EP0623025A1 - Vaccination et procedes contre des maladies resultant de reponses pathogenes de populations de lymphocytes t specifiques - Google Patents

Vaccination et procedes contre des maladies resultant de reponses pathogenes de populations de lymphocytes t specifiques

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Publication number
EP0623025A1
EP0623025A1 EP93902714A EP93902714A EP0623025A1 EP 0623025 A1 EP0623025 A1 EP 0623025A1 EP 93902714 A EP93902714 A EP 93902714A EP 93902714 A EP93902714 A EP 93902714A EP 0623025 A1 EP0623025 A1 EP 0623025A1
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European Patent Office
Prior art keywords
cells
cell
vaccine
sequence
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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EP93902714A
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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
Publication of EP0623025A1 publication Critical patent/EP0623025A1/fr
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    • 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
    • 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
    • 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
  • an antigen 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
  • lymphocytes or B cells, produce antibodies that
  • T lymphocytes 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
  • the immune system functions inappropriately and reacts to a component of the host as if it were, in fact, foreign. Such a response results in an autoimmune disease, in which the host's immune system attacks the host's own tissue. T cells, as the primary regulators of the immune system, directly or indirectly effect such autoimmune pathologies.
  • 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
  • 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.
  • Such a treatment should ideally control the inappropriate T cell response, rather than merely
  • the present invention satisfies this need and provides related advantages as well.
  • 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
  • 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 V ⁇ 3, V ⁇ 14 and V ⁇ 17, which are associated with the pathogenesis of autoimmune diseases, for example rheumatoid arthritis (RA) and multiple sclerosis (MS). Additional VDJ
  • CDR3 junctional
  • the present invention further relates to means for detecting,
  • 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 V ⁇ 3, V ⁇ 14 and V ⁇ 17.
  • the boxed segments depict the CDR1, CDR2 and CDR4 hypervariable regions of each V ⁇ 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 1 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 autoimmune disease and T cell lymphomas.
  • T cell mediated autoimmune diseases and diseases resulting from unregulated clonal T cell replication.
  • the term is intended to include both diseases directly
  • T cells 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
  • 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
  • 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
  • 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
  • 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
  • fragment 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
  • 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
  • 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
  • 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 V ⁇ without substantial cross-reactivity with other TCRs that lack the specific V ⁇ .
  • 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.
  • an antigen-specific immune response is 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
  • 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).
  • CD8+ T cells cytotoxic T cells that recognize and destroy cellular targets (for example, virus infected cells of the host).
  • CD4+ T cells 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. Since D segments and elements are found in only some of the TCR genetic loci, and polypeptides, further references herein to D segments and elements will be in parentheses to indicate the inclusion of these regions only in the appropriate TCR chains. Thus, V(D)J refers either to VDJ sequences of chains which have a D region or refers to VJ sequences of chains lacking D regions.
  • V ⁇ beta chain of the variable region referred to as a V ⁇
  • the nomenclature used herein to identify specific V ⁇ s follows that of Kimura et al., Eur. J. Immuno. 17:375-383 (1987), with the exception that the V ⁇ 14 herein corresponds to V ⁇ 3.3 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. After in vitro stimulation with MBP, these T cell clones rapidly induce EAE when adoptively transferred to healthy hosts.
  • EAE- inducing T cells are specific not only for the same antigen (MBP), but usually also for a single epitope on that antigen.
  • TCRs of EAE-inducing T cells have 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
  • superantigens means antigens or fragments thereof that bind preferentially to T cells at specific sites on the ⁇ 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 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 V ⁇ s.
  • the superantigen binding sites of various TCRs have been distinguished from the conventional hypervariable regions (CDRs) of TCRs. These CDRs
  • TCRs represent the regions of TCRs thought to be responsible for binding conventional antigens that are complexed to MHC.
  • 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. By vaccinating, rather than passively
  • 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.
