WO2001034649A2 - Compositions et methodes de traitement de maladies auto-immunes et des rejets de greffon - Google Patents

Compositions et methodes de traitement de maladies auto-immunes et des rejets de greffon Download PDF

Info

Publication number
WO2001034649A2
WO2001034649A2 PCT/US2000/030739 US0030739W WO0134649A2 WO 2001034649 A2 WO2001034649 A2 WO 2001034649A2 US 0030739 W US0030739 W US 0030739W WO 0134649 A2 WO0134649 A2 WO 0134649A2
Authority
WO
WIPO (PCT)
Prior art keywords
gene delivery
antagonist
delivery vector
polynucleotide
antibody
Prior art date
Application number
PCT/US2000/030739
Other languages
English (en)
Other versions
WO2001034649A3 (fr
Inventor
Keting Chu
Changyu Wang
Original Assignee
Chiron Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Corporation filed Critical Chiron Corporation
Priority to AU15902/01A priority Critical patent/AU1590201A/en
Publication of WO2001034649A2 publication Critical patent/WO2001034649A2/fr
Publication of WO2001034649A3 publication Critical patent/WO2001034649A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer

Definitions

  • This invention relates to compositions and methods for treating autoimmune diseases and transplant rejections and, more specifically, to the use of gene delivery vector technology to direct the expression of CD40 antagonist polynucleotides suitable for the treatment of autoimmune diseases and transplant rejections in a mammal.
  • the present invention provides, in one embodiment, polynucleotides that encode CD40 antagonists.
  • Inventive CD40 antagonists include, but are not limited to, antibodies or fragments thereof as well as fusion proteins, peptides and other molecules that bind to CD40.
  • Representative CD40 antagonist fusion proteins may comprise at least one anti-CD40 antibody Fv heavy chain and one anti-CD40 antibody Fv light chain.
  • one such fusion protein comprises portions of the humanized anti- CD40 antagonist monoclonal antibody 5H7 Fv heavy and light chains and is encoded by the polynucleotide depicted by SEQ ID NO:l or related polynucleotides having at least 70% sequence identity with or that hybridize under stringent conditions to the polynucleotide of SEQ ID NO : 1.
  • gene delivery vectors are provided that comprise polynucleotides encoding CD40 antagonists.
  • Suitable gene delivery vectors for use in expressing inventive CD40 antagonists may comprise transcriptional sequences from a retrovirus, such as Moloney murine leukemia virus, lentivirus or spumavirus; adenovirus; adeno-associated virus; or herpes virus as well as other viruses suitable for the construction of gene delivery vectors.
  • the gene delivery vector comprises adeno-associated virus 5' and 3' inverted terminal repeat (ITR) sequences as well as a CMV immediate early enhancer/promoter.
  • the vector can also comprise a signal sequence at the N-terminus, such as a PDGF signal sequence.
  • adeno-associated virus gene delivery vectors may also comprise a polyadenylation sequence for example, the bovine growth hormone (BGH) polyadenylation sequence.
  • All of the gene delivery vectors of the present invention comprise one or more polynucleotides that encode a CD40 antagonist.
  • Representative CD40 antagonists include polypeptides, proteins and other molecules sharing the CD40 antagonist functional activity.
  • the CD40 antagonists may specifically bind to CD40 and may block the interaction of CD40 with its ligand CD40L.
  • Other embodiments of the present invention provide gene delivery vectors that encode monoclonal or humanized monoclonal antibodies as well as Fab, F(ab) 2 and Fv fragments.
  • Still other embodiments provide gene delivery vectors for the expression of CD40 antagonists derived from the genetic fusion of polynucleotide fragments that encode heavy and light chain Fab and Fv antibody fragments.
  • the CD40 antagonist is derived from a cell line that expresses a CD40 monoclonal antibody such as 5H7 or a fragment thereof.
  • one inventive fusion protein is encoded by the polynucleotide as depicted by SEQ ID NO:l.
  • Other related embodiments provide fusion proteins encoded by polynucleotides that are at least 70% identical with SEQ ID NO:l or that hybridize under stringent conditions with the polynucleotide depicted in SEQ ID NO: 1.
  • compositions including pharmaceutical compositions, comprising a therapeutically effective amount of one or more gene delivery vectors that directs the expression of one or more polynucleotides encoding a CD40 antagonist.
  • Inventive compositions may further comprise a pharmaceutically acceptable carrier or stabilizer suitable for in vivo administration. These compositions may be further combined with additional agents efficacious against autoimmune diseases or transplant rejections. Further embodiments of the present invention provide methods for use of inventive compositions in the treatment of autoimmune diseases in a mammal.
  • inventive compositions can be formulated in amounts sufficient to reverse or diminish the severity of one or more autoimmune disease such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dermatomyositis, scleroderma and psoriasis.
  • autoimmune disease such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dermatomyositis, scleroderma and psoriasis.
  • compositions that comprise one or more gene delivery vectors encoding a CD40 antagonist.
  • Administration of such compositions by the present methods is expected to permit a diminution or reversal of organ, tissue or cellular transplant rejections.
  • inventive compositions may be administered in vivo to the mammal through, for example, intravenous, intraperitoneal or intradermal injection.
  • inventive compositions or gene delivery vectors may be administered directly to mammalian cells or tissues ex vivo prior to treatment of the mammal with the CD40 antagonist expressing cells or tissue.
  • one or more gene delivery vectors encoding a CD40 antagonist may be applied to peripheral blood mononuclear cells (PBMC) isolated from a subject in need of anti-autoimmune disease therapy prior to reintroducing the PBMC in vivo.
  • PBMC peripheral blood mononuclear cells
  • Still further embodiments provide in vitro methods for the identification of alternative CD40 antagonists including, but not limited to, antibodies and fusion proteins as well as other molecules efficacious in the treatment of autoimmune diseases and transplant rejections.
  • the present invention thus provides the art with polynucleotides, gene delivery vectors, compositions and methods that are generally effective in treating autoimmune diseases and transplant rejections.
  • Figure 1 is a schematic illustration of the adeno-associated virus based gene delivery vector D 10- 1.
  • Figure 2 is a schematic illustration of the adeno-associated virus based gene delivery vector D10p ⁇ CD40-l comprising the coding region of a CD40 antagonist antibody Fv heavy and light chain gene fusion derived from the monoclonal antibody 5H7.
  • the present invention is directed generally to polynucleotides, gene delivery vectors, compositions and methods for the treatment of autoimmune diseases and transplant rejections. More specifically, the invention disclosed herein provides polynucleotides encoding CD40 antagonists, gene delivery vectors comprising such CD40 antagonist encoding polynucleotides, gene delivery vector based compositions comprising CD40 antagonist encoding polynucleotides and methods for treating autoimmune diseases and transplant rejections by the administration of compositions comprising one or more of these gene delivery vectors and compositions.
  • CD40 is a 40-50 kDa type I membrane glycoprotem belonging to the
  • CD40 ligand referred to variously as CD40L, gp39 or CD 154, is a 33 kDa type II membrane glycoprotein that is transiently expressed primarily on the surface of activated CD4 + T cells. Datta, supra.
  • CD40 Owing to its expression on various cell types of the immune system, CD40 is thought to play a general role in immune regulation. For example, signaling between CD40L and CD40 is believed to be obligatory and nonredundant for helper T- cell and B-cell interactions leading to T-cell-dependent activation, proliferation or differentiation of B-cells as well as regulation of B-cell survival and apoptosis. See, e.g., Denfeld, R.W. et al, Eur. J. Immunol. 26(10):2329-34 (1996). Thus, the CD40- CD40L interaction is likely to be essential in humoral immunity, isotype switching and formation of germinal centers.
  • X-HIM X- linked hyper-IgM syndrome
  • CD40 and CD40L have also been implicated in autoimmune diseases including multiple sclerosis (MS) and systemic lupus erythematosus (SLE) as well as T-cell inflammatory skin diseases such as psoriasis.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • T-cell inflammatory skin diseases such as psoriasis.
  • administration of antibodies to CD40L has been shown to be effective in blocking autoimmunity in proteolipoprotein-induced experimental encephalomyelitis (EAE).
  • EAE is not induced in CD40L knockout mice carrying the T-cell receptor as a transgene. See van Kooten et al., Int. Arch. Allergy and Immunol. 113:393-399 (1997) and references cited therein.
  • Psoriasis is a T-cell mediated autoimmune disease believed to be linked to both genetic and environmental triggering factors such as bacterial superantigens. See, e.g., Valdimarsson, H. et al., Immunol. Today 16(3):X45-9 (1995); Boehncke, W.H. et al., Nature 379(6568):777 (29, 1996); Boehncke, W.H., Trends Microbiol.
  • This disease is characterized by complex alterations of various cell types including parakeratosis, the hyperproliferation and differentiation of the epidermal keratinocytes, and akanthosis, the increase in epidermal thickness resulting from keratinocyte hyperproliferation.
  • psoriatic lesions exhibit an infiltration of mixed leukocytes composed of activated T lymphocytes, neutrophils within the dermis and epidermal microabscesses, lining macrophages and dermal mast cells.
  • CD40 co-localizes with the expression of known markers of inflammatory skin disorders, such as ICAM-1 and Bcl-x, CD40 may have a functional role in the progression of these diseases, and an effective therapeutic regimen might involve disrupting the CD40/CD40-ligand pathway. Id.
  • CD40 has recently been found to play a critical role in tissue transplant rejection. This observation has significant implications for transplant therapy where the major goal is to induce, in the host mammal, immune tolerance to the donor organ, tissue or cells. That is, successful transplantation requires that a state be induced in the host in which the mammal does not mount an inflammatory response to the allo or xenograft. Gudmundsdottir, H. et al., J. Am. Soc. Nephrol. 70:1356-1365 (1999). It has become well established that T cells require two signals to generate a productive immune response. Janeway, CA. Jr., et al., Cell 76:275-285 (1994); Harding, F.A.
