2/3 or Vβ6.5 was used heavily, sequence studies have been conducted on material from 8 patients to determine (i) the degree of clonality based on homogeneity of sequences in the CDR3 junctional region and (ii) which of the many members of the Vβ6.2/3 and Vβ6.5 family is involved.

DNA Sequencing. To determine the DNA sequence of the expressed Vβ, the PCR reaction was repeated as described above for 30 cycles with Cβ-1 and the Vβ6 specific primer. Following amplification, the resulting PCR products were first made blunt-ended by addition of 5 units T4 DNA polymerase (Pharmacia Fine Chemicals; Piscataway, N.J.) for 15 minutes at 37°C, then extracted with chloroform to remove mineral oil, precipitated with ethanol, and digested with EcoRl according to manufacturer's specifications (New England Biolabs; Beverley, MA); the resulting DNA was separated on a 1.4% agarose gel (Ultra PURE Agarose, GIBCO BRL; Gaithersburg, MD) . The appropriate size product was Isolated using Prep-A-Gene (Bio-Rad; Richmond, CA) , ligated into the Hincll/EcoRl site of pBluescript II (Stratagene; San Diego, CA) , and the ligation mixture was then transformed into the bacterial strain DH5 (available from GIBCO, BRL) . Multiple ampicillin resistant colonies were selected and miniprep DNA was prepared by standard methods (Maniatas, T., E.F. Fritsch, and J. Sambrook, 1982. Molecular cloning, a laboratory manual, Cold

Spring Harbor Laboratory, New York which is incorporated herein by reference) . The plasmid DNA was then sequenced directly by dideoxy chain termination (Sanger, F., S. Nicklen, and A.R. Coulson, "DNA sequencing with chain- terminating inhibitors." Proc . Natl. Acad. Sci . USA. 78:5453 (1977), which is incorporated herein by reference, using the Sequenase sequencing method (U.S. Biochemical, Cleveland, OH) .

These studies established that Vβ utilization is indeed clonal, displaying shared CDR3 sequences on a majority of the PCR amplified material, and that a single Vβ6.2/3 and Vβ6.5 family member dominates. To date, of the 8 patients, 4 use Vβ6.5, 1 uses Vβ6.4, 2 use Vβ6.2, and 1 uses Vβδ.l. These results indicate that the peptide vaccine would be effective for 6 or 7 of the 8 patients . EXAMPLE IX ANALYSIS OF CSF SAMPLES WITH VB7 CLONES

A study was performed on CSF samples from MS patients in which Vβ7 was a dominant clone in 3 out of a total of 5 samples. The protocol used was parallel to that provided in Example VIII with the following modifications. The oligonucleotide primers have different amplification efficiencies and can amplify additional TCR Vβ subfamily members than those described above. In addition, the CSF samples were not depleted of CD8+ cells.

Unfractionated CSF were cultured in single or duplicate microtiter wells for 14-23 days in IL-2/IL-4 and Lymphocult (Biotest Diagnostics Corp.; Denville, N.J.) (hereafter referred to as the "culture") as described above. The T cell subset phenotyping was performed after this culture period and was analyzed as the percentage each of CD4+ and CD8+ T cells. For patients 88 and 94, the CSF samples were split into 2 wells prior to culturing which is noted as well 1 and well 2. It is clear from this that in nearly all CSF and PBL samples, over 50% of the cultured cells were CD4+, which is desirable since it is this population which is of most interest.

In patient 82 a TCR usage was dominated by Vβ7 and 18, which account for approximately 65% of the total signal (as assessed by .Zombis scanning) . This dominant or "restricted" Vβ usage was explored further by sequencing the CDR3 domain, the most variable region of the TCR β chain and one believed to be important in binding antigen. This analysis showed that the dominant Vβ7 of the PCR arose from one cell as assessed by the monoclonal (11/11 clones sequenced were identical) CDR3 domain. Vβl8 was oligoclonal in that 2 cells contributed to the PCR product as seen by 2 discrete CDR3 domains.

Surprisingly, when the material from patient 88 was equally split into 2 wells and treated identically, different dominant clones arose. Well 1 was dominated by Vβl4 while well 2 had dominant Vβ7 and 20. Analysis of the CDR3 domains of several of the clones growing in each well indicated that each was clonal. Restricted Vβ usage in the cultured CSF samples was not true for the patient's PBLs cultured at similar densities (Figure 3). The Vβ profile for the PBLs were more diverse, where more of the signal was contributed by a wider variety of T cells .

The CSF sample from patient 94 was again split into 2 replicate samples prior to culture. Like the previous samples, there is evidence of a restricted Vβ usage by clones expanded by the culture conditions. Both populations were over 75% CD4+. However, like CSF 88, there were different dominant clones growing in each well although Vβ3 was dominant in both wells. Although the CDR3 domains of Vβ3 for each clone are monoclonal, they are discrete rearrangements, indicating that they did not descend from one progenitor. This observation is presently being investigated. Cultured PBLs from patient 94 demonstrated a broad array of TCR Vβ usage. For these samples, different seeding cell densities (40,000 versus 200,000) were found to yield different Vβ profiles.

The cultured CSF and PBLs from patient 95 contained a dominant Vβ7, which accounted for approximately 65% of the total signal. The normal range in blood for Vβ7 is 10-11%. There is clearly a preferential expansion of Vβ7+ CSF cells under the present culture conditions. Although several other Vβ bands can be seen, they account for a relatively small percentage of the total. The PBLs from this patient, like the others, is quite diverse, with no dominance noted. Analysis of the CDR3 domain of Vβ7 from patient 95 demonstrated that a single cell arose during culturing .

The last sample, 101 was actually sorted for

CD4 and HLA DR positivity after culture but prior to PCR analysis. The CSF clearly contained 4-5 dominant Vβs . Interestingly, this was the first PBL sample to show a restricted profile which may be due to sorting the sample after culture.

Although a limited number of MS patients were examined it appears that Vβ7 is overrepresented in the CSF T cell population cultured with IL-2, IL-4, and Lymphocult .

EXAMPLE X

ANALYSIS OF CELLULAR FLORA AND TCR UTILIZATION

To assess the cellular flora present in the CSF of MS patients and to determine whether TCR utilization by CSF T-cells is biased in those patients, the T-cell populations present in the CSF of a group of 77 MS patients were characterized with respect to surface phenotype and state of activiation, TCRβ chain utlitzation, features of the CDR3 jucntional region, the extent of clonality and persistence of selected clonotypes over time. All 77 patients had definite clinical signs of MS (Poser et al., Ann. Neurol. 13:227- 31 (1983) ) and were either relapsing remitting (RR, 40%) and chronic progressive (CP, 60%) . The mean age of patients was 45.2 ± 10.7 years. The mean duration of MS was 12.8 ± 9 years. Patients had an average incapacity status scale (ISS) score of 11.5 ± 6.7 (range 2-20) and an average disability status scale (EDSS) score of 3.6 ± 2.2 (range 1-7). For each patient, 20 ml of CSF was obtained by lumbar puncture with minimal RBC contamination. In this study, no attempt was made to obtain CSF samples based on the stage of disease expression.

For flow cytometry analysis, aliquots of CSF cells (25 X 10³) from 29 different patients were stained, on ice for 30 min with PE conjugated mAb specific for human CD25 (IL-2R) and FITC conjugated mAbs specific for human CD4 or CD8 (PharMingen, San Diego) , then washed twice with PBS containing 1% FCS and 0.02% Na azide. Flow cytometry was performed on a FACScan (Becton Dickinson) or an EPICS profile (Coulter Electronics) .

CSF cells from 47 different patients were depleted of CD8+ cells with anti-CD8 coated magnetic beads (Dynal, Great Neck) according to the supplier's instructions. For culture, CD8-depleted populations were resuspended in 200 μl of T-cell culture medium consisting of RPMI 1640 media supplemented with 15% human AB serum (Gemini Bio-Products, Calabasas) , 2 mM glutamine, 10 mM HEPES buffer, 0.05 mM 2-mercaptoethanol, 50 IU/ml penicillin/streptomycin, 50 U/ml of rIL-2 and rIL-4 (R&D Systems, Minneapolis) and 20% Lymphocult T-Lectin free (Biotest Diagnostics, Denville) . Cultures were conducted in 1 well of a 96 well U-bottom plate and expanded to 6 wells over 10-14 days to generate approximately 10⁶ cells. Over 90% of cultured CSF cells were shown by flow cytometry to be CD3+ CD4+. To analyze TCRVβ expression, total cellular RNA was isolated from cultured cell populations using a kit (RNeasy Total RNA Kit. Qiagen, Chatsworth) according to the manufacturer's instructions. RNA from the equivalent of 0.5 X 10⁶ cells was then converted to cDNA using a Cβ- specific oligonucleotide primer (C&E, see Table VI) as described previously (Gold et al . , J. Exp . Med. 174:1467- 76 (1991); Offner et al . , J. Immunol. 151:506-17 (1993)). The Cβ-E oligonucleotide primer is complimentary to a sequence found at the C terminal of human Cβ-1 and Cβ-2 mRNA.

