WO1999027957A1 - Vaccination et procede de lutte contre la sclerose en plaques au moyen de peptides tcr vbeta specifiques - Google Patents

Vaccination et procede de lutte contre la sclerose en plaques au moyen de peptides tcr vbeta specifiques Download PDF

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Publication number
WO1999027957A1
WO1999027957A1 PCT/US1997/023147 US9723147W WO9927957A1 WO 1999027957 A1 WO1999027957 A1 WO 1999027957A1 US 9723147 W US9723147 W US 9723147W WO 9927957 A1 WO9927957 A1 WO 9927957A1
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cells
individual
amino acid
cell receptor
acid sequence
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PCT/US1997/023147
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English (en)
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Steven W. Brostoff
Darcy B. Wilson
Lawrence R. Smith
Daniel P. Gold
Dennis J. Carlo
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The Immune Response Corporation
Sidney Kimmel Cancer Center
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Priority to PCT/US1997/023147 priority Critical patent/WO1999027957A1/fr
Priority to AU55281/98A priority patent/AU5528198A/en
Publication of WO1999027957A1 publication Critical patent/WO1999027957A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to the immune system and, more specifically, to methods of modifying pathological immune responses.
  • lymphocytes a substance which protects them against invasion by potentially deleterious substances or microorganisms.
  • an antigen enters the body, and is recognized as foreign
  • the immune system mounts both an antibody-mediated response and a cell-mediated response.
  • B lymphocytes or B cells
  • B lymphocytes produce antibodies which specifically recognize and bind to the foreign substance.
  • T lymphocytes T cells, both effect and regulate the cell-mediated response resulting eventually in the elimination of the antigen.
  • 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.
  • T cells as the primary regulators of the immune system, directly or indirectly effect such autoimmune pathologies.
  • autoimmune diseases 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.
  • lymphocytes replicate inappropriately and without control. Such replication results in a cancerous condition known as a ly phoma.
  • the tumors are termed T cell lymphomas .
  • T cell lymphomas are difficult to treat effectively.
  • 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.
  • 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 ⁇ -chain variable regions of the T cell receptor, designated V ⁇ 6.2/3, V ⁇ 6.5, V ⁇ 6.7, V ⁇ 2, V ⁇ 5.1, V ⁇ l3 and V ⁇ 7, which are central to the pathogenesis of multiple sclerosis (MS) . Also provided are means to detect, prevent and treat MS.
  • MS multiple sclerosis
  • Figure 1 shows the frequency of the various TCR V ⁇ genes expressed in the cultures from CSF of MS patients.
  • Figure 2 shows the sequences of the V ⁇ gene family that are most frequently expressed in the cultures from CSF of the MS patients.
  • Figure 3 shows a summary of the V ⁇ 7 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • MHC Major Histocompatibility Antigen
  • ligand means any molecule that reacts to form a complex with another molecule.
  • selective binds means that a molecule binds to one type of molecule but not substantially to other types of molecules .
  • selective binding indicates binding to V ⁇ l7 containing TCRs but not substantially to other TCRs which lack V ⁇ l7.
  • the immune system is the primary biological defense of the host (self) against potentially pernicious agents (non-self) .
  • 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.
  • antigens 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.
  • CD4+ T Cells 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.
  • TCR T cell receptor
  • 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.
  • V variable
  • J junction
  • C constant
  • Beta and delta chains contain an additional element termed the diversity (D) gene segment.
  • 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.
  • 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.
  • 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.
  • EAE experimental allergic encephalomyelitis
  • MBP myelin basic protein
  • these T cell clones 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.
  • CD8+ T cells with suppressive activity for EAE-inducing T cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • T cell clones 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.
  • 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 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.
  • 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.
  • 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.
  • the host's own immune system is mobilized to suppress the autoaggressive T cells.
  • 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.
  • 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.
