EP0568623A1 - Vaccination et procedes de lutte contre des maladies causees par des reactions pathogenes de populations de lymphocytes t - Google Patents

Vaccination et procedes de lutte contre des maladies causees par des reactions pathogenes de populations de lymphocytes t

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Publication number
EP0568623A1
EP0568623A1 EP92904843A EP92904843A EP0568623A1 EP 0568623 A1 EP0568623 A1 EP 0568623A1 EP 92904843 A EP92904843 A EP 92904843A EP 92904843 A EP92904843 A EP 92904843A EP 0568623 A1 EP0568623 A1 EP 0568623A1
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EP
European Patent Office
Prior art keywords
vaccine
cell
sequence
cells
pathology
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP92904843A
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German (de)
English (en)
Inventor
Mark D. Howell
Steven W. Brostoff
Dennis J. Carlo
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Immune Response Corp
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Immune Response Corp
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Application filed by Immune Response Corp filed Critical Immune Response Corp
Priority to EP95250317A priority Critical patent/EP0722738A3/fr
Publication of EP0568623A1 publication Critical patent/EP0568623A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • 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 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 that specifically recognize and bind to the foreign substance.
  • B lymphocytes Other lymphocytes termed T lymphocytes, or 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 lymphoma.
  • the tumors are termed T cell lymphomas.
  • T cell lymphomas are difficult to treat effectively.
  • the present invention provides vaccines and a means of vaccinating a vertebrate so as to prevent or control specific T cell mediated pathologies.
  • the vaccine is composed of a substantially pure T cell receptor (TCR) or an immunogenic 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 invention additionally provides specific ⁇ - chain variable regions and their immunogenic segments, and in particular three T cell receptors, designated VB3, VB14 and VB17, which are associated with the pathogenesis of autoimmune diseases, for example rheumatoid arthritis (RA) and multiple sclerosis (MS) . Additional VDJ junctional (CDR3) regions associated with other autoimmune diseases are also provided.
  • the present invention further relates to means for detecting, preventing and treating RA, MS and other autoimmune diseases.
  • the invention further provides methods of preventing or treating T cell mediated pathologies, including RA and MS, by gene therapy.
  • pure DNA or RNA encoding for a TCR, an immunogenic fragment thereof or an anti-idiotype antibody having an internal image of a TCR or an immunogenic fragment is administered to an individual.
  • Vectors containing the DNA or RNA and compositions containing such vectors are also provided for use in these methods.
  • Figure 1 shows the variable region sequences of VB3, VB14 and VB17.
  • the boxed segments depict the CDR1, CDR2 and CDR4 hypervariable regions of each VB chain.
  • the sequences between the CDR2 and CDR4 regions represent an overlap between these two hypervariable regions.
  • Figure 2(A) shows the location of primers used in polymerase chain reaction amplification of T cell receptor ⁇ -chain genes
  • 2(B) shows primer sequences used in polymerase chain reaction.
  • Figure 3 shows the location and sequence of primers used in polymerase chain reaction amplification of HLA-DR B, genes. Also shown are HLA-DR allele specific oligonucleotides.
  • the invention generally relates to vaccines and their use for preventing, ameliorating or treating 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 encompass both T cell mediated autoimmune diseases and diseases resulting from unregulated clonal T cell replication.
  • 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.
  • substantially the amino 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 that 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 or segment of the described immunizing sequence can be used so long as it is sufficiently characteristic of the desired T cell receptor or fragment thereof to cause an effective immune response against desired T cell receptors, but not against undesired T cell receptors.
  • variations in the sequence can easily be made, for example by synthesizing an alternative sequence. The alternate sequence can then be tested, for example by immunizing a vertebrate, to determine its effectiveness.
  • fragment means an immunogenically effective subset of the amino acid sequence that comprises a TCR.
  • the term is intended to include 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.”
  • Reference herein to a "fragment,” “portion” or “segment” of a T cell receptor does not mean that the composition must be derived from intact T cell receptors.
  • Such "fragments,” portions” or “segments” can be produced by various means well-known to those skilled in the art, such as, for example, manual or automatic peptide synthesis, various methods of cloning or enzymatic treatment of a whole TCR.
  • corresponding to means that the peptide fragment has an amino acid sequence which is sufficiently homologous to a TCR sequence or fragment thereof to stimulate an effective regulatory response in the individual.
  • the sequence need not be identical to the TCR sequence as shown, for instance, in Examples II and III.
  • immunologically effective an amount of the T cell receptor or fragment thereof which is effectively elicits an immune response to prevent or treat a T cell mediated pathology or an unregulated T cell clonal replication in an individual. Such amounts will vary between species and individuals depending on many factors for which one skilled in the art can determine.
  • binding partner means a compound which is reactive with a TCR.
  • this compound will be a Major Histocompatibility Antigen (MHC) but can be any compound capable of directly or indirectly stimulating T cell activation or proliferation when bound to the TCR.
  • MHC Major Histocompatibility Antigen
  • Such compounds can also, for example, be a superantigen that binds to a superantigen binding site on the TCR.
  • “individual” means any vertebrate, including humans, capable of having a T cell mediated pathology or unregulated clonal T cell replication and is used interchangeably with “vertebrate.”
  • ligand means any molecule that reacts with another molecule to form a complex.
  • selective binding means that a molecule binds to one type of molecule or related group of molecules, but not substantially to other types of molecules.
  • selective binding indicates binding to TCRs or fragments thereof containing a specific VB without substantial cross-reactivity with other TCRs that lack the specific VB.
  • 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 that 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, joining (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 joining
  • 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.
  • VB beta chain of the variable region referred to as a VB
  • the nomenclature used herein to identify specific VBs follows that of Kimura et al., Eur. J. Immuno. 17:375-383 (1987), with the exception that the VB14 herein corresponds to VB3.3 of Kimura et al.
  • 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.
  • EAE experimental allergic encephalomyelitis
  • MBP myelin basic protein
  • 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 having 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.
  • 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 usually also for a single epitope on that antigen. These observations indicate that discrete populations of autoaggressive T cells are responsible for the pathogenesis of EAE.