  • 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 ⁇ -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 or CAIGSNTE of V ⁇ 4 and V ⁇ 12, respectively, for MS or
  • 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 V ⁇ 17, V ⁇ 14 or V ⁇ 3 can be used to immunize an individual having a T cell mediated pathology to treat or prevent the disease.
  • 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 V ⁇ 17, V ⁇ 14 or V ⁇ 3 and, thus, prevent or treat the deleterious effects of such V ⁇ -bearing T cells.
  • V ⁇ 17, V ⁇ 14 or V ⁇ 3 is common to T cell receptors on pathogenic T cells
  • such vaccines can also be effective in ameliorating such other autoimmune diseases.
  • V ⁇ 17 refers to a specific human ⁇ -chain variable region of three T cell receptors. V ⁇ 17 has the following amino acid sequence: MSNQVLCCWLC FLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQ IVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASS.
  • V ⁇ 14 refers to a specific human ⁇ -chain variable regions of another TCR.
  • V ⁇ 14 has the following amino acid sequence: MGPQLLGYWLCLLGAGPLEAQVTQNPRYLITV TGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEK RNFPLILESPSPNQTSLYFCASS.
  • V ⁇ 3 refers to a family of specific human ⁇ - chain variable region. Two members of the V ⁇ 3 family have been identified as V ⁇ 3.1 and V ⁇ 3.2.
  • V ⁇ 3.1 has the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKV TQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIP EGYSVSREKKERFSLILESASTNQTSMYLCASS.
  • V ⁇ 3.2 has the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKV TQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIP EGYSVSREKKERFSLILESASTNQTSMYLCASS.
  • V ⁇ 3.2 has the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKV TQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIP EGYSVSREKKERFSLILESASTNQTSMYLCASS.
  • V ⁇ 3.2 has the following amino acid
  • 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 V ⁇ 3, V ⁇ 14 and V ⁇ 17 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 RA studies
  • the results indicate a limited number of V ⁇ s among the activated T cells
  • peptides corresponding to the CDR2 region are viable alternatives for use as vaccines of the present invention.
  • DPGLGLRLIYFSYDVKMKEKG of V ⁇ 14, DPGLGLRQIYYSMNVEVTDKG, or of V ⁇ 17, DPGQGLRLIYYSQIVNKFQKG, can be used.
  • TCR fragment can be joined with any D and J segment of the TCR.
  • immunogenically representative fragments of V ⁇ 3, V ⁇ 14 and V ⁇ 17 are also included in the definition of "V ⁇ 3,” “V ⁇ 14” and “V ⁇ 17,” respectively.
  • substantially pure it is meant that the
  • TCR is substantially free of other biochemical moieties with which it is normally associated in nature. Such substantially pure TCRs or fragments thereof, for
  • TCRs 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 V ⁇ regions that contain sequences of high homology which are conserved among pathogenic TCRs. These regions of conserved homology include the
  • CDRs such as CDR1 and CDR2, which are common to T cells bearing the same V ⁇ , and also the superantigen binding site, which can be common to
  • 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
  • the vaccines are administered to a host
  • autoimmune mechanisms precede the onset of overt clinical disease (for example. Type I Diabetes).
  • prognostic indicators 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
  • 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
  • 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
  • T cell 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 , 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 T cells can be obtained from blood of affected individuals .
  • 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
  • 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
  • the invention also provides a method of
  • V ⁇ -containing T-cells particularly V ⁇ 3, V ⁇ 14 and V ⁇ 17
  • the V ⁇ -containing T cells are treated with a cytotoxic or cytostatic agent that selectively binds to the V ⁇ 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
  • Rheumatoid arthritis is a T cell mediated autoimmune disease.
  • the invention describes clonal infiltrates of activated V ⁇ 3, V ⁇ 14 and V ⁇ 17 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 V ⁇ s 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 ⁇ -chain genes containing virtually any desired V ⁇ gene element.
  • PCR polymerase chain reaction
  • V ⁇ 17 T cells are likely involved in the pathogenesis of RA.
