WO2006133286A2 - Traitement de troubles inflammatoires resistants aux antagonistes tnf et procedes associes - Google Patents

Traitement de troubles inflammatoires resistants aux antagonistes tnf et procedes associes Download PDF

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WO2006133286A2
WO2006133286A2 PCT/US2006/022099 US2006022099W WO2006133286A2 WO 2006133286 A2 WO2006133286 A2 WO 2006133286A2 US 2006022099 W US2006022099 W US 2006022099W WO 2006133286 A2 WO2006133286 A2 WO 2006133286A2
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vla
antagonist
patient
tnfα
treatment
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PCT/US2006/022099
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WO2006133286A3 (fr
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Ilan Bank
Itamar Goldstein
Shomron Ben Horin
Leonard Chess
Koltakov Alexander
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Tel Hashomer Medical Research Infrastructure And Services Ltd.
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Publication of WO2006133286A2 publication Critical patent/WO2006133286A2/fr
Publication of WO2006133286A3 publication Critical patent/WO2006133286A3/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
    • C07K14/70546—Integrin superfamily
    • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention provides methods of treatment in which a VLA-I antagonist is administered to a patient who suffers from one or more inflammatory disorders, wherein: (1) the patient does not respond adequately to, or is unlikely to respond adequately to, TNF ⁇ antagonist therapy; or (2) the patient evidences tachyphylaxis and/or refractory effects during TNF ⁇ antagonist therapy; or (3) the VLA-I antagonist is administered as an adjuvant to
  • VLA-I antagonist is effective in treating the patient's inflammatory disorder(s).
  • Integrins are transmembrane heterodimeric receptors for extracellular matrix molecules. Integrin ⁇ l ⁇ l (VLA-I) is expressed on mesenchymal cells, some epithelial cells, activated T cells, and macrophages, and interacts, via the I- domain of the extracellular domain of the alpha 1 subunit, with collagen molecules in the extracellular matrix (ECM). Ben-Horin, et ah, Clin Immunol. 2004 Nov;113(2):119-29.
  • VLA-I By “outside-in” transmembranal signaling to the interior of the cell, VLA-I mediates adhesion, migration, proliferation, and remodeling of the ECM, and cytokine secretion by endothelial cells, mesangial cells, fibroblasts, and immunocytes.
  • VLA-I expression and function is enhanced by inflammatory cytokines including interferon gamma (IFN ⁇ ) and tumor necrosis factor alpha (TNF ⁇ ), thus augmenting angiogenesis and fibrosis linked, in particular, to inflammation.
  • IFN ⁇ interferon gamma
  • TNF ⁇ tumor necrosis factor alpha
  • VLA-I marks effector memory CD4+ and CD8+ T cells that are retained in extralymphatic tissues by interactions of the integrin with collagen and produces high levels of IFN ⁇ .
  • immune-mediated inflammation in vivo is inhibited by blockade of the VLA-I -collagen interaction in experimental animal models of arthritis, colitis, nephritis, and graft versus host disease.
  • VLA-I is expressed in a large fraction of CD45RO+ memory cells localizing to inflamed tissues, including inflamed synovium. Hemler et al., 1986, /. Clin. Invest. 78:692-702.
  • PB peripheral blood
  • RA rheumatoid arthritis
  • TNF-necrosis factor (TNF)-related cytokines are mediators of host defense and immune regulation. TNF secretion can amplify an event leading to well-described changes in the vasculature lining and the inflammatory state of cells. In inflammatory diseases such as rheumatoid arthritis (RA) and inflammatory bowel disease, pro-inflammatory cytokines including TNF ⁇ are expressed and act to induce cell proliferation and effector function, thereby exacerbating the disease.
  • Anti- TNF ⁇ antibody therapies have proven useful in the treatment of TNF ⁇ -implicated diseases. For example, the TNF ⁇ antagonists infliximab, etanercept and adalimumab have been used to treat RA and Crohn's Disease (CD).
  • TNF ⁇ antagonists have proven useful in the treatment of inflammatory disorders such as RA, there are a substantial number of patients who have been treated with a wide range of diseases and conditions.
  • Anti-TNF ⁇ treatment of inflammatory disorders can also be associated with tachyphylaxis and/or refractory effects.
  • TNF ⁇ antagonist infliximab is highly effective in the treatment of CD, attenuated response to infliximab may develop over time in a subgroup of
  • TNF ⁇ antagonists may preferentially target the outgrowth of VLA-I and IFN ⁇ -producing T cells and a subset of anti-TNF ⁇ -resistant VLA-I and T cells may contribute to the persistence of inflammatory disease in TNF ⁇ antagonist therapy non-responders.
  • VLA-I antagonists are useful in treating patients who suffer from an inflammatory disorder and who have either failed to respond adequately to TNF ⁇ antagonist therapy or who no longer benefit from a previously effective TNF ⁇ antagonist therapy.
  • our invention provides methods of treatment in which a VLA-I antagonist is administered to a patient who suffers from one or more inflammatory disorders, wherein: (1) the patient does not respond adequately to, or is unlikely to respond adequately to, TNF ⁇ antagonist therapy; or (2) the patient evidences tachyphylaxis and/or refractory effects during TNF ⁇ antagonist therapy; or (3) the VLA-I antagonist is administered as an adjuvant to TNF ⁇ antagonist monotherapy; and
  • VLA-I antagonist is effective in treating the patient's inflammatory disorder(s).
  • a TNF ⁇ antagonist therapy non-responder is treated successfully with one or more pharmaceutical compositions comprising one or more VLA-I antagonist proteins, e.g., one or more VLA-I antagonist antibodies.
  • a patient who suffers from rheumatoid arthritis and who has failed to respond adequately to TNF ⁇ antagonist therapy is treated successfully by administration of one or more VLA-I antagonists.