  • TCR T cell receptor
  • CD28 The costimulatory signal provided through the T cell surface molecule CD28 has been most thoroughly characterized experimentally.
  • CD28 interacts with two ligands, CD80 (B7-1) and CD86 (B7-2). These ligands are expressed on activated antigen presenting cells (APC) and some other cell types.
  • APC activated antigen presenting cells
  • CD28 costimulation enables T cells to respond to low levels of TCR ligation and supports sustained T cell responses; that is, in the absence of CD28 signaling, T cell responses are transient.
  • CD40 and CD40L play an essential role in T cell costimulation and in transplantation.
  • CD40 ligation on APC, and other cells promotes their ability to induce and support T cell responses.
  • engagement of CD40 induces the expression of B7-1.
  • the CD40/CD40L pathway may support T cell immune responses through induction of CD28 ligands and resultant activation of the CD28 pathway.
  • ICAM-1 and CD44H are also induced in CD40-activated APC. Shinde, S. et al., J. Immunol. 757:2764-2768 (1996).
  • Blockade of T cell costimulatory signals may improve long-term allograft survival by inducing transplant tolerance.
  • Blockade of co-stimulatory molecules is effective in both mouse and rat models of cardiac, hepatic, islet, renal, lung and bone marrow transplantation. See, e.g., Sayegh, M.H. et al., J Am. Soc. Nephrol. 6:1143-1150 (1995) and Harlan, D.M. et al., Graft 7:63-70 (1998). This was shown, for example, with blocking antibodies to CD40L (CD154). Linsley, PS. et al., J Exp. Med. 774:561-569 (1991). Hancock, W.H.
  • CD40 Antagonists are Effective in the Treatment of Autoimmune Diseases and Transplant Rejections
  • the present invention provides gene transfer vectors, compositions and methods for treating autoimmune diseases and transplant rejections in a mammal.
  • Each of the inventive gene transfer vectors expresses one or more CD40 antagonist polynucleotide.
  • the present compositions comprise one or more gene transfer vectors having one or more polynucleotide sequence that encodes a CD40 antagonist. Methods encompassed within the scope of the present invention utilize the gene delivery vectors to administer CD40 antagonist polynucleotides to various tissues, organs and cells.
  • the term "antagonist” generally refers to the property of a molecule, compound or other agent to, for example, interfere with the binding of one molecule with another molecule or the stimulation of one cell by another cell either through steric hindrance, conformational alterations or other biochemical mechanism.
  • the term antagonist relates to the property of an agent to prevent the binding of a receptor to its ligand, e.g., the binding of CD40 with CD40L, thereby inhibiting the activation of the respective B- or T-cell population.
  • the term antagonist is not limited to any specific action mechanism, but, rather, refers generally to the functional property presently defined.
  • Antagonists of the present invention include, but are not limited to, antibodies or fragments thereof as well as fusion proteins, peptides and other molecules that bind to CD40.
  • the CD40 antagonists may inhibit the up-regulation of activation markers, e.g., CD25 and CD69, on CD4 + T-cells to between about 10 and 30% the levels of the untreated control cells.
  • inventive CD40 antagonists are effective in inhibiting the morphological characteristics of psoriasis such as epidermal thickening and hyperproliferation, i.e., akanthosis and parakeratosis, respectively, in the SCID mouse xenogeneic transplant animal model system.
  • autoimmune disease refers generally to those diseases characterized by the failure of one or more B- and/or T-cell populations, or gene products thereof, to distinguish between self and non-self antigenic determinants.
  • Autoimmune diseases are often characterized by the infiltration of the target cells with inflammatory lymphoid cells, for example, mononuclear phagocytes, lymphocytes and plasma cells as well as secondary lymphoid follicles.
  • Exemplary autoimmune diseases include, but are not limited to, organ specific disorders such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, and insulin- dependent diabetes mellitus as well as non-organ specific disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, dermatomyositis, scleroderma and psoriasis.
  • organ specific disorders such as Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis, pernicious anemia, Addison's disease, and insulin- dependent diabetes mellitus
  • non-organ specific disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, dermatomyositis, scleroderma and psorias
  • CD40 antagonists will also find use in minimizing or eliminating the onset of graft rejection following tissue transplantation.
  • CD40 antagonists may be effective in treating, for example, cardiac, hepatic, islet, renal, lung and bone marrow transplantation.
  • the present invention provides gene delivery vector systems for the expression of a variety of CD40 antagonists.
  • the phrase "gene delivery vector” refers generally to a nucleic acid construct that carries and, within certain embodiments, is capable of directing the expression of a polynucleotide of interest.
  • gene delivery vector systems see Molecular Biotechnology: Principles and Applications of Recombinant DNA, Ch. 21, pp. 555-590 (ed. B.P. Glick and J.J. Pasternak, 2 nd ed. 1998); Jolly, Cancer Gene Ther. 7:51-64 (1994); Kimura, Human Gene Ther. 5:845-852 (1994); Connelly, Human Gene Ther. 6:185-193 (1995); and Kaplitt, Nat. Gen. 6:148-153 (1994).
  • Virus based gene delivery systems include, but are not limited to retro virus, such as Moloney murine leukemia virus, spumaviruses and lentiviruses; adenovirus; adeno-associated virus; and herpes-simplex virus vector systems. Viruses of each type are readily available from depositories or collections such as the American Type Culture Collection (ATCC; Rockville, Maryland) or may be isolated from known sources using commonly available materials and techniques.
  • retro virus such as Moloney murine leukemia virus, spumaviruses and lentiviruses
  • adenovirus adeno-associated virus
  • herpes-simplex virus vector systems herpes-simplex virus vector systems.
  • ATCC American Type Culture Collection
  • the gene delivery vector systems of the present invention will find applications both in in vivo as well as ex vivo therapeutic regimens. Each of these applications is described in further detail below.
  • retroviral gene delivery vectors are provided which are constructed to carry or express a CD40 antagonist polynucleotide.
  • CD40 antagonist polynucleotide refers generally to a nucleic acid sequence that encodes a polypeptide, protein, fusion protein, antibody, antibody fragment or fusion of antibody fragments having CD40 antagonist activity.
  • Retroviral gene delivery vectors of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses. See RNA Tumor Viruses, Cold Spring Harbor Laboratory (2 nd ed.1985).
  • retroviral gene delivery vectors may be readily utilized in order to assemble or construct retroviral gene delivery vectors given the disclosure provided herein, and standard recombinant DNA techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed. 1989) and Kunkle, Proc. Natl. Acad. Sci. U.S.A. 82:488 (1985).
  • portions of the retroviral gene delivery vectors may be derived from different retroviruses.
  • a retroviral vector suitable for the expression of a CD40 antagonist polynucleotide, must include at least one transcriptional promoter/enhancer or locus defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
  • Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
  • the retroviral vector may also include a signal which directs polyadenylation, selectable markers such as Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • selectable markers such as Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR
  • retroviral gene delivery vector constructs comprising a 5' LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR, wherein the vector construct lacks gaglpol or env coding sequences.
  • retroviral gene delivery vectors may likewise be utilized within the context of the present invention, including, for example, those disclosed in the following each of which is incorporated herein by reference: EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 93/10218; Vile et al., Cancer Res. 55:3860-3864 (1993); Vile et al., Cancer Res. 55:962-967 (1993); Ram et al., Cancer Res. 55:83-88 (1993); Takamiya et al, J. Neurosci. Res.
  • Packaging cell lines suitable for use with the above described retroviral gene delivery vector constructs may be readily prepared. See, e.g., U.S. Patent Nos.
  • packaging cell lines may be utilized to create producer cell lines (also termed vector cell lines or "NCLs") for the production of recombinant vector particles. It may be preferred to use packaging cell lines made from human (e.g., HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that avoid inactivation in human serum.
  • Adeno-associated viruses possess a number of qualities that make them particularly suitable for the development of gene delivery vectors generally and for the delivery of polynucleotides encoding CD40 antagonists in particular.
  • AAV is a non-pathogenic, defective human parvovirus that is non-infective without an adeno or herpes helper virus.
  • helper virus AAV becomes integrated latently into the host genome.
  • AAV has the advantage over the retroviruses, discussed above, in being able to transduce a wide range of both dividing and quiescent cell types.
  • a variety of AAV gene delivery vectors may be utilized to direct the expression of one or more CD40 antagonists.
  • Representative examples of such vectors include the AAV vectors disclosed by Srivastava in WO 93/09239; Samulski et al., J. Virol. 65:3822-3828 (1989); Mendelson et al., Virol. 166:154-165 (1988); and Flotte et al., Proc. Natl. Acad. Sci. U.S.A. 90f22 :10613-10617 (1993), incorporated herein by reference.
  • an AAV gene delivery vector of the present invention should include, in order, a 5' adeno-associated virus inverted terminal repeat; a signal sequence for secretion, such as a PDGF sequence; a polynucleotide encoding VH and VL of the CD40 antagonist wherein the VH and VL regions are separated by a linker; a sequence operably linked to the CD40 antagonist polynucleotide which regulates its expression in a target tissue, organ or cell; and a 3' adeno-associated virus inverted terminal repeat.