  • MBP antigen
  • TCRs of EAE-inducing T cells has revealed restricted heterogeneity in the structure of these disease-associated receptors.
  • 33 MBP-reactive T cells only two alpha chain V region gene segments and a single alpha chain J region gene segment were found. 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.
  • 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.
  • superantigens means antigens or fragments thereof that bind preferentially to T cells at specific sites on the B chain of a TCR and stimulate T cells at very high frequency rate. Such superantigens can be endogenous or exogenous. "Frequency” refers to the proportion of T cells responding to antigens and ranges from about 1/5 to 1/100 in response to superantigens. Thus, superantigens are distinguishable from ⁇ conventional antigens, which have a much lower T cell response frequency rate ranging from about 1/10 4 to 1/10 6 . Superantigens activate T cells by binding to specific VBs. The superantigen binding sites of various TCRs have been distinguished from the conventional hypervariable regions (CDRs) of TCRs. These CDRs represent the regions of TCRs thought to be responsible for binding conventional antigens that are complexed to MHC.
  • CDRs hypervariable regions
  • the present invention provides an effective method of immunotherapy for T cell mediated pathologies, including autoimmune diseases, which avoids many of the problems associated with previously suggested methods of treatment.
  • T cell mediated pathologies including autoimmune diseases
  • the host's own immune system is mobilized to suppress the autoaggressive T cells.
  • the suppression is persistent and may involve any or 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 ex vivo-derived regulatory T cell clones which requires a highly individualized therapeutic approach because of MHC non-identity among humans in order to avoid graft versus host reactions.
  • the methods of the present invention are also more effective than vaccination with attenuated disease-inducing T cells that lack specificity for the protective antigen on the surface of a particular T cell as well as the variable induction of immunity to that antigen.
  • vaccination with attenuated T cells is plagued by the same labor intensiveness and need for individualized therapies as noted above for ex vivo derived regulatory T cell clones.
  • the vaccine peptides of the present invention comprise TCRs or immunogenic fragments thereof from specific 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 different TCRs that contribute to the T cell mediated pathology.
  • These vaccine peptides can be of variable length so long as they can elicit a regulatory response. Preferably, such peptides are between about 5 - 100 amino acids in length, and more preferably between about 6 - 25 amino acids in length.
  • the immunizing peptide can have the amino acid sequence of a B-chain VDJ region when the subject has MS or RA.
  • Any immunogenic portion of these peptides can be effective, particularly a portion having substantially the sequence SGDQGGNE (SEQ ID No. 1) or CAIGSNTE (SEQ ID No. 2) of VB4 and VB12, respectively, for MS or substantially the sequence ASSLGGAVSYN (SEQ ID No. 3), ASSLGGEETQYF (SEQ ID No. 4), ASSLGGFETQYF (SEQ ID No. 5) or ASSLGGTEAFF (SEQ ID No. 6) or RA.
  • amino acid substitutions can be made which do not destroy the immunogenicity of the peptide.
  • this peptide can be linked to a carrier to further increase its immunogenicity.
  • whole T cell receptors or TCR fragments that include these sequences can be used to vaccinate directly.
  • T cell receptors whole T cells or fragments of TCRs that contain VB17, VB14 or VB3 can be used to immunize an individual having a T cell mediated pathology to treat or prevent the disease.
  • rheumatoid arthritis can be so treated.
  • the immune response generated in the individual can neutralize or kill T cells having VB17, VB14 or VB3 and, thus, prevent or treat the deleterious effects of such VB-bearing T cells.
  • VB17, VB14 or VB3 is common to T cell receptors on pathogenic T cells mediating other autoimmune diseases or autoimmune diseases in general, such vaccines can also be effective in ameliorating such other autoimmune diseases.
  • VB17 refers to a specific human B-chain variable region of three T cell receptors. VB17 has the following amino acid sequence: MSNQVLCCWLC FLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQ IVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASS (SEQ ID No. 7).
  • VB14 refers to a specific human B-chain variable regions of another TCR.
  • VB14 has the following amino acid sequence: MGPQLLGYWLCLLGAGPLEAQVTQNPRYLITV TGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEK RNFPLILESPSPNQTSLYFCASS (SEQ ID No. 8) .
  • VB3 refers to a family of specific human ⁇ - chain variable region. Two members of the VB3 family have been identified as VB3.1 and VB3.2.
  • VB3.1 has the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKV TQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIP EGYSVSREKKERFSLILESASTNQTSMYLCASS (SEQ ID No. 9).
  • VB3.2 has the following amino acid sequence: MGIRLLCRVAFC FLAVGLVDVKVTQSSRYLVKRTGEKVFIECVDMDHENMFWYQRQDPGLGLRLIYF
  • the hypervariable or junctional regions are useful for the vaccines of the present invention.
  • Hypervariable regions useful in the present invention include CDR1, CDR2, CDR3 and CDR4.
  • the amino acid sequences of the CDR1, CDR2 and CDR4 hypervariable regions for VB3, VB14 and VB17 are shown in Figure 1.
  • the CDR3, also known as the V(D)J region is useful as a vaccine of the present invention since T cell immunity elicited by peptides corresponding to this region is expected to be highly specific for a particular antigen. Due to the recombination of the V, D and J region genes prior to maturation, the amino acid sequence across these regions is virtually unique to each T cell and its clones.
  • the CDR2 region is also useful in human diseases such as MS and in particular RA.
  • MS human diseases
  • RA studies, the results indicate a limited number of VBs among the activated T cells infiltrating the synovial target tissue with only a few incidences of sequence homology in the BVDJ region.
  • peptides corresponding to the CDR2 region are viable alternatives for use as vaccines of the present invention.
  • the CDR2 region of VB3, DPGLGLRLIYFSYDVKMKEKG (SEQ ID No.75), of VB14, DPGLGLRQIYYSMNVEVTDKG (SEQ ID No.76), or of VB17, DPGQGLRLIYYSQIVNKFQKG (SEQ ID No.77), can be used.
  • TCR fragment Modifications in these sequences that do not affect the ability of the receptor or an immunogenic fragment thereof to act as an immunogen to stimulate the desired immune response are contemplated and are included in the definition of TCR fragment.