  • the invention provides methods for assessing whether an inflammatory disorder patient, based on VLA-I expression levels in patient cells implicated in the inflammatory disorder, has responded adequately to, or is likely to respond adequately to, TNF ⁇ antagonist therapy.
  • Related diagnostic methods and kits are also provided.
  • the invention provides methods for enhancing the responsiveness of an inflammatory disorder patient to anti-TNF ⁇ therapy by down- regulating expression of VLA-I in patient cells implicated in the inflammatory disorder.
  • FIGURE 1 illustrates the percentage of VLA- 1+ CD3+ T cells within the total PB CD3+ T cells of patients studied in the experiment of Example 1.
  • FIGURE 2 illustrates that neutralizing TNF ⁇ with a humanized mAb to TNF ⁇ during polyclonal activation of VLA-I -depleted fresh PB lymphocytes (PBL) results in reduced numbers of VLA- 1+ T cells in the culture, as determined in accordance with the experiment of Example 2.
  • PBL VLA-I -depleted fresh PB lymphocytes
  • FIGURE 3 illustrates high levels of ⁇ l integrin VLA-4 expression on the cell surface of greater than 82% of T cells in both infliximab-treated and untreated cultures, as determined in accordance with the experiment of Example 3.
  • FIGURE 4(a)-(b) illustrate that the addition of infliximab to activated peripheral blood mononuclear cells from patients with arthritis also resulted in reduced secretion of interferon (IFN ⁇ ), as determined in accordance with the experiment of Example 4.
  • IFN ⁇ interferon
  • FIGURE 5 illustrates that VLA- 1+ T cells were identified in the synovial fluid of a patient relapsing after TNF ⁇ antagonist therapy, and in a patient relapsing during anti- TNF ⁇ therapy, as dete ⁇ nined in accordance with the experiment of Example 5.
  • FIGURE 6 illustrates that ex vivo adhesion to collagen of the VLA-I + T cells persisting in a patient's peripheral blood after TNF ⁇ neutralization was blocked by a mAb against the ⁇ l-I domain of the VLA-I molecule, as determined in accordance with the experiment of Example 6.
  • VLA-I antagonist refers to a composition, e.g., a protein including an antibody or antibody fragment, that binds to the T-cell ⁇ l-I integrin.
  • VLA-I antagonist refers to any composition that at least partially inhibits an activity of a VLA-I integrin, particularly a binding activity of a VLA-I integrin or a signaling activity, e.g., ability to transduce a VLA-I mediated signal.
  • a VLA-I antagonist may inhibit binding of VLA-I to a cognate ligand of VLA-I, e.g., a cell surface protein, or to an extracellular matrix component, such as fibronectin or osteopontin.
  • a VLA-I antagonist that binds to VLA-I may bind to either the ⁇ l subunit or the ⁇ l subunit, or to both.
  • a VLA-I antagonist may also interact with other ⁇ l subunit containing integrins or with other ⁇ l containing integrins.
  • a VLA-I antagonist includes but is not limited to ⁇ l ⁇ l antibodies such as, mAb AQC2 (ATCC PTA-3580)(see United States Patent Application Document No. 20040081651); mAb AJHlO(ATCC PTA-3580)(jee United States Patent Application Document No. 20020146417); mAb 1B3.1 (ATCC No. HB10336) and mAb TS2/7.1 (ATCC No. HB-245).
  • Preferred ⁇ l ⁇ l antibodies and ⁇ l ⁇ l antibody homologs which can be used in the methods of treatment of the invention include human antibodies, antibody homologs, humanized antibody homologs, chimeric antibody homologs, Fab, Fab', F(ab')2 and F(v) antibody fragments, and monomers or dimers of antibody heavy or light chains, or mixtures thereof.
  • Monoclonal antibodies, or homologs or fragments of such antibodies, which bind to an ⁇ l ⁇ l integrin epitope are particularly preferred for use in methods of treatment of the invention.
  • an antibody refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or immunoglobulin variable domain sequence.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • VH heavy chain variable region
  • L light chain variable region
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab') 2 fragments, Fd fragments, Fv fragments, and dAb fragments) as well as complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).
  • the light chains of the immunoglobulin may be of types kappa or lambda.
  • the antibody is glycosylated.
  • An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be non-functional for one or both of these activities.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” ("CDR"), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is typically composed of three CDR's and four PR's, arranged from amino-terminus to carboxyl-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • Immunoglobulin domain refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two ⁇ -sheets formed of about seven ⁇ -strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6:381-405).
  • an "immunoglobulin variable domain sequence” refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain.
  • the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain.
  • the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations.
  • a polypeptide that includes an immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or "antigen binding site"), e.g., a structure that interacts with VLA-I.
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains.
  • the heavy and light immunoglobulin chains can be connected by disulfide bonds.
  • the heavy chain constant region typically includes three constant domains, CHl, CH2 and CH3.
  • the light chain constant region typically includes a CL domain.
  • the variable region of the heavy and light chains contains a binding domain that interacts with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • One or more regions of an antibody can be human, effectively human, or humanized.
  • one or more of the variable regions can be human or effectively human.
  • one or more of the CDRs e.g., HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3, can be human.
  • Each of the light chain CDRs can be human.
  • HC CDR3 can be human.
  • One or more of the framework regions can be human, e.g., FRl, FR2, FR3, and FR4 of the HC or
  • all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell.
  • the human sequences are germline sequences, e.g., encoded by a germline nucleic acid.
  • One or more of the constant regions can be human, effectively human, or humanized.
  • At least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FRl, FR2, and FR3, collectively, or FRl, FR2, FR3, and FR4, collectively) or the entire antibody can be human,, effectively human, or humanized.
  • FRl, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical to a human sequence encoded by a human germline segment.
