EP0605606A1 - Diagnostic et therapie de maladies auto-immunitaires - Google Patents

Diagnostic et therapie de maladies auto-immunitaires

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Publication number
EP0605606A1
EP0605606A1 EP92920872A EP92920872A EP0605606A1 EP 0605606 A1 EP0605606 A1 EP 0605606A1 EP 92920872 A EP92920872 A EP 92920872A EP 92920872 A EP92920872 A EP 92920872A EP 0605606 A1 EP0605606 A1 EP 0605606A1
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European Patent Office
Prior art keywords
seq
sequence
beta
tcr
composition
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EP92920872A
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German (de)
English (en)
Inventor
Edward P. Amento
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Genentech Inc
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Genentech Inc
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Priority claimed from US07/779,444 external-priority patent/US5203631A/en
Application filed by Genentech Inc filed Critical Genentech Inc
Priority claimed from PCT/US1992/008094 external-priority patent/WO1993006135A1/fr
Publication of EP0605606A1 publication Critical patent/EP0605606A1/fr
Withdrawn legal-status Critical Current

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Definitions

  • This invention relates to autoimmune diseases.
  • it relates to methods for treating rheumatoid arthritis and for diagnosing and treating autoimmune disorders.
  • Autoimmune disorders are characterized by an immune attack mounted against one or more self antigens. They include arthritis (osteo- or rheumatoid), Issac's syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus, Sjogren's syndrome, rheumatic fever, ankylosing spondylitis, Reiter's disease, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), Guillain-Barré-Strohl syndrome, primary biliary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris and Graves' disease.
  • arthritis osteo- or rheumatoid
  • Issac's syndrome psoriasis
  • insulin dependent diabetes mellitus multiple sclerosis
  • This invention is especially concerned with T cell-mediated autoimmune disorders, i.e. chronic autoimmune responses in which T-cells are primarily involved in the direct immune response (in contrast to such disorders as some forms of glomerulonephritis which primarily result from the generation of excessive amounts of antibody-antigen complexes).
  • T cell-mediated autoimmune disorders i.e. chronic autoimmune responses in which T-cells are primarily involved in the direct immune response (in contrast to such disorders as some forms of glomerulonephritis which primarily result from the generation of excessive amounts of antibody-antigen complexes).
  • T-mediated autoimmune disorders i.e. chronic autoimmune responses in which T-cells are primarily involved in the direct immune response (in contrast to such disorders as some forms of glomerulonephritis which primarily result from the generation of excessive amounts of antibody-antigen complexes).
  • myasthenia gravis myasthenia gravis, inflammatory bowel disease, insulin dependent diabetes mellitus and rheumatoid arthritis
  • Rheumatoid arthritis is a chronic heterogeneous disorder in which a variety of etiological agents may be responsible for initiating a series of events leading to inflammation in multiple joints.
  • the cause of the disease remains unknown, although by analogy with other forms of arthritis such as that accompanying Lyme disease, it has been postulated that infection with as yet unidentified bacteria or viruses in a genetically susceptible host is an initiating event. Persistence could result from the presence of viral or bacterial antigens that generate an immune response or cross-react with host tissues together with amplification effects of cellular products of the host.
  • RA While many patients have systemic manifestations in RA, many of the most serious consequences of RA stem from its effects on articular connective tissues, which are characterized by alterations of the synovial membrane with proliferation of lining cells and infiltration by chronic inflammatory cells. Erosions of bone occur in areas contiguous with the inflammatory cell mass as well as in regions adjacent to bone marrow distant from the inflammation. The bone erosions are probably produced through induction of differentiation and activation of osteoclast progenitors.
  • the erosion of soft connective tissues results from direct release of proteolytic enzymes from cells of the inflammatory cell mass or from polymorphonuclear leukocytes that are typically abundant in rheumatoid synovial fluids, although rare in the synovial membrane.
  • proteolytic enzymes from cells of the inflammatory cell mass or from polymorphonuclear leukocytes that are typically abundant in rheumatoid synovial fluids, although rare in the synovial membrane.
  • the major macr omolecules contributing to the structural integrity of the extracellular matrix are the interstitial collagens.
  • Other matrix components include glycoproteins and proteoglycans.
  • Degradation of articular cartilage could not only result from release of proteases from cells (synovial fibroblasts and monocyte/macrophages) outside the cartilage, but chondrocytes within the cartilage may be induced to degrade their own matrix. This process is relevant not only to rheumatoid arthritis, but also to polychondritis and osteoarthritis.
  • Human mononuclear phagocytes release relatively little collagenase, but these cells contribute to matrix degradation by producing other enzymes, such as elastase, as well as active oxygen species.
  • lymphocytes directly or indirectly can stimulate synovial fibroblasts to produce enzymes that destroy joint tissue.
  • IL-1 and other growth/activation factors including TNF/cachectin, stimulate proliferation of the synovial cells and induce biosynthesis of prostaglandins and proteinases by the synovial cells.
  • Adherent synovial cells respond by mitogenesis to factors that do not stimulate production of prostaglandins and/or proteinases.
  • Synovial cells exposed to IL-1 react with a battery of responses, including the expression of prostaglandins, metalloproteinases, the proto-oncogene products fos and jun, and melanoma growth stimulatory activity (MGSA).
  • the responses of synovial cells to IL-1 are modified by the action of other lymphokines and monokines such as TNF- ⁇ and TNF- ⁇ , produced by the inflammatory cells.
  • the cytokines act primarily through cell surface receptors to activate transcription of the procollagenase gene, probably mediated by complexes of nuclear oncoprote ⁇ ns.
  • T lymphocytes that are present at the disease site. Activated T lymphocytes are present in rheumatoid synovial tissue and frequently are found adjacent to macrophages. Clonai dominance of T lymphocytes has been observed using Southern blot analysis of DNA obtained from synovial T-cells of patients with rheumatoid arthritis (Stamenkovic et al., Proc. Natl. Acad. Sci. 85:1 179-1 183 [1988]).
  • Stage 1 where a postulated antigen is presented to T-cells with no obvious arthritic symptoms, is not treated.
  • Stage 2 involves T-cell and B-cell proliferation and angiogenesis in synovial membrane, resulting in malaise, mild joint stiffness, and swelling.
  • Stage 3 neutrophils accumulate in synovial fluid and synovial cells proliferate without polarization or invasion of cartilage, resulting in joint pain and swelling, morning stiffness, malaise, and weakness.
  • Current therapy for Stages 2 and 3 includes bed rest, application of heat, supplemental icosapentaenoic and docosahexanoic acid, and drugs.
  • Nonsteroidal anti-inflammatory drugs continue to be the foundation of drug therapy in treating Stages 2 and 3 of the disease.
  • Those anti-inflammatory drugs other than aspirin include indomethacin, phenylbutazone, phenylacetic acid derivatives such as ibuprofen and fenoprofen, naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, zomepirac, and diflunisal.
  • Second-line drugs for RA Stages 2 and 3 include anti-malarial drugs such as hydroxychloroquine, sulfasalazine, gold salts, and penicillamine, and low-dose methotrexate. These alternatives frequently produce severe side effects, including retinal lesions and kidney and bone marrow toxicity.
  • cyclic oligosaccharides which, when combined with a noninflammatory steroid (cortexolone), inhibit angiogenesis in vivo.
  • cyclodextrins cyclic oligosaccharides
  • cortexolone noninflammatory steroid
  • Antibodies against crucial components of the early phase of the immune response include anti-Class II MHC antibodies [Gaston et al., Arthritis Rheum., 31 : 21 -30 (1988); Sany et al., Arthritis Rheum., 25: 17-24 (1982)1, anti-interleukin-2 receptor antibodies [Kyle et al., Ann. Rheum.
  • Cytokines or their inhibitors have properties that could down-regulate inflammatory and proliferative pathways in RA.
  • TGF- ⁇ transforming growth factor- ⁇
  • Cytokines or their inhibitors have properties that could down-regulate inflammatory and proliferative pathways in RA.
  • TGF- ⁇ transforming growth factor- ⁇
  • EMBO J., 6 1899-1904 (1987).
  • the T-Cell Receptor is a glycoprotein with a molecular weight of 89 to 90 kdal. It is a dimer consisting of one alpha polypeptide chain and one beta polypeptide chain, each about 40 to 50 kdal, linked by disulfide and noncovalent bonds. In addition, two more TCR chains have been identified, the gamma and delta chains. The TCR exhibits structural and amino acid sequence homology with immunoglobulins. Each TCR chain contains, in N- to C- terminal order, a leader sequence, a variable domain (made up of at least V and J sequences), a constant domain, a transmembrane sequence, and an endoplasmic domain.
  • the genome contains a multiplicity of V, P, and J segments that must be assembled by DNA recombination to produce a particular TCR. This assembly occurs during thymic development of the T-cell repertoire, at least for alpha and beta chains. As a result, a clone or group of T-cells will bear the same TCR, and this clone or group will be found in the presence of a number of other clones bearing differently rearranged TCR.
  • TCR beta chain V region there are approximately 24 presently known families of the TCR beta chain V region, each family consisting of a number of alleles of a distinctive V domain (there are over 40 such alleles embraced within the 24 presently known families).
  • Beta chain V region families are henceforth designated V ⁇ X, where X is the family designation.
  • the joining or "J" domains of the beta chains are grouped into 2 large classes, 1 and 2, and these in turn are subdivided between them into about 14 presently known families. Finally, 2 classes of beta chain constant domains of have been identified.
  • V ⁇ 8 genes (note that the beta chains of more than one species bearing the same numerical designations should not be assumed to be homologous) are preferentially used by encephalitogenic T-cells in mice with experimental allergic encephalomyelitis (EAE), and that immunization with a fragment of V ⁇ 8 protects against induction of the disease (Howell et al., "Science” 3 November 1989, pp 668- 670; Vandenbark et al., "Nature” 341:541 -544 [19891; and Offner et al., "J. Immun.” 146:4165-4172 [1989]). Howell et al., WO 90/1 1294, published Oct.
  • Vandenbark et al., WO 91.01 133 contains disclosure relating to the screening of TCR peptides by DH response, administration of pharmaceutical compositions (anti-TCR and TCR compositions), and notes a bias to use of V beta 5.2 in humans in MS.
  • Wucherpfenning et al., W0 91 /15225 teach that MS in humans is linked to V beta 12 or 17.
  • compositions for the treatment of autoimmune disorders which comprise consensus and/or hybrid sequences of TCR domains amplified in such disorders.
  • Another object is to facilitate and simplify the diagnosis of TCR subdomain usage in an autoimmune disease.
  • Another object of this invention is to improve administration of the TCR or its fragments. It is another object of this invention to provide improved methods for the diagnosis and treatment of RA and other autoimmune disorders.
  • RA is treated in human patients by administering a therapeutically effective amount of a polypeptide comprising an amino acid sequence of a human TCR V, J or C domain selected from the group of V ⁇ 7, V ⁇ 8, V ⁇ 12, V ⁇ 13, V ⁇ 14, V ⁇ 15, J ⁇ 2.1, J ⁇ 2.2, V ⁇ 2.3, J ⁇ 2.5, J ⁇ 2.7 or C ⁇ 2, or of an antibody capable of binding specifically to such sequence.
  • TCR domains appearing with greatest frequency in RA patients share a great deal of amino acid sequence.
  • consensus or hybrid TCR domains are employed for patients having a given autoimmune disorder.
  • a "universal" J ⁇ polypeptide family of consensus or hybrid sequences, and their amino acid sequence variants, are provided for the treatment of RA.
  • A is hydrogen, a sequence of about from 1 to 10 residues found in a TCR at the analogous site, a nonpeptidyl polymer, a non-TCR polypeptide, a TCR polypeptide selected from the group of a V beta 7, 8, 12, 13, 14 or 15 sequence, or a blocking group;
  • Xaa1 is Glu or an amino acid residue having a hydroxy-substituted alkyl or hydroxy-substituted heteroalkyl side-chain;
  • Xaa2 is Gin or an amino acid residue having a hydrophobic side- chain;
  • Xaa3 is an amino acid residue having a cycloalkyl or hydroxy-substituted cycloalkyl side-chain; each of a, b and c are independently 1 or zero, provided that neither b nor c are zero if a is 1 and c is not zero if b is 1 ;
  • Xaa4 is Pro or Glu;
  • Xaa5 is
  • V beta consensus or hybrid sequences are designed by selecting residues from V beta 12, 13, 14, 15, 3, 7 and 8.
  • This invention also relates to methods for determining the specific V ⁇ or J ⁇ usage of T-cells in autoimmune diseases which does not require outgrowth of T-cells from affected tissue.
  • the patient's T-cell population is screened by exposing the population to a bank of TCR polypeptides to identify the existence of T-cells that respond to each polypeptide.
  • a T-cell proliferative response is taken as evidence of the existence of T-cells directed against T-cells bearing the test TCR, and therefore that such TCR-bearing T-cells are targets for therapy.
  • the peripheral T-cell population is assayed either in situ, for example by use of a multiple skin prick test using the bank of TCR polypeptides and observing for a DTH reaction or by use of an in vitro environment, e.g. by measuring the proliferative response in culture wells of the patient's peripheral blood T-cells (PBLs) to the TCR polypeptides.
  • PBLs peripheral blood T-cells
  • This invention also relates to a method for treating T-cell mediated autoimmune conditions in a patient by administering a V ⁇ polypeptide corresponding to a TCR expressed on clonally amplified T-cells (as determined by the method of this invention) in the patient or expressed on T-cells persistent at the site of inflammation.
  • the J ⁇ polypeptide for use in RA is set forth in Formula I.
  • Other J and V ⁇ domains preferentially used by T-cells in other autoimmune disorders such as those described supra are identified in the same fashion as is described herein for RA.
  • treatment with the TCR or a domain thereof is facilitated by simply administering a therapeutically effective dose of the TCR or its domain into the inflammatory lesion, e.g., the synovial fluid in RA, or directly into the vascular system - without using an immune adjuvant such as Freunds adjuvant or the like to stimulate the patient's response to the TCR or its selected subdomain.
  • an immune adjuvant such as Freunds adjuvant or the like
  • Fig. 1 illustrates the relative frequency of appearance of V beta usage in synovial infiltrating lymphocytes from RA patients.
  • a suitable process for identifying preferential TCR use in RA or other autoimmune disorders comprises first obtaining a sample of synovium or other inflamed T-cell infiltrated tissue ("affected tissue") from the patient concerned.
  • affected tissue synovium or other inflamed T-cell infiltrated tissue
  • synovium samples are obtained at the time of joint replacement or other surgical procedures such as
  • synovectomy involving an affected joint of the patient, although it is possible to obtain the samples by biopsy at the same time synovial fluid is expirated for analysis.
  • the sample of the affected tissue is then placed in culture medium in the presence of IL-2 and the T-cells are allowed to proliferate from the tissue. Thereafter the proliferating T-cells are transferred to culture in the presence of IL-2. and exposed to cross-linked antiCD3 antibodies every 7-10 days. They are grown until adequate amounts of T-cells are obtained for analysis. Any method for determining the TCR families in these cells is acceptable, for example. Southern blotting will determine clonai rearrangement of TCR.
  • PCR analysis (using individual priming oligonucieotides capable of hybridizing only to one of the 14 families of J ⁇ or the 24 families of V ⁇ ) is preferably used to identify the TCR domain (J ⁇ or V ⁇ ) family member. Nucleic acid sequencing will confirm the identity of the specific V ⁇ family member and serve as another means to determine J ⁇ and C ⁇ usage. Alternatively, individual priming oligonucieotides can be used to determine the TCR domain usage. A beta chain C domain primer is used as a control for the V ⁇ or J ⁇ assay. The PCR method is semiquantitative, and differs from that of the prior art (Skibbens et al., supra) in its treatment of background TCR usage.
  • the peripheral blood lymphocyte (PBL) usage of TCR regions is determined, but in a different fashion than that of the art.
  • the amount of mRNA or cDNA obtained in conventional fashion from PBLs from pooled blood (or from the individual patient being analyzed) is titered so that the quantity used is at the detectability threshold of the PCR amplification conditions to be employed in the affected tissue analysis. That is, under the conditions selected, the PCR analysis will not detect any peripheral TCR use but will detect at least one TCR domain in the affected tissue.
  • the titered amount of mRNA or cDNA to be used will vary depending upon the PCR conditions selected used, but this can be determined by routine methods. In contrast, the Skibbens et al.
  • the method laboriously amplifies each TCR domain from PBLs and from tissue and prepares a ratio of the degree of usage in tissue to that in PBLs for each TCR domain, and then identifies T-cell clones responsible for the autoimmune response on the basis of those possessing the highest numerical ratio of tissue: PBL usage. Accordingly, the method herein does not determine individual TCRs tissue PBL ratios. Stated differently the baseline control in the method of this invention is the highest relevant TCR domain (e.g. all V ⁇ or J ⁇ ) usage in PBLs. As noted this difference produces more accurate results than those reported by Skibbens et al. and the method is much simpler to use in practice. The foregoing method is described in more detail in the examples below.
  • the RA patient's preferential Beta chain usage is determined by a DTH (Delayed Type Hypersensitivity) test using the otherwise conventional skin prick method with a bank of V ⁇ and/or J ⁇ polypeptides as set forth herein. Those polypeptides to which the patient exhibits a DTH response are administered to the patient for treatment of the patient's autoimmune disorder.
  • DTH Delayed Type Hypersensitivity
  • a (RA or other autoimmune disease) patient's peripheral blood T-cell proliferation response to a bank of V ⁇ or J ⁇ peptides may be used to determine the specific V ⁇ or J ⁇ peptides to be used for further treatment.
  • PBLs from a peripheral source e.g., blood from the patient
  • TCR or TCR domain polypeptides that stimulate PBL growth are selected as candidate therapeutic agents for treating that patient.
  • Normal donor PBLs preferably from individuals who do not carry the MHC class I or II susceptibility determinants for RA or the autoimmune disease to be treated) are used as controls.
  • V ⁇ 14 is also designated V ⁇ 3.3 in the literature; they have the same sequence. All patients used C ⁇ 2 with virtually each V beta (Table III). Most patients used more than one domain preferentially. Since some TCRs may have been derived from CD8 + cells, subtraction of the contributions from these cells can be expected to remove some of the indicted TCRs from consideration, particularly those which are found in low frequency (1 or 2 instances). Further studies have demonstrated that the most prevalent V ⁇ are V ⁇ 12 and 13, then V/I 4 followed by V ⁇ 8, 7, 3 and 15. The remaining V ⁇ 's are found in minor proportions of patients.