  • a CD4+, V ⁇ 17 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 V ⁇ 17 T cells in RA.
  • RNAs from activated (IL2-R+) synovial T cells were analyzed by PCR-amplification with a panel of V ⁇ - specific PCR primers. In this analysis, V ⁇ 17 transcripts were found in four of the five patients tested,
  • V ⁇ 14 was found in four of the five RA patient samples and V ⁇ 3 and V ⁇ 9 were detectable in three of five patients.
  • V ⁇ 6 is most closely homologous, followed by hV ⁇ 3, hV ⁇ 12.1, hV ⁇ 14, and mV ⁇ 7.
  • V ⁇ 12.1 was negligible in the synovium despite considerable overall homology with V ⁇ 17.
  • V ⁇ 9 was found in three of five synovial samples, yet is only weakly homologous to V ⁇ 17.
  • V ⁇ 9 V ⁇ 9
  • Table 2 The 15 amino acid sequences of these V ⁇ s as well as other human and mouse V ⁇ s are shown in Table 2. Within the ⁇ -chain, this region of conservation corresponds positionally to that previously shown to contain superantigen binding sites.
  • the exogenous superantigen, SEC 2 stimulates human V ⁇ 13.2 T cells as a result of binding to a site on V ⁇ 13.2 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 V ⁇ 13.2.
  • a binding site for Mls-1a, 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.
  • V ⁇ 8.2a the V ⁇ 8.2 isoform common to laboratory mice
  • V ⁇ 8.2c a ⁇ -chain found in wild mice.
  • These ⁇ -chain polypeptides are distinguished functionally by their differential reactivities with Mis- la and structurally by a difference of five amino acids. Of particular importance are the residues at position 70 and 71.
  • the responder ⁇ -chain, V ⁇ 8.2c has lysine and glutamic acid, respectively, at these positions.
  • both these exogenous and endogenous superantigens can bind in the vicinity of the 15 amino acid sequence homology identified in Table 2.
  • the lysine- glutamic acid pair charge motif is implicated in Mls-1a reactivity. Involvement of this charge motif in Mls-1a reactivity is confirmed by its presence in mouse V ⁇ 6 and V ⁇ 7, two other Mls-1a reactive murine ⁇ -chains.
  • the V ⁇ 8.2c, V ⁇ 6 and V ⁇ 7 charge motif of a lysine or arginine followed by a glutamic acid residue is underlined in
  • the present invention is directed to the unexpected discovery that human V ⁇ 3, V ⁇ 14 and V ⁇ 17 have a region that corresponds to the Mls-1a binding site.
  • V ⁇ 12 while displaying a high degree of overall and local homology with V ⁇ 3, V ⁇ 14 and V ⁇ 17, lacks the charge motif, perhaps accounting for its presence in the synovium of only one of five RA patients.
  • V ⁇ 9 shows no overall or local homology to V ⁇ s 3, 14 or 17 and lacks the charge motif.
  • V ⁇ 3, V ⁇ 14 and V ⁇ 17-bearing T cells have been demonstrated among the activated synovial T cells in RA.
  • These three ⁇ -chain polypeptides possess overall and local sequence homolo-gy and an apparent superantigen binding charge motif.
  • V ⁇ 3, V ⁇ 14 and V ⁇ 17 are the only known human V ⁇ chains to possess this apparent superantigen binding site characterized by this local sequence homology and the identified charge motif.
  • it is possible that other V ⁇ s may become known that contain such binding sites.
  • V ⁇ 3, V ⁇ 14 or V ⁇ 17 sequence containing the charge motif can be used as an immunogen in the vaccines of the present invention.
  • the sequences or fragments thereof shown in Table 2 for V ⁇ 3, V ⁇ 14 and V ⁇ 17 can be used.
  • Vaccines containing any combination of these three V ⁇ sequences, including all three sequences, can be used effectively to ameliorate T cell associated diseases.