  • a suitable regulatory sequence for the expression of CD40 antagonists is, e.g., the enhancer/promoter sequence of cytomegalovirus (CMV).
  • the AAV vector may preferably have a polyadenylation sequence such as the bovine growth hormone (BGH) polyadenylation sequence.
  • AAV vectors should have one copy of the AAV ITR at each end of the polynucleotide encoding the CD40 antagonist, to allow replication, packaging, efficient integration into the host cell genome and rescue from the chromosome.
  • the 5' ITR sequence consists of nucleotides 1 to 145 at the 5' end of the AAV DNA genome, and the 3' ITR includes nucleotides 4681 to 4536 of the AAV genome.
  • the AAV vector may also include at least 10 nucleotides following the end of the ITR (i.e., a portion of the so-called "D region").
  • FIG. 1 One exemplary AAV based gene delivery vector encompassed within the scope of the present invention is the DlO-1 vector depicted in Figure 1.
  • this vector comprises a 145 bp 5' ITR and a 145 bp 3' ITR that flank, in order from 5' to 3', a CMV immediate early (IE) enhancer/promoter (ENH/PRO) sequence for high level transcription of an inserted CD40 antagonist polynucleotide; a multiple cloning site, to permit the convenient subcloning of a desired CD40 antagonist polynucleotide, downstream of the CMV ENH PRO sequence; a signal sequence; and a polyadenylation (pA) sequence from bovine growth hormone (BGH).
  • Optimal packaging of an adeno-associated virus gene delivery vector requires that the 5' and 3' ITRs be separated by approximately 2-5 kb. It will be apparent, however, that the ideal spacing between ITR sequences may vary depending on the particular packaging system utilized. This spacing may be achieved by incorporating a "stuffer” or “filler" polynucleotide fragment to bring the total size of the nucleic acid sequence between the two ITRs to between 2 and 5 kb.
  • a non-coding stuffer polynucleotide may be incorporated, for example, 3' to the 5' ITR sequence and 5' of the CD40 antagonist polynucleotide.
  • the precise nucleotide sequence of the stuffer fragment is not an essential element of the final construct.
  • the stuffer polynucleotide is a 1.35 kb Haelll fragment from ⁇ X174.
  • each CD40 antagonist polynucleotide may be separated by a ribosome readthrough or, alternatively, by an Internal Ribosome Entry Site or "IRES.” It may also be preferred to incorporate several different CD40 antagonist polynucleotides separated by a ribosome readthrough or an IRES. In the latter instance, it may be preferred to organize the polynucleotides as two or more separate transcription units each with its own promoter and polyadenylation signal.
  • Recombinant AAV vectors of the present invention may be generated from a variety of adeno-associated viruses, including for example, serotypes 1 through
  • ITRs from any AAV serotype are expected to have similar structures and functions with regard to replication, integration, excision and transcriptional mechanisms.
  • expression of the CD40 antagonist polynucleotide may be accomplished by a separate promoter (e.g., a viral promoter).
  • suitable promoters include a CMV promoter, an RSV promoter, an SV40 promoter, or a MoMLV promoter.
  • Other promoters that may similarly be utilized within the context of the present invention include cell or tissue specific promoters or inducible promoters.
  • Representative inducible promoters include tetracycline-response promoters (e.g., the "Tet" promoter) as described in Gossen et al., Proc. Nat/. Ac ⁇ d. Sci. U.S.A.
  • the AAV gene delivery vector may also contain additional sequences, for example from an adenovirus, which assist in effecting a desired function for the vector.
  • additional sequences include, for example, those which assist in packaging the AAV gene delivery vector in adenovirus particles.
  • Packaging cell lines suitable for producing adeno-associated viral vectors may be routinely prepared given readily available techniques. See, e.g., U.S. Patent No. 5,872,005, incorporated herein by reference. At a minimum, suitable packaging systems for AAV gene delivery systems of the present invention will include the AAV replication and capsid genes. Preferred packaging cell lines may contain both an AAV helper virus as well as an AAV gene delivery vector containing the CD40 antagonist polynucleotide.
  • Representative packaging cell line systems see, e.g. Holscher, C. et al., J Virol. 65:7169-7177 (1994); Clark, K.R et al., Hum. Gene Ther. 6:1329-1341 (1995); and Tamayosa, K. et al, Hum. Gen. Ther. 7:507-513 (1996) which are incorporated herein by reference.
  • packaging of AAV may be achieved in vitro in a cell free system to obviate transfection protocols or packaging cell lines.
  • Such in vitro systems incorporate an AAV gene delivery vector bearing the CD40 antagonist polynucleotide and a source of Rep-protein, capsid-protein and Adenovirus proteins that supply helper- viral functions. The latter proteins are typically supplied in the form of a cell extract. Representative in vitro systems are further described in Ding, L. et al., Gen. Ther. 4:1167-1172 (1997) and Zhou, Z. et al, J. Virol. 72:3241-3247 (1998) which are incorporated herein by reference.
  • adenoviral vectors may be employed. Representative examples of such vectors include those described by, for example, Berkner, Biotechniques 6:616-627 (1988); Rosenfeld et al., Science 252:431-434 (1991); WO 93/9191; Kolls et al, Proc. Natl. Acad. Sci.
  • Gene delivery vectors of the present invention also include herpes vectors. Representative examples of such vectors include those disclosed by Kit in Adv.
  • Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO 95/04139 (Wistar Institute), pHSVlac described in Geller, Science 247:1667-1669 (1988), and in WO 90/09441 and WO 92/07945; HSV Us3::pgC-lacZ described in Fink, Human Gene Therapy 5:11-19 (1992); and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.
  • Gene delivery vectors may also be generated from a wide variety of other viruses including, for example, poliovirus (Evans et al., Nature 559:385-388 (1989); and Sabin, J. Biol. Standardization 7:115-118 (1973)); rhinovirus; pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Natl. Acad. Sci. U.S.A. 56:317-321 (1989); Flexner et al., Ann. N Y. Acad. Sci. 569:86-103 (1989); Flexner et al., Vaccine 5:17-21 (1990); U.S. Patent Nos.
  • CD40 antagonists such as, for example, nucleic acid expression vectors; polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example, see Curiel, Hum Gene Ther 5:147-154 (1992); ligand linked DNA, for example, see Wu, J Biol Chem 264:16985-16987 (1989); eucaryotic cell delivery vectors; deposition of photopolymerized hydrogel materials; hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; ionizing radiation as described in U.S. 5,206,152 and in WO 92/11033; nucleic charge neutralization or fusion with cell membranes.
  • nucleic acid expression vectors polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example, see Curiel, Hum Gene Ther 5:147-154 (1992); ligand linked DNA, for example, see Wu, J Biol Chem 264:16985-16987 (1989); eucaryotic cell delivery
  • CD40 antagonist polynucleotide of interest can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu, et al., J. Biol. Chem.
  • Naked DNA may also be employed.
  • Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads.
  • DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO 94/23697, and WO 91/144445, and European Patent Publication No. 524,968.
  • Nucleic acid sequences can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously- active promoters.
  • Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. U.S.A. 97(24):11581-11585 (1994).
  • the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials.
  • Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Patent Nos. 5,422,120 and 4,762,915, in PCT Patent Publication Nos.
  • CD40 antagonists within the scope of the present invention include, but are not limited to antibodies, or fragments thereof, as well as fusion proteins or other molecules that bear the CD40 antagonist functional activity.
  • CD40 antagonists are monoclonal antibodies prepared essentially as described in de Boer et al. U.S. Patent No. 5,677,165 (1997) (de Boer '165) which patent is incorporated herein by reference.
  • DNA encoding CD40 or a fragment thereof is PCR amplified from a mixture of cellular cDNAs.
  • the PCR product is digested with one or more restriction endonucleases to create appropriate ends and ligated into a baculovirus plasmid or other expression system.
  • the plasmid encoding CD40, or a fragment thereof is introduced into, e.g., Sf9 cells to facilitate protein production.
  • Clones of Sf9 cells expressing CD40 are identified, e.g., by enzyme linked immunosorbant assay (ELISA) as discussed in de Boer '165 and injected, intraperitoneally, into BALB/c mice to induce antibody production. Serum is tested for the production of specific antibodies and spleen cells from animals having a positive specific antibody titer are used for cell fusions with myeloma cells to generate hybridoma clones. Supernatants derived from hybridoma clones are tested, via fluorescent cell staining of EBV-transformed B-cells, for the presence of monoclonal antibodies having specificity against CD40.
  • CD40 antagonists are humanized anti-CD40 monoclonal antibodies.
  • humanized antibody refers to an antibody derived from a non-human antibody - typically a mouse monoclonal antibody.
  • a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans.
  • chimeric antibody refers to an antibody containing sequences derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567), which typically originate from different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally human constant and mouse variable regions.
  • humanized antibodies are far less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis. Thus, these antibodies may be preferred in therapeutic applications that involve in vivo administration to a human afflicted with an autoimmune disease or, alternatively, experiencing, or at risk of experiencing, a transplant rejection.
  • Another approach for the isolation of fully humanized anti-CD40 antibodies is afforded by the so-called xenomouse technology. See Jakobovits et al., Adv. Drug Deliv. Rev. 31:33-42 (1998); Jakobovits et al., Exp. Opin. Invest.
  • mice are genetically engineered to suppress mouse antibody gene expression.
  • the mouse antibody repertoire is functionally replaced with human antibody gene expression, while the rest of the mouse immune system is left intact.
  • These xenomice are capable of generating human antibodies to human antigens, such as CD40, which antigens are recognized as foreign by the mouse such that an immune response is elicited.