  • the variable region can be joined with any D and J segment of the TCR.
  • immunogenically representative fragments of VB3, VB14 and VB17 are also included in the definition of "VB3,” "VB14” and "VB17,” respectively.
  • TCR is substantially free of other biochemical moieties with which it is normally associated in nature.
  • Such substantially pure TCRs or fragments thereof, for instance, can be synthesized, produced recombinantly by means known to those skilled in the art.
  • whole TCRs can be enzymatically treated to produce such fragments.
  • vaccine peptides can correspond to the VB regions that contain sequences of high homology which are conserved among pathogenic TCRs. These regions of conserved homology include the conventional CDRs, such as CDR1 and CDR2, which are common to T cells bearing the same VB, and also the superantigen binding site, which can be common to pathogenic TCRs bearing different VBs. The superantigen binding site is also known to be in or around the CDR4 hypervariable region.
  • the vaccines of the present invention comprise peptides of varying lengths corresponding to the TCR or immunogenic fragments thereof.
  • the vaccine peptides can correspond to regions of the TCR which distinguish that TCR from other nonpathogenic TCRs. Such specific regions can, for example, be located within the various region(s) of the respective TCR polypeptide chains, for example, 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 (for example, Type I Diabetes) .
  • 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 peptides can be administered in many possible formulations, including pharmaceutically acceptable mediums.
  • the peptide can be conjugated to a carrier, such as KLH, in order to increase its immunogenicity.
  • the vaccine can include or be administered in conjunction with an adjuvant, of which several are known to those skilled in the art. After initial immunization with the vaccine, further boosters can be provided.
  • the vaccines are administered by conventional methods, in dosages which are sufficient to elicit an immunological response. Such dosages 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.
  • T cells are obtained preferably from the site of active autoaggressive activity such as a lesion in the case of pemphigus vulgaris, the central nervous system (CNS) in the case of multiple sclerosis or the synovial fluid or tissue in the case of rheumatoid arthritis.
  • CNS central nervous system
  • T cells can be obtained 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.
  • An alternative method for isolating pathogenic T cells is provided by Albertini in PCT Publication No. WO88/10314, published on December 29, 1988.
  • 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.
  • methods of preventing the proliferation of T cells associated with a T cell mediated pathology include determining a T cell receptor binding partner according to the above methods and administering an effective amount of such binding partner in an appropriate form to prevent the proliferation of the T cells.
  • the methods can be used, for example, to build a tolerance to self antigens as in the case of an autoimmune disease.
  • the present invention also relates to other methods of preventing or treating a T cell pathology by inhibiting the binding of a T cell receptor to its TCR binding partner in order to prevent the proliferation of T cells associated with the T cell pathology.
  • Ligands that are reactive with the T cell receptor or its binding partner at binding sites that inhibit the T cell receptor attachment to the binding partner can be used.
  • Such ligands can be, for example, antibodies having specificity for the T cell receptor or its binding partner.
  • the invention also provides a method of preventing or treating a T cell mediated pathology in an individual comprising cytotoxically or cytostatically treating a VB-containing T-cells, particularly VB3, VB14 and VB17, in the individual.
  • the VB-containing T cells are treated with a cytotoxic or cytostatic agent that selectively binds to the VB region of a T cell receptor that mediates a pathology, such as RA or MS for example.
  • 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.
  • Rheumatoid arthritis is a T cell mediated autoimmune disease.
  • the invention describes clonal infiltrates of activated VB3, VB14 and VB17 T cells in the synovium of rheumatoid arthritis patients. The presence of these T cells in the diseased tissue of most of patients examined, their clonality, and the cytotoxic activity of one such T cell for synovial adherent cells, demonstrate a central role for T cells bearing these VBs in the pathogenesis of RA.
  • TCR T cell receptor
  • IL-2R+ IL-2 receptor positive
  • TCR mRNAs were amplified using a polymerase chain reaction (PCR) protocol designed to amplify human TCR B-chain genes containing virtually any desired VB gene element.
  • PCR polymerase chain reaction
  • a CD4+, VB17 bearing T cell clone has been isolated from one of the synovial tissue specimens and its in vitro cytotoxicity for synovial adherent cells supports the direct involvement of VB17 T cells in RA. Additional studies were conducted to ensure that the prevalence of VB17 T cells in the initial studies did not result from an amplification bias for the VB consensus primer, and to examine the involvement of other TCR VB gene families in RA. As described in
  • RNAs from activated (IL2-R+) synovial T cells were analyzed by PCR-amplification with a panel of VB- specific PCR primers.
  • VB17 transcripts were found in four of the five patients tested, confirming the association of VB17 with RA and validating the utility of the VB consensus primer.
  • VB14 was found in four of the five RA patient samples and VB3 and VB9 were detectable in three of five patients.
  • mouse VB6 is most closely homologous, followed by hVB3, hVBl2.1, hVBl4, and mVB7.
  • VB12.1 was negligible in the synovium despite considerable overall homology with VB17.
  • VB9 was found in three of five synovial samples, yet is only weakly homologous to VB17
  • a surprising discovery is the greater homology found among all of the VBs detected in the synovia of RA patients, except VB9, in a contiguous stretch of 15 amino acids located carboxy to the CDR2 region.
  • the 15 amino acid sequences of these VBs as well as other human and mouse VBs are shown in Table 2. Within the B-chain, this region of conservation corresponds positionally to that previously shown to contain superantigen binding sites.
  • %H % homology compared with VB17
  • SEC 2 The exogenous superantigen, SEC 2 , stimulates human VB13.2 T cells as a result of binding to a site on VB13.2 as described in Choi et al., Nature 346:471-473 (1990) , which is incorporated herein by reference.
  • the sequence of this binding site is shown in Table 2 as the last 11 amino acids for VB13.2.
  • a binding site for Mls-la, an endogenous superantigen, has also been mapped to this region as described in Pullen et al.. Cell 61:1365-1374 (1990), which is incorporated herein by reference.
  • VB8.2a the VB8.2 isoform common to laboratory mice
  • VB8.2c a B-chain found in wild mice.