Table VI Oligonucleotide primers for human TCRVβ genes

Primer 5 '-3'

HuVβl GAGATGCTCCCCTAGGTCTGGA

HuVβ2 TCAGGCCACAACTATGTTTTGGT

HuVβ3 GGAATGTGTCGATATGGACCATG

HUVβ4 GACAGAGCCTGACACTGATCGC

HuVβ5.1 and 5.4 TCAGTTCCTCTTTGAATACTTCAG

HuVβ5.2 and 5.3 CCAGTTTATCTTTCAGTATTATGAG

HuVβ6 CTCAGGTGTGATCCAATTTC

HuVβ7 GGAATGACAAATAAGAAGTCTTTG

HuVβδ.l and 8.2 TTTACTTTAACAACACGTTCCGA

HuVβ8.3 CTTACTTCCGCAACCGGGCTCCTC

HuVβ9 ACAGATGGGAAACGACAAGTCC

HuVβlO ACTTCTGGTCAAAGCAAGTGAAC Table VI (continued) Oligonucleotide primers for human TCRVβ genes

Primer 5'-3'

HuVβll GTTCTCAAACCATGGGCCATGA HuVβl2.1 GGAAGGCAGGTGACCTTGGCGT

HuVβl2.2 CAGGAACACCAGTGACTCTGAGA

HuVβl3.1 -> 13.4 GTGTCACTCAGACCCCAAAATTC

HuVβl3.5 GATCACCCAGGCACCAACATCT

HuVβl4 CAGAACCCAAGATACCTCATCAC HuVβl5 CTGGAATGTTCTCAGACTAAGGGT

HuVβlδ CCACAGCGTAATAGAGAAGGGC

HuVβl7 GAACAGAATTTGAACCACGATGCC

HuVβlδ GCAGCCCAATGAAAGGACACAG

HuVβl9 ACAAAGATGGATTGTACCCCCG HuVβ20 TGTGGAGGGAACATCAAACCCC

HuV21.1 CTGGTTCAATTTCAGGATGAGAGT

HuVβ21.2 GATTCGATATGAGAATGAGGAAGC

HuVβ21.3 TCTGATTCAGTTTCAGAATAACGG

HuVβ22 AAAGAGGGAAACAGCCACTCTG HuVβ23 CGCTGTGTCCCCATCTCTAATC

HuVβ24 CAGTGACCCTGAGTTGTTCTCA

HuCβ-E AGGCAGTATCTGGAGTCATTGA

HuCβ-I GGGCGGGCTGCTCCTTGAGG

HuCβ-P CTCTGCTTCTGATGGCTCAAAC

cDNA was amplified by PCR as a semi-quantitative assessment of levels of TCRVβ chain expression. cDNA (12 μl; 10⁵ cell equivalents) was transferred to a tube containing the following: a Cβ primer (Cβ-I, 0.6 μM) corresponding to a sequence internal to the one used for cDNA synthesis, dNTPs (200 μM) and Taq DNA polymerase (30 U, Perkin Elmer Cetus, Norwalk), in Taq polymerase buffer (Perkin Elmer Cetus). 50 μl of this mixture was added to each of 30 individual v-bottom wells of a Concord-25 microassay plate (MJ Research, Watertown) , each of which contained 2 μl of different Vβ oligonucleotide primers (Table VI) (0.6 μM, final concentration) or no Vβ primer as a control. Wells were overlaid with light mineral oil (Sigma, St. Louis), heated to 94°C for 5 min to denature DNA/RNA duplexes and then subjected to 28 amplification cycles of 1 min at 94°C for melting, 2 min at 55°C for annealing and 2 min at 72°C for extension in a 96 well thermal cycler (MJ Research) .

For quantitation of Vβ expression following PCR amplification, 15 μl of PCR product was denatured for 20 min at room temperature by the addition of 15 μl of 1 N NaOH. Samples were then neutralized by the addition of 15 μl 1 N HC1 and 15 μl 20 X SSC. 15 μl of the neutralized samples was spotted onto nitrocellulose filters (BA85, Schleicher & Schuell, Keene) using a

Bio-Dot microfiltration apparatus (Bi-Rad Laboratories, Richmond) and then cross-linked to the filter with UV light (UV Stratalinker 1800, Stratagene, San Diego) according to manufacturer's recommendations. Relative levels of amplification were assessed by probing with a gamma ³²P (DuPont, Boston) end-labelled Cβ specific oligonucleotide (Cβ-P, Table VI) corresponding to a sequence located 5' to the one used for PCR. Filters were prehybridized at 37°C for 1 h in a mixture containing 6 X SSC, 1 X Denhardt 's solution, 0.5% SDS, 0.05% sodium pyrophosphate and 100 μg/ml sonicated herring sperm DNA (Sigma, St. Louis). The filters were then hybridized with the radiolabeled oligonucleotide Cβ probe overnight at 37°C in a mixture containing 6 X SSC, 1 X Denhardt ' s solution, 0.1% SDS, 0.05% Na pyrophosphate and 20 μg/ml wheat germ tRNA (Type V, Sigma) . Following hybridization, the filters were washed twice at 37°C for 30 min in 6 X SSC containing 0.05% Na sodium pyrophosphate and once at 47°C for 10 min. The level of hybridization for each Vβ was measured using an AMBIS radioisotope detector (Ambis, San Diego) . All values are corrected by subtracting counts incorporated into the water blank control well. Relative Vβ expression was calculated by summing all counts detected and dividing this value into the net counts for any given well.

Oligonucleotide primer efficiencies were compared by cloning each of 18 different Vβ TCR rearrangements into pBluescript (Stratagene, La Jolla) . 1 μg aliquots of each clone were combined and distributed into separate wells, each containing a different Vβ primer and the common Cβ-I primer. PCR was performed and the products quantified as described above. If the primer efficiencies for each of the oligonucleotides were identical, an average signal of 100/18 = 5.55% would be expected. In this study, the observed average was 5.2% (range 2.4-7.7) and the expression level for the Vβ6 primer was 4.1%.

CDR3 nucleotide sequencing of the mRNA samples from patients having high levels of Vβ6 were accomplished by repeating the RT-PCR reaction, as described above, for 30 cycles with a Cβ-specific oligonucleotide primer. This primer, 5 ' -CATAGAAtTcCACTTGGCAGCGGAAGTGGT-3 ' , anneals to human TCR Cβl and Cβ2 mRNA; the bases indicated in small letters denote changes in the Cβ sequence made to create an EcoRl restriction endonuclease site for cloning. Following amplification, the resulting PCR products were first made blunt-ended by addition of 6 units T4 DNA polymerase (New England Biolabs, Beverly) for 15 min at 37°C, extracted with chloroform to remove mineral oil, purified with Prep-A-Gene (Bio-Rad, Richmond) and digested with EcoRl (New England Biolabs, Beverly); the resulting DNA was separated on a 1.4% agarose gel. The appropriate size product was isolated using Prep-A-Gene (Bio-Rad) , ligated into the HincII/JEcoRl site of pBluescript II (Stratagene, San Diego) and the ligation mixture was then transformed into the bacterial strain DH5 (Gibco-BRL, Gaithersburg) . Multiple (10-15 per patient) ampicillin resistant colonies were selected and miniprep DNA was prepared by standard methods (Maniatis et al., Molecular cloning, a Laboratory Mannual, Cold Spring, Harbor, N.Y. (1982)). The plasmid DNA was then sequenced directly by the dideoxy chain termination method (Sanger et al . , Proc. Natl. Acad. Sci. USA 78:5453-57 (1977)) using Sequenase (Amersham, Arlington Heights) .

Table VII summarizes the flow cytometry analysis of surface phenotype of CSF cells from 29 MS patients analyzed and correlates them with the number of cells recovered from 20 ml of CSF, the disease diagnoses, disability and incapacity status scores and IgG synthesis rates. Several findings emerge from these results. First, thirty different taps from 26 of these patients displayed a relatively high percentage of activated IL-2R+ (CD25+) cells in their CSF (39 ± 11%) and high levels of CD4+ (63 ± 15%) and CD8+ (22 ± 8%) cells. Approximately 40% of the CD4+ cells were also IL-2R+ (27 ± 10% of total) , which shows the presence in the CSF of MS patients of a population of activated CD4+ T-cells much larger than is found in the peripheral lymphocyte pool, which is generally only a few percent (Hafler et al., N. Engl. J. Med. 312:1405-11 (1985); Bellamy et al . , Clin. Exp. Immunol. 61:248-56 (1985); Zhang et al . , J.

Ex. Med. 179:973-84 (1994)). In contrast, only about 10% of the CD8+ cells also expressed CD25 (2.3 ± 1.5% of total) . The three other patients showed relatively few (3.3 ± 1.6%) IL-2R+ cells in their CSF; these. also had significantly fewer CD4+ and CD8+ cells (4.4 ± 1.2% and 2.3 ± 1.2%, respectively). The phenotype of the remaining majority of these cells is unknown. Values for the disability and incapacity status scales, IgG synthesis rates and diagnoses did not correlate with CSF cellular recovery levels nor with the grouping of patients with respect to high or low levels of IL-2R+ CSF cells. Cell numbers and phenotypes obtained from the same patient in repeat taps separated in time by as much as a year tended to be similar. These findings are in accord with the notion that activated T-cells infiltrating the CNS participate in the progressive pathogenesis of MS (Waksman and Reynolds, Proc. Soc. Exp. Biol. Med. 175:282-294 (1984); Seboun et al . , Cell. 57:1095-1100 (1989); Oksenberg et al . , Nature 362:68-70 (1993) ) .