  • variable regions of the ⁇ -chains of three TCRs designated V ⁇ 3, V ⁇ 14, and V ⁇ 17, are closely
  • 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
  • T cells having the ⁇ -chain variable region from V ⁇ 3, V ⁇ 14 or V ⁇ 17 in a sample from the individual the presence of abnormal levels of such V ⁇ -containing T cells indicating the pathology or susceptibility to the
  • the V ⁇ -containing T cells can be any V ⁇ -containing T cells.
  • Such diagnosis can be performed, for example, by detecting a portion of the V ⁇ s that does not occur on non-rheumatoid arthritis associated ⁇ -chain variable region T-cell receptors.
  • the V ⁇ s of the present invention can be detected, for example, by contacting the V ⁇ s with a detectable ligand capable of specifically binding to the individual V ⁇ s.
  • detectable ligands are known in the art, e.g. an enzyme linked antibody.
  • nucleotide probes are known in the art, e.g. an enzyme linked antibody.
  • V ⁇ - containing T cells can be utilized to detect such V ⁇ - 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 V ⁇ 3-, V ⁇ 14- or V ⁇ 17-containing T-cell receptor to its binding partner.
  • attachment is prevented by binding a ligand to V ⁇ 3, V ⁇ 14 or V ⁇ 17.
  • attachment is prevented by binding a ligand to the V ⁇ 3, V ⁇ 14 or V ⁇ 17 binding partner. Attachment can be prevented by known methods, e.g. binding an antibody to the individual V ⁇ s or to its binding partner in order to physically block attachment.
  • encephalitogenic T cells show striking conservation of ⁇ - chain V(D)J amino acid sequence, despite known
  • 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 ⁇ -chain with a V(D)J amino acid sequence of V ⁇ 4 homologous with that of ⁇ -chains from MBP-reactive T cells mediating pathogenesis in experimental allergic encephalomyelitis (EAE), an animal model of MS, as shown in Table 4.
  • EAE experimental allergic encephalomyelitis
  • Table 4 This finding demonstrates the involvement of MBP-reactive T cells in the pathogenesis of MS and demonstrates that TCR peptides similar to those described herein for the prevention of EAE can be appropriate in treating MS.
  • EAE experimental allergic encephalomyelitis
  • pathologies including MS.
  • the invention is directed to the discovery that ⁇ -chain VDJ fragments homologous to VDJ sequences found in rodent EAE, such as SGDQGGNE and CAIGSNTE, are closely associated with multiple sclerosis in human subjects. This discovery allows for the
  • the invention provides a method of diagnosing or predicting susceptibility to multiple sclerosis in an individual comprising detecting T cells having various V ⁇ s, such as V ⁇ 4 or V ⁇ 12, and particularly having substantially the sequence SGDQGGNE or CAIGSNTE, 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
  • 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 V ⁇ s, including V ⁇ 4, V ⁇ 12 or fragments thereof, such as those having substantially the SGDQGGNE or
  • CAIGSNTE sequence to its binding partner in one
  • 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 V ⁇ s, and in particular to the SGDQGGNE or CAIGSNTE sequences, to physically block attachment.
  • the invention also provides a method of preventing or treating multiple sclerosis in an
  • T-cells are treated with a cytotoxic or cytostatic agent which selectively binds to these V ⁇ s or their immunogenic fragments.
  • the agent can be, for example, an antibody attached to a
  • radioactive or chemotherapeutic moiety is radioactive or chemotherapeutic moiety.
  • T cell lymphoma is another T cell pathology which would be amenable to this type of treatment.
  • the TCR genes of the T lymphomas are seguenced, appropriate regions of those TCRs are
  • the vaccines can comprise single or multiple peptides, and can be
  • the present invention further relates to an alternative method of treating or preventing a T cell mediated pathology by gene therapy.
  • the nucleic acid can be DNA or RNA encoding for TCRs, immunogenic
  • 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
  • the vector is subsequently administered
  • the DNA or RNA-containing vector is injected into the tissue of an individual.