  • These B-chain polypeptides are distinguished functionally by their differential reactivities with Mls- la and structurally by a difference of five amino acids. Of particular importance are the residues at position 70 and 71.
  • the responder ⁇ -chain, VB8.2c has lysine and glutamic acid, respectively, at these positions.
  • Specific mutagenesis of the non-responder gene to encode a lysine-glutamic acid pair at positions 70 and 71 rendered that non-responder B-chain Mls-la-reactive.
  • VB8.2c, VB6 and V ⁇ 7 charge motif of a lysine or arginine followed by a glutamic acid residue is underlined in Table 2.
  • the superantigen binding site for Mls-la is characterized by this charge motif contained within the region of local homology.
  • the present invention is directed to the unexpected discovery that human VB3, VB14 and VB17 have a region that corresponds to the Mls-la binding site. These three human VBs display a significant degree of overall homology within the entire 94 amino acid sequence and of local homology within the 15 amino acid sequence with mVB6 and mVB7.
  • Each of these VBs possess a lysine or arginine/glutamic acid pair, which are underlined in Table 2 and represent what is meant by the term "charge motif.”
  • VB12.1 while displaying a high degree of overall and local homology with VB3, VB14 and VB17, lacks the charge motif, perhaps accounting for its presence in the synovium of only one of five RA patients.
  • VB9 shows no overall or local homology to VBs 3, 14 or 17 and lacks the charge motif.
  • VB3, VB14 and VBl7-bearing T cells have been demonstrated among the activated synovial T cells in RA.
  • These three B-chain polypeptides in contrast to other known VBs, possess overall and local sequence homology and an apparent superantigen binding charge motif. These results indicate that VB-specific T cell activation by superantigen plays a role in RA.
  • VB3, VB14 and VB17 are the only known human VB chains to possess this apparent superantigen binding site characterized by this local sequence homology and the identified charge motif. However, it is possible that other VBs may become known that contain such binding sites. Thus, a substantially pure VB3, VB14 or VB17 sequence containing the charge motif can be used as an immunogen in the vaccines of the present invention. For example, the sequences or fragments thereof shown in Table 2 for VB3, VB14 and VB17 can be used. Vaccines containing any combination of these three VB sequences, including all three sequences, can be used effectively to ameliorate T cell associated diseases. In addition, other common V(D)J sequences of the B-chain observed in RA patients are listed in Table 3. The results taken from two different RA studies show sequence homologies in the BVDJs from four different clones, which indicates the usefulness of peptides corresponding to the CDR3 region as appropriate vaccine candidates.
  • the invention provides the discovery that specific variable regions of the B-chains of three TCRs, designated VB3, VB14, and VB17, are closely associated with T cell mediated pathologies, especially rheumatoid arthritis in human subjects.
  • This discovery allows for the detection, prevention and treatment of rheumatoid arthritis using the methodology set out in this invention. Similar therapeutic approaches set out above for EAE can be applied to rheumatoid arthritis by those skilled in the art.
  • the invention provides a method of diagnosing or predicting susceptibility to T cell mediated pathologies in an individual comprising detecting T cells having the B-chain variable region from VB3, VB14 or VB17 in a sample from the individual, the presence of abnormal levels of such VB-containing T cells indicating the pathology or susceptibility to the pathology.
  • the VB-containing T cells can be qualitatively or quantitatively compared to that of normal individuals.
  • diagnosis can be performed, for example, by detecting a portion of the VBs that does not occur on non-rheumatoid arthritis associated B-chain variable region T-cell receptors.
  • the VBs of the present invention can be detected, for example, by contacting the VBs with a detectable ligand capable of specifically binding to the individual VBs.
  • detectable ligands are known in the art, e.g. an enzyme linked antibody.
  • nucleotide probes, complementary to the individual VB of interest, encoding nucleic acid sequences can be utilized to detect such VB- containing T cells, as taught, for instance, in Examples X and XI.
  • the invention also provides a method of preventing or treating a T cell mediated pathology comprising preventing the attachment of a VB3-, VB14- or VBl7-containing T-cell receptor to its binding partner.
  • attachment is prevented by binding a ligand to VB3, VB14 or VB17.
  • attachment is prevented by binding a ligand to the VB3, VB14 or VB17 binding partner.
  • Attachment can be prevented by known methods, e.g. binding an antibody to the individual VBs or to its binding partner in order to physically block attachment.
  • MBP-reactive T cells have been proposed to play a role due to the clinical and histologic similarities between MS and EAE.
  • MBP-reactive, encephalitogenic T cells show striking conservation of B- chain V(D)J amino acid sequence, despite known differences in MHC restriction and MBP-peptide antigen specificity.
  • One embodiment of the 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 B-chain with a V(D)J amino acid sequence of VB4 homologous with that of B-chains from MBP-reactive T cells mediating pathogenesis in experimental allergic encephalomyelitis (EAE), an animal model of MS, as shown in Table 4.
  • EAE allergic encephalomyelitis
  • the invention is directed to the discovery that B-chain VDJ fragments homologous to VDJ sequences found in rodent EAE, such as SGDQGGNE (SEQ ID No.l) and CAIGSNTE (SEQ ID No. 2), are closely associated with multiple sclerosis in human subjects.
  • SGDQGGNE SEQ ID No.l
  • CAIGSNTE SEQ ID No. 2
  • the invention provides a method of diagnosing or predicting susceptibility to multiple sclerosis in an individual comprising detecting T cells having various VBs, such as VB4 or VB12, and particularly having substantially the sequence SGDQGGNE (SEQ ID No.l) or CAIGSNTE (SEQ ID No.2), in a sample from the individual, the presence of the sequence indicating multiple sclerosis or susceptibility to multiple sclerosis.
  • the sequences can be detected, for example, by contacting T cells or TCRs with a detectable ligand.
  • nucleotide probes complementary to the nucleic acid encoding the sequence can be utilized as taught, for instance, in Example IX.
  • the invention also provides a method of preventing or treating multiple sclerosis comprising preventing the attachment of a T-cell receptor containing various VBs, including VB4, VB12 or fragments thereof, such as those having substantially the SGDQGGNE (SEQ ID No.l) or CAIGSNTE (SEQ ID No.2) sequence to its binding partner.