  • an “effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • An “effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
  • Humanized version of an antibody produced by an AQC2 hybridoma or cell line means all humanized versions of antibodies produced by the AQC2 hybridomas and cell lines disclosed in U.S. Patent Application Serial No.
  • humanized version of an antibody produced by an AQC2 hybridoma or cell line includes, but is not limited to, humanized versions of antibodies produced by the hybridoma mAQC2 (ATCC PTA3273).
  • a "humanized” immunoglobulin variable region is an immunoglobulin variable region that is modified such that the modified form elicits less of an immune response in a human than does the non-modified form, e.g., is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human.
  • humanized immunoglobulins include those, for example, in United States Patent No. 6,407,213 and United States Patent No. 5,693,762.
  • humanized immunoglobulins can include a non-human amino acid at one or more framework amino acid positions. All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof.
  • Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2- terminus (about
  • Full-length immunoglobulin "heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
  • antigen-binding fragment of a full length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest, e.g., VLA-I.
  • binding fragments encompassed within the term "antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab') 2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
  • scFv single chain Fv
  • Antibodies that bind to VLA-I can be generated by immunization, e.g., using an animal, or by in vitro methods such as phage display. All or part of VLA-I can be used as an immunogen. For example, the extracellular region of the ⁇ l subunit can be used as an immunogen.
  • the immunized animal contains immunoglobulin producing cells with natural, human, or partially human immunoglobulin loci.
  • the non-human animal includes at least a part of a human immunoglobulin gene. It is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected.
  • Non-human antibodies to VLA-I can also be produced, e.g., in a rodent.
  • the non-human antibody can be humanized, e.g., as described in United States
  • Patent No. 6,602,503 EP 239400; United States Patent No. 5,693,761; and United States Patent No. 6,407,213.
  • EP 239400 (Winter et al.) describes altering antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those from another.
  • CDR-substituted antibodies can be less likely to elicit an immune response in humans compared to true chimeric antibodies because the CDR-substituted antibodies contain considerably less non- human components.
  • CDRs of a murine antibody substituted into the corresponding regions in a human antibody by using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody.
  • Human constant region gene segments of the desired isotype usually gamma I for CH and kappa for CL
  • the humanized heavy and light chain genes can be co- expressed in mammalian cells to produce soluble humanized antibody.
  • Tempest et al. 1991, Biotechnology 9:266-271, utilize, as standard, the V region frameworks derived from NEWM and REI heavy and light chains, respectively, for CDR-grafting without radical introduction of mouse residues.
  • An advantage of using the Tempest et al. approach to construct NEWM and REI based humanized antibodies is that the three dimensional structures of NEWM and REI variable regions are known from x-ray crystallography and thus specific interactions between CDRs and V region framework residues can be modeled.
  • Non-human antibodies can be modified to include substitutions that insert human immunoglobulin sequences, e.g., consensus human amino acid residues at particular positions, e.g., at one or more (preferably at least five, ten, twelve, or all) of the following positions: (in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 7OL, 7 IL, 73L, 85L, 87L, 98L, and/or (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 6OH, 67H, 68H, 69H, 7OH, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or
  • Fully human monoclonal antibodies that bind to VLA-I can be produced, e.g., using in vitro-primed human splenocytes, as described by Boerner et al., 1991, /. Immunol., 147, 86-95. They may be prepared by repertoire cloning as described by Persson et al., 1991, Proc. Nat. Acad. Sci. USA,
  • Antibodies can be produced in prokaryotic and eukaryotic cells.
  • the antibodies e.g., scFv's
  • the antibodies are expressed in a yeast cell such as
  • Pichia see, e.g., Powers et al. (2001) J Immunol Methods. 251:123-35), Hanseula, or Saccharomyces.
  • antibodies are produced in mammalian cells.
  • mammalian host cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) MoI. Biol.
  • lymphocytic cell lines e.g., NSO myeloma cells and SP2 cells, COS cells, K562, and a cell from a transgenic animal, e.g., a transgenic mammal.
  • the cell is a mammary epithelial cell.
  • the recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., United States Patent Nos. 4,399,216; 4,634,665; and 5,179,017).
  • Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr ' host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, to transfect the host cells, to select for transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
  • Antibodies may also include modifications, e.g., modifications that alter Fc function, e.g., to decrease or remove interaction with an Fc receptor or with CIq, or both.
  • modifications e.g., modifications that alter Fc function, e.g., to decrease or remove interaction with an Fc receptor or with CIq, or both.
  • the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the numbering in United States Patent No. 5,648,260.
  • Other exemplary modifications include those described in United States Patent No. 5,648,260.
  • the antibody production system may be designed to synthesize antibodies in which the Fc region is glycosylated.
  • the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain.
  • This asparagine is the site for modification with biantennary-type oligosaccharides. This glycosylation participates in effector functions mediated by FcY receptors and complement CIq (Burton and Woof (1992) Adv. Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76).
  • the Fc domain can be produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297.
  • Fc domain can also include other eukaryotic post-translational modifications.
  • Antibodies can also be produced by a transgenic animal.
  • United States Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal.
  • a transgene is constructed that includes a milk-specific promoter and nucleic acid sequences encoding the antibody of interest, e.g., an antibody described herein, and a signal sequence for secretion.
  • the milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest, e.g., an antibody described herein.
  • the antibody can be purified from the milk, or for some applications, used directly.
  • Antibodies can be modified, e.g., with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, bronchoalveolar lavage, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold.
  • a VLA-I binding antibody can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide.
  • a polymer e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide.
  • Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
  • a VLA-I binding antibody can be conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone.
  • a water soluble polymer e.g., a hydrophilic polyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone.
  • a non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides that comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g.
  • polymannuronic acid or alginic acid
  • D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparan.
  • Pharmaceutical Compositions such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.