  • the DNA or RNA-containing vector is injected into the tissue of an 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
  • 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 desired TCR, immunogenic fragment or anti-idiotype antibody can be effectively expressed by the cells of the individual as an alternative to
  • 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.
  • EXAMPLE II SELECTION AND PREPARATION OF VACCINES Vaccinations were conducted with a T cell receptor peptide whose sequence was deduced from the DNA sequence of a T cell receptor beta gene predominating among EAE-inducing T cells of BIO.PL mice.
  • the DNA sequence was that reported by Urban, et al., supra, which is incorporated herein by reference.
  • 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
  • IFA incomplete Freund's adjuvant
  • CFA complete Freund's adjuvant
  • 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
  • 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
  • VDJ peptide used in the previous examples was synthesized according to the sequence of TCR ⁇ chain molecules found on EAE-inducing T cells in B10.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 B10.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.
  • 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 IR1, 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.
  • V ⁇ 8.2 (residues 39-59) provided less protection than IR7 as shown in Table 8.
  • the 21 amino acid sequence of V ⁇ 8.2 is DMGHGLRLIHYSYDVNSTEKG.
  • CFA complete Freund's adjuvant
  • 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 IR1, 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 V ⁇ 16mer primer and nested C ⁇ primers for 55 cycles as described in Example X.
  • TCR ⁇ -chain sequences of human MBP-reactive T cells V ⁇ 16mer amplified TCR ⁇ -chain genes from human
  • MBP-reactive T cell lines were sequenced using the C ⁇ seq primer. Amplification products were gel purified, base denatured and sequenced from the C ⁇ seq primer. Readable DNA sequence was obtained from 5 of these lines,
  • RNAs were reversed transcribed and amplified in 20 cycle stage I reactions with the V ⁇ 16mer and C ⁇ ext primers. One ⁇ l aliquots of these stage I reactions were reamplified for 35 cycles with the V ⁇ Re and C ⁇ int primers. One ⁇ l aliquots of these reactions were
  • SGDQGGNE can be synthesized as shown in Example II and used to immunize human subjects by methods demonstrated in Example III. Such immunizations can result in an effective immune response.
  • 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
  • IL2-R human IL2-R
  • Synovial tissue was digested for 4 hrs at 37°C in RPMI + 10% Fetal Bovine Serum (FBS) containing 4 mg/ml
  • a T cell clone was derived from the Ficoll pellet of patient 1008.
  • the cells in the pellet were cultured at 2 x 10 6 /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
  • Cells were typsinized, washed and plated at 2000 cells per well of a 96-well round bottom microtiter plate.
  • TCR ⁇ -chain genes were amplified with several combinations of the primers shown in Figure 2.
  • T cell receptor ⁇ -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 C ⁇ ext 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 V ⁇ 16mer primer, nucleotides and reaction buffer as above but without MgCl 2 to give a final Mg +2 concentration of 3.6 mM. Samples were denatured for 15 minutes at 95°C, 1 unit of heat stable recombinant DNA polymerase (Cetus Corporation, Emeryville, CA, Ampli-taqTM) was added and 20 cycles of PCR conducted.
  • 1 unit of heat stable recombinant DNA polymerase (Cetus Corporation, Emeryville, CA, Ampli-taqTM) was added and 20 cycles of PCR conducted.
  • Each cycle consisted of a 1 min denaturation at 95°C, a two minute annealing step and a two minute extension at 72°C.
  • the first two cycles were annealed at 37°C and 45°C, respectively, and the remainder at 50°C.