  • attachment is prevented by binding a ligand to the sequence.
  • attachment is prevented by binding a ligand to the binding partner. Attachment can be prevented by known methods, such as binding an antibody to these VBs, and in particular to the SGDQGGNE (SEQ ID No.l) or CAIGSNTE (SEQ ID No.2) sequences, to physically block attachment.
  • the invention also provides a method of preventing or treating multiple sclerosis in an individual comprising cytotoxically or cytostatically treating T cells containing various VBs, including VB4, VB12 and fragments thereof, particularly those having substantially the SGDQGGNE (SEQ ID No.l) or CAIGSNTE (SEQ ID No.2) sequence in the individual.
  • T-cells are treated with a cytotoxic or cytostatic agent which selectively binds to these VBs or their immunogenic fragments.
  • the agent can be, for example, an antibody attached to a radioactive or chemotherapeutic moiety.
  • T cell lymphoma is another T cell pathology which would be amenable to this type of treatment.
  • Application of this technology in the treatment of T lymphoma would be conducted in virtually identical fashion. In one respect, however, this technology is more readily applied to T cell proliferative disease since the isolation of the pathogenic T cells is more easily accomplished.
  • the technology is applied in the manner described herein. Specifically, the TCR genes of the T lymphomas are sequenced, appropriate regions of those TCRs are identified and used as vaccines.
  • the vaccines can comprise single or multiple peptides, and can be administered in pharmaceutically acceptable formulations, with or without adjuvants, by conventional means.
  • 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 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).
  • the treated cells will respond to the direct injection of DNA or RNA by expressing the encoded polypeptide for at least about 60 days.
  • 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.
  • 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 (SEQ ID No.23). (Amino acids are represented by the conventional single letter codes.)
  • the equivalent sequence in the rat has been reported to be: SSD-SSNTE (SEQ ID No.
  • 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) .
  • 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 icroliters 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 10 ⁇ 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 5. 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 The VDJ peptide used in the previous examples was synthesized according to the sequence of TCR B 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) :
  • VB8 is the most common B 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 VB8 gene, and which is not found among other rat VB genes whose sequences were reported by Morris, et al., Immunogenetics 27:174-179 (1988) .
  • the sequence of this VB8 peptide, designated IR7, is:
  • the results of vaccinations conducted with the rat VB8 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.
  • VB8.2 (residues 39-59) provided less protection than IR7 as shown in Table 8.
  • the 21 amino acid sequence of VB8.2 is DMGHGLRLIHYSYDVNSTEKG (SEQ ID No.30) .
  • Each peptide was used at 100 ⁇ g doses and dissolved in saline prior to being emulsified in an equal volume of complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • Animals were challenged after 42 days with 50 ⁇ g of guinea pig myelin basic protein in CFA.
  • the CFA contained 10 mg/ml of mycobacteria tuberculosis. Injections and evaluation of clinical signs and histology were performed as previously described.
  • Other animals (five per group) were immunized with peptides in the same way and their splenocytes were removed after 14 days to test for lymphocyte proliferation as described in Olee et al., J. Neuroimmunol. 21:235-240 (1989).
  • the sequences of the 20 amino acid peptide and the 21 amino acid peptide are designated in Table 8 as VB8.2 20 and VB8.2 21 respectively.
  • 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:
  • Vaccinations were conducted with a mixture of 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) .
  • MBP-reactive T cell lines were established from peripheral blood mononuclear cells (PBMC) of nine chronic progressive MS patients and two healthy controls. Cells were maintained in culture by regular stimulation with purified human MBP and irradiated-autologous PBMC for three days followed by four days in IL-2 containing medium.
  • PBMC peripheral blood mononuclear cells
  • T cells were harvested from log phase cultures and RNA was prepared, amplified with the VBl6mer primer and nested CB primers for 55 cycles as described in Example X.
  • MBP-reactive T cell lines were sequenced using the C ⁇ seq primer. Amplification products were gel purified, base denatured and sequenced from the CBseq primer. Readable DNA sequence was obtained from 5 of these lines, indicating that predominant T cell clones had been selected by long term in vitro passage.
  • One of these sequences, from the MS-Re cell line (Table 11) possessed a B-chain VDJ amino acid sequence that shared five of the first six and six of nine total residues with the B-chain VDJ amino acid sequence conserved among MBP reactive, encephalitogenic T cells in the BIO.PL mouse model of EAE. This sequence was not present among the predominant TCR rearrangements found in the remaining four human MBP reactive T cell lines.
  • RNAs were reversed transcribed and amplified in 20 cycle stage I reactions with the VB16mer and CBext primers. One ⁇ l aliquots of these stage I reactions were reamplified for 35 cycles with the VBRe and CB int primers. One ⁇ l aliquots of these reactions were analyzed by Southern blot hybri .dization wi.th a 32P-labeled human CB probe.
  • SGDQGGNE SEQ ID No.l
  • SEQ ID No.l SEQ ID No.l
  • Synovial tissue specimens were obtained from radiographically proven rheumatoid arthritis patients undergoing joint replacement therapy.
  • Activated T cells were selected using magnetic beads and antibodies reactive with the human IL2-R ( ⁇ IL2-R) as follows.
  • Synovial tissue was digested for 4 hrs at 37°C in RPMI + 10% Fetal Bovine Serum (FBS) containing 4 mg/ml collagenase (Worthington Biochemical, Freehold, NJ) and 0.15 mg/ml DNAse (Sigma, St. Louis, MO.). Digests were passed through an 80-mesh screen and single cells were collected by Ficoll density gradient centrifugation. Cells at the interface were washed and were incubated at 10 /ml for 30 min at 0°C with 5 ⁇ g/ml control mouse IgG (Coulter Immunology, Hialeah, FL) in PBS containing 2% FBS (PBS-FBS) .
  • FBS Fetal Bovine Serum
  • a T cell clone was derived from the Ficoll pellet of patient 1008.
  • the cells in the pellet were cultured at 2 x 10 /ml in media without IL-2 for two weeks.
  • Non-adherent cells from this culture were cloned by limiting dilution onto autologous synovial cell monolayers.