  • a VLA-I binding agent such as a VLA-I binding antibody can be formulated as a pharmaceutical composition.
  • a pharmaceutical composition includes a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial g
  • compositions of the invention may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injec tables, as do natural pharmaceutically-accep table oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • compositions of the invention may be administered orally in any orally-acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers such as mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, and water.
  • the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Topically-transdermal patches may also be used.
  • compositions of the invention can be administered topically by other methods, e.g., encapsulation in either a temperature and/or pressure sensitive matrix or in film or in a solid carrier which is soluble in body fluids to enable subsequent release, preferably sustained-release, of the active component.
  • encapsulation in either a temperature and/or pressure sensitive matrix or in film or in a solid carrier which is soluble in body fluids to enable subsequent release, preferably sustained-release, of the active component.
  • creams, genies, dressings, shampoos, tinctures, pastes, ointments, salves, powders, liquid or semi-liquid formulation can be used.
  • compositions of the invention can be administered as an aerosol which uses propellants such as nitrogen, carbon dioxide, or freons, or without a propellant, e.g., as a pump spray, drops, lotions, or a semisolid such as a thickened composition which can be applied by a swab.
  • propellants such as nitrogen, carbon dioxide, or freons
  • a propellant e.g., as a pump spray, drops, lotions, or a semisolid such as a thickened composition which can be applied by a swab.
  • Nasal aerosols, nebulizers, dry powder inhalers, or metered dose inhalers can be used.
  • compositions used in the methods of the present invention comprise an anti- VLA-I antibody formulated in vesicles such as liposome-containing compositions, e.g., as described in EP-A-253,619.
  • "Inadequate response”, “does not respond adequately to”, or “unlikely to respond adequately to” refer to an actual or probable response by a patient which indicates that TNF ⁇ antagonist therapy has been, or is likely to be, ineffective, toxic, or poorly tolerated insofar as the patient is concerned.
  • a clinician e.g., a healthcare provider such as a physician
  • a patient may assess whether TNF ⁇ antagonist therapy has been, or is likely to be, ineffective, toxic, or poorly tolerated on the basis of a variety of objective or subjective clinical indicators, including but not limited to those described below.
  • "adequate response", “effective”, “treated successfully”, and “enhancing the responsiveness” mean that a therapy (e.g., a VLA-I antagonist therapy) has been, or is likely to be, effective, non-toxic, or well-tolerated on the basis of a variety of objective or subjective clinical indicators, including but not limited to those described below.
  • an anti-arthritic TNF ⁇ antagonist therapy may be deemed ineffective based on its likely or actual failure to manifest a decrease in either arthritic score after TNF ⁇ antagonist therapy (e.g., by anywhere from about 10% to about 60% or more from pre-treatment values) or one or more other clinical markers (e.g., reduced swollen tender joints, lowered levels of C-reactive protein, and/or mitigated morning stiffness). Elevated levels of VLA-1+ T cells in the PB of an arthritic patient and other indicators (e.g., increased secretion of interferon
  • TNF ⁇ can also be used to assess whether the patient is likely to respond to TNF ⁇ antagonist therapy.
  • Clinical indicators may be evaluated before, during, and after TNF ⁇ antagonist therapy and may be applied and evaluated in different ways for different patients. For example, clinical histories of individual patients or groups of patients may influence the interpretation of particular clinical markers. The duration of TNF ⁇ antagonist therapy (if any) prior to an assessment of efficacy, toxicity, or tolerance may also vary in individual patients or groups of patients.
  • TNF ⁇ antagonist therapy may be deemed ineffective based on its failure to reduce abdominal pain, diarrhea, hematochezia, weight loss and/or complications such as bowel obstruction, fistula formation and extraintestinal manifestations.
  • TNF ⁇ antagonist therapy may be deemed ineffective based on its failure to reduce choroidal neovacularisation (CNV) secondary to AMD in a patient or group of patients.
  • CNV choroidal neovacularisation
  • TNF ⁇ antagonist therapy may be deemed ineffective based on its failure to improve the psoriasis area and severity index (PASI) of a patient or group of patients.
  • PASI psoriasis area and severity index
  • ineffective TNF ⁇ antagonist therapies are purely illustrative, and an assessment of whether an inflammatory disease patient has responded adequately to a particular treatment regimen may be determined using other clinical indicators known to those of ordinary skill in the art.
  • Intolerable or unacceptable toxicity implicated in TNF ⁇ antagonist therapy may include actual or potential hepatic injury or dysfunction, severe allergic reaction, severe depression or suicidal ideation, anaphylaxis, or injection site necrosis.
  • methods of treatment of the invention are used to treat an inflammatory disorder patient who is refractory to TNF ⁇ antagonist therapy.
  • the refractory patient has either not shown an acceptable improvement in relevant clinical indicators in response to TNF ⁇ antagonist therapy, or an initial improvement in relevant clinical indicators proves unsustainable over the course of TNF ⁇ antagonist therapy.
  • methods of treatment of the invention are used to treat an inflammatory disorder patient who exhibits tachyphylaxis to TNF ⁇ antagonist therapy.
  • the tachyphylaxis patient requires increasingly larger dosages of a TNF ⁇ antagonist in order to achieve an acceptable improvement in relevant clinical indicators.
  • an acceptable improvement in relevant clinical indicators could be either a decrease in arthritic score after a prescribed duration of treatment (e.g., a decrease in arthritic score of about 10% to about 60% or more from pre-treatment values), or reduced swollen tender joints, lowered levels of C- reactive protein, and/or mitigated morning stiffness.
  • methods of treatment of the invention are used to treat a breakthrough patient who exhibits: (a) at least one relapse as evidenced by relevant clinical indicators (i.e., an increase in arthritic score); and (b) progression of an inflammatory disease during a specific duration (e.g., four weeks to two years) of TNF ⁇ antagonist therapy.