To determine whether TCR utilization by CSF T-cells is biased in MS patients, two assumptions were made. Based on the results of Table VII, it was assumed that the CSF cell population of most MS patients contains significant levels of activated CD4+ T-cells and that the CD4+ T-cell subset is the most relevant in the pathogenesis MS. Based on these two assumptions, a dual strategy was employed for enriching and expanding this subset for analysis of TCRVβ expression. CSF populations were first depleted of CD8+ T-cells and then cultured in IL2/IL4 supplemented media to select the subset of activated IL-2R+ CD-4 T-cells. Various T-cell mitogens were not used in expansion cultures since these agents would also stimulate the non-activated lymphocyte by-stander population presumed to be irrelevant to the disease process. Table VII

Cells/20

Sequential Spinal taps

007.3 921116 60 74 20 53 45 2.8 RR 5 9

007.4 930118 81 64 19 53 44 3.8 RR 5 11

026.2 921110 73 44 16 29 20 0.8 RR 2 4

026.3 931118 32 53 23 33 19 1.1 RR 2 4

027.1 920929 40 60 10 50 30 1.8 CP 6 13

027.2 930108 46 75 20 54 43 1.9 CP 6 13

030.1 921110 72 33 21 18 15 5.2 CP 3 7

030.2 930127 60 71 32 41 31 4.0 CP 3 7

ro

014.3 65 61 28 41 35.0 3.0 3 9

015.2 30 68 24 36 20.0 3.7 CP 7 24

016.2 56 62 14 44 30.0 1.5 CP 7 17

018.2 72 24 26 49 13.0 3.3 CP 6 10

021.2 80 46 15 14 8.4 0.7 5 9

Table VII continued

Cells/20 ml CFS

Patient Date (000) CD CD8 (^! CD25 (%) CD 8 CD25 (% ) MS DSS ISS

022.2 32 72 19 44 34.0 2.0 CP 6 11 024.3 16 60 28 36 26.0 5.2 CP 6 22 025.2 100 60 28 29 16.0 1.3 031.1 35 72 11 18 12.0 0.7 CP 3 18 034.1 130 81 15 53 37.0 2.5 CP 7 23

(75 cr ro 035.1 170 69 26 30 19.0 1.3 SP 1 5 co 036.1 160 76 20 44 28.0 1.4 CP 1 2 037.1 130 72 22 52 38.0 1.1 RR 2 13 038.1 1400 72 21 43 31.0 0.9 RR 2 4 041.1 340 74 24 46 32.0 2.4 1 6 042.1 210 75 20 47 31.0 1.1 RR 2 14 ro 043.1 52 28 25 30 15.0 2.3 2 13 044.1 430 54 51 27 20.0 6.0 RR 5 24 045.1 320 82 15 44 32.0 0.9 RR 1 6^" 046.1 210 74 18 36 28.0 3.0 2 12 050.1 450 80 12 54 43.0 1.6 CP 2 13

Table VII continued Phenotype of CSF cells from patients with MS

Cells/20 ml CFS

Patient Date (000) CD$(%) CD8(%) CD25(%) CD8 CD25 (%) MS DSS ISS

Individual spinal taps: low frequency of CD25+cells

009.2 36 5.2 2.7 5.1 4.8 2.2 CP 5 20

028.1 74 3.0 3.2 2.2 1.5 0.6 CP 7 20

033.1 22 5.0 1.0 2.5 1.5 0.1 RR 1 2

σ^*>

This protocol for selecting activated CD4+ T-cells was applied to CSF cell populations from 47 of the 77 MS patients. CD8 depleted CSF cells (30-200 X 10³) were maintained in cytokine supplemented cultures for 10-14 days, a period required to generate approximately 10⁶ cells, the mRNA from which was then analyzed by RT-PCR for expression of the known human TCR β chain families. The results shown in Table VIII indicate a highly biased pattern of TCR β chain expression, in some patients the level of a particular TCR β chain mRNA exceeded 70% of the total Vβ message level. Among the 47 patients, we found numerous examples of TCR β chain gene usage equal to or exceeding 20% of the total β chain message level. A comparison of paired cultures of CD8 depleted peripheral blood lymphocytes (PBL) and CSF cells from 5 patients expressing high levels of CSF Vβ6 mRNA indicated that CSF levels were significantly greater (CSF: mean 36.4%; range 21-72%; PBL: mean 7.81%; range I -14%) (Table IX) . This finding argues against the possibility that high levels of Vβ6 expression noted here is an antifact due to biased survival of such cells in cytokine supplemented cultures .

Table IX focusses on the CSF data in a slightly different way. It identifies 39 of the 47 MS patients (83%) whose TCR β chain profiles are characterized by disproportionate expression of Vβ6 by 20% or more, or of any of the other β chains by 15% or more, a finding similar to the rat EAE model with its marked involvement of Vβ8 T-cells in the disease process. The data in Table X indicate that the most common β chain genes used in a disproportionate manner are the various members of the Vβ6 family (21/47), Vβ2 (9/47), the Vβ5 family (6/47) and Vβ4 (4/47). One or more of these four β chain families Table VIII

Number of samples with indicated %TCR Vβ expression in CD8 depleted IL-2/IL-4 expanded cultures of CFS cells from

MS patients

%

0-9 10-19 20-29 30-39 40-49 50-59 60-69 >70

Vβ

1 45 1

2 31 12 1

3 40 5 1 1

4 39 5 1 2

5.1 41 4 1 1

5.2 44 1 1

6 15 11 13

7 42 4 1

8.1 46 1

8.3 45 2

11 45

12.1 41

Table VIII continued

0- 9 10- 19 20-29 30 -39 40-49 50- 59 60-69 >70

12.2 47

13 42

13.5 47

14 44

15 46

16 47

17 45 1 1

18 44 2 1

19 47

20 45 2

21.1 46 1

21.2 47

21.3 46

22 47

23 44

This table summarizes the mRNA levels of TCRVβ chain expression from one culture taken from each of 47 different patients: of these 47 cultures, 21 revealed various levels of Vβ6 mRNA expression equal to or greater than 20%.

Table IX. A Comparison of Vβ levels in paired CFS and PBL cultures

Patient 007 037 040 041 044

% Vβ6 in CSF 21.0 28.0 23.0 38.0 72.0 % Vβ6 in PBL 1.0 8.7 4.0 13.6 10.8

Table X

TCR β chain gene usage (%) by CD4 + T-cells cultired from CSF of MS patients³

Patient TCR Vβ

5.1 5.2 11 12.1 17 18 20 21.3 23

007 21

013 26

018 46

027 82

037 28

040 23

044 72

049 29

057 49

060 26

072 26

078 26

019 15

026 25 41

048 54 22

050 16

Table X continued

TCR β chain gene usage (%) by CD4 + T-cells cultired from CSF of MS patients³

Patient TCR Vβ

5.1 5.2 11 12.1 17 18 20 21.3 23

065 34 21

076 17 16 24

041 17 38

020 18 23

017 61 17

035 2 17 2

045 1 64 7

012 55

036 71

063 18 39

042 29

025 68

061 64

030 25 20

Table X continued

TCR β chain gene usage (%) by CD4 + T-cells cultired from CSF of MS patients³

Patient TCR Vβ

1 2 3 4 5.1 5.2 6 7 11 12.1 17 18 20 21.3 23 021 43

064 21

055 18 19

080 28 15

014 25

028 79

070 20

005 26 29

043 18

Total 2 9 2 4 3 3 21 2 2 2 2 1 1 1 1

^■Patients indicated displayed a level of TCRVβ6 expression ≥20% of toal Vβ message or for any other TCRVβ member ≥15%.

were disproportionately over expressed in 31/47 (67%) of MS patients. Some of these patients, for example patients 026 and 048, expressed high levels of more than one of these β chain genes simultaneously.

Table XI shows the distribution of the various members of the Vβ6 family. It is derived from sequence analyses of the TCR β6 chains of those 21 patients (see Tables 3 and 5) showing ≥ 20% Vβ6 in their cultured CSF cells. The TCRVβ6 chain family in humans consists of 8 functional members (Rowen, Koop and Hood, personal communication): Vβ6.1, 6.2, 6.4, 6.5, 6.7, 6.8, 6.11, and 6.14. The oligonucleotide primers used for PCR amplification were designed to accommodate each of these members, with the possible exception of Vβδ.ll. .Among the 21 patients with high levels of Vβ gene expression indicated in Table X, we detected 6 of the 8 different members of the Vβ6 family in various frequencies. Of 205 Vβ6 sequence determinations from these 21 patients (approximately 10 per patient), the Vβ6.5 and 6.7 genes were expressed most frequently (each approximately 30% of the time) .