  • V ⁇ 6 TCR subunits were sequenced from 8 patients. From these 8 patients three-quarters (6 of 8) were identified as members of the V ⁇ 6.2/3 and V ⁇ 6.5 subfamily shown in Figure 2. These two subfamilies of the V ⁇ 6 gene family show considerable homology in the CDR2 region between residues 39 and 58. It appears that these two particular members of the V ⁇ 6 family are particularly associated with multiple sclerosis.
  • V ⁇ 6.7 also appears to be particularly associated with multiple sclerosis.
  • CSF cells were analyzed for V ⁇ expression.
  • elevated levels of V ⁇ 6 expression were observed in CD4+ T-cells.
  • the V ⁇ 6 family members most frequently detected were
  • V ⁇ 6.5 and V ⁇ 6.7 As shown in Figure 2, the CDR2 region sequences, encompassing amino acids 39-58, are very similar between these two V ⁇ family members. .
  • T cell receptors whole T cells or fragments of the TCR which contain the V ⁇ chains designated V ⁇ 6.2/3, V ⁇ 6.5, V ⁇ 6.7, V ⁇ 2, V ⁇ 5.1, V ⁇ l3, V ⁇ 7 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 V ⁇ subunit and, thus, prevent or treat the deleterious effects of the V ⁇ -bearing T cells.
  • these V ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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 rheuma
  • 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.
  • MBP-reactive T cells have been proposed to play a role due to the clinical and histologic similarities between MS and EAE.
  • MBP-reactive, encephalogenic T cells show striking conservation of ⁇ - 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 ⁇ -chain with a VDJ amino acid sequence homologous with that of ⁇ -chains from MBP-reactive T cells mediating pathogenesis in experimental allergic encephalomyelitis (EAE) , an animal model of MS.
  • EAE allergic encephalomyelitis
  • peptides derived from the TCR selected from V ⁇ 6.2/3, V ⁇ 6.5, V ⁇ 6.7, V ⁇ 5.1, V ⁇ 7, V ⁇ l3 or V ⁇ 2 can be used as vaccines to prevent or reduce the severity of MS.
  • Such peptides can be used alone or together in various combinations.
  • the invention provides a method of diagnosing or predicting susceptibility to T cell mediated pathologies in an individual comprising detecting T cells having the ⁇ -chain variable regions designated V ⁇ 6.2/3, V ⁇ 6.5, V ⁇ 6.7, V ⁇ 5.1, V ⁇ 7, V ⁇ l3, or V ⁇ 2 in a sample from the individual, the presence of abnormal levels of these V ⁇ -containing T cells indicating the pathology or susceptibility to the pathology.
  • the V ⁇ containing T cell can be qualitatively or quantitatively compared to that of normal individuals.
  • diagnosis can be performed for example by detecting a portion of the V ⁇ l7 which does not occur on multiple sclerosis associated ⁇ -chain variable region T-cell receptors.
  • the V ⁇ l7 can be detected, for example, by contacting the V ⁇ l7 with a detectable ligand capable of specifically binding to V ⁇ l7.
  • detectable ligands are known in the art, e.g. an enzyme linked antibody.
  • nucleotide probes complementary to the V ⁇ subunit- encoding nucleic acid sequences can be utilized to detect T cells containing the corresponding V ⁇ 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 V ⁇ subunit containing T-cell receptor to its binding partner.
  • attachment is prevented by binding a ligand to the V ⁇ subunit.
  • 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 V ⁇ subunit in the individual.
  • the V ⁇ containing T- cells are treated with a cytotoxic or cytostatic agent which selectively binds V ⁇ l7.
  • 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.
  • 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.
  • the present invention further relates to an alternative method of treating or preventing a T cell mediated pathology by gene therapy.
  • 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.
  • 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.
  • the DNA or RNA-containing vector is injected into the skeletal muscle of the individual.
  • 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 ⁇ g 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 ⁇ l of hypotonic, isotonic or hypertonic sucrose solutions or sucrose solutions containing 2 mM CaCl 3 .
  • 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).
  • TCRs which can be used for the treatment of multiple sclerosis include, for example, nucleic acids encoding TCR amino acid sequences corresponding to V ⁇ 6.2/3, V ⁇ 6.5, V ⁇ 6.7, N ⁇ 2 , V ⁇ 5.1, V ⁇ 7 and V ⁇ l3 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.