  • CD40 suitable for inoculation may be prepared by recombinant DNA methods as discussed, supra, and as otherwise well known in the art. See, e.g., Sambrook et al, supra and de Boer '165, supra.
  • a strong CD40 antigen-specific antibody response may be detected by ELISAs performed on sera from immunized mice.
  • Hybridomas may be derived from spleen or lymph node by standard hybridoma technology and screened for secretion of antigen-specific human monoclonal antibodies by ELISA.
  • Monoclonal Antibodies filed October 2, 2000, as Serial No. , which is incorporated herein by reference.
  • the application discloses CD40 antibodies raised in mice transgenic for human immunoglobulin loci.
  • the antibodies specifically bind to CD40 expressed by a variety of cells, and are suitable for use in the methods of the present invention.
  • Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as “humanizing”), or, alternatively, (2) transplanting the entire non-human variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”).
  • humanized antibodies will include both “humanized” and “veneered” antibodies. These methods are disclosed in, e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al, Proc. Natl. Acad. Sci., U.S.A.
  • the complementarity determining regions (CDRs) of a mouse antibody are inserted into human framework regions to generate antibodies in which only the CDR sequences are derived from the original mouse antibody.
  • CDRs complementarity determining regions
  • the CDRs are identified by comparing the amino acid sequence of the variable regions of the mouse monoclonal antibody to the sequences of all known mouse variable regions, and the sequences of all known human variable regions. See Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office, Washington, D.C (4th ed. 1987).
  • PCR methodology may be used to graft CDRs from mouse, or other rodent, monoclonal antibodies onto human FRs to make fully humanized antibodies or antibody fragments. Lewis, A.P. and Crowe, J.S., Gene 101 (2):29 -302 (1991). PCR primers that bind to the FR regions flanking the CDRs have been developed; these permit amplification of the CDR from hybridoma cells secreting the CD40 antagonist monoclonal antibody of interest. The PCR product is cloned and sequenced, and joined to the framework and constant regions of choice.
  • complementarity determining region refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. See, e.g., Chothia et al., J. Mol. Biol. 796:901-917 (1987); Kabat et al., U.S. Dept. of Health and Human Services NIH Publication No. 91-3242 (1991).
  • constant region refers to the portion of the antibody molecule which confers effector functions. In the present invention, mouse constant regions are substituted by human constant regions.
  • the constant regions of the subject humanized antibodies are derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu.
  • One method of humanizing antibodies comprises aligning the non- human heavy and light chain sequences to human heavy and light chain sequences, selecting and replacing the non-human framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region which disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-human CDRs of the parent non-human antibody.
  • Such humanized antibodies may be further derivatized to facilitate uptake and clearance, e.g., via Ashwell receptors. See, e.g., U.S. Patent Nos.
  • humanized antibodies may be prepared essentially as described in de Boer, U.S. Patent No. 5,874,082 (1999) (de Boer '082) which patent is incorporated herein by reference. Briefly, mRNA is prepared from a hybridoma which expresses an anti-CD40 monoclonal antibody. cDNA encoding the variable regions of the heavy and light chains is amplified using RT-PCR employing degenerate oligonucleotide primers.
  • PCR products are cloned into a sequencing plasmid from which clones the nucleotide sequence of the variable heavy and light chain cDNAs are determined and from which sequence a consensus amino acid sequence for the variable heavy and light chains is derived.
  • the deduced amino acid sequences are used to search databases for human antibody sequences having the highest degree of sequence similarity to the monoclonal antibody (de Boer '082). Based on the identified homologous human sequence, mutagenesis primers are designed and used to change the indicated residues from mouse to human.
  • cDNAs encoding the humanized variable heavy and light chains are expressed off a baculovirus expression plasmid including a portion of the constant region of human IgG heavy chain and the complete human constant light chain. Humanized heavy and light chains are co-expressed in Sf9 insect cells and the resulting culture supernatants are analyzed for antibody expression using Western blot and fluorescence-activated cell sorting (FACS) analysis (de Boer '082).
  • FACS fluorescence-activated cell sorting
  • CD40 antagonists may be readily obtained by other methods commonly known in the art. Because of the desirability of having fully humanized sequences, depending on the precise application involved, any of the following methods may be preferred.
  • CD40 antagonists within the scope of the present invention may be isolated by screening Fab phage display libraries using recombinant CD40 as a probe.
  • CD40 antagonist antibodies may be derived from hybridoma cells produced from spleen cells of one or more xenomouse inoculated with CD40. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) for a discussion of the hybridoma technology.
  • Another methodology that may be employed to obtain humanized CD40 antagonists suitable for expression in a gene delivery vector of the present invention is the CDR grafting technology. These methods are described in further detail below.
  • Phage display libraries for the production of high-affinity antibodies have been the subject of several recent reviews. See, e.g., Hoogenboom, H.R. et al., Immunotechnology 4(l):X-20 (1998); Hoogenboom, H.R., Trends Biotechnol. 75:62-70 (1997) and McGuinness, B. et al, Nature Bio. Technol. 74:1149-1154 (1996) each of which is incorporated herein by reference.
  • Among the advantages of the phage display technology is the ability to isolate antibodies of human origin that cannot otherwise be easily isolated by conventional hybridoma technology. Furthermore, phage display antibodies may be isolated in vitro without relying on an animal's immune system.
  • Antibody phage display libraries may be accomplished, for example, by the method of McCafferty et al., Nature 348:552-554 (1990) which is incorporated herein by reference.
  • the coding sequence of the antibody variable region is fused to the amino terminus of a phage minor coat protein (pill).
  • pill phage minor coat protein
  • CD40 suitable for screening a phage library may be expressed on baculovirus Sf9 cells as detailed in deBoer '165, see supra.
  • the CD40 coding region may be PCR amplified using primers specific to the CD40 extracellular domain.
  • the resulting fragment may then be isolated by, e.g., agarose gel purification and ligated in frame with a suitable tag protein such as 6-His, glutathione-S-transferase (GST) or other such readily available affinity tag.
  • GST glutathione-S-transferase
  • the resulting fusion protein may then be adsorbed to a solid matrix, e.g., a tissue culture plate or bead.
  • a solid matrix e.g., a tissue culture plate or bead.
  • Phage expressing antibodies having the desired anti- CD40 binding properties may subsequently be isolated by successive panning, in the case of a solid matrix, or by affinity adsorption to a CD40 antigen column.
  • Phage having the desired CD40 antagonist properties may be reintroduced into bacteria by infection and propagated by standard methods known to those skilled in the art. See Hoogenboom, H.R., Trends Biotechnol, supra for a review of methods for screening for positive antibody-pill phage.
  • Identification of additional CD40 antagonists may be achieved by using any of a number of known methods for identifying and obtaining proteins that specifically interact with other proteins or polypeptides, for example, a yeast two- hybrid screening system such as that described in U.S. Patent No. 5,283,173 and U.S. Patent No., 5,468,614.
  • a cDNA encoding CD40, or a fragment thereof may be cloned into a two-hybrid bait vector and used to screen a complementary target library for a protein having CD40 binding activity.
  • RNA may be isolated from one or more hybridoma cell lines derived from spleen cells of immunized animals that express CD40 antagonist antibodies and the respective polynucleotide encoding that antibody or antibody Fab heavy and light chains may be isolated, e.g., by the polymerase chain reaction (PCR) using oligonucleotides specific to the CD40 antagonist antibody sequence.
  • PCR polymerase chain reaction
  • the CD40 antagonists of the present invention are said to be immunospecific or specifically binding if they bind to CD40 with a K a of greater than or equal to about 10 4 M "1 , preferably of greater than or equal to about 10 5 M "1 , more preferably of greater than or equal to about 10 6 M “1 and still more preferably of greater than or equal to about 10 7 M "1 .
  • K a of greater than or equal to about 10 4 M "1 , preferably of greater than or equal to about 10 5 M "1 , more preferably of greater than or equal to about 10 6 M “1 and still more preferably of greater than or equal to about 10 7 M "1 .
  • Such affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using 125 I-labeled CD40; or by another method known to the skilled artisan.
  • the affinity data may be analyzed, for example, by the method of
  • CD40 antagonists of the present invention include, where applicable, functional equivalents.
  • molecules may differ in length, structure, components, etc. but may still retain one or more of the defined functions.
  • functional equivalents of the antibodies, antibody fragments or peptides of the present invention may include mimetic compounds, i.e., constructs designed to mimic the proper configuration and/or orientation for antigen binding.
  • Preferred CD40 antagonists may comprise one or more conservative amino acid substitutions.
  • conservative amino acid substitutions is meant those changes in amino acid sequence that preserve the general charge, hydrophobicity/hydrophilicity and or steric bulk of the amino acid substituted. For example, substitutions between the following groups are conservative: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Ser/Cys,/Thr, and Phe/Trp/Tyr. Such modifications will not substantially diminish the efficacy of the CD40 antagonists and may impart such desired properties as, for example, increased in vivo half-life or decreased toxicity.