  • stage II amplification reactions were added to 100 ⁇ l stage II amplification reactions (Cetus, Gene-Amp KitTM) containing 100 pmols of the C ⁇ int primer and 100 pmols of the V ⁇ 8, V ⁇ 17 or 5'C ⁇ primers or 700 pmols of the V ⁇ 16mer 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 1012lL2.d5 and 1008.8 cultures were amplified with the V ⁇ 16mer and C ⁇ ext primers in stage I reactions and with the V ⁇ 16mer and the C ⁇ int primer in 35 cycle stage II reactions. Reaction
  • V ⁇ 17 rearrangement predominance of a single V ⁇ 17 rearrangement in this sample reflects in vivo clonal expansion of V ⁇ 17+ T cells in this patient.
  • V ⁇ 17 TCR DNA could be amplified from magnetic bead samples derived from the 4 patients examined. Ethidium bromide staining of
  • V ⁇ 17 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 V ⁇ 16mer and C ⁇ ext primers and then reamplified for twenty cycles with the C ⁇ int primer and each of the V ⁇ 17, BB8 and 5'C ⁇ primers.
  • Amplification reactions were serially diluted in 20X SSC, denatured by boiling and chilled in an ice slurry.
  • amplified was increased in the IL-2R+ fraction only in patient 1015.
  • V ⁇ 17 rearrangements from the IL-2R+ RNAs of the three patients showing enrichment were amplified with the V ⁇ 17 and C ⁇ int primer pair and the reaction products sequenced with the C ⁇ seq primer.
  • sample 1012 IL-2.d5 1014 and 1015 contained single sequences (Table 14), indicative of clonal expansion of V ⁇ 17 T cells in vivo.
  • direct sequencing of the rearrangements amplified with the V ⁇ 8 specific primer was not possible due to significant heterogeneity in the ⁇ - chain product.
  • the resulting negative strand probes were hybridized to slot blots containing 10 pmol of the HLA-DR allele specific oligos (positive strands) using conditions previously described by Amar et al., J. Immunol. 138:1947 (1987), which is incorporated herein by reference.
  • the slots were washed twice for 20 minutes with
  • Each of the patients in this study possessed at least one allele of the HLA-DR genes, DR4w4, DR1, DR4w14 or DR4w15, that are known to predispose for RA (Table 16). Also shown in Table 16 are HLA-DR allele specific oligonucleotides.
  • T cell receptors containing V ⁇ 17 or fragments thereof which are immunogenic or can be made immunogenic can be used to immunize human subjects by methods
  • Synovial tissue specimens were obtained from proven RA patients undergoing joint replacement surgery. HLA-DR analysis was conducted as described in Example IX(D).
  • PCR Polymerase chain reaction
  • T cell receptor ⁇ -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 C ⁇ ext primer (40 pmol) in 12 ⁇ l of reaction buffer (Hart et al.,
  • Samples were denatured (15 minutes, 95°C) and 20 cycles of PCR were conducted using Tag polymerase (1 unit, Cetus Ampli-taq). Each cycle consisted of a 1 minute denaturation at 95°C, a two minute annealing step and a two minute extension at 72°C. The first two cycles were annealed at 37°C and 45°C and the remainder at 50°C.
  • Stage II amplification reactions (Cetus Gene-Amp Kit) containing the C ⁇ int primer (100 pmol) and either the V ⁇ 8, V ⁇ 12, V ⁇ 17, or 5'C ⁇ primers (100 pmol) or the V ⁇ cons 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 45oC ramping.
  • V ⁇ 17 T cells are cytotoxic for synovial adherent cells
  • 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 4 cells/well in flat- bottomed, 96-well microtiter plates and cultured
  • 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
  • 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. Cytotoxicity assays were conducted as described
  • 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+. One culture designated 1008.8, grew especially vigorously and, as assessed microscopically promoted the destruction of the monolayer cells. CTL assays confirmed the cytotoxicity of 1008.8 for these monolayer targets as discussed above.
  • Cytolysis was specific for synovial cell targets, as no lysis of autologous Epstein-Barr virus-transformed B cells was demonstrable. Neither of the targets was lysed by a CD4+, myelin basic protein-reactive human CTL clone. MS3, excluding the possibility that the monolayer cells were susceptible to lysis by any activated T cell. In repeated assays with 1008.8, specific lysis never
  • synovial target cell comprises approximately that proportion of the total monolayer culture, which based on morphology is a mixture of multiple cell types.