  • a CD4+ T cell clone 1008.8 was obtained and adapted to culture by regular stimulation with autologous synovial monolayers for 3 days in media without IL-2 followed by a 4 day culture in medium with LAK supernatant.
  • TCR B-chain genes were amplified with several combinations of the primers shown in Figure 2.
  • This primer has been used to amplify TCR B-chains from more than 25 different human T cell clones, lines or primary tissue preparations.
  • a spectrum of VB genes has been sequenced from these amplified DNAs, argues against a significant bias of the primer for certain VB families.
  • PCR amplification with the VBl ⁇ mer primer facilitates analysis of T cell populations for which a priori knowledge of VB gene usage is unavailable.
  • T cell receptor B-chain genes were amplified in two-stage amplification reactions with nested pairs of the primers shown in Figure 2.
  • the primer sequences used in the polymerase chain reactions are listed in Table 13.
  • RNAs were reverse transcribed for 1 hour at 42 C with 40pmol of the CBext primer in a 12 ⁇ l reaction using conditions described by Hart et al.. The Lancet, p. 596 (1988) , included by reference herein. Reactions were diluted with a master mix containing 40 pmols of the VBl6mer primer, nucleotides and reaction buffer as above but without MgCl 2 to give a final Mg + concentration of
  • TM reactions (Cetus, Gene-Amp Kit ) containing 100 pmols of the CBint primer and 100 pmols of the VB8, VB17 or 5'CB primers or 700 pmols of the VBl ⁇ mer primer.
  • Stage II amplifications were conducted as above with a 50°C annealing temperature and without the 37°C and 45°C ramping.
  • RNA samples from 1012IL2.d5 and 1008.8 cultures were amplified with the VB16mer and CBext primers in stage I reactions and with the VBl ⁇ mer and the CBint primer in 35 cycle stage II reactions.
  • Reaction products purified from low melting agarose gel slices with Gene Clean glass beads (Bio 1D1, San Diego, CA) , were base denatured and sequenced from the CBseq primer with T7 polymerase (Sequenase, United States Biochem, Cleveland, OH) .
  • a predominate VB sequence, corresponding to a single VB17 rearrangement (Table 14) , was clearly readable in the 1012IL2.d5 sample.
  • TCR B-chain genes were amplified with a VBl7-specific primer in the second stage amplification after an initial amplification with the VBl ⁇ mer.
  • VB17 TCR DNA could be amplified from magnetic bead samples derived from the 4 patients examined. Ethidium bromide staining of electrophoresed reaction products revealed greater VB17 amplification in some of the IL-2R+ samples than in the corresponding controls. Accordingly, the relative amounts of VB17 TCRs in each control and IL-2R+ sample were quantified by slot blot hybridization analysis as follows.
  • RNAs from magnetic bead preps were amplified in the first stage with the VBl ⁇ mer and CBext primers and then reamplified for twenty cycles with the CBint primer and each of the VB17, BB8 and 5'CB primers.
  • Amplification reactions were serially diluted in 2OX SSC, denatured by boiling and chilled in an ice slurry. Samples were loaded onto nitrocellulose membranes, hybridized to a human TCR B-chain constant region probe and washed with 0.1X SSC, 0.1% SDS at 56°C. Bound radioactivity was quantified by liquid scintillation spectroscopy and endpoint dilutions were those samples with fewer than 200 cpm bound. The amounts of product produced by forty total cycles with each of the respective primer combinations falls in the linear portion of a product versus cycle number quantification curve.
  • Amplifications with the 5'CB and CBint primer pair were used to estimate the total B-chain amplified from each sample, providing benchmarks for normalizing the results of VB17 and VB8 quantification in the respective IL-2R+ and control sample pairs (Table 15) .
  • the quantity of VB17 DNA amplified was increased in the IL-2R+ samples, relative to the control samples, in 3 of the 4 patients.
  • the magnitude of the increase ranged from 5-fold in patient 1015 to 40-fold in patient 1014 (Table 15) . This enrichment was not a product of the isolation procedure, since the quantity of VB8 DNA amplified was increased in the IL-2R+ fraction only in patient 1015.
  • Sample 7 1015 IL-2R+, Sample 8 1015 mlgG.
  • VB17 rearrangements from the IL-2R+ RNAs of the three patients showing enrichment were amplified with the VB17 and CBint primer pair and the reaction products sequenced with the CBseq primer.
  • 1012 IL-2.d5 1014 and 1015 contained single sequences (Table 14) , indicative of clonal expansion of VB17 T cells in vivo.
  • direct sequencing of the rearrangements amplified with the VB8 specific primer was not possible due to significant heterogeneity in the B- chain product.
  • HLA-DR analysis in rheumatoid arthritis patients was performed as follows. DNA from each patient was prepared by boiling 10 synovial cells in 200 ⁇ l dH 2 0. Ten ⁇ l were amplified for 35 cycles in a 100 ⁇ l reaction
  • Each of the patients in this study possessed at least one allele of the HLA-DR genes, DR4w4, DR1, DR4wl4 or DR4wl5, that are known to predispose for RA (Table 16) . Also shown in Table 16 are HLA-DR allele specific oligonucleotides.
  • T cell receptors containing VB17 or fragments thereof which are immunogenic or can be made immunogenic can be used to immunize human subjects by methods demonstrated by Example VIII. Such immunizations can result in an effective immune response.
  • Synovial tissue specimens were obtained from proven RA patients undergoing joint replacement surgery. HLA-DR analysis was conducted as described in Example IX(D) .
  • T cell receptor B-chain genes were amplified in two-stage amplification reactions with nested pairs of HPLC-purified oligonucleotide primers (Midland Certified Reagents, Midland, TX.) shown in Figure 3.
  • RNAs were reverse transcribed (1 hour, 42°C) with the CBext primer (40 pmol) in 12 ⁇ l of reaction buffer (Hart et al.,
  • Stage II amplification reactions (Cetus Gene-Amp Kit) containing the CBint primer (100 pmol) and either the VB8, VB12, VB17, or 5'CB primers (100 pmol) or the VBcons primer (100-700 pmol) .