  • the present invention provides, in certain embodiments, single-chain antibody fusion proteins comprising one or more Fv heavy chain and one or more Fv light chain.
  • Single-chain antibodies may be used for a variety of therapeutic and diagnostic applications in which Fc effector functions are not required and when a small size is advantageous. It may also be desirable to link additional protein-coding sequences onto CD40 antagonist polynucleotides to create dual-function molecules that can bind to CD40 and carry with the CD40 antagonist an independent functional activity.
  • An exemplary CD40 antagonist is depicted by the amino acid sequence of SEQ ID NO:2 which is the protein product of the polynucleotide of SEQ ID NO:l.
  • SEQ ID NO:2 is the protein product of the polynucleotide of SEQ ID NO:l.
  • this single-chain anti-CD40 antagonist antibody was derived from the hybridoma 5H7 cell line by fusion of the coding region of an anti-CD40 antagonist Fv heavy chain gene and an anti-CD40 antagonist Fv light chain gene.
  • RNA was isolated from the 5H7 hybridoma cell line, amplified by reverse transcriptase polymerase chain reaction (RT- PCR) using two pairs of oligonucleotide primers specific to the Fab heavy and light chain sequences, the resulting DNA fragments were connected by a linker sequence and were ligated into the AAV-derived gene delivery vector DlO-l, Figure 1, to create the vector D10p ⁇ CD40-l, Figure 2.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • VH and VL chains may be reversed or otherwise fused to alternative heavy and/or light chain sequences to create CD40 antagonist molecules having the desired functional properties.
  • length and nucleotide sequence of the linker region may be varied as necessary in order to achieve optimal CD40 binding affinity or other property appropriate for a given therapeutic or diagnostic application.
  • nucleotide sequence of SEQ ID NO:l may be varied to modify the CD40 antagonist's biological properties, in vivo stability or other functionality in accordance with the particular application desired.
  • the present invention contemplates modifications of the sequence of SEQ ID NO:l wherein the resulting polynucleotide is not less than 70% identical with the nucleic acid sequence of SEQ ID NO:l.
  • the resulting polynucleotide is not less than 90% identical with the nucleic acid sequence of SEQ ID NO:l. More preferably, the resulting polynucleotide is not less than 98% identical with the nucleic acid sequence of SEQ ID NO: 1.
  • amino acid sequence identity may be determined by standard methodologies, including use of the National Center for Biotechnology Information BLAST search methodology available at www.ncbi.nlm.nih.gov using default parameters. The identity methodologies most preferred are those described in U.S. Patent 5,691,179 and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997) both of which are incorporated herein by reference.
  • Global DNA sequence identity may also be determined using the Smith- Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty: 12, gap extension penalty: 1.
  • polynucleotides comprising a portion or portions of the nucleic acid sequence of SEQ ID NO:l may provide the desired CD40 antagonist activity.
  • the present invention provides polynucleotides or the complements thereof that hybridize under stringent conditions to the polynucleotide depicted by SEQ ID NO:l.
  • hybridize under conditions of a specified stringency is commonly used to describe the stability of hybrids formed between two single-stranded nucleic acid molecules. Stringency of hybridization is typically expressed in conditions of ionic strength and temperature at which such hybrids are annealed and washed.
  • high, medium and low stringency encompass the following conditions or equivalent conditions thereto: (1) By high stringency is meant 0.1 x SSPE or SSC, 0.1% SDS, 65°C; (2) by medium stringency is meant 0.2 x SSPE or SSC, 0.1% SDS, 50°C; and (3) by low stringency is meant 1.0 x SSPE or SSC, 0.1% SDS, 50°C
  • Compounds potentially useful in treating autoimmune diseases and transplant rejections may be screened in a number of systems. Animal models are used to identify those compounds having therapeutic activity in vivo as well as possessing acceptable levels of host toxicity. The models are useful for identifying compounds that are efficacious in the treatment of autoimmune diseases such as, e.g., systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and psoriases as well as against transplant rejections, for example, those occurring after transplantation of cardiac, hepatic, islet, renal, lung and bone marrow tissue.
  • autoimmune diseases such as, e.g., systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and psoriases
  • transplant rejections for example, those occurring after transplantation of cardiac, hepatic, islet, renal, lung and bone marrow tissue.
  • Efficacy of a given CD40 antagonist gene delivery vector can be tested in any of the animal model systems familiar to those skilled in the art.
  • Animal model systems for autoimmune diseases are described in Roitt, I. et al., "Autoimmunity and Autoimmune Disease," Immunology, Ch. 28 (1998); animal model systems available for the study of psoriasis, in particular, are described in Schon, M.P, supra.
  • the skilled artisan will appreciate that the selection of an appropriate animal model system will depend on the particular disease being treated. The following animal model systems are, therefore, provided by way of example not limitation.
  • autoimmunity can be induced in experimental animals by injecting autoantigen (i.e., self antigen) together with Freund's adjuvant.
  • autoantigen i.e., self antigen
  • such an animal model system may be used, for example, by injecting thyroglobulin to induce an inflammatory disease of the thyroid.
  • thyroglobulin to induce an inflammatory disease of the thyroid.
  • This animal model has been used to model the human condition known as Hashimoto's thyroiditis.
  • myelin basic protein, or T-cells specific for myelin basic protein may be injected in mice or rats to induce autoallergic encephalomyelitis.
  • Alternative animal model systems that may be used to test compounds and treatment regimens within the scope of the present invention include animals exhibiting spontaneous autoimmune diseases.
  • the Obese strain (OS) of chicken is characterized by the spontaneous occurrence of autoantibodies and by the progressive destruction and chronic inflammation of the thyroid.
  • the OS chicken parallels human autoimmune thyroid disease in displaying thyroid lesions as well as the production of antibodies to various thyroid components.
  • a number of animal model systems for psoriasis have been described including transplantation of human psoriatic skin onto nude mice, the asebia (ab/ab) strain of mice or the HLA-B27 transgenic rat as well as transplantation of skin from the flaky skin mouse onto nude mice.
  • the asebia mouse model features epidermal akanthosis, increased dermal vascularity and dermal infiltrate of macrophages and mast cells, but does not contain T-cell and neutrophil infiltrates.
  • the skin alterations in the ab/ab mouse do not precisely mirror every biological characteristic of psoriatic lesions.
  • the SCID mouse is widely used as an in vivo model of psoriasis. Id.
  • a standard measure of efficacy in the SCID model is the ability to lessen the severity of akanthosis and parakeratosis as well as to reduce mononuclear cell infiltrate in animals transplanted with psoriatic skin.
  • SCID severe combined immunodeficiency
  • the SCID mouse is also amenable to the adoptive transfer of components of the human immune system. See, e.g., Boehncke, W.-H. et al., Arch. Dermatol. Res., 286:325-330 (1994).
  • the autosomal recessive mutation responsible for the SCID phenotype in mice prevents antigen receptor gene rearrangements resulting in an intrinsic defect of T- and B-cells. Botsma, M.J. et al., Annu. Rev. Immunol., 9:323-350 (1991).
  • PP psoriatic plaque skin
  • NN normal human skin from healthy individuals
  • PN symptomless skin from a patient with psoriasis
  • corneal transplants have been reported in a Lewis and Fisher rat model system where the efficacy of, for example, neutralizing antibodies was tested (Yatoh, S. et al., Transplantation 66:1519-1524 (1998); see also, Ayliffe, W. et al., Br. J. Ophthalmol. 76:602 (1992); and Williams K.A., et al., Invest. Ophthalmol. Vis. Sci. 26:23 (1985)) and in a rabbit model system where an immunosuppressive agent was examined (Kobayashi, C. et al., Transplant Proc. 27:3156 (1989)).
  • An alternative model for transplant rejection is the murine popliteal lymph node (PLN) assay, a graft vs. host model that predicts activity of compounds in blocking human transplant rejection and that has been used routinely to evaluate compounds that are in use clinically.
  • PPN murine popliteal lymph node
  • cyclosporin and cyclophosphamide are both active in the murine PLN animal model system and are both used clinically.
  • compositions for and methods of treating autoimmune diseases and transplant rejections may utilize one or more gene delivery vector used singularly or in combination with other therapeutics to achieve the desired diminution of the autoimmune disease or transplant rejection of interest.
  • the gene delivery vectors of the present invention may be administered by intravenous delivery or by other methods described herein in order to induce long term in vivo expression of a variety of CD40 antagonists.
  • a retroviral, adenoviral, adeno-associated viral, herpes viral or other gene delivery vector comprising a CD40 antagonist polynucleotide intravenously results in sustained long-term systemic expression of therapeutic proteins or antibodies.
  • methods for obtaining long-term systemic expression in vivo of a variety of CD40 antagonists is encompassed by the instant invention.
  • the action of human complement on the retroviral vector is suppressed. This can be achieved by a number of techniques known to those skilled in the art.
  • human packaging cell lines are used in order to inhibit the action of human complement on the retroviral vector particles.
  • the present invention provides the production of high titer preparations of, for example, recombinant retroviral or adeno-associated viral vectors.
  • high titer viral vector preparation refers to a viral vector preparation that has a titer greater than 10 6 cfu/ml, more preferably greater than 10 7 cfu/ml, still more preferably greater than 10 cfu/ml, more preferably greater than 10 cfu/ml, yet more preferably greater than 10 10 cfu/ml, and most preferably greater than 10 11 cfu/ml.