  • Methods of treatment of the invention are also used to treat patients who fail to demonstrate an acceptable decrease in relapses during TNF ⁇ antagonist therapies of various durations.
  • An acceptable decrease in relapse rate could be a
  • 5%-40% relapse decrease over about six months to about four years of TNF ⁇ antagonist therapy For example, an acceptable decrease in relapse rate would be evidenced by a patient who, after beginning VLA-I antagonist therapy, achieves around 40% to around 70% decrease in the rate of relapses suffered while undergoing TNF ⁇ antagonist therapy.
  • treating refers to administering a therapy in an amount and manner (e.g., schedule of administration, and/or route of administration) which is effective to improve an inflammatory disorder or a symptom thereof, or to prevent or slow the progression of an inflammatory disorder or a symptom thereof.
  • amount and manner e.g., schedule of administration, and/or route of administration
  • Effective dosages and dose rates for VLA-I antagonists used in methods of the invention will depend on a variety of factors, such as the nature of the VLA-I antagonist, the size of the patient, the goal of the treatment, the nature of the pathology to be treated, the specific pharmaceutical composition used, and the judgment of the treating physician.
  • Dosage levels of between about 0.001 and about 100 mg/kg body weight per day, preferably between about 0.1 and about 50 mg/kg body weight per day of a VLA-I antagonist are useful.
  • a dosage of about 0.1 mg/kg/day to about 10 mg/kg/day of a VLA-I antagonist antibody or antibody fragment is administered to a patient between one to seven times per week.
  • a VLA-I antagonist antibody or antibody fragment is administered to a patient: (a) around once or twice approximately every seven days (b) in an amount of between about 0.3 mg/kg/day to about 5 mg/kg/day.
  • a TNF ⁇ antagonist and/or an anti-inflammatory which may or may not be a TNF ⁇ antagonist (e.g., a NSAID) is administered prior to, concurrently with, or after the administration of the VLA-I antagonist.
  • a TNF ⁇ antagonist e.g., a NSAID
  • the term "biologic" refers to a protein-based therapeutic agent. In a preferred embodiment, the biologic is between about ten to about one hundred amino acid residues in length.
  • TNF ⁇ antagonist therapy means administering to a patient suffering from an inflammatory disorder one or more TNF ⁇ antagonists including but not limited to etanercept (Enbrel®), infliximab (Remicade®), methotrexate, and adalimumab (Humira®).
  • a "TNF ⁇ antagonist” includes any substance that inhibits TNF ⁇ -induced adhesion of mactophages to endothelial cells, or inhibits the TNF ⁇ -induced release of superoxide anions from neutrophils, or which inhibits the mitogenic activity of TNF ⁇ with respect to the fibroblasts of the dermis.
  • “Inflammatory disorders” include, but are not limited to, arthritis (including rheumatoid arthritis and osteoarthritis), psoriasis, eczema, burns and dermatitis, asthma, bronchitis, menstrual cramps, tendinitis, bursitis, pain and headaches, fever, gastrointestinal conditions such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis, vascular diseases, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, rheumatic fever, type I diabetes, myasthenia gravis, multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity, conjunctivitis, swelling occurring after injury, myocardial ischemia, allergic rhinitis, respiratory distress syndrome, endotoxin
  • Patient means a human or non-human mammal.
  • An adult patient is diagnosed with RA and is treated with adalimumab monotherapy (40 mg every other week). After eight weeks of adalimumab monotherapy, the patient presents with swollen tender joints, elevated levels of C- reactive protein, and prolonged morning stiffness. Adalimumab therapy is discontinued and the patient is treated with a VLA-I antagonist antibody (5 mg/kg twice per week). After eight weeks of VLA-I antagonist monotherapy, the patient presents with reduced swollen tender joints, lowered levels of C-reactive protein, and mitigated morning stiffness. Improvement of these clinical indicators establishes that VLA-I antagonist therapy is effective in a patient for whom adalimumab monotherapy proved ineffective.
  • a PB sample is taken from an adult patient who has been diagnosed with RA. The sample evidences an elevated percentage of VLA-1+ ThI cells. TNF ⁇ antagonist therapy is excluded as a treatment option based on the elevated percentage of PB
  • VLA-1+ ThI cells VLA-1+ ThI cells and the patient is treated with a VLA-I antagonist antibody (5 mg/kg weekly). After eight weeks of VLA-I antagonist therapy, the patient presents with reduced swollen tender joints, lowered levels of C-reactive protein, and mitigated morning stiffness. Improvement of these clinical indicators establishes that VLA-I antagonist therapy is effective in a patient for whom TNF ⁇ antagonist may have proven ineffective.
  • An adult patient is diagnosed with RA and is treated with etanercept monotherapy (25 mg twice weekly). After eight weeks of etanercept monotherapy, the patient presents with swollen tender joints, elevated levels of C- reactive protein, and prolonged morning stiffness.
  • Etanercept monotherapy is discontinued and the patient is treated concurrently with a VLA-I antagonist antibody (5 mg/kg weekly) and an NSAID (200 mg/day).
  • VLA-I antagonist-NSAID co- therapy the patient presents with reduced swollen tender joints, lowered levels of C-reactive protein, and mitigated morning stiffness.
  • VLA-I antagonist antibody therapy is discontinued and NSABD therapy is maintained. Four weeks later, the patient presents with swollen tender joints.
  • VLA-I antagonist-NSAID co-therapy is resumed and, after four weeks, the patient presents with reduced joint swelling, reduced C-protein levels, and decreased morning stiffness. Improvement of these clinical indicators establishes that VLA- 1 antagonist therapy is effective in a patient for whom etanercept and NSAID monotherapy proved ineffective. 4.
  • An adult patient is diagnosed with RA and is treated with etanercept monotherapy (25 mg twice weekly). After eight weeks of etanercept monotherapy, the patient presents with swollen tender joints, elevated levels of C- reactive protein, and prolonged morning stiffness.
  • a sample of the patient's PB contains elevated VLA- 1+ ThI cell levels. Etanercept therapy is discontinued and VLA-I antagonist antibody (5 mg twice weekly) is initiated. After four weeks of
  • VLA-I antagonist therapy the patient presents with reduced swollen tender joints, lowered levels of C-reactive protein, and mitigated morning stiffness. Improvement of these clinical indicators establishes that VLA-I antagonist therapy is effective in a patient for whom etanercept monotherapy proved ineffective.
  • An adult patient is diagnosed with RA and is treated initially concomitantly with etanercept (25 mg twice weekly) and a VLA-I antagonist antibody (5 mg/kg twice weekly).
  • etanercept 25 mg twice weekly
  • VLA-I antagonist antibody 5 mg/kg twice weekly
  • etanercept monotherapy 25 mg twice weekly
  • etanercept monotherapy 5 mg/kg twice weekly
  • VLA-I antagonist antibody monotherapy After four weeks of VLA-I antagonist antibody monotherapy, the patient presents with reduced swollen tender joints, lowered levels of C-reactive protein, and mitigated morning stiffness. Improvement of these clinical indicators establishes that VLA-I antagonist therapy is effective in a patient for whom etanercept monotherapy proved ineffective.
  • a juvenile patient is diagnosed with Crohn's Disease and is treated with infliximab monotherapy (1 mg/kg, three doses over eight weeks). After eight weeks of infliximab monotherapy, the patient presents with bowel obstruction, fistula formation and extraintestinal manifestations. Infliximab therapy is discontinued and the patient is thereafter treated with a VLA-I antagonist antibody (1 mg/kg once per week). After four weeks, the patient presents with reduced abdominal pain, reduced diarrhea, reduced hematochezia, and weight gain. Improvement of these clinical indicators establishes that VLA-I antagonist therapy is effective in a patient for whom infliximab monotherapy proved ineffective.
  • An adult patient is diagnosed with psoriasis and is treated initially with etanercept monotherapy (25 mg twice weekly). After two months of etanercept treatment, the patient shows improvement based on the psoriasis area and severity index (PASI). However, PASI results from an examination eight weeks later show that etanercept treatment is no longer effective (e.g., skin lesions are worsening).
  • Etanercept monotherapy is discontinued and VLA-I antagonist antibody (5 mg/kg twice per week) is initiated. After four weeks of VLA-I antagonist monotherapy, the patient presents with an improved PSAI.
  • An adult patient is diagnosed with both RA and age-related macular degeneration (AMD) and is treated initially with infliximab monotherapy (5 mg/kg, three doses over eight weeks).
  • infliximab monotherapy 5 mg/kg, three doses over eight weeks.
  • the patient presents with reduced swollen tender joints, lowered levels of C-reactive protein, and decreased morning stiffness.
  • CNV choroidal neovacularisation
  • the patient is thereafter treated concomitantly with infliximab (5 mg/kg, three doses over eight weeks) and a VLA-I antagonist antibody (5 mg/kg, twice per week).
  • the responsiveness of an inflammatory disease patient to TNF ⁇ antagonist treatment is enhanced in accordance with the invention by suppressing VLA-I expression in patient cells implicated in the inflammatory disorder by:
  • introducing a vector into the cell genome in vitro the vector comprising a suppressor sequence and nucleic acid encoding one or more amplifiable markers, wherein the vector does not comprise a targeting sequence, is integrated into the genome of the cell by non-homologous recombination, and wherein the supressor sequence becomes operably-linked to the endogenous VLA-I gene such that expression of the VLA-I gene is suppressed in the cell following integration of the vector construct;
  • the responsiveness of a RA patient to TNF ⁇ antagonist treatment is enhanced as described above by suppressing VLA-I expression in ThI cells in the PB of the patient.
  • the invention provides a method for assessing whether an inflammatory disorder patient has responded adequately to, or is likely to respond adequately to, TNF ⁇ antagonist therapy based on VLA-I levels in patient cells implicated in the inflammatory disorder, wherein levels of VLA-I expression in patient cells implicated in the inflammatory disorder are compared to VLA-I expression reference levels and TNF ⁇ antagonist therapy is selected or excluded based on the comparison.
  • VLA-I expression reference levels can include, e.g., pre-disease VLA-I expression levels in patient cells implicated in the inflammatory disorder and/or VLA-I expression levels determined by examination of relevant cells of TNF ⁇ antagonist therapy-responsive and non- responsive patients. Assessments made in accordance with the invention may be used to decide upon a variety of courses of action, including recommendations of, and reimbursement for, specific medical treatments.
  • levels of VLA-I expression in ThI cells in the PB of a RA patient are compared to PB VLA-1+ ThI reference levels and anti- TNF ⁇ therapy is selected or excluded based on the comparison.
  • the invention provides a test kit for determining whether an inflammatory disorder patient has responded adequately to, or is likely to respond adequately to, TNF ⁇ antagonist therapy.
  • the kit comprises a probe/primer comprising nucleic acid molecules which hybridize under stringent hybridization conditions with a nucleic acid molecules derived from a sample of cells that have been isolated from a patient, that are implicated in the inflammatory disorder (e.g., T-cells), and that have a sequence comprising the nucleotide sequence of a VLA-I gene, or a sequence complementary thereto, or an equivalent thereof, or a functional equivalent thereto, wherein the hybridizing nucleic acid molecules are at least about 80% or about 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides up to the full length of a VLA-I gene, or a sequence complementary thereto, or an equivalent thereof, or a sequence complementary thereto.
  • the kit may further include instructions for using the kit, solutions for suspending or fixing the cells, detectable tags or labels, solutions for rendering a nucleic acid susceptible to hybridization, solutions for lysing cells, or solutions for the
  • Diagnostic methods and kits of the present invention may also use as a probe/primer a substantially purified oligonucleotide containing a region of nucleotide sequences which hybridize under stringent conditions to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides of sense or antisense sequence selected from the VLA-I gene, or a sequence complementary thereto, or an equivalent thereof, up to the full length of the VLA- 1 gene, or a sequence complementary thereto, or an equivalent thereof, or a sequence complementary thereto, or up to the full length of VLA-I gene of which said sequence is a fragment.
  • the probe selectively hybridizes with a target nucleic acid.
  • the probe may include a detectable label group selected from radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • the invention further provides arrays of at least about 10, at least about 25, at least about 50, or at least about 100 different probes as described above attached to a solid support.
  • RA rheumatoid arthritis
  • PB mononuclear cells of the patient groups indicated on the abscisse were stained with mAb to VLA-I and CD3 and analyzed by FACS. Results are depicted as the percent of VLA-1+CD3+ cells in the total CD3+ population. Number of patients per group (n) is indicated on the abscisse. Horizontal bars indicate the mean values, and the numerical values of the means ⁇ 1 std are indicated on the right of each bar. P values comparing the means of groups of patients as indicated by the arrows are shown.
  • PB mononuclear cells were activated with PHA and IL-2 in medium alone or in medium with 50 ug/ml of anti TNF ⁇ . After 8 days, cells were stained with mAb to VLA-4 (Y axis) and VLA-I (X axis) and analyzed by FACS. Numbers represent percent of cells in each quadrant. Whereas the percent of VLA- 1+ cells was reduced by anti TNF ⁇ . the ⁇ l integrin VLA-4 was highly expressed on the cell surface of greater than 83% of T cells in both anti TNF ⁇ -treated and untreated cultures (Figure 3). These results show that the effect of anti TNF ⁇ is specific for inhibition of the VLA- 1+ but not the VLA-4+ T cell subset when T cells are activated.
  • FIG. 4(a) illustrates a dot plot FACS analysis, after staining with mAb to VLA-I and CD3, of synovial fluid mononuclear cells from a knee joint in a patient relapsing after anti TNF ⁇ therapy.
  • the numbers indicate the percent of cells in each quadrant.
  • VLA-1+CD3+ T cells are in the enclosed quadrangle, and the calculated percentage of VLA-1+CD3+ within the total CD3+ population in the synovial fluid is bolded.
  • Figure 4(b) illustrates the percentage of VLA-I +CD3+ of total CD3+ T cells in the PB of two patients (M and B) with rheumatoid arthritis in remission and subsequent relapse during therapy.
  • the bar on the right shows percent of VLA-1+CD3+ cells in a sample from synovial fluid drawn during the relapse.
  • Anti-TNF ⁇ Decreases IFN v Along With VLA-I
  • CD4+ T cells from PB of a patient with RA were activated with anti CD3 and cultured in medium alone, or with 50 ug/ml of anti- TNF ⁇ or non-specific Ig.
  • the dot plots in Figure 5 represent FACS analysis of the expression of VLA-I on the CD4+ T cells in each culture. Percent positive cells in relevant quadrants are indicated. The cells were washed of all medium, then re-triggered with anti CD3 and autologous irradiated PBMC. After 72 hours, supernatants were analyzed for their content of IFN ⁇ and TNF ⁇ , and the concentration of each cytokine in pg/ml is indicated within the respective cytofluorograms. It is demonstrated that the cells cultured with anti
  • the dot plots on the left side of Figure 6 represent FACS analysis of lymphocytes obtained from a patient with RA in whom TNF ⁇ therapy had been stopped due to inefficacy. FACS analysis of fresh PB mononuclear cells, after staining with anti CD3 and VLA-I is shown in the upper panel of Figure 6.
  • the middle (left) panel of Figure 6 shows a similar analysis after PBMC fractionation into VLA-1+ and VLA-I- subsets and the lower (left) panel, of the respective subsets that were cultured for 18 days after activation with PHA and IL-2.
  • the percent positive cells in respective quadrants, and in bold, the VLA-1+CD3+ T cells as percent of total CD3+ cells are indicated in each dot plot.
  • cells from the 18 day cultures of VLA-I + and VLA-I- PBMC were added to collagen IV coated or control BSA coated tissue culture wells, in triplicate experiments, for 40 minutes, in the presence of control non binding mAb TEPC- 15, or anti VLA-I mAb IB 3.1 as indicated in the legend.
  • Non adherent cells were removed, and adherent cells were stained with a blue dye.
  • the cells were lysed in triton to release the dye, and its concentration in the detergent, which is proportional to the number of adherent cells, was measured by spectrometry at OD 550 nm (Y axis). Bars indicate OD in the detergent for each group as indicated in the legend.
  • the p value compares the adhesion of the cells of VLA-1+ and VLA-I- derived cultures.

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Abstract

Dans certains modes de réalisation, l'invention concerne des procédés de traitement dans le cadre desquels un antagoniste VLA-I est administré à un patient qui souffre d'un ou plusieurs troubles inflammatoires. Selon l'invention: (1) le patient ne répond pas de façon adéquate à, ou n'est pas susceptible de répondre de façon adéquate à la thérapie par antagoniste TNFa; ou (2) on observe chez le patient des effets réfractaires et/ou de tachyphylaxie au cours de la thérapie par antagoniste TNFa; ou (3) l'antagoniste VLA-I administré est un adjuvant à la monothérapie par antagoniste TNFa; et (4) l'administration d'antagoniste VLA-I est efficace pour traiter le(s) trouble(s) inflammatoire(s) dont souffre le patient.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014505703A (ja) * 2011-02-03 2014-03-06 サンタラス， インコーポレイテッド 対象の選択および治療
US9644030B2 (en) 2001-04-13 2017-05-09 Biogen Ma Inc. Antibodies to VLA-1
US9902774B2 (en) 1999-06-01 2018-02-27 Biogen Ma Inc. Method for the treatment of inflammatory disorders
US10119979B2 (en) 2006-05-25 2018-11-06 Biogen Ma Inc. Methods of treating stroke and traumatic brain injury using humanized AQC2 anti-VLA-1 antibodies
US10160808B2 (en) 2012-02-16 2018-12-25 Santarus, Inc. Anti-VLA1 (CD49A) antibody pharmaceutical compositions
WO2020010118A1 (fr) * 2018-07-03 2020-01-09 Novartis Inflammasome Research, Inc. Méthodes de traitement ou de sélection d'un traitement pour un sujet résistant à un inhibiteur de tnf à l'aide d'un antagoniste de nlrp3
WO2021002887A1 (fr) * 2019-07-02 2021-01-07 Novartis Inflammasome Research, Inc. Antagonistes de nlrp3 ciblant l'intestin et leur utilisation en thérapie

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020146417A1 (en) * 1999-06-01 2002-10-10 Philip Gotwals Method for the treatment of inflammatory disorders
US20040081651A1 (en) * 2001-04-13 2004-04-29 Michael Karpusas Antibodies to vla-1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020146417A1 (en) * 1999-06-01 2002-10-10 Philip Gotwals Method for the treatment of inflammatory disorders
US20040081651A1 (en) * 2001-04-13 2004-04-29 Michael Karpusas Antibodies to vla-1

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BEN-HORIN S ET AL: "The role of very late antigen-1 in immune-mediated inflammation" CLINICAL IMMUNOLOGY, ACADEMIC PRESS,, US, vol. 113, no. 2, November 2004 (2004-11), pages 119-129, XP004576813 ISSN: 1521-6616 cited in the application *
BEN-HORIN SHOMRON ET AL: "Spontaneous ex vivo proliferation of synovial memory CD4+ T cells in auto-immune arthritis is oligoclonal, dependent upon VLA-1+ memory T cells, and inhibited by anti TNF alpha" FASEB JOURNAL, vol. 19, no. 4, Suppl. S, Part 1, March 2005 (2005-03), page A915, XP008072754 & EXPERIMENTAL BIOLOGY 2005 MEETING/35TH INTERNATIONAL CONGRESS OF PHYSIOLOGICAL SCIENCES; SAN DIEGO, CA, USA; MARCH 31 -APRIL 06, 2005 ISSN: 0892-6638 *
FIORUCCI STEFANO ET AL: "VLA-1 immunoneutralization protects against colon damage in a mouse model of colitis" GASTROENTEROLOGY, vol. 120, no. 5 Supplement 1, April 2001 (2001-04), page A.47, XP008072753 & 102ND ANNUAL MEETING OF THE AMERICAN GASTROENTEROLOGICAL ASSOCIATION AND DIGESTIVE DISEASE WEEK; ATLANTA, GEORGIA, USA; MAY 20-23, 2001 ISSN: 0016-5085 *
IANARO A ET AL: "Anti-very late antigen-1 monoclonal antibody modulates the development of secondary lesion and T-cell response in experimental arthritis" LABORATORY INVESTIGATION, UNITED STATES AND CANADIAN ACADEMY OF PATHOLOGY, BALTIMORE,, US, vol. 80, no. 1, 2000, pages 73-80, XP002974262 ISSN: 0023-6837 *
MARIETTE X: "Emerging biological therapies in rheumatoid arthritis" JOINT BONE SPINE, ELSEVIER, PARIS, FR, vol. 71, no. 6, November 2004 (2004-11), pages 470-474, XP004678683 ISSN: 1297-319X *
SU C ET AL: "Are there predictors of Remicade treatment success or failure?" ADVANCED DRUG DELIVERY REVIEWS, AMSTERDAM, NL, vol. 57, no. 2, 6 January 2005 (2005-01-06), pages 237-245, XP004650707 ISSN: 0169-409X *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9902774B2 (en) 1999-06-01 2018-02-27 Biogen Ma Inc. Method for the treatment of inflammatory disorders
US9644030B2 (en) 2001-04-13 2017-05-09 Biogen Ma Inc. Antibodies to VLA-1
US10119979B2 (en) 2006-05-25 2018-11-06 Biogen Ma Inc. Methods of treating stroke and traumatic brain injury using humanized AQC2 anti-VLA-1 antibodies
JP2014505703A (ja) * 2011-02-03 2014-03-06 サンタラス， インコーポレイテッド 対象の選択および治療
EP2670438A4 (fr) * 2011-02-03 2015-05-20 Santarus Inc Sélection et traitement de patients
JP2017078075A (ja) * 2011-02-03 2017-04-27 サンタラス， インコーポレイテッド 対象の選択および治療
AU2012212194B2 (en) * 2011-02-03 2017-05-04 Santarus, Inc. Selection and treatment of subjects
CN107252483A (zh) * 2011-02-03 2017-10-17 桑塔鲁斯股份有限公司 受试者的选择和治疗
US10160808B2 (en) 2012-02-16 2018-12-25 Santarus, Inc. Anti-VLA1 (CD49A) antibody pharmaceutical compositions
WO2020010118A1 (fr) * 2018-07-03 2020-01-09 Novartis Inflammasome Research, Inc. Méthodes de traitement ou de sélection d'un traitement pour un sujet résistant à un inhibiteur de tnf à l'aide d'un antagoniste de nlrp3
WO2021002887A1 (fr) * 2019-07-02 2021-01-07 Novartis Inflammasome Research, Inc. Antagonistes de nlrp3 ciblant l'intestin et leur utilisation en thérapie

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