These same sequence data were used to assess utilization frequencies of the various Jβ gene elements (see Table XI). As expected (Grunewald et al . , Int. Immunol. 4:643-50 (1992)), these were distributed approximately 25% Jβl and 75% Jβ2. Jβl.l, 2.1, 2.3 and 2.5 were most frequently used, but Jβl.3 and 1.6 were not seen.

In view of previous findings of the predominant usage of DS (AspSer) as the first two amino acids in the (N)D(N) rearrangement of the CDR3 region of Vβ8.2 by T-cells reactive to MBP 68-86 in the rat EAE model (Gold et al., J. Exp. Med. 174:1467-1476 (1991)), the existence of a biased amino acid distribution was explored in the (N)D(N) region among the most commonly used Vβ6 family members (Vβ6.5 and 6.7) by CSF T-cells in our MS patient population. Of the total of 205 sequence determinations of the various Vβ6 family members (Table XI), 72 and 64 of these were assigned to the Vβ6.5 and 6.7 family members, respectively. Of these, 33 for Vβ6.5 and 39 for Vβ6.7 were different independent rearrangements of the CDR3 region of these two family members. The remainder were a variety of repeat sequences representing expanded clones .

Table XII records the distribution of amino acid residues in the first three (N) D (N) positions of the 72 different CDR3 junctional rearrangements for Vβ6.5 and 6.7. For both these Vβ genes, the most frequent amino acid residues in the first two positions are L and G. This fact might indicate that LG . . . is a dominant motif of this junctional region in T-cells associated with MS. This seems not to be the case, however. A compilation of (N)D(N) sequences for the CDR3 region of Vβ6.5 and Vβ6.7 reveals that LG occurred 7/33 and 7/39 times, respectively. These observed frequencies approximate frequencies to be expected if these two amino acids are used independently of one another. It seems likely that the frequent independent appearance of L and G is due to the fact that the terminal 3' codon, tta, for the Vβ6 family encodes L (Li et al . , J. Exp. Med. 174:1537-47 (1991)) and due to the fact that G can be encoded in each of the three different reading frames of Dβl and Dβ2 (Toyonaga et al . , Proc. Natl. Acad. Sci . USA 82:8624-28 (1985) ) . Sequences through the CDR3 region were also used to determine the degree of clonality of CSF T-cells from the 21 MS patients with disproportionate expression of Vβ6. A total of 45 Vβ6 PCR isolates was prepared from the 21 MS patients. Between 10-15 subclones (ampicillin resistant colonies) were prepared from each isolate and sequenced. The extent of clonal dominance, expressed as a percentage of total colonies in each of the 45 PCR isolates is shown in Table XIII. Nearly two-thirds (29/45) of the sequences from the PCR isolates showed a dominant clone (≥ 50% of sequences) and a single clone (homogeneous sequences) was seen 6/45 times. Overall, among the 21 patients with elevated levels of Vβ6 expression, Vβ6.5 and Vβ6.7 were the Vβ6 family member most frequently detected (Table X) , and dominant clones expressing Vβ6.5 were twice as frequent as those expressing V6.7.

Examples of the clonal dominance in the CSF of 5 MS patients are shown in Table XIV. Each shows a dominant sequence representing a majority of the PCR isolates and a few less frequent sequences. These 5 patients are not representative examples; they are the same 5 that also showed clonal persistence over time (see below) .

Just how long a given T-cell clone that is presumed to be involved in the pathogenesis of MS will persist in the CNS is an important question to consider in any attempt to control this disease by immune regulation of selected clones. Table XV shows a comparison of TCRVβ profiles in 5 patients that underwent repeat spinal taps. While the duration between CSF taps varied from 3-16 months, the TCR β chain profiles were similar over time. This supports the notion that the subpopulation of T-cells that may be involved in the disease process of MS has a significant life span.

More definitive evidence for the longevity of activated T-cell clones found in the CSF of MS patients is shown in Table XVI. Here, in all 5 of these same patients, Vβ6 T-cell clones identified by their CDR3 sequence could be detected over periods of time. For example, patient 019 had a homogeneous clone with the CDR3 sequence SHSRDVK that was still present (5/17 sequences) in CSF 17 months later; a minority clone LTSGGRKD in patient 037 revealed itself as a majority clone over a year later. While the quantitative aspects of clonal duration leave much to be desired in this assessment, there was some evidence indicating clonal waning. A very dominant clone of patient 041 (TKSEGTH) was a minority clone 4 months later; dominant clones in patient 044 (FGE) and patient 057 (PSSG) were minority clones 15-18 months later.

Table XI

Distribution (No.) of Vβ6 family mambers and Jβ elements

Vβ6.1 6.2 6.4 6.5 6.7 6.14 Total (%)

Jβl . l 18 28 (14)

CΛ 1 . 2 8(4)

W 1 . 3 0 Λ

1 . 4 1 3 (1)

1— *

H 1 . 5 4 9(4)

1 . 6 0 Λ

Jβ2.1 13 24 16 1 59(29) 2.2 3 8 11(5) a 2.3 14 12 22 8 56(27) 2.4 2(1) 2.5 16 3 23(11) 2.6 1(0.5) 2.7 5(2)

Total (%) 32 (16) 10(5) 15(7) 72 (35) 64 (31) 12(6) 205

Approximately 10 subclones of PCR amplified Vβ6 cDNA were sequencesd for each of 21 different patients expressing disproportionately high levels of Vβ6 mRNA.

Table XII

Amino acid usage in the first three positions of CDR3

TCRVβ 6.5 6.7

No.

Seq. 33 39

CΛ d w aa pos . 1 2 1 2

CΛ

H A 1 4 2

>-

H R 2 2 5 d

H N 3 W D 1

CΛ

C

Q 1 1

E 1

^•n

G 1 10 12

I H 2

C^*\

I

L 21 19

K 1

M

F

Table XII continued

Amino acid usage in the first three positions of CDR3

TCRVβ 6.5 .7

"1 ϊ-i

Table XIII

Extent of clonal dominance in Vβ6 T-cells from CSF of MS patients

% Expression of most frequent clone (%) <25 25-49 50-74 75-99 100 _j No. PCR isolates 7 9 11 12 6 ₄* Fourty five PCR isolates were prepared from the CSF of 21 MS patients showing disproportionate usage of Λ Vβ6 TCR genes. Ten-15 subclones were prepared from each isolate and sequenced.

Λ

O

*5^" ***.

Table XIV

CDR3 sequences of Vβ6 T-cells in MS CFS: Analysis of clonality

Patient Date ϋVβ6 No. PCR isolates Vβ member (N)D(N) Jβ

019 921211 39 12/12 6.5-CASS SHSRDVK FFGPGTR TVL-2.1 Λ

037 930315 25 13/20 6.1-CASS ROSY TEAFFGQGTR TW- 1.1 Λ 4/20 6.4-CASS LTSGGRKD QPQHFGDGTR SI - 1.5 2/20 6.7-CASS DPGRL STDTQYFGPGTRLTV -2.3 1/20 6.7 CASS LSASGLRG EQFFGPGTRLTV -2.1 Λ

041 940527 38 15/16 6.7-CASS TKSEGTH SNEQFFGPGTR TV -2.1 1/16 6.5 -CASS RGVT EK FFGSGTQ SV - 1.4

044 930219 72 6/12 6-7-CASS FGE NTGELFFGEGSRLTV-2.2 p^" 1/12 6.7-CASS LDWN SYEQYGPGTR TVT-2.7 3/12 6.5-CASS LSLAGQK SNQPQHFGDGTRLSIL- 1.5 2/12 6.5 -CASS VEGN GYTFGSGTRLTW- 1.2

057 930628 49 16/17 6.1-CASS PSSG ETQYFGPGTRLLVL- 2 . 5 1/17 6.4 -CASS RLSL.AD NEQFFGPGTRLTVL- 2 . 1

Table XV

Persistence of Vβ profiles in CD4 - T-cells from MS patients

Patient (date)

019 037 041 044 057

6.0 60 25 25 28 21 18 19 18 49 23 . 7.0 1 1 2 2 1 3 1 1 2 0

8.1 1 3 2 2 0 0 4 4 7 5

8.3 0 0 2 0 0 1 2 0 1 0

9.0 3 6 0 1 0 0 5 2 3 0

10.0 3 1 4 1 0 0 1 2 0 0

11.0 0 1 0 1 0 0 1 1 0 0

12.1 0 3 1 1 12 1 7 6 1 6

Table XV continued

Persistence of Vβ profiles in CD4 - T-cells from MS patients

Patient (date)

019 037 041 044 057

930827 940509 930315 930722 930205 940527 931005 940315 930628 930917

CΛ d β w X) 12.2 0 3 0 2 0 0 2 1 1 0

H

H 13.0 0 4 4 2 4 2 7 4 3 8 d 13.5 0 1 0 1 0 1 1 1 2 0

14.0 0 2 13 11 5 2 6 10 0 1 Λ

15.0 0 1 0 1 0 1 3 2 1 6

16.0 0 4 0 0 0 0 0 7 0 3 cr

17.0 0 4 0 0 2 7 0 3 0 0

C 18.0 0 2 1 2 12 12 1 3 0 5 f^*D

19.0 0 0 0 0 1 0 0 0 0 0

20.0 0 1 0 3 0 2 1 2 1 0

21.1 0 0 0 0 0 0 2 1 4 0

21.2 0 0 0 0 2 0 1 1 0 0

21.3 0 2 1 1 0 2 1 2 0 0

22.0 0 0 0 2 0 3 0 0 1 1

Table XV continued

Persistence of Vβ profiles in CD4 - T-cells from MS patients

Patient (date)

019 037 041 044 057

930827 940509 930315 930722 930205 940527 931005 940315 930628 930917

CΛ d Vβ w

CΛ 23.0 17 14 4 3 0 3 0

H

H 24.0 0 1 0 0 0 0 d

H Λ

σ

"I

5 n.^"

Table XVI

CDR3 Sequences of Vβ6 T-cells in MS CSF: Analysis of clonal persistence

Patient Date irVβδ No. PCR isolates Vβ member (N)D(N) Jβ

019 921211 39 12/12 6.5 -CASS SHSRDVK FFGPGTRLTVL-2.1

041 940304 18 1/8 6.7-CASS TRQGPLL TQYFGPGTRLTVL-2.3

940527 38 15/16 6.7-CASS TKSEGTH SNEQFFGPGTRLTVL- 2.1

940930 18 1/17 6.7-CASS TKSEGTH SNEQFFGPGTRLTVL- 2.1 9/17 6.7-CASS TRQGPLL TQYFGPGTRLTVL-2.3

Table XVI continued

CDR3 Sequences of Vβ6 T-cells in MS CSF: Analysis of clonal persistence

Patient Date %Vβ6 No. PCR isolates Vβ member (N)D(N) Jβ 044 930219 72 6/12 6.7 -CASS FGE NTGELFFGEGSRLTVL-2.2

930820 15 7/8 6.7 -CASS FGE NTGELFFGEGSRLTVL-2.2

CΛ

940513 7 1/16 6.7-CASS QGTREG QPQHFGDGTRLSIL- 1.5

940909 10 1/13 6.7 -CASS QGTREG QPQHFGDGTRLSIL-1.5

1/13 6.7-CASS FGE NTGELFFGEGSRLTVL-2.2

057 930628 49 16/17 6. .1-CASS PSSG ETQYFGPGTRLLVL-2.5 Λ 930917 23 3/12 6 .1-CASS PSSG ETQYFGPGTRLLVL-2.5 940315 16 3/16 6 , .2 -CASS LGVGGVQR TQYFGPGTRLLVL-2.5 s

1/16 .1-CASS PSSG ETQYFGPGTRLLVL-2.5 ^***.

5/16 6. .7 -CASS S GG Ga.πStVLTFGAGSRLTVL-2.6 ^"

940511 30 1/17 6. .7-CASS S GG GANVLTFGAGSRLTVL-2.6 ^*\ 940909 19 2/14 6. .2 -CASS LGVGGVQR TQYFGPGTRLLVL-2.5

1/14 6. .7 -CASS SWGG GANVLTFGAGSRLTVL-2.6

EXAMPLE XI VACCINATION WITH VB6.5 PEPTIDE

Ten of tne 21 MS patient described m Example X that expressed disproportionately high levels of Vβ6 T- cells in their CSF were vaccinated with a CDR2 region peptide of Vββ.5 emulsified in IFA. The purpose of this study was to access toxicity, lmmunogenicity and whether a response to the peptide would be accompanied by alterations in the T-cell flora of the CSF of these patients . This study involved administration of two doses of 100 or 300 μg TCRVβ6.5 peptide vaccine (Table XVII) at weeks 0 and 4 to patients with clinically definite MS. Patients were monitored for safety, cnanges in clinical status, immune responses to the peptide and TCRVβ utilization among CSF cells for 24 weeks.

The 10 patients participating in this study (Table XVIII) all had clinically definite MS for at least a year as defined by Poser criteria (Poser et al . , Ann. Neurol . (1983)) w th diagnoses confirmed by at least two neurologists. All were Caucasian females, age 41. 9 + 12 (25.8-67.6) years; mean disease duration from onset to study entry was 12.8 ± 9.0 (3.7-26.2) years. Six patients were diagnosed relapsing/remitting disease (RR) and 4 chronic/progressive (CP; 1 primary CP and 3 secondary CP) . The baseline EDSS and ISS Kurtzke scores for these 10 patients were 4.5 ± 2.9 ( 1.0-8.0) and 20.7± 12.9 (1-43), respectively. Mean baseline EDSS Kurtzke scores for the 100 and 300 μg vaccination groups were 3.9 and 5.1, respectively. These 10 patients were selected from among those described in Example X on tne basis of their biased usage of TCRVβ6.2 and/or 6.5 genes by CSF T-cells. They also met at least one of tne following 4

S ie 26 criteria: presence of T2 hypeπntensities in MRI scans or the presence of gadolinium enhancement; increased IgG synthesis rates (Ig-GSR m spinal fluid; presence of oligoclonal bands m spinal fluid; and abnormal visual, auditory, or somatosensory-evoked potentials. Criteria for exclusion from this study included: low (< 300 /mm-) CD4 cell counts or CD4/CD8 ratios (< 0.6); clinical laboratory values for hematology, unnalysis, platelet counts or liver enzymes > ± 1.0 S.D. from normal levels; positive serum test for HIV; active systemic infection within one month of entry; pregnancy or lactation or sexually active females opting not to use birth control; ano use of immunosuppressant or lmmunomooulatory drugs during the previous 3 months prior to enrollment.

Table XVII

.Amino acid 39-58 sequences of the TCRVβ6 family members and the V 6.5 TCR peptide

Vβ6.5 39-58 GQGPEFLTYFQNEAQLEKS peptide vaccine

Vβ6.2 39-58 D.. VB6.8 39-58 P

Vβ6.4 39-58 NY...Q...

Vβ6.14 39-58 NY ... P ...

Vββ.l 39-58 I ... GTGAADD .

Vβ6.7 39-58 ....L... I... GNSAPD .. Vββ.ll 39-58 .... S . V ... S . SD .. R...

Pseudogenes

Vβδ.10 39-58 ....L..PI...GKDAA... Y at C23

VB6.12 39-58 ....Q .... S ..D . T . Q ... Deletion at 77 A CDR2 region 20-mer peptide of Vβ6.5 39-58 (IR208) was synthesized as the vaccine m this study (see Table XVII). A fragment of tne TCR β6.5 chain was cnosen since this was the most abundantly expressed β chain of the VB6 subfamily in 21 different MS patients, especially on dominant T-cell clone. Selection of the particular CDR2 fragment of the TCR β6.5 chain was based on analysis of deduced ammo acid sequences of human TCRVβ6 chains by two different methods for predicting T-cell epitopes (Margalit et al, J. Immunol. 138:2213-24 (1987); Rothbaro and Taylor, EMBO J. 7: 93-100 (1988)) and the finding by otners that this same stretch tne rat Vβ8.2 chain is immunogenic and provokes immune resistance to EAE in LEW rats Vandenbark et al . , Nautre 341:541-44 (1989)). The human Vβ6.5 peptide sequence differs by a single ammo acid in Vβ6.2 and Vβ6.8 and 3 ammo acids Vβ6.4 and V6.14 (Rowen, Koop and Hood, personal communication) . An immune response to this single Vβ6.5 39-58 peptide might provoKe an immune response effective against other members of the Vβ6 family.

Table XVIII

Demographic and disease characteristics of the study population

Pt Sex MS¹ EDSS Years HLA

Diagnosed

Drβl DQ l DQβl ø Λ

005 F CP 7 .0 21 1302, 1302 0102, 0102 0604, 0604

019 F CP 7 .5 20 07, 07 0201, 0201 0201, 0201

041 F RR 1 .0 6 0701, 15/16 0102, 0201 ND Λ 044 F RR 2 , .5 10 0301, 1501 0102, 0501 0201, 0602

057 F RR 1 , .5 14 0103, 1305 0501, 0501 0201, 0301

017 F CP 8. .0 24 1301, 1302 0102, 0103 0603, 0604 ^*.

018 F CP 6 a .0 30 0301, 1501 0102, 0501 0201, 0602 T

037 F RR 2 a .0 8 0701, 1501 0102, 0201 0201, 0602 N

048 F RR l a .5 15 0101, 15/16 0101, 0501 0301, 0602

065 F RR 8. .0 26 1302, 1501 0102, 0102 0602, 0604/ϊ

CP = chronic progressive; RR = relapsing remitting.

The TCRVB6 peptide 39-58 was synthesized by Merrifield solid-phase procedures (Merrifield, 1978) on a phenylacetamidomethyl polystyrene (PAM) resin support (Mitchell et al . , J. Org . Chem. 43:2845-52 (1978)) and purified by preparative reverse-phase HPLC (Bachem,

Torrance) . Single dose, 1 ml aliquots of an oil-m-water emulsion were prepared consisting of 100 or 300 μg peptide, 0.9% saline (0.5 ml), light mineral oil (DraKeol 6 VR; 0.45 ml Penrico, Karns¹ and Montamde 80 (0.05 ml Seppic, Paris) .

Patients received 1 ml injections of emulsion l.m. at week 0 and week 4. The first group of 5 patients received the first of two injections of 100 μg Vβ6 vaccine and were closely monitored for 4 weeks. In the absence of indications of toxicity and serious adverse events, they were then given a second injection of 100 μg . In the absence of toxicity this low dose group, the higher dose vaccinations of 300 μg Vβ6 vaccine was given twice l.m. at 0 and 4 weeks to 5 additional individuals.

The polymorphic HLA-DRB, -DQB1 and -DQA1 alleles were typed using nonradioactive sequence specific oligonucleotide ^SSO) prooes to screen polymerase chain reaction (PCR) amplified DNA from peripheral blood lymphocytes as described by Begovich and colleagues

(Begovich et al . , J. Immunol. 148:249-58 (1992); Erlich et al., Eur. J. Immunogenet 18:33-35 (1991)).

Within 1 month prior to vaccination and at weeks 6, 12 and 24 post vaccination, DTH skin responses were assessed 48 h after mtradermal inoculation of 10, 1 or 0 μg of the Vβ6.5 TCR peptide in 0.1 ml saline. Skin reactions were considered positive if any measurable induration accompanied erythema of > 5mm.

At time periods within 1 month prior tc vaccination and at weeks 0, 2, 4, 6, 12 and 24 post vaccination, peripheral blood mononuclear cells ^'PBMC) were isolated from whole blood by density centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway) . Cultures (200 X 10' cells per well, in quadruplicate, in round-bottom microtiter plates) were conducted for 5 days in RPMI medium supplemented with heat inactivated (10 %) human AB serum (Interstate Blood Bank, Chicago) and containing PHA, various dilutions of the TCR peptide, or no peptide, Proliferative responses were assessed with ^JHTdR. Maximal responses were seen with the peptide at a concentration of 100-200 μM. Positive responses were defined as a stimulation index (S.I.) > 3.0.

Patient sera were screened by ELISA for the presence of TCR peptide specific antibodies. 96-well PVC icroplates (Titertech, Becton Dickinson, Sar. Jose) were coated with TCR peptides conjugated to KLH 5 μg\ml in PBS, pH 7.2). Plates were blocked with BSA ■ . k ) in PBS. Patient sera were added in four-fold dilutions starting at 1:10 biotinylated sheep anti-hu Ig .'The Binging Site, San Diego) and Strep-Avidin-HRP (Pierce, Rockford) were used to detect binding of human Ig. As a positive control, an immune serum was prepared ir. LEW rats inoculated with the TCR Vβ6.5 peptide in CFA and boosted 2 weeks later with peptide in IFA; reactions with this serum were visualized with biotinylated sheep anti- rat Ig. After tne first vaccination, patients were monitored for a variety of safety and clinical status variables at 2, 4, 6, 12 and 24 weeks. This included physical examinations and clinical lab tests involving blood counts, a multi-channel chemistry panei, urmaiysis and assessments cf adverse events. Lumbar puncture (LP for assessment cf TCR utilization in CSF T-cells was done at time 0 and at weeKs 6 and 24. As part of this safety panel, gadolinium enhanced MRI scans were conducted before and at 24 weeks post vaccination. Adverse events and Kurtzke disaoility scores were evaluated serially with each clinic visit.

With regard to safety, a total of 35 adverse events were reported by the 10 patients over the 24 weeK study period. A minority (6/35), reported as injection site pain, were classified as having a probable relationship to tne study drug. The majority (24/35) of adverse events reported during the first 8 weeKs of the trial were mild and were deemed to be unrelated to the treatment. Nineteen were reported after the first injection and 5 after the second. No adverse events were classified as severe or very severe, as defined dy tne WHO Adverse Event Classification of Toxicity Grading. No patient discontinued the study due to an adverse event.

No meaningful changes were noted in physical examinations, vital sign measurements or in clinical laboratory values . Disability and incapacity status scores remained stable during the course of the study (see Table XIX and summary Table XX) . Gadolinium enhanced MRI scans indicated no increase m plaque ourden or of inflammatory activity the two vaccination groups . Table XIX

Longitudinal summary of MS patients vaccinated with TCR Vβ6 peptide

Vacc Pt Lp^a EDSS ISS IgGSR^b Cells/ Days^d Post Vβ6

(w) 20ml Cultured Culture (%)

CSF (%) fluid CD₃+ CD3 +

(000) ^{CD4+ CD8+}

100 x 0 00055 0 7 7.. 00 1 199 2 200.. .88 5 500 16 15 15

+6 18 17. .7 70 14 50 18

+24 7. ,0 20 21. .9 60 33 no growth

019 -27 6. 0 25 22 26 ND ND 60

0 7. .5 34 0. .0 33 13 76 1 25

+6 0. .0 43 16 89 3 32

+24 7. ,5 28 Pt refused

LP

041 -56 1. .0 6 6. .2 340 14 62 2 21

0 1. .0 7 2, .7 220 12 31 2 18

+6 3 , .0 245 12 42 1 38

+24 1, .0 6 3 .8 192 13 73 6 18

044 -55 5. .0 24 430 14 50 4 72

-28 5, .0 23 12. .3 48 24 58 2 15

-22 6, .0 28 11 .2 170 14 34 3 19

0 2. .5 22 11 .9 160 10 29 1 18

+6 18 .0 390 10 22 1 7

+24 6 .0 27 28 .9 175 11 56 34 10

057 -38 2. .0 10 3 .2 65 14 78 1 49 -26 10 86 13 46 1 23

0 1 .5 9 1 .6 66 24 48 1 16

+6 2 .4 59 14 88 2 30

+24 9 6 .5 120 12 73 1 19 Table XIX Continued

Longitudinal summary of MS patients vaccinated with TCR Vβ6 peptide

Vacc Pt Lp^» EDSS ISS IgGSR^c Cells/ Days^d Post Vβ6

( ) 20ml Cultured Culture ('■ )

CSF (%) fluid CD3 + CD3 +

(000) CD4 + CD8 +

300 x 2 017 0 8.0 36 0.0 19 20 57 3 61

+ 6 0.0 38 51 no growth

+ 24 8.5 36 0.0 16 32 no growth

018 -22 6.0 10 64 14 70 6 46

0 6.0 21 o.c 24 16 74 2 20

+ 6 0.0 22 18 94 2 3

+ 24 6.0 22 0.0 24 19 92 1 9

037 -56 2.0 18 95 14 91 1 25

-38 2.0 17 82 14 91 0 28

0 2.0 15 1.9 77 14 86 5 16

+ 6 0.9 40 19 88 8 10

+ 24 3.0 25 0.0 57 23 95 5 11

048 -65 1.0 4 6.9 630 16 70 2 22

0 1 0.0 200 14 15 2 22

+ 6 5.6 62 14 74 4 1

+24 2.5 8 3.7 80 14 68 1 6

065 0 8.0 43 0.0 14 39 35 11 21

+ 6 0.0 8 44 no growth

+24 8.0 42 0.0 9 37 no growth

^* Timing (weeks) of serial lumbar punctures: 0 = baseline timing point at trial entry.

^D Normal values for IgGSR: 0-8.8 mg/day. ^z Gadolinium enhanced MRI scans: inflammation and active lesions (+) present or (-) absent.

^: CSF cells were CD8 depleted in cultured in cytokine supplemented expansion cultures for the indicated number of days . Table .XX

Comparison of mean (±SD) EDSS and ISS scores before and after vaccination

Group EDSS ISS (weeks) (weeks)

0 24 24

100 μg 3.9 (3.1) 4.5 ^■.3.2) 18.2 (10.9) 18. C (10.1)

300 μg 5.1 (3.2) 5.6 : 2.8 ) 23.2 (16.7^'. 26.6 {13.2)

A comparison cf several parameters before and after immunization of the group 1 (100 μg X 2) and group 2 (300 μg X 2) patients with the TCRVβ6.5 peptide is shown in Table XIX. Details of post -vaccination changes in the percent Vβ6 T-cells in CSF populations and immunogenicity assessments of the TCR peptide are shown in Tables Q and R. After vaccination with the V66 TCR peptide, all patients showed proliferative resonses to the eptide in culture (Table XXII) . Thus the TCR peptide is clearly immunogenic.

Table .XXI

Qpmparison of % Vβ6 T-cells in CSF of MS patients before and after

Group No . Before vaccination After vaccination Comparison Pts

No . Lp growth mean ; S D ) No. growth in mean % (SD) /} in Vβ6 Lp^d culture" Vβ6

H culture¹' H

100 μg 5 12 12/12 29.3 (19.6) 9 8/9 21.5 (10.9) 1.13 0.27 300 μg 5 9 9/9 29.0 (14.8) 10 6/10 6.6 (4.0) 4.31 0.002 Λ

^■' Total number of lumbar punctures performed on the 5 patients before and after immunization. ^h Number of cytokine supplemented expansion cultures with surviving cells/total number conducted

' P-values determined from 2-tailed Student's t-test. -p"

For the group 1 (100 μg) patients, 2/5 of the patients expressed the DR2 phenotype (DRβl 1501 or 15/16) . Four patients demonstrated positive T-cell proliferative responses on at least 2 occasions to the peptide m culture and erythematous skin reactions, but only 2 patients showed a positive DTH reaction with induration at these time periods (Table XXII) . The cellularity of the CSF, m terms of the number of cells recovered from 20 ml CSF, increased slightly (average increase = 24.8 ± 40.4%, range = -13 to 82, N=4 ) over the 24 week study period (Table XIX) and the levels of Vβ6 gene bias remained about the same 21.5%) i.e. equivalent to prevaccmation levels (29.3%) 'see Table XXI) .

The 5 patients m Group 2 (300 μg) showed the following similarities and differences. Four patients were typed DR2. Three of the 5 patients snowed positive T-cell proliferative responses to the peptide on two or more occasions. All 5 showed erythematous skin reaction, but only 2/5 showed strong DTH skin reactions with induration (Table XXII) . CSF cellularity was slightly reduced (average decrease = 27.6 - 22.5%, range = 0 to 60, N = 5) (Table XIX) and levels of Vβ6 gene usage diminisned markedly. Concerning this last point, CSF cells of 4/5 patients showed a dramatic reduction m Vβ6 gene usage at 6 and at 24 weeks post vaccination (Tables XIX and XXI) . Table XXII

Immunological assessments of MS patients vaccinated with TCR peptide

Vacc Pt PBL T-cell proliferation assays (S.I. . ) DTH skin tests (mm) screen week week week week week week screen week week week

300 x 2 011 0 0 ND 1 f f 0 0 0 0 (++) (++) ++ +

018 0 0 0 0 0 4 + 0 0 + 0 0 p^"

037 0 0 ND +++ 0 + + 0 0 ++ 4 +

048 0 0 ++ 4-4 +++ ++ 0 0 (++) (++) + +

065 0 + 0 0 +++ ND + 0 ++ ND +++

T-cell response assays: S.I.: 0 = <3, 4- = 3-4.9, 4-+ = 5-9.9, 4-++ = >10.

DTH skin test scoring: diameter (mm): 0 = <5, 4- = 5-9.9, ++ = 10-14.9, -f+4- = >15; (bold) positive induration.

In addition to the decrease in biased usage of Vβ6 TCR, another significant finding revealed in this comparison is the failure of CSF cells to survive in cytokine supplemented cultures on 5 occasions for 3 post-vaccination patients, two of them from the high dose group at both post vaccination time points (Tables 0 and XXI) . Of a total of 200 attempts to establish CSF cells in cytokine supplemented expansion cultures from 77 MS patients, including those prior to vaccination, our experience has shown that the only cultures that repeatedly fail to flourish were those from patients with minimal levels of activated, IL-2R bearing T-celis ir. their CSF. Lack of ceil growth in cytokine supplemented cultures implies a significant absence of a subset of activated, IL-2R positive, T-cells among the cells present in the CSF of these vaccinated patients.

Table .XXIII

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ6 vaccination (100 μg x 2)

Pt LP No. Vβ (N)D(N) Jβ

Seq.

005 -ou

-20

0 6/13 6.2 RRGGALAWD 2.3

4/13 6.2 SIRDRET 1.5

1/13 6.2 TLSQGL 1.2

2/13 o . / HRTGGR

4-6 7/17 6.5 LVAGGAG 2.1

1/17 6.5 LARTTS 1.3

1/17 6.2 LTEI 2.5

5/17 6.7 EGR 2.5

1/17 6.7 LPGA 2.1

1/17 6.14 SRRT 2.3

+24 no growth

019 --6600 1122//1122 66..55 S SHHSSRRDDVVKK 2.1

-20 6/10 6.5 SHSRDVK 2.1

2/10 6.5 LVEGN 1.2

2/10 6.2 FTWAGG 1.5

0 5/10 6.5 LAPPGFFG 2.1

3/10 6.2 RASGLAGS 2.1

2/10 6.7 LRG 1.5

+6 11/17 6.7 LGRD 2.7

1/17 6.7 PRQA 2.5

5/17 6.5 SHSRDVK 2.1

4-24 no growth

SUBSTITUTE SHEET ( ruie 26 ) Table .XXIII Continued

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ 6 vaccination ( 100 μg x 2 )

Pt LP No . Vβ (N) D (N) Jβ

Seq .

041 - -6600 5 5//1111 6 6..55 L LAAAATTGGEE 2.3

5/11 6.5 KLAGGSH 2.1

1/11 6.4 SHGVT 2.1

-20

0 1/8 6.5 LPLDQV 1.1

1/8 6.5 NNALEN 2.1

1/8 6.4 LASGH 2.1

1/8 6.4 LAGTNL 2.5

2/8 6.7 SPASGRS 2.7

1/8 6.7 TRQGPLL 2.3

1/8 6.7 LLRGAAH 2.7

4-6 15/16 6.7 TKSEGTH 2.1

1/16 6.7 RGVT 1.4

4-24 9/17 6.7 TRQGPLL 2.3

1/17 6.7 TKSEGTH 2.1

1/17 6.7 PHGGE 2.3

1/17 6.7 PRGE 1.5

4/17 6.5 LVGTSV 2.7

1/17 6.5 FS 2.3 044 - -6600 6 6//1122 6 6..77 F FGGEE 2.2

1/12 6.7 LDWN 2.7

3/12 6.5 LSLAGQK 1.5

2/12 6.5 LVEGN 1.2

-20 7/8 6.7 FGE 2.2 Table XXIII Continued

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ6 vaccination (100 μg x 2 )

Pt LP No. Vβ (N)D(N) Jβ

Seq.

1/8 6.7 PSGN 2.7

5/11 6.7 PGQGWGS 2.1

2/11 6.7 PSIAGG 2.1

2/11 6.5 LVAGTGEH 1.5

1/11 6.5 LVG 2.1

1/11 6.4 LD 2.1 +6 2 2//1166 6 6..77 S SRRGGGGAA 2.3

1/16 6.7 QGTREG 1.5

1/16 6.7 LGE 2.2

1/16 6.7 LGN 1.2

1/16 6.7 LAQG 2.7

2/16 6.5 ITGGA 2.6

1/16 6.5 HVGG 2.5

1/16 6.5 LGQRLA 2.7

1/16 6.5 GGGA 1.2

1/16 6.5 RLPGQGI 2.5

1/16 6.1 LNVGLAGER 2.5

1/16 6.1 FAGTS 2.1

1/16 6.1 RVASG 1.3

1/16 6.4 LAVSS 2.1 4-24 2 2//1133 6 6..77 V VSSPPGGTTTTRRSS 2.1

1/13 6.7 FGE 2.2

1/13 6.7 QGTREG 1.5

1/13 6.7 LAAGREG 2.3

1/13 6.7 LVVGG 2.7

SUB TIT ie 26 Table XXIII Continued

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ6 vaccination (100 μg x 2)

Pt LP No. Vβ (N)D(N) Jβ

Seq.

1/13 6.5 SRSSEDT 2.3

1/13 6.5 SED 2.1

1/13 6.5 SQTEGG 2.3

1/13 6.2 YEGGE 3.7

1/13 6.2 LEALAGTD 2.3

1/13 6.2 LGAGTPLR 1.5

1/13 6.4 5IPGS 1.1

057 -60 1 166//1177 6 6..11 P PSSSSGG 2.5

1/17 6.4 RLSLAD 2.1 -20 3 3//1122 6 6..11 P PSSSSGG 2.5

2/12 6.5 LGDVSG 2.7

1/12 6.5 LGQGRM 1.1

2/12 6.7 LLADGG 2.4

2/12 6.7 PGQGR 2.4

1/12 6.7 LGE 2.2

1/12 6.7 SRGTRK 2.1

4/16 6.5 GV 2.7

3/16 6.2 LGVGGVQR 2.5

1/16 6.1 PSSG 2.5

LREGLDRD 2.1

SWGG 2.6

LPSGQRD 2.5 +6 3 3//1177 6 6..55 L LGG 2.7

2/17 6.5 LGGG 8.5

1/17 6.5 LGGRG 1.1

SUBSTITUT ruie 26 Table .XXIII Continued

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ 6 vaccination (100 μg x 2)

Pt LP No. Vβ (N)D(N) Jβ

Seq.

2/17 6.2 VDQGP 2.7

4/17 6.7 SQGGPIGN 2.5

1/17 6.7 SWGG 2.6

1/17 6.7 PTSGI 2.7

1/17 6.1 LYRG 2.7

2/17 6.1 DLAGG 2.1 4-24 2 2//1144 6 6..22 L LGGVVGGGGVVQQRR 2.5

2/14 6.2 RLKGTDKRT 2.7

1/14 6.2 LTGTGVLSL 1.3

1/14 6.5 SWRGAD 2.7

1/14 6.5 SGVGSGVE 2.5

1/14 6.7 SWGG 2.6

1/14 6.7 LGTK 2.5

1/14 6.7 VTSG 2.2

1/14 6.7 SGTS 2.1

1/14 6.7 KASGGAV 2.1

1/14 6.1 LRRD 2.1

1/14 6.4 LAETGVA 2.2

S Tables S and T present a summary of the clonal behavior of TCRVB6 cells as a consequence of vaccination with tne Vβ6 peptide. A CDR3 sequence establishes the identity of a given clone, and the number of times this sequence appears is related ti its clone size. For group 1 patients, receiving the lower dose of peptide vaccine (Table XXIII), 4/5 showed persistence of one or more clones present oefore vaccination. For example, a dominant clone, 6.5-SHSRDVK, found over a year prior to vaccination m patient 019 was still dominant 6 weeks post vaccination. Two of the patients (044 and 057) snowed an interesting evolution m the clonal pattern of Vβ6 T-cells. Prior to immunization the Vβ6 repertoire in the CSF was oligoclonal, consisted of predominating clonotypes, but after immunization it was more polyclonal; there were fewer dominant clones and numerous different clones of smaller size.

For the 5 group 2 patients that received the higher vaccine dose (Table XXIV) , the data concerning clonality is somewhat meager since no cells could be recovered from 6 week and 24 week expansion cultures from two of the patients and only low levels of Vβ6 cells from two otners were recovered. For the two patients where the data are available, we note that no clone pre-existing prior to vaccination appeared after vaccination .

This study shows that the Vβ6 TCR peptide is lmunogenic and causes minimal discomfort and no toxicity. The peptide also provokes a major change in the CSF cellular flora of MS patients. Table XXIV

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ6 vaccination (300 μg x 2)

Pt LP No. Vβ (N)D(N) Jβ Seq.

005 -60 -20

0 6/10 6.5 STLIGKD 1.2

4/10 6.7 LNTVH 1.1

+ 6 no growth

4-24 no growth

018 -60

-20 6/13 6.5 LGQTS 2.3

3/13 6.7 PRQGS 2.3

2/13 6.7 FGLE 2.3

2/13 6.5 LSA 2.4

0 11/17 6.1 SPGGG 2.1

1/17 6.1 LKGGAE 2.1

2/17 6.7 LAGGPS 2.1

1/17 6.7 LGRD 2.7

1/17 6.7 RQ 1.2

1/17 6.2 LNEGI 1.5

+ 6 No seq 6% Vβ6

4-24 8/13 6.1 LTSGS 2.7

2/13 6.5 LALAV 2.2

1/13 6.5 LDNE 1.5

1/13 6.5 QGLR 2.5

1/13 6.7 LGGRGG 2.7

S Table XXIV Continued

Clonal comparison of CSF T-cells from MS patients before and after TCRVβ6 vaccination (300 μg x 2)

Pt LP No. Vβ (N)D(N) Jβ Seq.

037 -60 13/20 6.1 RDSY 1.1

4/20 6.4 LTSGGRKD 1.5

2/20 6.7 DPGRL 2.3

1/20 6.7 LSASGLRG 2.1

-20 6/12 6.1 SRQGRR 2.3

2/12 6.1 RDSY X . _

3/12 6.7 SRRDFI 2.3

1/12 6.7 DPGRL 2.3

0 9/14 6.4 LTSGGRKD 1.5

2/14 6.5 WDG 2.1

2/14 6.2 LRDGFVS 2.2

1/14 6.1 KRGPGD 1.4

+6 No seq 10% Vβ6

4-24 9/14 6.7 FTSGG 2.5

1/14 6.7 FTSGG 2.1

2/14 6.5 LGCDRD 1.5

1/14 6.5 LAGSSA 1.1

1/14 6.1 RAGGRG 1.7

048 -60 -20

0 3/15 6.7 PGTGGV 2.7

2/15 6.7 LASGS 2.7

2/15 6.1 LTY 2.5

2/15 6.1 RRE 2.6

2/15 6.14 SVVP 2.3 Table .XXIV Continued

Clonal comparison of CSF T-cells from MS patients before and after TCRVββ vaccination (300 μg x 2)

Pt LP No. Vβ (N)D(N) Jβ Seq.

1/15 6.1 LSPTGT 2.2

1/15 6.1 RPGEGG 1.2

1/15 6.5 LVGA 2.5

1/15 6.7 GGG 2.6

4-6 No seq 1% Vβ6

+24 No seq 6% Vβ6

065 -60 -20

0 12/12 6.5 LVTGI 1.1

4-6 no growth

4-24 no growth

Although the invention has been described with regard to present embodiments, the invention is not limited except by the claims.

Claims

WE CLAIM :

1. A vaccine for preventing or treating multiple sclerosis in a mammal, comprising an immunologically effective amount of a T cell receptor peptide, or a fragment thereof, corresponding to the amino acid sequence of a V╬▓6.7 T cell receptor present on the surface of T cells mediating said pathology, and a pharmaceutically acceptable medium.

2. The vaccine of claim 1, wherein said the amino acid sequence is LGQGLEFLIYFQGNSAPDKS or a portion thereof .

3. A vaccine for preventing or treating multiple sclerosis in a mammal, comprising an immunologically effective amount of a T cell receptor peptide, or a fragment thereof, corresponding to at least a portion of the amino acid sequence of more than one type of T cell receptor present on the surface of T cells mediating said pathology and a pharmaceutically acceptable medium, wherein said T cell receptors are selected from V╬▓6.2/3, VB6.5, V╬▓6.7, V╬▓2, VB5.1, V╬▓7 or V╬▓l3 T cell receptors.

4. The vaccine of claim 3, wherein one said T cell receptor is a V╬▓6.7 T cell receptor.

5. The vaccine of claim 4, wherein said amino acid sequence contains LGQGLEFLIYFQGNSAPDKS or a portion thereof .

6. The vaccine of claims 1-5, further comprising an adjuvant.

7. A method of diagnosing or predicting susceptibility to multiple sclerosis in an individual comprising detecting T cells having the ╬▓-chain variable region designated V╬▓6.7 or a fragment thereof in a sample from the individual, the presence of abnormal expression of V╬▓6.7-containing T cells indicating said pathology or susceptibility to said pathology.

8. A method of preventing or treating multiple sclerosis comprising preventing the attachment of a

V╬▓6.7 -containing T cell receptor to its binding partner.

9. A method of preventing or treating multiple sclerosis in an individual comprising cytotoxically or cytostatically treating V╬▓6.7-containing T cells in the individual .

10. A method of preventing or treating multiple sclerosis in an individual, comprising administering to the individual a nucleic acid encoding a T cell receptor or an immunogenic fragment thereof in a form capable of being expressed in said individual .

11. The method of claim 10, wherein said nucleic acid is DNA.

12. The method of claim 10, wherein said nucleic acid is RNA.

13. The method of claim 10, wherein said nucleic acid is administered into a muscle of the individual .

14. The method of claim 10, wherein said immunogenic fragment comprises the amino acid sequence of a variable region of said T cell receptor.

SU ie 26

15. The method of claim 10, wherein said immunogenic fragment comprises the amino acid sequence of a ╬▓-chain V(D)J region of said T cell receptor.

16. The method of claim 10, wherein said immunogenic fragment comprises the amino acid sequence of a CDR2 region of said T cell receptor.

17. The method of claim 10, wherein an amino acid sequence is substantially the amino acid sequence of V╬▓6.2/3, V╬▓6.5, V╬▓6.7, V╬▓2 , V╬▓5.1, V╬▓7 and V╬▓l3 or any combination thereof.

AMENDED CLAIMS

[received by the International Bureau on 22 October 1998 (22.10.98); original claims 10 and 17 amended ; remaining claims unchanged (2 pages)]

7. A method of diagnos.ing^' or predicting susceptibility to multiple sclerosis in an individual comprising detecting T cells having the ╬▓-chain variable region designated V╬▓6.7 or a fragment thereof in a sample from the individual, the presence of abnormal e.xpression of V╬▓6.7-containing T cells indicating said pathology or susceptibility to said pathology.

8. A method of preventing or treating multiple sclerosis comprising preventing the attachment of a V╬▓6.7-containing T cell receptor to its binding partner.

9. A method of preventing or treating multiple sclerosis in an individual comprising cytotoxically or cytostatically treating V╬▓6.7-containing T cells in the individual .

10. A method of preventing or treating multiple sclerosis in an individual, comprising administering to the individual a nucleic acid encoding an amino acid sequence which is substantially the amino acid sequence of a V╬▓6.2/3, V╬▓6.5, V╬▓6.7, V╬▓2, V╬▓5.1, V╬▓7 or V╬▓l3 T cell receptor or an immunogenic fragment thereof in a form capable of being e.xpressed in said individual .

11. The method of claim 10, wherein said nucleic acid is DNA.

12. The method of claim 10, wherein said nucleic acid is UNA.

13. The method of claim 10, wherein said nucleic acid is administered into a muscle of the individual .

14. The method of claim 10, wherein said immunogenic fragment comprises the amino acid sequence of a variable region of said T cell receptor.

AMENDED SHEET (AP.T!C I 1§)

15. The method of claim 10, wherein said immunogenic fragment comprises the amino acid sequence of a ╬▓-chain V(D)J region of said T cell receptor.

16. The method of claim 10, wherein said immunogenic fragment comprises the amino acid sequence of a CDR2 region of said T cell receptor.

17. The method of claim 10, wherein said amino acid sequence is substantially the amino acid sequence of any combination of said V╬▓6.2/3, V╬▓6.5, V╬▓6.7, V╬▓2, V╬▓5.1, V╬▓7 or V╬▓l3 T cell receptors or immunogenic fragments thereof.