  • the level of expression will vary depending on the packaging cell line employed, e.g., human HT1080 cells for retroviral packaging.
  • cfu refers to colony forming units when vectors contain a selectable marker.
  • the term refers to identifiable colonies when a phenotypically observable marker is used, such as blue colonies when the marker is beta-galactosidase.
  • cfu may be determined, for example, by PCR-based titer.
  • packaging cells are transduced with the markerless vector and the number of pro viral DNA copies are measured by quantitative PCR.
  • phrases "long term systemic expression” or “sustained systemic expression” as used herein in reference to in vivo expression of a CD40 antagonist encoded by a gene delivery vector mean measurable or biologically active expression into the bloodstream for 30 days, more preferably for 60 days, yet more preferably for 90 days, more preferably for six months, still more preferably for 1 year and most preferably for at least 5 years after administration of the gene delivery vector to a host. Expression may be achieved for the life of the patient either by a single administration or by one or more additional "booster" injections.
  • systemic expression is meant that CD40 antagonist proteins are expressed into the circulation and are thus useful for treatment of certain diseases. Expression levels may be measured, for example, by an ELISA assay specific for the given CD40 antagonist administered by the gene delivery vector system. As discussed, supra, a number of autoimmune diseases or transplant rejections are amenable to treatment by this type of gene delivery vector system.
  • CD40 antagonists of the present invention may be advantageous to administer CD40 antagonists of the present invention in conjunction with additional co-stimulatory blockade molecules.
  • additional co-stimulatory blockade molecules for example, in the case of transplant rejections, depending on the particular transplant regimen envisioned, increased therapeutic efficacy including long-term graft survival may be enhanced by co-administration of a CD40 antagonist gene delivery vector in conjunction with transfusion of donor-specific lymphocytes or CTLA4Ig.
  • a CD40 antagonist gene delivery vector in conjunction with transfusion of donor-specific lymphocytes or CTLA4Ig.
  • CTLA4Ig administration alone may be inadequate therapeutically, but that the combination of CTLA4Ig and antiCD40L antibody together have an additive effect.
  • Kirk, A.D. et al Proc. Natl. Acad. Sci. U.S.A. 94:8789-8794 (1997); Levisetti, M. et al., J Immunol. 759:5187-5191 (1997); and Chang, A.C. et al., Proc. 23 rd Am. Soc. Transplant Surgeons Ann. Meeting, Thorofare, N.J., American Society of Transplant Surgeons (1997).
  • compositions comprising one or more CD40 antagonist gene delivery vector may be administered to persons or mammals suffering from, or predisposed to suffer from, an autoimmune disease or transplant rejection.
  • CD40 antagonists are believed to minimize the severity of autoimmune diseases by reducing the infiltration of target cells with inflammatory lymphoid cells such as mononuclear phagocytes, lymphocytes, plasma cells and secondary lymphoid follicles and, in the specific case of psoriasis, by diminishing the severity of akanthosis and parakeratosis.
  • compositions of the invention may be sterilized by conventional, well known sterilization techniques.
  • the resulting solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and stabilizers (e.g., 1-20% maltose, etc.).
  • the CD40 antagonist gene delivery vectors of the present invention may also be administered via liposomes.
  • Liposomes which include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, can target the CD40 antagonist gene delivery vectors to a particular tissue as well as increase the half-life of the composition.
  • a variety of methods are available for preparing liposomes, as described in, e.g., U.S. Patent Nos. 4,837,028 and 5,019,369, which patents are incorporated herein by reference.
  • Determination of an effective amount of a composition of the invention to treat an autoimmune disease or transplant rejection in a patient can be accomplished through standard empirical methods which are well known in the art. For example, in the case of psoriasis, reversal of akanthosis and parakeratosis as well as diminution in lymphocyte infiltration in the keratinocytes can be measured.
  • compositions of the invention are administered to a mammal already suffering from an autoimmune disease or predisposed to an autoimmune disease in an amount sufficient to prevent or at least partially arrest the development of the autoimmune disease.
  • compositions for the treatment of transplant rejections may be administered in such a dose so as to reduce or eliminate the onset of graft rejection.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Effective amounts of a gene delivery vector will vary and depend on the severity of the disease and the weight and general state of the patient being treated. Administration is daily, weekly or less frequently, as necessary depending on the response to the disease and the patient's tolerance of the therapy. Maintenance dosages over a prolonged period of time may be needed, and dosages may be adjusted as necessary.
  • compositions of the present invention can be administered alone or as an adjunct therapy in conjunction with other therapeutics well known in the art for the treatment of autoimmune diseases or transplant rejections.
  • the methods of the invention can also be employed for ex vivo or extracorporeal therapy against autoimmune diseases by performing the therapeutic manipulations on peripheral blood mononuclear cells (PBMC) outside of the body.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • These cells may be subsequently administered to the subject to block or substantially reduce the activation of CD4 + T-cells.
  • significantly higher concentrations of the gene delivery vector may be employed than would be tolerated through in vivo administration.
  • Ex vivo applications of the present methods may further comprise the administration of additional agents which together provide enhanced therapeutic activity against autoimmune diseases and transplant rejections.
  • compositions of the present invention also find use in vitro.
  • gene delivery vectors encoding CD40 antagonists may be used to inhibit superantigen activation of PBMCs in vitro.
  • inventive compositions can be used in screening assays to assess the effective levels of therapeutics or other treatments for autoimmune diseases and transplant rejections.
  • the present compositions may be used in the design or screening of various potential treatment modalities, such as methods for the treatment of psoriasis or other autoimmune disease as well as transplant rejections.
  • a diagnostic method for assessing the efficacy of, e.g., autoimmune therapeutics is also provided by the present invention.
  • the DlO-1 AAV vector was constructed by replacing the AAV gene encoding sequences of pD-10, see Wang, X. et al., J. Virol. 77:3077 (1997), incorporated herein by reference, with the 5' and 3' AAV-2 inverted terminal repeats (ITRs), CMV immediate early promoter/enhancer, multiple cloning site and bovine growth hormone (BGH) polyadenylation sequences from pKm201CMV.
  • ITRs inverted terminal repeats
  • CMV immediate early promoter/enhancer CMV immediate early promoter/enhancer
  • BGH bovine growth hormone
  • pKm201CMV is an AAV cloning vector in which an expression cassette, consisting of a CMV immediate early promoter/enhancer and a bovine growth hormone (BGH) polyadenylation site, is flanked by inverted terminal repeat (ITR) sequences from AAV-2.
  • ITR inverted terminal repeat
  • D10p ⁇ CD40-l was derived from D10-1 by inserting a CD40-scFv cassette at the Hind III and Xbal sites of the D10-1 multiple cloning site.
  • a 1.35 kb ⁇ X174 Haelll restriction fragment was ligated between the 5' ITR and CMV enhancer/promoter sequence, at the Notl site, to increase the size of the resulting plasmid in order to increase the sequence length between the 5' and 3' ITRs so as to optimize viral packaging.
  • the CD40-scFv cassette encodes a fusion protein comprised of an anti- CD40 antagonist antibody heavy chain and light chain Fab sequence of antibody 5H7.
  • the hybridoma cell line 5H7 produces a humanized mouse monoclonal antibody which binds CD40 and exhibits a CD40 antagonist activity. 5H7 is effective in inhibiting the manifestations of autoimmune diseases such as psoriasis and is described in detail in co-pending application no. 60/157,461.
  • RNA was isolated from the 5H7 hybridoma and used as template in a reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers that hybridize to the corresponding heavy and light chain Fab coding regions. These primers have the nucleotide sequence as indicated in SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Amplified cDNA fragments of the heavy and light variable regions were connected by a polynucleotide linker. The resulting fusion construct was cloned into the pBS-KS plasmid vector and subcloned into the DlO-1 AAV gene delivery vector to create D10p ⁇ CD40-l as described above.
  • RT-PCR reverse transcription-polymerase chain reaction
  • CD40 antagonist expressed in the D10p ⁇ CD40-l gene delivery vector system was confirmed by competition assay whereby the CD40 antagonist blocks the binding of the native 5H7 antibody to CD40 expressing cells.
  • D10p ⁇ CD40-l and D10-1 were transiently transfected into COS-cells and culture supematants were collected after 36 hours growth at 37°C in Dulbecco's modified eagle's medium (DMEM).
  • DMEM Dulbecco's modified eagle's medium
  • Ramos-S cells which cells express CD-40, were used to assess the anti- CD40 binding activity of the COS-cell culture supematants. 1 x 10 5 Ramos-S cells were pre-blocked on ice with FcR prior to incubating with either DlO-l or D10p ⁇ CD40-l transfected COS-cell supernatant. After removing unbound supernatant, the Ramos-S cells were subsequently incubated in the presence of either culture medium or 5H7 antibody. Binding of 5H7 to CD40 was assessed by complex formation with FITC conjugated anti-mouse-IgG2b (available from Pharmagen, San Diego, CA), with detection by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Diabetes (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Dermatology (AREA)
  • Virology (AREA)
  • Endocrinology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Transplantation (AREA)
  • Emergency Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