  • Stage II amplifications were conducted for the indicated number of cycles with a 50°C annealing temperature and without the 37°C and 45°C ramping.
  • B. VB17 T cells are cytotoxic for synovial adherent cells
  • Two parallel cultures were established from single cell suspensions, derived by enzymatic digestion as described below from the synovial tissue of patient 1008.
  • the first a bulk culture of total synovial cells, was plated at 2 x 10 6 /ml in RPMI 1640, 5% FBS, 20% HL-1 (Ventrex Laboratories, Portland, ME) , 25mM HEPES, glutamine and antibiotics. The culture was grown undisturbed for two weeks in the absence of exogenous antigen or growth factors.
  • the second a synovial cell monolayer culture, was initiated as above and regularly trypsinized and passaged during this two-week period.
  • Monolayer cells were plated at 5 x 10 cells/well m flat- bottomed, 96-well microtiter plates and cultured overnight. Non-adherent cells from the total synovial cell culture were then plated at 10 cells/well onto the monolayers.
  • Wells positive for T cell growth were expanded and adapted to culture by regular stimulation with autologous synovial monolayers for 3 days in media without IL-2 followed by a 4 day culture in medium containing 20% supernatant from lymphokine activated killer (LAK) cells, a source of IL-2 (Allegretta et al., Science 247:718-721 (1990)). Later passages were adapted to weekly stimulation with allogeneic PBLs and anti-CD3 antibody (Coulter Immunology, Hialeah, FL) in place of synovial cell monolayers.
  • LAK lymphokine activated killer
  • Cytotoxicity assays were conducted as described Stedman et al., Immunol. Meth. 119:291-294 (1989), incorporated herein by reference, using S-labeled synovial monolayer cells or EBV-transformed B cells as targets.
  • Target cells were labeled overnight, trypsinized (adherent cells only) , washed and plated at 2000 cells per well in 96-well round bottom microtiter plates.
  • T cells were activated with allogenic PBLs and anti-CD3 antibody in medium containing LAK supernatant for 7 days prior to the assay and added to the targets at the indicated effector:target ratios. Cultures were incubated overnight at 37°C, centrifuged at 300xg for 2 minutes and radioactivity in 50 ⁇ l of the supernatant quantified.
  • the per cent specific lysis was calculated relative to detergent-lysed targets as described in Townsend et al., Cell 44:959-968 (1986), incorporated herein by reference.
  • effector:target ratios of 1.0, 2.5 and 5.0
  • the percent lysis of synovial adherent cells by 1008.8 T cells was approximately 4%, 14% and 33%, respectively.
  • 1008.8 T cells had no demonstrated effect on EBV- transformed B cell targets.
  • the percent lysis of synovial adherent cells by MS3 cells was approximately 0%, 0% and 3%, respectively.
  • T cells were isolated from the synovial tissue of patient 1008 by co-cultivation with synovial cell monolayers. Since the antigens recognized by pathogenic T cells in RA are unknown, synovial cell monolayers were used as stimulators of in vitro synovial T cell growth. The relevant target cells were assumed to be present in a bulk -adherent cell culture from diseased synovium. Of 192 microwell co-cultures plated, 7 were positive for T cell growth within 10-14 days. T cells were expanded and maintained in vitro by alternately stimulating with synovial cell monolayers and medium containing LAK supernatant. Flow cytometry revealed that each of these seven cultures was 100% CD4+.
  • VBcons consensus VB primer
  • This primer was designed to amplify B-chain rearrangements containing any of the known VB genes, thus allowing analysis of T cell clones for which a . priori knowledge of V ⁇ gene usage is unavailable. Sequencing of the VBcons-amplified ⁇ -chain gene of 1008.8 revealed a single VB17-JB1.1 rearrangement as shown in Table 16. Subsequently, the remaining six cultures derived from patient 1008 were analyzed by PCR amplification using a VBl7-specific primer shown in Table 13. TCR genes from three of those six were amplifiable, indicating that, of the 7 T cell cultures obtained from the synovium of this patient, 4 had rearranged and expressed the VB17 gene. C. VB17 T cells are enriched among activated T cells in RA synovium
  • Synovial tissue was digested with agitation for 4 hrs at 37°C in RPMI-1640 and 10% fetal bovine serum (FBS) containing 4 mg/ml collagenase (Worthington
  • RNAs from magnetic bead preparations were reverse transcribed and amplified for 20 cycles in stage I reactions with the VBcons and CBext primers. One ⁇ l of each reaction was reamplified for 20 cycles in individual stage II reactions containing the CBint primer in conjunction with the VB17, VB8, VB12 and 5'CB primers. Aliquots of each reaction were diluted in 20X SSC, denatured by boiling and chilled in an ice slurry. Samples were loaded onto nitrocellulose membranes, hybridized to a human TCR ⁇ -chain constant region probe and washed with 0.1X SSC, 0.1% SDS at 56°C. Bound radioactivity was quantified by liquid scintillation spectroscopy.
  • RNAs were directly extracted from cells in the IL-2R+ and control samples without in vitro culture and, therefore, are expected to accurately reflect T cell distributions in synovial tissue at the time of surgical removal.
  • the initial PCR amplifications of these magnetic bead RNA preparations revealed greater amounts of VB17 PCR product in the IL2-R+ samples than in the corresponding mlgG controls.
  • the presence of TCR mRNA in the mlgG samples indicates that T cells non-specifically adhered to the magnetic beads.
  • the apparent increase in the IL2-R+ sample suggests that the activated T cell compartment contained more VB17 T cells than the unselected synovial T cell compartment.
  • a quantitative PCR analysis was used to formally examine the relative proportions of VB17 T cells in the IL-2R+ and mlgG control samples (Table 17) .
  • Magnetic bead RNAs were reverse transcribed, preamplified with VBcon and CBext and reamplified in separate reactions with a constant region primer (CBint) and each of the VB- specific primers, VB17, VB8 and VB12.
  • the second stage amplification was also performed with two CB primers (5'CB and CBint) in order to estimate the total B-chain present in each sample and to provide benchmarks for normalizing the results of specific VB quantification in the respective IL-2R+ and control sample pairs.