  • T cell receptor (TCR) ⁇ -chain gene of TCR The T cell receptor (TCR) ⁇ -chain gene of TCR
  • PCR polymerase chain reaction
  • V ⁇ ons V ⁇ ons
  • n 16 nucleotide primer
  • V ⁇ l7 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 V ⁇ cons and C ⁇ ext primers. One ⁇ l of each reaction was reamplified for 20 cycles in individual stage II reactions containing the C ⁇ int primer in
  • V ⁇ 17, V ⁇ 8, V ⁇ 12 and 5'C ⁇ primers were diluted in 20X SSC, denatured by boiling and chilled in an ice slurry.
  • V ⁇ 17 T cells in the synovial tissue of other RA patients was determined. Since the rheumatoid synovium contains a mixture of activated and non-activated T cells, the activated T cells were identified as the most relevant for the initiation and perpetuation of the disease pathogenesis. Thus, activated T cells from single-cell suspensions of synovial tissue were selected using magnetic beads and antibodies reactive with the human interleukin-2 receptor (IL-2R). Cell suspensions from each patient were
  • 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.
  • V ⁇ 17 T cells were reverse transcribed, preamplified with V ⁇ con and C ⁇ ext and reamplified in separate reactions with a constant region primer (C ⁇ int) and each of the V ⁇ - specific primers, V ⁇ 17, V ⁇ 8 and V ⁇ 12.
  • C ⁇ int constant region primer
  • the second stage amplification was also performed with two C ⁇ primers (5'C ⁇ and C ⁇ int) in order to estimate the total ⁇ -chain present in each sample and to provide benchmarks for normalizing the results of specific V ⁇ quantification in the respective IL-2R+ and control sample pairs.
  • the proportion of V ⁇ 17 DNA, relative to total C ⁇ was
  • RNAs were reverse transcribed and amplified with the V ⁇ cons and C ⁇ ext primers in 20 cycle stage I reactions and with the C ⁇ int and V ⁇ cons (1008.8) or V ⁇ 8, VT312, and V ⁇ 17 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.
  • V ⁇ 17 repertoire in the RA synovium is of limited heterogeneity, indicative of clonal or oligoclonal expansion of V ⁇ 17 T cells in vivo. This is in contrast to the V ⁇ 8 and V ⁇ 12
  • V ⁇ 8 and V ⁇ 12 samples analyzed were directly sequenceable and plasmid cloning of V ⁇ 8 rearrangements from patient 1012 revealed 4 different sequences in 5 clones analyzed.
  • V ⁇ 17 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 V ⁇ 17 primer, as for the synovial sample, and the products cloned into plasmid.
  • T cell receptor ⁇ -chain genes were amplified in two-stage reactions with individual V ⁇ -specific primers as described in Wucherfpennig et al., Science, 248:1016- 1019 (1990), incorporated herein by reference, and nested C ⁇ primers.
  • Stage II amplifications were conducted for 20 cycles with a 50°C annealing temperature and without the 37oC and 45°C ramping. Five ⁇ l of each reaction was
  • V ⁇ 17 , V ⁇ 14 , VB9 and V ⁇ 3 transcripts are common among activated synovial T cells
  • TCR transcripts in the IL2-R+ samples were analyzed with a panel of 19 PCR primers , specific for known human V ⁇ gene families (Table 18).
  • the V ⁇ cons primer of Example XI is equivalent to the V ⁇ 1 ⁇ mer primer.
  • the number of V ⁇ genes detectable in these samples was variable, ranging from two to twelve.
  • V ⁇ 17 was found in four of the five patients, confirming the previous analyses using the V ⁇ 16mer primer.
  • V ⁇ 14 transcripts also were found in four of the five patients and V ⁇ 3 and V ⁇ 9 transcripts were detectable in three of the five samples.
  • T cells bearing these 3 V ⁇ polypeptides may also contribute to synovial inflammation.
  • Rats were immunized with 100 ⁇ g of a synthetic peptide containing the sequence VPNGYKVSRPSQGDFFLTL found in the fourth hypervariable or CDR4 region of the rat TCR ⁇ chain identified as V ⁇ 8.2.
  • the peptide dissolved in saline, was emulsified in an equal volume of complete Freund's adjuvant (CFA) containing 10 mg/ul of CFA
  • Synthetic peptides representing amino acid sequences from the CDR4 region of both rat B ⁇ 8.2 T cells and human V ⁇ 17 T cells can protect rats from the
  • the rat V ⁇ 8.2 CDR4 peptide has the sequence VPNGYKVSRPSQGDFFLTL (residues 61-79) and the human V ⁇ 17 CDR4 peptide has the sequence EKKESFPLTVT (residue 68-79).
  • Lewis rats were immunized with emulsions containing equal volumes of peptide in saline and of IFA intradermally in 0.1 ml doses divided equally between the two hind footpads (0.05 ml/footpad).
  • Each animal of Groups A and B received 100 ⁇ g of peptide.
  • Group C 50 ⁇ g of peptide.
  • Group D received IFA alone and Group E, no treatment.
  • each animal was challenged with 50 ⁇ g guinea pig myelin basic protein (GP-MBP) in saline emulsified with an equal volume of complete
  • Table 20 shows that animals receiving either human CDR4 (Group A) or rat CDR4 (Group B) had mild or nonclinical signs of disease after challenge whereas animals receiving control peptide (C), IFA (D) or no treatment (E) had a normal disease course.
  • C control peptide
  • D IFA
  • E no treatment
  • Table 21 shows that animals receiving human CDR4 peptides (Group A) from V ⁇ 17 or V ⁇ 14 had mild or no disease and 3 of 5 animals receiving a similar V ⁇ 3 peptide had no disease. In contrast, animals receiving IFA (Group D) or no pretreatment (Group E) had a normal disease course.

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Abstract

L'invention concerne des vaccins ainsi qu'un moyen de vaccination d'un vertébré de manière à prévenir ou à réguler des pathologies induites par des lymphocytes T spécifiques, y compris des maladies auto-immunes ainsi que la réplication non régulée de lymphocytes T. Le vaccin se compose d'un récepteur de lymphocytes T (TCR) ou d'un fragment de celui correspondant à un TCR présent à la surface de lymphocytes induisant la pathologie. Le fragment du vaccin peut être un peptide correspondant à des séquences de TCR caractéristique des lymphocytes T induisant ladite pathologie. Ledit peptide peut se fixer à des antigènes classiques dont la transformation en complexe permet d'obtenir des cellules présentant des antigènes de complexe majeur d'histocompatibilité ou des superantigènes. L'invention concerne également des moyens de détermination des séquences d'acides aminés appropriés destinés à ces vaccins. Le vaccin est administré aux vertébrés d'une manière induisant une réponse immunitaire dirigée contre le TCR de lymphocytes T induisant la pathologie. Cette réponse immunitaire réduit ou supprime les lymphocytes T pathogènes, stoppant ainsi la pathogénèse de la maladie. L'invention concerne également des régions variables à chaînes beta spécifiques des récepteurs de lymphocytes T, appelés Vbeta3, Vbeta4, Vbeta12, Vbeta14 et Vbeta17, lesquels sont associés à la pathogénèse de maladies auto-immunes, telle que la polyarthrite rhumatoïde et la sclérose en plaques. En outre, l'invention concerne des procédés d'administration d'ADN ou d'ARN codant les polypeptides utiles en tant que vaccins de l'invention, dans les cellules tissulaires d'un individu.
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WO1993012814A3 (fr) 1993-09-30
WO1993012814A2 (fr) 1993-07-08

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