On utilise des vecteurs d'apport de gène pour diriger l'expression de polynucléotides codant des antagonistes de CD40. On utilise ces vecteur d'apport de gène d'antagoniste de CD40 pour préparer des compositions permettant de traiter des maladies auto-immunes ainsi que le rejet du greffon chez le mammifère. Ces compositions d'antagoniste de CD40 se révèlent des plus utiles pour inverser le processus de maladies auto-immunes ou pour les atténuer sensiblement. Au nombre de ces maladies auto-immunes, figurent le lupus érythémateux systémique, l'arthrite rhumatoïde, la sclérose en plaque et le psoriasis. Ces compostions sont également utilisées pour atténuer la gravité d'un rejet de greffon après une transplantation cellulaire.
PCT/US2000/030739 1999-11-09 2000-11-08 Compositions et methodes de traitement de maladies auto-immunes et des rejets de greffon WO2001034649A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15902/01A AU1590201A (en) 1999-11-09 2000-11-08 Compositions and methods for treating autoimmune diseases and transplant rejections

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16450399P 1999-11-09 1999-11-09
US60/164,503 1999-11-09

Publications (2)

Publication Number Publication Date
WO2001034649A2 true WO2001034649A2 (fr) 2001-05-17
WO2001034649A3 WO2001034649A3 (fr) 2002-01-10

Family

ID=22594784

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/030739 WO2001034649A2 (fr) 1999-11-09 2000-11-08 Compositions et methodes de traitement de maladies auto-immunes et des rejets de greffon

Country Status (2)

Country Link
AU (1) AU1590201A (fr)
WO (1) WO2001034649A2 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003011324A1 (fr) * 2001-07-31 2003-02-13 Genset S.A. Agonistes et d'antagonistes du moxifin pour le traitement de troubles metaboliques
WO2005044306A2 (fr) * 2003-11-04 2005-05-19 Chiron Corporation Utilisation d'anticorps antagonistes anti-cd40 pour le traitement de maladies autoimmunes et inflammatoires et le rejet d'organes transplantes
US6899879B2 (en) 1992-07-09 2005-05-31 Chiron Corporation Method for treating an IgE-mediated disease in a patient using anti-CD40 monoclonal antibodies
US7288252B2 (en) 2000-10-02 2007-10-30 Novartis Vaccines And Diagnostics, Inc. Methods of therapy for B-cell malignancies using antagonist anti-CD40 antibodies
US7678822B2 (en) * 2002-11-21 2010-03-16 Genzyme Corporation Induction of immune tolerance
US7687530B2 (en) 2002-11-21 2010-03-30 Genzyme Corporation Inhibition of chronic tissue transplant rejection
US8277810B2 (en) 2003-11-04 2012-10-02 Novartis Vaccines & Diagnostics, Inc. Antagonist anti-CD40 antibodies
US8518999B2 (en) 2000-05-12 2013-08-27 Genzyme Corporation Modulators of TNF-αsignaling
US8828396B2 (en) 2010-11-15 2014-09-09 Novartis Ag Silent Fc variants of anti-CD40 antibodies
US8926979B2 (en) 2005-11-01 2015-01-06 Novartis Ag Treatment of cancer or pre-malignant conditions using anti-CD40 antibodies
US8945564B2 (en) 2006-04-21 2015-02-03 Novartis Ag Antagonist anti-CD40 antibody pharmaceutical compositions
US10174121B2 (en) 2015-05-29 2019-01-08 Abbvie, Inc. Anti-CD40 antibodies
CN112409486A (zh) * 2020-11-26 2021-02-26 杭州百凌生物科技有限公司 一种抗cd40抗体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031025A1 (fr) * 1996-02-23 1997-08-28 Chiron Corporation Anticorps monoclonaux humanises de type anti-cd40 et fragments capables de bloquer l'activation des lymphocytes b
WO1999042075A2 (fr) * 1998-02-19 1999-08-26 Bristol-Myers Squibb Company Anticorps contre le cd40 humain
EP0945465A1 (fr) * 1992-07-09 1999-09-29 Chiron Corporation Anticorps monoclonaux antagonistes contre la molécule humaine CD40
WO2001024823A1 (fr) * 1999-10-04 2001-04-12 Chiron Corporation Antagonistes de cd40 permettant de traiter le psoriasis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0945465A1 (fr) * 1992-07-09 1999-09-29 Chiron Corporation Anticorps monoclonaux antagonistes contre la molécule humaine CD40
WO1997031025A1 (fr) * 1996-02-23 1997-08-28 Chiron Corporation Anticorps monoclonaux humanises de type anti-cd40 et fragments capables de bloquer l'activation des lymphocytes b
WO1999042075A2 (fr) * 1998-02-19 1999-08-26 Bristol-Myers Squibb Company Anticorps contre le cd40 humain
WO2001024823A1 (fr) * 1999-10-04 2001-04-12 Chiron Corporation Antagonistes de cd40 permettant de traiter le psoriasis