  • 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.
  • 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.
  • 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.
  • KLH keyhole limpet hemocyanin
  • PBS phosphate buffered saline
  • 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 ⁇ l in each of the hind footpads) . 5 ⁇ g of unconjugated VDJ peptide were administered per rat.
  • IFA incomplete Freund's adjuvant
  • CFA complete Freund's adjuvant
  • KLH-VDJ conjugate was administered at a dose equivalent to lO ⁇ g of KLH per rat. Twenty-nine days later each rat was challenged with 50 ⁇ g 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.
  • VDJ peptide used in the previous examples was synthesized according to the sequence of TCR ⁇ chain molecules found on EAE-inducing T cells in BIO. PL mice.
  • 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) .
  • V ⁇ 8 is the most common ⁇ 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 V ⁇ 8 gene, and which is not found among other rat V ⁇ genes whose sequences were reported by Morris, et al . , Immunogenetics 27:174-179 (1988).
  • the sequence of this V ⁇ 8 peptide, designated IR7, is:
  • the results of vaccinations conducted with the rat V ⁇ 8 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.
  • IR5 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:
  • TCR peptides This mixture contained 50 ⁇ g of each of the peptides IRl, 2, 3 and 5 (the three rat VDJ peptides and the rat J TA39 peptide) .
  • 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 ⁇ l 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) .
  • HTC human T cell media
  • 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) .
  • IL-2R CD25 marker
  • 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 ⁇ /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.
  • RNA 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 ⁇ g) 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 MgCl 2 , and 0.01% gelatin) in the presence of RNasin (20 units, Promega, Madison, WI), ⁇ -mercaptoethanol (40mM), dNTPs (0.5mM, Pharmacia), C ⁇ specific oligonucleotide primer (C ⁇ -1; l ⁇ M) and AMV (Avian myeloblast
  • C ⁇ oligonucleotide primer (C ⁇ - 1)
  • C ⁇ -2 primer 0.6 ⁇ M
  • dNTPs 200 ⁇ M
  • Taq DNA polymerase 23u, Perkin Elmer Cetus, Norwalk, CT
  • V ⁇ expression Following amplification, 15 ⁇ l of the PCR product was denatured for 20 minutes at room temperature by the addition of 15 ⁇ l IN NaOH. The samples were then neutralized by the addition of 15 ⁇ l IN HC1 and 15 ⁇ l 20X SSC. 15 ⁇ l 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) .
  • nitrocellulose filters BA85, Schleicher & Schuell; Keene, NH
  • Bio-Dot microfiltration apparatus Bio-Rad Laboratories; Richmond, CA
  • the relative level of amplification in each well was assessed by probing with a gamma 32 P (DuPont; Boston, MA) end- labelled C ⁇ specific oligonucleotide, C ⁇ -3, which was further 5' to the C ⁇ 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 ⁇ g/ml sonicated salmon sperm DNA (Salmon sperm DNA Cat. #1626; Sigma; St. Louis, MO) .
  • the filters were then hybridized with the radiolabeled oligonucleotide C ⁇ primer overnight at 37°C in a mixture containing 6X SSC, IX Denhardt' s, 0.1% SDS, 0.05% sodium pyrophosphate and 20 ⁇ g/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 V ⁇ 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 V ⁇ expression was calculated by summing all counts detected and dividing this value into the net counts for any given well.
  • AMBIS radioisotope detector Ambis; San Diego, CA
  • Figure 1 indicates the disproportionate usage (greater than 20%) of at least 4 different V ⁇ members among the T cells of CSF from MS patients. In order of most frequent usage (greater than 20%) V ⁇ 6.2/3 or V ⁇ 6.5 are used disproportionately in 13 samples, V ⁇ 2 in 6 samples, V ⁇ 5.1 and V ⁇ l3 in 4 samples each. Thus, among 39 samples, 27 of them show a disproportionate usage of one or more of these 4 different TCR V ⁇ members.
  • a peptide of the CDR2 region of V ⁇ 6.5 encompassing amino acids 39-58 was made 39- LeuGlyGlnGlyProGluPheLeuThrTyrPheGlnAsnGluAlaGlnLeuGluLys Ser-58 (LGQGPEFLTYFQNEAQLEKS) .
  • This region is nearly identical to the corresponding sequences found in V ⁇ 6.2/3, V ⁇ 6.8 and V ⁇ 6.9, and differs only slightly from sequences found in V ⁇ 6.4, V ⁇ 6.7, and V ⁇ 6wl. This peptide is effective in provoking an immune response against TCR of most of the V ⁇ 6 family.
  • 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
  • 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.
  • 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.
  • V ⁇ 7 and 18 account for approximately 65% of the total signal (as assessed by .Zombis scanning) .
  • This dominant or "restricted" V ⁇ usage was explored further by sequencing the CDR3 domain, the most variable region of the TCR ⁇ chain and one believed to be important in binding antigen. This analysis showed that the dominant V ⁇ 7 of the PCR arose from one cell as assessed by the monoclonal (11/11 clones sequenced were identical) CDR3 domain. V ⁇ l8 was oligoclonal in that 2 cells contributed to the PCR product as seen by 2 discrete CDR3 domains.
  • 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 V ⁇ 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 V ⁇ 3 was dominant in both wells. Although the CDR3 domains of V ⁇ 3 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 V ⁇ usage. For these samples, different seeding cell densities (40,000 versus 200,000) were found to yield different V ⁇ profiles.
  • the cultured CSF and PBLs from patient 95 contained a dominant V ⁇ 7, which accounted for approximately 65% of the total signal.
  • the normal range in blood for V ⁇ 7 is 10-11%.
  • V ⁇ 7+ CSF cells under the present culture conditions.
  • the PBLs from this patient like the others, is quite diverse, with no dominance noted.
  • Analysis of the CDR3 domain of V ⁇ 7 from patient 95 demonstrated that a single cell arose during culturing .
  • CD4 and HLA DR positivity after culture but prior to PCR analysis The CSF clearly contained 4-5 dominant V ⁇ s . Interestingly, this was the first PBL sample to show a restricted profile which may be due to sorting the sample after culture.
  • V ⁇ 7 is overrepresented in the CSF T cell population cultured with IL-2, IL-4, and Lymphocult .
  • 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, TCR ⁇ 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).
  • ISS incapacity status scale
  • EDSS average disability status scale
  • CD8-depleted populations were resuspended in 200 ⁇ l 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) .
  • RNA from the equivalent of 0.5 X 10 6 cells was then converted to cDNA using a C ⁇ - 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 C ⁇ -E oligonucleotide primer is complimentary to a sequence found at the C terminal of human C ⁇ -1 and C ⁇ -2 mRNA.
  • HuV ⁇ l4 CAGAACCCAAGATACCTCATCAC HuV ⁇ l5 CTGGAATGTTCTCAGACTAAGGGT
  • HuV ⁇ l9 ACAAAGATGGATTGTACCCCCG HuV ⁇ 20 TGTGGAGGGAACATCAAACCCC
  • HuV ⁇ 22 AAAGAGGGAAACAGCCACTCTG HuV ⁇ 23 CGCTGTGTCCCCATCTCTAATC
  • cDNA was amplified by PCR as a semi-quantitative assessment of levels of TCRV ⁇ chain expression.
  • cDNA (12 ⁇ l; 10 5 cell equivalents) was transferred to a tube containing the following: a C ⁇ primer (C ⁇ -I, 0.6 ⁇ M) corresponding to a sequence internal to the one used for cDNA synthesis, dNTPs (200 ⁇ M) and Taq DNA polymerase (30 U, Perkin Elmer Cetus, Norwalk), in Taq polymerase buffer (Perkin Elmer Cetus).
  • V ⁇ oligonucleotide primers Table VI (0.6 ⁇ M, final concentration) or no V ⁇ 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) .
  • 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 ⁇ g/ml sonicated herring sperm DNA (Sigma, St. Louis). The filters were then hybridized with the radiolabeled oligonucleotide C ⁇ 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 ⁇ g/ml wheat germ tRNA (Type V, Sigma) .
  • 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 V ⁇ 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 V ⁇ expression was calculated by summing all counts detected and dividing this value into the net counts for any given well.
  • CDR3 nucleotide sequencing of the mRNA samples from patients having high levels of V ⁇ 6 were accomplished by repeating the RT-PCR reaction, as described above, for 30 cycles with a C ⁇ -specific oligonucleotide primer.
  • This primer 5 ' -CATAGAAtTcCACTTGGCAGCGGAAGTGGT-3 ' , anneals to human TCR C ⁇ l and C ⁇ 2 mRNA; the bases indicated in small letters denote changes in the C ⁇ sequence made to create an EcoRl restriction endonuclease site for cloning.
  • 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) .
  • 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.
  • CD4+ 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.
  • 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 10 3 ) were maintained in cytokine supplemented cultures for 10-14 days, a period required to generate approximately 10 6 cells, the mRNA from which was then analyzed by RT-PCR for expression of the known human TCR ⁇ chain families.
  • the results shown in Table VIII indicate a highly biased pattern of TCR ⁇ chain expression, in some patients the level of a particular TCR ⁇ chain mRNA exceeded 70% of the total V ⁇ message level.
  • TCR ⁇ chain gene usage equal to or exceeding 20% of the total ⁇ chain message level.
  • Table IX focusses on the CSF data in a slightly different way. It identifies 39 of the 47 MS patients (83%) whose TCR ⁇ chain profiles are characterized by disproportionate expression of V ⁇ 6 by 20% or more, or of any of the other ⁇ chains by 15% or more, a finding similar to the rat EAE model with its marked involvement of V ⁇ 8 T-cells in the disease process.
  • the data in Table X indicate that the most common ⁇ chain genes used in a disproportionate manner are the various members of the V ⁇ 6 family (21/47), V ⁇ 2 (9/47), the V ⁇ 5 family (6/47) and V ⁇ 4 (4/47).
  • Table VIII One or more of these four ⁇ chain families Table VIII
  • This table summarizes the mRNA levels of TCRV ⁇ chain expression from one culture taken from each of 47 different patients: of these 47 cultures, 21 revealed various levels of V ⁇ 6 mRNA expression equal to or greater than 20%.
  • Table XI shows the distribution of the various members of the V ⁇ 6 family. It is derived from sequence analyses of the TCR ⁇ 6 chains of those 21 patients (see Tables 3 and 5) showing ⁇ 20% V ⁇ 6 in their cultured CSF cells.
  • the TCRV ⁇ 6 chain family in humans consists of 8 functional members (Rowen, Koop and Hood, personal communication): V ⁇ 6.1, 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 V ⁇ .ll. .
  • Table X shows that the 21 patients with high levels of V ⁇ gene expression indicated in Table X.
  • 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 V ⁇ 6.5 and 6.7.
  • 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 V ⁇ 6.5 and V ⁇ 6.7 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.
  • V ⁇ 6 PCR isolates A total of 45 V ⁇ 6 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 V ⁇ 6 expression, V ⁇ 6.5 and V ⁇ 6.7 were the V ⁇ 6 family member most frequently detected (Table X) , and dominant clones expressing V ⁇ 6.5 were twice as frequent as those expressing V6.7.
  • Table XIV 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) .
  • Table XV shows a comparison of TCRV ⁇ profiles in 5 patients that underwent repeat spinal taps. While the duration between CSF taps varied from 3-16 months, the TCR ⁇ 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.
  • V ⁇ 6 cDNA Approximately 10 subclones of PCR amplified V ⁇ 6 cDNA were sequencesd for each of 21 different patients expressing disproportionately high levels of V ⁇ 6 mRNA.