  • antagonists are either 1 ) the target TCR domain or a biologically active fragment thereof or 2) an antibody or receptor capable of specifically binding to and neutralizing the identified TCR domain.
  • TCR beta chain containing one of the target V, J or C sequences
  • an isolated domain of the TCR such as a J or ⁇ / ⁇ region or fragment thereof.
  • the TCR polypeptides are synthesized by recombinant or in vitro methods using the known amino acid sequences of the domain in question.
  • the TCR domain sequence chosen is exactly that of the patient to be treated.
  • therapy is more convenient if the sequence chosen is a consensus sequence and thus does not represent the sequence of any naturally occurring domain per se. This may reduce the number of polypeptides in the therapeutic "bank".
  • V ⁇ polypeptide will contain the sequence of about residues 39-59 of the rat CDNA clone V ⁇ 510 (Vandenbark et al., Nature 341 :541-544 [1988]) or to the rat VDJ2 sequence SSDSGNTE (SEQ.ID.2) OR ASSDSGNTE (SEQ.ID.3).
  • Preferred for use herein are the V beta 7, 8, 12, 13, 14 or 15 sequences shown in Table I, together with their naturally occuring alleles falling within the same families.
  • the sequences in Table I represent the CDR2 domains of the various human V ⁇ s.
  • the polypeptide antagonists for use in the therapy of RA are selected from within the V beta domains. They will contain about from 5 to 15 residues in most instances, but may constitute the entire CDR2 domain. Polypeptides of less than full V beta length shown in Table I generally will have N or C-terminal deletions of from 1 to about 5 resides.
  • fragments are the series of fragments containing about from 6 to 12 residues, preferably about 10 residues, commencing at residue 1, 2, 3, 4, 5, or 6 of each V beta domain, in particular the V beta 7, 8, 13, 14 and 15 domains.
  • Fragments ordinarily comprise the sequences DPG, LGL, RLl, YYS, YGV, KDT, DKG, PGL, GLR, LIY, YSY, GVK, DTD, KGE, GLG, LRL, IYY, SYG, VKD or TDK.
  • V beta fragments optionally are fused to form inter-V beta hybrid domains, as is described more fully below.
  • consensus sequences are derived from the autoimmune (particularly RA)-linked V beta sequences.
  • the residues selected for any site within the V beta sequence are, for RA, ranked on the basis of the frequency of usage as shown in Fig. 1 , with the optimal residue being that which is shared at the given site by the greatest number of V beta sequences falling within the foregoing group.
  • the same analysis is applied to other TCR V beta domains associated with other autoimmune disorders.
  • Hybrid V beta sequences comprise regions of about from 4 to 15 residues selected from at least 2 of each of the relevant V beta sequences.
  • the component sequences used in the hybrid preferably are arranged in homologous order, e.g., for RA a exemplary hybrid would contain selected N-terminal sequence of V beta 13 fused at its C-terminus to the N-terminus of the residues making up the C-terminal domain of V beta 8 which is homologous to the remaining C-terminal residues of V beta 13.
  • An exemplary hybrid comprises residues 1-6 of V beta 14 and residues 7-22 of V beta 7. In general, however, the hybrids will contain no less than about 7 residues of each V beta domain.
  • hybrids which contain more than 2 V beta sequences, and up to 5 or more, at least several of which are not homologousiy positioned. These are more properly characterized as heteropolymeric V beta domains or fragments of V beta domains.
  • the hybrid V beta sequences also optionally include consensus sequences.
  • V beta consensus polypeptides intended for RA treatment comprise the following sequence of residues, from the N- to C- terminus (residues at each site are listed in order of preference; the sites are from Table I and are numbered from left to right of the sequences shown in Table I): 1 : D, K, T; 2: P, A, M; 3: G, K, M; 4: L, H, M, K, R; 5: G, P; 6: L, P; 7: R, E; 8: -, L; 9: L, Q, M; 10: I, F; 1 1 : Y, H, V; 12: Y, F; 13: S, N; 14: Y, V, M, N, F; 15: G, N, D, E; 16: V, A, K; 17: K, G, E, L, P; 18: I, D, V, S, M; 19: T, l, D, K; 20: -, N; 21
  • V beta sequences herein whether fragments, consensus sequences or hybrids, and whether or not the RA-linked TCR domains described above, have N- or C-terminii which are the same as A and B defined in Formula I.
  • the V ⁇ polypeptides optionally are covalently labelled as is described elsewhere herein for the J ⁇ peptides.
  • A preferably is hydrogen and B is hydroxyl.
  • the terminal portions of the compound are not believed to be important for biological activity and thus can be substituted with virtually any radical.
  • substances which are clinically and unacceptably toxic to patients or which interfere with suppression of the target TCR-bearing T-cell clone preferably are not included with A or B, though it will be appreciated that such radicals can be present if the intended use for the
  • polypeptide is as an in vitro diagnostic reagent.
  • a or B also may be blocking groups usable in the in vitro synthesis of the polypeptides.
  • a or B include nonpeptid ⁇ l polymers such as polysaccharides, polyethylene glycol, pluronic copolymers or other
  • polyoxyalkylenes or non-TCR polypeptides such as an antigen from a microbial or non- human animal source, an immunoglobulin chain (wherein the TCR polypeptide replaces a CDR, hypervariable region, Fv, Fc or fragment thereof), a receptor chain (replacing a transmembrane and/or cytoplasmic domain), a cytotoxic polypeptide such as ricin A chain, and the like.
  • Polysaccharides include dextrans or other carbohydrates useful, for example, as matrices for insolubilization of the sequence for use in the purification of anti-TCR domain antibodies.
  • the A or B groups are substituted onto other TCR fragments or domains associated with other autoimmune disorders.
  • Xaa1 may be either Glu or an amino acid residue having a hydroxy substituted side chain, e.g. Thr, Hydroxypro or Tyr. Generally, the side chain will be a hydroxy-substituted alkyl or hydroxy-substituted heteroalkyl group.
  • alkyl or heteroalkyl means a radical comprising a linear, secondary or tertiary saturated alkyl group containing from 1 to about 10 carbon atoms and, optionally, one or more S or O heteroatoms (in the form of esters or ethers, typically).
  • amino acid residue refers to the structure - (H)N-C(R)(H)-C(O)- wherein R is a side chain as noted.
  • Xaa2 generally is Gin or a amino acid residue having an alkyl side chain such as Leu.
  • other hydrophobic residues such as those having aryl or alkyl aryl side chains can be employed, e.g. Phe.
  • Xaa3 is preferably Phe or Tyr.
  • the cycloalkyl group is bonded to the alpha carbon directly or indirectly by alkyl of from 1 to 3 carbon atoms.
  • the hydroxyl, if present, is generally in the para position.
  • Xaa1 , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 or Xaa7 need to be naturally occurring amino acid residue, although it is preferable if they are.
  • Xaa4 is preferably Pro.
  • Xaa5 is a residue having a hydroxy substituted side chain, e.g. Thr, Hydroxypro or Tyr.
  • the side chain will be a hydroxy-substituted linear, secondary or tertiary saturated alkyl group containing from 1 to about 10 carbon atoms and, optionally, one or more S or 0 heteroatoms (in form of esters or ethers, typically).
  • Xaa5 is Thr.
  • Xaa6 and Xaa7 preferably are Thr or Leu, although either also can be any residue having an alkyl, heteroalkyl, hydroxy-substituted alkyl or hydroxy substituted heteroalkyl side chain.
  • Xaa1 , Xaa2, Xaa3, Xaa7 are not believed to critical and may be omitted, although Xaa3 and Xaa7 are preferentially present if any of these four residues is deleted.
  • the polypeptides are useful as diagnostic reagents.
  • the J ⁇ polypeptides of formula 1 or the V beta sequences are covalently substituted with, fluorescent groups, chemiluminescent groups, haptens, radioisotopes, enzymes, stable free radicals and the like detectable functionalities.
  • the N or C-termininus of the polypeptide is the preferred substitution site, although amino acid side chains alternatively may be covalently substituted with the detectable groups.
  • Antibodies capable of binding specifically to TCR domains and neutralizing their activity are known or may be prepared in conventional fashion. These antibodies are produced by hybridomas or recombinant host cell culture. The antibodies may be homologous to the patient, i.e., human antibodies are used with humans, or are
  • the antibodies may be substituted with toxic moieties, e.g. the ricin A chain, or they may be in the form of polyvalent antibodies which are capable of binding other antigens, e.g. CD3, in addition to the target TCR.
  • the IG class chosen preferably is IgG rather than IgD, IgA IgM or IgE.
  • the IgG class optimally should be capable of binding complement and initiating ADCC.
  • the antibodies will be capable of specifically binding the V beta 7, 8, 12, 13, 14 or 15 sequences, J beta 2.1, 2.2, 2.3, 2.4, 2.5, 2.7 sequences, or C beta 2 sequences, and preferably, V beta 12, 13, 7, 8 or 15 and J beta 2.3 or 2.7.
  • a cocktail of antibodies having specificities for each target TCR domain is provided if desired, and heteropolyfunctional antibodies for multiple, different TCR domains also are within the scope hereof.
  • TCR antagonist polypeptides or antibodies are formulated into conventional pharmaceutical vehicles.
  • the formulations are sterile and isotonic and contain any substances needed to maintain the stability and activity of the antagonist.
  • the TCR antagonists or antibodies are formulated generally by mixing them at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides. It also is preferably free of immune adjuvants or stimulants, in particular Freunds adjuvant.
  • the formulations are prepared by contacting the TCR antagonists or antibodies uniformly and intimately with liquid carriers.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
  • Phosphate buffered saline or other isotonic buffers are preferable.
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as giycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorb
  • the TCR antagonist polypeptide or antibody to be used for therapeutic purposes is the TCR antagonist polypeptide or antibody to be used for therapeutic purposes.
  • Therapeutic TCR antagonist polypeptide or antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • TCR antagonist polypeptide or antibody ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • the initial consideration here must be that the carrier itself, or its degradation products, is nontoxic in the target tissue and will not further aggravate the disease. This can be determined by routine screening in animal models of the target disorder.
  • TCR polypeptides have been administered so as to provoke an immune response by immunization with adjuvant or by subcutaneous administration.
  • they may be administered without adjuvants and intravenously or intralesionally, and that immunostimulation is not necessary to achieve clinical
  • the products herein are used with adjuvants they may be fused to immunogenic polypeptides or haptens, or may be administered with a conventional adjuvant such as muramyl dipeptide, alum, a chemoattractant such as IL-8 or a cytokine such as TNF.
  • a conventional adjuvant such as muramyl dipeptide, alum, a chemoattractant such as IL-8 or a cytokine such as TNF.
  • the TCR antagonist or antibody is formulated and delivered at a dosage capable of inducing the suppressive or anergic immune response.
  • Antibodies and polypeptides are formulated at concentrations compatible with physical integrity and consistent with convenient administration of a dosage by injection.
  • the antagonist or polypeptide concentration will range about from 25 to 4,000 micrograms/m I, ordinarily 50 to 500 m ⁇ crograms/ml and preferably 100 to 300
  • the formulated, appropriate polypeptides or antibodies are administered to patients having autoimmune diseases.
  • a candidate dosage to be used may be extrapolated from that which has been used previously in connection with the EAN model, in the case of humans taking into account the difference in pharmacokinetics between animals and humans. This means that the effective single iv. dose of 50 micrograms of V beta 8 polypeptide in the EAN rat (without adjuvant) which I have shown to be effective would extrapolate to approximately 2,000 micrograms in a human of typical 70 kg weight.
  • this dosage will be affected by the use of adjuvants, the stage of the autoimmune disorder, the potency of the polypeptide, the use of other-therapies and other factors known to the skilled artisan.
  • the dosage of antibody also will be a matter to be determined by the clinician based on the affinity of the antibody for its TCR, the population of T cell clones bearing that TCR, the population of TCR on each clone, the stage of the autoimmune disorder, the use of other therapies (including a V beta or J beta polypeptide, at an earlier or later point in the therapy with antibody), and other factors known to the clinician.
  • the dosage will be selected at a low level and gradually increased until the desired therapeutic effect (decline in inflammation and other symptoms) is achieved.
  • the frequency of dosing will be dependent on the effective biological half life of the TCR antagonist and the duration of the subsequent immune response that ameliorates the disease process. Dosing frequency is readily determined by following the clinical condition of the patient or by assaying body fluids to detect deletion of the identified TCR-bearing lymphocytes if antibody is used or an elevated lymphocyte response in vivo or in vitro to the administered TCR peptides if TCR domains are used. This will be within the ordinary skill in the art. It is within the scope hereof to combine the TCR antagonist or antibody therapy with other novel or conventional therapies for autoimmune diseases.
  • the TCR antagonist or antibody therapy may be delivered in concert with other RA therapies, including bed rest, physical therapy, reconstructive joint surgery, application of heat, total lymphoid irradiation, synovectomy by irradiation with macroaggregates of ferric hydroxide labeled with a short-lived isotope (dysprosium-165) [Sledge et al., Arthritis Rheum., 29: 153-159 (1986); Vella et al., Arthritis Rheum., 31 : 789-792 (1988)], supplemental icosapentaenoic and docosahexanoic acid, and drugs.
  • RA therapies including bed rest, physical therapy, reconstructive joint surgery, application of heat, total lymphoid irradiation, synovectomy by irradiation with macroaggregates of ferric hydroxide labeled with a short-lived isotope (dysprosium-165) [Sledge e
  • Suitable cotreatment drugs include nonsteroidal anti-inflammatory agents (drugs) such as aspirin, indomethacin,
  • phenylbutazone phenylacetic acid derivatives such as ibuprofen and fenoprofen, naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, zomepirac, and diflunisal; salicylates; anti-malarial agents such as hydroxychloroquine, sulfasalazine, gold colloids or salts, and penicillamine; immunosuppressive agents such as methotrexate and cyclosporine; intravenous methylprednisolone; cyclic oligosaccharides such as cyclodextrins, preferably in combination with a noninflammatory steroid such as cortexolone as described by Folkman et al., Science.
  • a noninflammatory steroid such as cortex
  • TCR antagonist compositions per se, although this may be convenient where such drugs are proteinaceous such as in the case of, e.g., Hnterferon, TGF- ⁇ , PDGF, and antibodies, including amino acid sequence variants.
  • proteins acting through different mechanisms may intervene at different points along the continuum of the immune disease process proteolytic enzyme cascade, so that the cytokines might be used in simultaneous treatment with the TCR antagonist or antibody or used earlier or later, depending on the nature and stage of the disease and the mechanism by which the particular cytokine operates.
  • T-cell activation was accomplished using the OKT3, an IgG2a mAB directed against CD3.
  • the following mABs were used at saturating concentrations; Leu 4 (CD3), Leu 3a (CD4), Leu 2a (CD8), Leu 18 (CD45RA), HLA-DR, TCR-1 (TCR ⁇ ), and mouse lgG2 negative controls (Becton Dickinson, Mountain View,
  • TCR ⁇ 1 TCR ⁇
  • 1 C1 VB5
  • OT145 OT145
  • 16G8 VB8
  • S511 VB12, T-cell Sciences, Cambridge, MA
  • PE conjugated goat anti mouse IgG Caltag Laboratories, So. San Francisco, CA
  • T-cells were allowed to expand until they became confluent, then were removed from the tissue culture, replated at 8x10 5 cells/ml, and fed triweekly with fresh media and IL-2. See Example 9.1.3 of PCT WO 90/06758, supra.
  • the T-cells were stimulated every 7 to 14 days by seeding them in plastic wells that had been precoated at 37°C for at least two hours with a 10 ug/ml solution of purified OKT3 ( ⁇ CD3) in PBS (pH 7.4).
  • T-cell lines were analyzed by flow cytometry for surface antigen composition one to two weeks after removing them from the tissue, at which time each line was found to be greater than 95% positive for CD3 expression.
  • TCR C ⁇ probe was obtained by using PCR; C-region primers in regions corresponding to the Eco RV and Hind III restriction sites amplified a 600 bp fragment from the constant region of JurB1 that was subcloned into a bluescript plasmid (pBS-). PCR Analysis of TCR Beta Chain Variable Regions
  • RNAzol (Cinna/Biotecx, Friendswood, TX)
  • -cDNA was prepared from T-cell mRNA and amplified by PCR as described by, e.g.,
  • V beta-J beta-C beta usage SIL36: 12.2-2.2-2, 12.2-2.1-2, 15.1-2.7-2; SIL37: 8-2.5-2, 15-2.1-2, 15-2.3-2, 15-2.7-2; SIL46B: 3.1-2.3-2, 7.2-2.7-NA, 14.1-2.7-NA; SIL47B: 8-2.1-2, 8-2.2-2, 8-2.4-2, 8-2.5-2, 12.2-2.3-2, 12.2-2.7-2, 12.3-1.2-1 , 12.3-2.1-2, 12.3-1.1-1, 12.4-2.7-2; SIL49: 14-2.2-2, 14-2.7-2, 15-2.1 -2, 15-2.7-2. D beta usage was not restricted in these patients, in the same fashion as noted for the other patients above.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Les patients souffrant de polyarthrite chronique évolutive utilisent de préférence les domaines Vbeta14, Vbeta7, Vbeta8, Vbeta12, Vbeta13, Vbeta15, Vbeta2.2, Vbeta2.3, Jbeta2.1, Jbeta2.5, Jbeta2.7 et Cbeta2 des récepteurs des cellules T. On traite ces patients avec des polypeptides contenant des séquences de ces domaines ou avec des anticorps capables de se lier spécifiquement à ces domaines. Des procédés améliorés de diagnostic de l'utilisation de Vbeta dans des maladies auto-immunitaires sont décrits. L'utilisation de polypeptides et d'anticorps Jbeta pour traiter des maladies auto-immunitaires est nouvelle.
EP92920872A 1991-09-23 1992-09-23 Diagnostic et therapie de maladies auto-immunitaires Withdrawn EP0605606A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US765222 1985-08-13
US76522291A 1991-09-23 1991-09-23
US779444 1991-10-18
US07/779,444 US5203631A (en) 1991-10-18 1991-10-18 Narrow spectral band pyrometry
US85336692A 1992-03-18 1992-03-18
US853366 1992-03-18
PCT/US1992/008094 WO1993006135A1 (fr) 1991-09-23 1992-09-23 Diagnostic et therapie de maladies auto-immunitaires

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9306135A1 *

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