  • the proportion of VB17 DNA, relative to total C ⁇ , was increased in the IL-2R+ samples over that found in the mlgG control samples for each of the 5 patients examined.
  • Synovial VB17 T cells display limited heterogeneity RNAs were reverse transcribed and amplified with the VBcons and CBext primers in 20 cycle stage I reactions and with the CBint and VBcons (1008.8) or VB8, VB12, and VB17 primers (magnetic bead RNA preparations) in 35 cycle stage II reactions. Double stranded reaction products were electrophoresed in 2% Nu-Sieve agarose gels. After purification from gel slices with Gene Clean (Bio 101, San Diego, CA) , samples were base denatured and either directly sequenced or cloned into plasmid for sequencing of multiple independent rearrangements. In all cases, samples were sequenced from the CBseq primer ( Figure 2) with T7 polymerase (Sequenase, United States Biochemicals, Cleveland, OH) .
  • VB17 rearrangements present in the IL-2R+ RNAs were amplified from the VBcons and CBext preamplification with the VB17 and CBint primer pair and the reaction products sequenced. PCR products from patients 1014 and 1015 were directly sequenced and the results obtained were consistent with the presence of a single VB17 rearrangement in each of these amplified samples (Table 17) .
  • the PCR product of patient 1013 was cloned into plasmid and the thirteen isolates that were sequenced contained identical VB17 rearrangements. Sequencing of plasmid clones from patient 1012 revealed the presence of two dominant rearrangements (5 isolates of each) and a single isolate of another.
  • the VB17 repertoire in the RA synovium is of limited heterogeneity, indicative of clonal or oligoclonal expansion of VB17 T cells in vivo. This is in contrast to the VB8 and VB12 repertoires, from these same synovial preparations, which showed significant heterogeneity. None of the PCR- amplified VB8 and VB12 samples analyzed were directly sequenceable and plasmid cloning of VB8 rearrangements from patient 1012 revealed 4 different sequences in 5 clones analyzed.
  • VB17 rearrangements in PBLs from patient 1012 also revealed greater diversity than that seen in the synovial IL-2R+ sample.
  • RNA from a 3 day culture of PHA/PMA stimulated 1012 PBLs was amplified with the VB17 primer, as for the synovial sample, and the products cloned into plasmid.
  • T cell receptor B-chain genes were amplified in two-stage reactions with individual VB-specific primers as described in Wucherfpennig et al., Science , 248:1016- 1019 (1990) , incorporated herein by reference, and nested CB primers.
  • RNAs were reverse transcribed (1 hour, 42°C) with the CBext primer (40 pmol) in 12 ⁇ l of reaction buffer (Hart et al. , supra) .
  • Stage II amplification reactions were conducted for 20 cycles with a 50°C annealing temperature and without the 37°C and 45°C ramping.
  • TCR transcripts in the IL2-R+ samples were analyzed with a panel of 19 PCR primers, specific for known human VB gene families (Table 18) .
  • the VBcons primer of Example XI is equivalent to the VBl6mer primer.
  • the number of VB genes detectable in these samples was variable, ranging from two to twelve.
  • VB17 was found in four of the five patients, confirming the previous analyses using the VBl6mer primer.
  • VB14 transcripts also were found in four of the five patients and VB3 and VB9 transcripts were detectable in three of the five samples.
  • T cells bearing these 3 VB polypeptides may also contribute to synovial inflammation.
  • Rats were immunized with 100 ⁇ g of a synthetic peptide containing the sequence VPNGYKVSRPSQGDFFLTL (SEQ ID No.46) found in the fourth hypervariable or CDR4 region of the rat TCR & chain identified as VB8.2.
  • the peptide dissolved in saline, was emulsified in an equal volume of complete Freund's adjuvant (CFA) containing 10 mg/ul of mycobacterial tuberculosis.
  • CFA complete Freund's adjuvant
  • the animals were challenged 30 days later with 50 ⁇ g of guinea pig myelin basic protein in CFA.
  • the CDR4 peptide vaccination resulted in a reduced incidence of disease as well as a reduced severity as measured both clinically and histologically as shown in Table 19.
  • the histology was performed essentially as described in Hughes & Powell, J. Neuropath. EXP. Neurol. 43:154 (1984), which is incorporated herein by reference.
  • rat CDR4 peptide sequence VPNGYKVSRPSQGDFFLTL (SEQ ID No.46) graded on a 3-point scale as described in Example I graded on a 4-point scale

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Abstract

L'invention décrit des vaccins et un moyen de vaccination d'un vertébré, afin de prévenir ou de réguler des pathologies spécifiques provoquées par les lymphocytes T, y-compris des maladies autoimmunes et la réplication démesurée des lymphocytes T. Le vaccin se compose d'un récepteur de lymphocyte T (TCR) ou d'un fragment dudit vaccin correspondant à un TCR présent sur la surface des lymphocytes T à l'origine de la pathologie. Le fragment du vaccin peut être un peptide correspondant à des séquences de TCR caractéristiques des lymphocytes T à l'origine de ladite pathologie. Ledit peptide peut se lier à des antigènes classiques combinés pour former des cellules de présentation d'un antigène du complexe majeur d'histocompatibilité (CMH) ou des superantigènes. L'invention décrit également des moyens de détermination de séquences d'aminoacides appropriées correspondant auxdits vaccins. Le vaccin s'administre au vertébré de façon à provoquer une réaction immune dirigée contre le TCR des lymphocytes T à l'origine de la pathologie. Cette réaction immune réduit ou supprime les lymphocytes T papathogènes, anihilant ainsi la pathogénèse de la maladie. L'invention décrit, de plus, des régions variables à chaîne beta spécifique des récepteurs de lymphocytes T, désignées Vbeta3, Vbeta4, Vbeta12, Vbeta14 et Vbeta17, , associées à la pathogénèse de maladies autoimmunes telles que l'arthrite rhumatoïde (RA) et des scléroses multiples (MS). Sont également décrits des moyens de détection, de prévention et de traitement de RA et MS. Sont également décrits des moyens d'administration de l'ADN ou de l'ARN codant les polypeptides s'utilisant en tant que vaccins objets de l'invention, dans les cellules tissulaires d'un individu.
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