  • Example X that expressed disproportionately high levels of V ⁇ 6 T- cells in their CSF were vaccinated with a CDR2 region peptide of V ⁇ .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 ⁇ g TCRV ⁇ 6.5 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 TCRV ⁇ utilization among CSF cells for 24 weeks.
  • 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 ⁇ g 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 TCRV ⁇ 6.2 and/or 6.5 genes by CSF T-cells. They also met at least one of tne following 4
  • V ⁇ 6.5 peptide sequence differs by a single ammo acid in V ⁇ 6.2 and V ⁇ 6.8 and 3 ammo acids V ⁇ 6.4 and V6.14 (Rowen, Koop and Hood, personal communication) .
  • An immune response to this single V ⁇ 6.5 39-58 peptide might provoKe an immune response effective against other members of the V ⁇ 6 family.
  • 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,
  • the first group of 5 patients received the first of two injections of 100 ⁇ g V ⁇ 6 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 ⁇ g . In the absence of toxicity this low dose group, the higher dose vaccinations of 300 ⁇ g V ⁇ 6 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
  • DTH skin responses were assessed 48 h after mtradermal inoculation of 10, 1 or 0 ⁇ g of the V ⁇ 6.5 TCR peptide in 0.1 ml saline. Skin reactions were considered positive if any measurable induration accompanied erythema of > 5mm.
  • peripheral blood mononuclear cells ' PBMC 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 J HTdR. Maximal responses were seen with the peptide at a concentration of 100-200 ⁇ M. Positive responses were defined as a stimulation index (S.I.) > 3.0.
  • TCR peptide specific antibodies 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 ⁇ g ⁇ 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.
  • an immune serum was prepared ir.
  • Timing (weeks) of serial lumbar punctures: 0 baseline timing point at trial entry.
  • CSF cells were CD8 depleted in cultured in cytokine supplemented expansion cultures for the indicated number of days .
  • Table .XX
  • Table XIX A comparison cf several parameters before and after immunization of the group 1 (100 ⁇ g X 2) and group 2 (300 ⁇ g X 2) patients with the TCRV ⁇ 6.5 peptide is shown in Table XIX. Details of post -vaccination changes in the percent V ⁇ 6 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.
  • Vacc Pt PBL T-cell proliferation assays S.I. . )
  • DTH skin tests mm
  • Tables S and T present a summary of the clonal behavior of TCRVB6 cells as a consequence of vaccination with tne V ⁇ 6 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.
  • 4/5 showed persistence of one or more clones present oefore vaccination.
  • a dominant clone, 6.5-SHSRDVK found over a year prior to vaccination m patient 019 was still dominant 6 weeks post vaccination.
  • V ⁇ 6 T-cells Two of the patients (044 and 057) snowed an interesting evolution m the clonal pattern of V ⁇ 6 T-cells.
  • the V ⁇ 6 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.

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Abstract

L'invention concerne des vaccins et un moyen de vaccination d'un mammifère de manière à prévenir ou lutter contre des pathologies spécifiques induites par les lymphocytes T ou à traiter la reproduction non régulée des lymphocytes T. Le vaccin est composé d'un récepteur des lymphocytes T (TCR) ou d'un fragment de celui-ci correspondant à un TCR présent sur la surface des lymphocytes T induisant la pathologie. Le fragment servant de vaccin peut être un peptide correspondant à des séquences de TCR caractéristiques des lymphocytes T induisant ladite pathologie. Le vaccin est administré au mammifère de manière à qu'il induise une réponse immunologiquement efficace pour enrayer l'évolution de la maladie. L'invention fournit en outre des régions variables spécifiques de chaîne β du récepteur des lymphocytes T, appelées Vβ6.2/3, Vβ6.5, Vβ6.7, Vβ2, Vβ5.1, Vβ13 et Vβ7 qui sont au coeur de la pathogenèse de la sclérose en plaques (MS).
PCT/US1997/023147 1997-12-03 1997-12-03 Vaccination et procede de lutte contre la sclerose en plaques au moyen de peptides tcr vbeta specifiques WO1999027957A1 (fr)

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