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CROXFORD J LUDOVIC ET AL: "Local gene therapy with CTLA4-immunoglobulin fusion protein in experimental allergic encephalomyelitis." EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 28, no. 12, December 1998 (1998-12), pages 3904-3916, XP000981675 ISSN: 0014-2980 *
HARTOG DEN M ET AL: "GENERATION OF A HUMANIZED ANTI-CD40 MAB FOR TREATMENT OF IMPORTANCE OF SIZE, SULFATION, AND ANTICOAGULANT ACTIVITY IN THE AUTOIMMUNE DISEASES. POTENTATION OF ACIDIC FIBROBLAST GROWTH FACTOR BY HEPARIN" IMMUNOTECHNOLOGY,NL,ELSEVIER SCIENCE PUBLISHERS BV, vol. 2, no. 4, 1 November 1996 (1996-11-01), pages 299-689, XP002032468 ISSN: 1380-2933 *
HAWKINS R E ET AL: "Antibodies: from genes to targeted cancer gene therapy ÄeditorialÜ" GENE THERAPY,GB,MACMILLAN PRESS LTD., BASINGSTOKE, vol. 5, 1998, pages 1581-1583, XP002106269 ISSN: 0969-7128 *
M DE BOER ET AL: "Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins" JOURNAL OF IMMUNOLOGICAL METHODS,NL,ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, vol. 152, no. 1, 31 July 1992 (1992-07-31), pages 15-23, XP002108669 ISSN: 0022-1759 *
PELEGRIN MIREIA ET AL: "Genetically engineered antibodies in gene transfer and gene therapy." HUMAN GENE THERAPY, vol. 9, no. 15, 10 October 1998 (1998-10-10), pages 2165-2175, XP000996554 ISSN: 1043-0342 *
WU HERREN ET AL: "Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues." JOURNAL OF MOLECULAR BIOLOGY, vol. 294, no. 1, 1 November 1999 (1999-11-01), pages 151-162, XP002166821 ISSN: 0022-2836 *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7790166B2 (en) 1992-07-09 2010-09-07 Novartis Vaccines And Diagnostics, Inc. Anti-CD40 antibodies capable of blocking B-cell activation
US6899879B2 (en) 1992-07-09 2005-05-31 Chiron Corporation Method for treating an IgE-mediated disease in a patient using anti-CD40 monoclonal antibodies
US7361345B2 (en) 1992-07-09 2008-04-22 Novartis Vaccines And Diagnostics, Inc. Anti-CD40 antibodies capable of blocking B-cell activation
US9579325B2 (en) 2000-05-12 2017-02-28 Genzyme Corporation Modulators of TNF-α signaling
US8921547B2 (en) 2000-05-12 2014-12-30 Genzyme Corporation Modulators of TNF-α signaling
US8518999B2 (en) 2000-05-12 2013-08-27 Genzyme Corporation Modulators of TNF-αsignaling
US7445780B2 (en) 2000-10-02 2008-11-04 Novartis Vaccines And Diagnostics, Inc. Antagonistic anti-CD40 antibodies
US7611708B2 (en) 2000-10-02 2009-11-03 Novartis Vaccines And Diagnostics, Inc. Methods of therapy for B-cell malignancies using antagonist anti-CD40 antibodies
US7820170B2 (en) 2000-10-02 2010-10-26 Novartis Vaccines And Diagnostics, Inc. Methods of therapy for B-cell malignancies using antagonist anti-CD40 antibodies
US7288252B2 (en) 2000-10-02 2007-10-30 Novartis Vaccines And Diagnostics, Inc. Methods of therapy for B-cell malignancies using antagonist anti-CD40 antibodies
US8088383B2 (en) 2000-10-02 2012-01-03 Novartis Vaccines And Diagnostics, Inc. Methods of therapy for B-cell malignancies using antagonist anti-CD40 antibodies
WO2003011324A1 (fr) * 2001-07-31 2003-02-13 Genset S.A. Agonistes et d'antagonistes du moxifin pour le traitement de troubles metaboliques
US7678822B2 (en) * 2002-11-21 2010-03-16 Genzyme Corporation Induction of immune tolerance
US7687530B2 (en) 2002-11-21 2010-03-30 Genzyme Corporation Inhibition of chronic tissue transplant rejection
US8277810B2 (en) 2003-11-04 2012-10-02 Novartis Vaccines & Diagnostics, Inc. Antagonist anti-CD40 antibodies
EP2248830A1 (fr) * 2003-11-04 2010-11-10 Novartis Vaccines and Diagnostics, Inc. Utilisation d'anticorps antagonistes anti-CD40 pour le traitement de maladies autoimmunes et inflammatoires et le rejet d'organes transplantés
US8637032B2 (en) 2003-11-04 2014-01-28 Novartis Vaccines And Diagnostics, Inc. Antagonist anti-CD40 monoclonal antibodies and methods for their use
WO2005044306A2 (fr) * 2003-11-04 2005-05-19 Chiron Corporation Utilisation d'anticorps antagonistes anti-cd40 pour le traitement de maladies autoimmunes et inflammatoires et le rejet d'organes transplantes
WO2005044306A3 (fr) * 2003-11-04 2005-09-15 Chiron Corp Utilisation d'anticorps antagonistes anti-cd40 pour le traitement de maladies autoimmunes et inflammatoires et le rejet d'organes transplantes
US8926979B2 (en) 2005-11-01 2015-01-06 Novartis Ag Treatment of cancer or pre-malignant conditions using anti-CD40 antibodies
US8945564B2 (en) 2006-04-21 2015-02-03 Novartis Ag Antagonist anti-CD40 antibody pharmaceutical compositions
US9221913B2 (en) 2010-11-15 2015-12-29 Novartis Ag Silent Fc variants of anti-CD40 antibodies
US8828396B2 (en) 2010-11-15 2014-09-09 Novartis Ag Silent Fc variants of anti-CD40 antibodies
US9688768B2 (en) 2010-11-15 2017-06-27 Novartis Ag Silent Fc variants of anti-CD40 antibodies
US9828433B2 (en) 2010-11-15 2017-11-28 Novartis Ag Nucleic acids encoding silent Fc variants of anti-CD40 antibodies
US10323096B2 (en) 2010-11-15 2019-06-18 Novartis Ag Nucleic acids encoding silent Fc variants of anti-CD40 antibodies
US11124578B2 (en) 2010-11-15 2021-09-21 Novartis Ag Method of treating transplant rejection with silent Fc variants of anti-CD40 antibodies
US10174121B2 (en) 2015-05-29 2019-01-08 Abbvie, Inc. Anti-CD40 antibodies
CN112409486A (zh) * 2020-11-26 2021-02-26 杭州百凌生物科技有限公司 一种抗cd40抗体及其应用

Also Published As

Publication number Publication date
AU1590201A (en) 2001-06-06
WO2001034649A3 (fr) 2002-01-10

Similar Documents

Publication Publication Date Title
KR100239607B1 (ko) 인화 항체 및 이것의 사용 방법
US7317087B2 (en) Members of the FC receptor homolog gene family (FCRH1-3, 6), related reagents, and uses thereof
EP1221973B1 (fr) Antagonistes de CD40 permettant de traiter le psoriasis
WO2001034649A2 (fr) Compositions et methodes de traitement de maladies auto-immunes et des rejets de greffon
MXPA04008937A (es) Metodos para modular los receptores cd200.
KR20210041631A (ko) TL1A-Ig 융합 단백질을 사용한 T 조절 세포의 조절을 위한 조성물 및 방법
US20050002935A1 (en) Use of B7-H3 as an immunoregulatory agent
CZ2001725A3 (cs) Použití CD2 vazebného činidla pro výrobu léku k modulaci paměťových T buněk
EP2596023A2 (fr) Compositions et méthodes comprenant des antagonistes de l''il-6 et de l'il-21
WO1998003551A1 (fr) Ligand de cd6
EP1294874B1 (fr) Variants cd154 et leurs utilisations
WO1998009638A1 (fr) INHIBITION DE L'ACTIVATION DES MASTOCYTES PAR DES MECANISMES ET DES REACTIFS A BASE DE gp49
US20200339687A1 (en) Compositions and methods for targeting gamma delta t cells with chimeric antigen receptors
WO1999003999A1 (fr) Procedes et compositions servant a inhiber la reaction proinflammatoire
EP4310188A1 (fr) Gène codant pour un récepteur chimérique pour auto-anticorps anti-récepteur de l'acétylcholine
AU2021219886A1 (en) Optimization of chimeric antigen receptor
US20030212025A1 (en) Cd154 variants
US20060165689A1 (en) Compositions and methods for modulation of effects on phagocyte and lymphoid cell populations employing tirc7
KR20230143135A (ko) Cd19+, cd20+ 또는 cd22+ 종양 또는 b-세포 유래 자가-면역질환을 치료하기 위한 car t-세포
KR101268562B1 (ko) Ｔｌｔ-6 단백질에 대한 항체 및 그 응용
WO2024036303A2 (fr) Lymphocytes t universels et compositions et leurs méthodes d'utilisation
AU2007201053A1 (en) CD154 Variants
EP1839674A1 (fr) Antagoniste CD40 pour le traitement du psoriasis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase