WO1999051258A1 - Utilisation d'anticorps anti-gp39 pour traiter et/ou faire regresser un lupus et une maladie renale associee - Google Patents

Utilisation d'anticorps anti-gp39 pour traiter et/ou faire regresser un lupus et une maladie renale associee Download PDF

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Publication number
WO1999051258A1
WO1999051258A1 PCT/US1999/007321 US9907321W WO9951258A1 WO 1999051258 A1 WO1999051258 A1 WO 1999051258A1 US 9907321 W US9907321 W US 9907321W WO 9951258 A1 WO9951258 A1 WO 9951258A1
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antibody
cells
cell
lupus
cd40cr
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PCT/US1999/007321
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English (en)
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Randolph J. Noelle
Christopher M. Burns
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Trustees Of Dartmouth College
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Priority to HU0101689A priority Critical patent/HUP0101689A3/hu
Priority to EP99915237A priority patent/EP1067954A4/fr
Priority to KR1020007011045A priority patent/KR20010072564A/ko
Priority to JP2000542029A priority patent/JP2002510643A/ja
Priority to AU33796/99A priority patent/AU743824B2/en
Publication of WO1999051258A1 publication Critical patent/WO1999051258A1/fr
Priority to NO20004966A priority patent/NO20004966L/no

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/2875—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/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a counter- receptor, referred to alternatively in the literature as CD40CR, gp39, or most recently CD154 for the CD40 B-cell antigen, and to soluble ligands for this receptor, including fusion molecules comprising at least a portion of CD40 protein. It is based, at least in part, on the discovery that a soluble CD40/immunoglobulin fusion protein was able to inhibit helper T-cell mediated B- cell activation by binding to a novel 39 kD protein receptor on helper T-cell membranes.
  • the present invention provides for a substantially purified CD40CR receptor; for soluble ligands of CD40CR, including anti- gp39 antibodies and fragments thereof, as well as fusion molecules comprising at least a portion of CD40 protein; and for methods of controlling B-cell activation which may be especially useful in the treatment of allergy or autoimmune disease. More specifically, the present invention relates to the use of anti-gp39 antibodies for treating systemic lupus erythematosus (SLE) or drug induced lupus .
  • SLE systemic lupus erythematosus
  • T h formed physical conjugates with class II major histocompatibility complex (MHC) compatible, antigen- presenting B-cells (Vitetta et al . , Immunol . Rev. , 99:193-239 (1987)) and that it was the B-cells within these conjugates that responded to T h (Barrett et al . , J " . Immunol . , 143:1745-1754 (1989)).
  • MHC major histocompatibility complex
  • T h -derived lymphokines exerted potent growth and differentiative effects on B-cells
  • soluble factor (s) released in proximity by activated T h mediated the activation of the interacting B-cell.
  • none of the molecularly cloned lymphokines, alone or in combination manifested the ability to induce B-cell cycle entry.
  • plasma membrane fractions from activated T h induced B- cell cycle entry Hodgkin et al . , J. Immunol . , 145:2025- 2034 (1990); Noelle et al . , J " . Immunol . , 146:1118-1124 (1991)
  • PM ACT from activated T h , but not resting T h expressed an activity that induced B- cell cycle entry in an antigen-nonspecific, class II- unrestricted manner.
  • PM REST resting T h
  • the activity expressed by PM ACT required 4-6 hours of activation, de novo RNA synthesis and was protein in nature (Bartlett et al . , J. Immunol , 145:3956-3962 (1990) ) .
  • the present invention relates to a counter- receptor, termed CD40CR, for the CD40 B-cell antigen, and to soluble ligands for this receptor, including fusion molecules comprising at least a portion of CD40 protein. It is based, at least in part, on the discovery that a soluble CD40/immunoglobulin fusion protein was able to inhibit helper T-cell mediated B- cell activation by binding to a novel 39 kD receptor protein (termed "CD40CR" for CD40 counter-receptor) on helper T-cell membranes, and on the discovery that a monoclonal antibody, termed MRl, directed toward this 39 kD receptor was able to inhibit helper T-cell mediated activation of B-cells.
  • CD40CR a counter- receptor
  • MRl monoclonal antibody
  • the present invention provides for a substantially purified CD40CR receptor for soluble ligands of CD40CR, including antibodies, as well as fusion molecules comprising at least a portion of CD40 protein; and for methods of controlling B-cell activation.
  • B-cell activation in a subject may be inhibited by contacting helper T cells of the subject with therapeutically effective amounts of a soluble ligand or CD40CR. Such inhibition of B-cell activation may be especially useful in the treatment of allergy or autoimmune disease.
  • the present invention provides a method of treating lupus in a subject in need of such treatment, e.g. a patient with ongoing systemic lupus erythematosus, or drug-induced lupus, even in the advanced stages of the disease process (wherein kidney damage is often observed) by the administration of a therapeutically effective amount of an anti-gp39 antibody, e.g. the anti-human gp39 antibodies or fragments thereof disclosed in commonly assigned U.S. Serial No. 08/475,847, filed June 7, 1995, now allowed.
  • an anti-gp39 antibody e.g. the anti-human gp39 antibodies or fragments thereof disclosed in commonly assigned U.S. Serial No. 08/475,847, filed June 7, 1995, now allowed.
  • One advantage of the present invention is that it enables intervention in an aspect of the immune response which is not antigen specific.
  • the present invention may be used to particular advantage in the treatment of allergic or autoimmune conditions in which the im unogen is not known, or has multiple components, for example, in hay fever, procainamide induced lupus or systemic lupus erythematosus (SLE) . It should also be useful in acute treatment of immune activation, for example, in therapy for anaphylaxis.
  • Panel A Resting B-cells were cultured with Pmtest or PM Act from T h l . 25 g/ml of anti-CD4, anti-LFA- 1 or anti-ICAM-1 or a combination of each of these (each at 25 ⁇ g/ml) was added to wells containing PM Act and B- cell RNA synthesis was measured by incorporation of [ 3 H] - uridine . B-cell RNA synthesis was addressed from 42 to 48 hours post-culture. Results presented are the arithmetic means of triplicate cultures +/- s.d., and are representative of 5 such experiments. Panel B. Resting B-cells are cultured with
  • CD40-Ig inhibited B-cell differentiation and proliferation.
  • Panel A Resting B-cells were cultured with PM Ac , rIL4 (10 ng/ml) and rIL5 (5 ng/ml) . Either at the initiation of culture, or on days 1, 2 or 3 post- initiation of culture, CD40-Ig or CD7E-Ig (25 ⁇ g/ml) were added. On day six of culture, SN from individual wells were harvested and quantitated for IgM ( ⁇ ) and IgG x (•) using an anti-isotype specific ELISA, as described in (Noelle et al . , J. Immunol . , 146:1118-1124 (1991)).
  • Panel B Thl were rested or activated with anti-CD3 for 16 hours, irradiated and cultured (lxl0 4 /well) with resting B-cells (4xl0 4 /culture) in the presence of IL4 (10 ng/ml) . Between 0 and 25 ⁇ g/ml of
  • CD40-Ig ( A ) or CD7E-Ig (•) were added to cultures. From
  • Results presented ar the arithmetic mean of triplicate cultures +/- s.d., and are representative of 2 such experiments .
  • CD40-Ig detected a molecule expressed on activated, but not resting Th. Resting and activated Th were harvested and incubated with fusion proteins for 20 minutes at 4°C, followed by FITC-conjugated goat anti- hlgG (25 ⁇ g/ml) . Percentage positive cells and MFI were determined by analysis of at least 5000 cells/sample. Results are representative of 6 such experiments. CD40- Ig binding is indicated by a filled-in profile.
  • Thl was rested or activated with insolubilized anti-CD3 for 16 hours.
  • [ 35 S] -labelled proteins from resting or activated Th were immunoprecipitated with purified antibodies or fusion proteins (l-10 ⁇ ) .
  • the gel profile is representative of 3 such experiments .
  • FIG. 5 A monoclonal antibody (mab) , specific to the induced 39 Kd Th membrane protein, inhibited induction of B-cell RNA synthesis by PM ACT . Resting B- cells and PM ACT were cultured with 10 ⁇ g/ml each of anti- / ⁇ , anti-CD3, CD40-Ig or MRl. RNA synthesis was determined as described in Figure 1. Results presented are the arithmetic means of triplicate cultures +/- s.d., and are representative of 3 such experiments. Figure 6. MRl and CD40-Ig recognized the same molecule expressed on activated Th.
  • mab monoclonal antibody
  • Panel A Activated Th were fluorescently stained with MRl or control Ig .
  • graded concentrations of MRl or control hamster Ig anti- ⁇ / ⁇ TCR
  • anti-CD40 20 ⁇ g/ml
  • Panel B Proteins from [ 35 S] -methionine- labelled, activated Th were immunoprecipitated with MRl (10 ⁇ g/sample) or CD40-Ig (10 ⁇ g/sample) and resolved by PAGE and fluorography. Results presented are representative of 2 such experiments.
  • FIG. 1 Binding of CD40-Ig to human cell lines. A variety of human T-cell lines were exposed to biotin- ⁇ 10 -
  • Panel B Schematic diagram of a plasmid that may be used to express CD40-Ig. The amino acid sequences at the site of fusion of ⁇ CD40 is shown below the diagramed portion of CD40.
  • Open circles ( o ) represent mice treated with MR-1 from 4 through 10 months of age; open triangles ( ⁇ -
  • mice receiving MR-1 after developing proteinuria that did not respond represent mice receiving MR-1 after developing proteinuria that did respond
  • closed squares ( ⁇ ) represent mice receiving MR-1 after developing proteinuria that did respond
  • closed circles ( • ) represent mice receiving no treatment. Values are mean of three titers from each group +/- SEM.
  • Open circles ( o ) represent mice treated with MR-1 from 4 through 10 months of age; open triangles ( ⁇ ) represent mice receiving MR-1 after developing proteinuria that did not respond, - 11 -
  • closed squares ( ⁇ ) represent mice receiving MR-1 after developing proteinuria that did respond, and closed circles ( • ) represent mice receiving no treatment .
  • the present invention provides for a substantially purified CD40CR receptor; for soluble ligands of CD40CR, including anti-gp39 antibodies and fragment thereof, as well as fusion molecules comprising CD40; and for methods of controlling B-cell activation using soluble ligands .
  • the present invention provides for soluble ligands of CD40CR, including (i) fusion molecules comprising at least a portion of CD40 protein and (ii) antibodies or antibody fragments that specifically bind CD40CR, or gp- 39, or CD154 as such antigen is also known.
  • soluble ligands of CD40CR including (i) fusion molecules comprising at least a portion of CD40 protein and (ii) antibodies or antibody fragments that specifically bind CD40CR, or gp- 39, or CD154 as such antigen is also known.
  • Soluble ligands of the invention are not permanently associated with a cell plasma membrane. Soluble ligands of the invention may, however, be affixed to a non- cellular solid support, including a lipid, protein, or carbohydrate molecule, a bead, a vesicle, a magnetic particle, a fiber, etc. or may be enclosed within an implant or vesicle.
  • CD40-Ig infra
  • MRl infra
  • another antibody that binds CD40CR such as disclosed in commonly assigned U.S. Serial No. 08/475,847.
  • the ligands of the invention may be comprised in pharmaceutical compositions together with a suitable carrier.
  • the present invention provides for soluble fusion molecules that are ligands of CD40CR.
  • Such fusion molecules comprise at least a portion of C040 protein attached to a second molecule.
  • the portion of CD40 preferably lacks the CD40 transmembrane domain.
  • a portion of CD40 protein which may be used according to the invention is defined as any portion which is able to bind to CD40CR, for example, such a portion may be shown to bind to the same protein as MRl or CD40-Ig.
  • Second molecules which may be used include peptides and proteins, lipids, and carbohydrates, and, in preferred embodiments of the invention, may be an - 13 -
  • the second molecule may be derived from either a non-human or a human source, or may be chimeric.
  • the second molecule may also be an enzyme, toxin, growth factor, lymphokine, antiproliterative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound.
  • the fusion molecules of the invention may be produced by chemical synthesis or, preferably, by recombinant DNA techniques .
  • a nucleic acid sequence encoding at least a portion of CD40 protein may be combined with a nucleic acid sequence encoding a second molecule in a suitable expression vector, and then expressed in a prokaryotic or, preferably, eukaryotic expression system, such as a yeast, baculovirus, or mammalian expression system, including transgenic animals.
  • a prokaryotic or, preferably, eukaryotic expression system such as a yeast, baculovirus, or mammalian expression system, including transgenic animals.
  • at least a portion of CD40 protein may be expressed using electrophoretic techniques or affinity chromatography using ligand that binds to either CD40 or to the second molecule.
  • Ligands that bind to CD40 include, but are not limited to, anti-CD40 antibodies such as G28-5, as produced by the hybridoma having accession number HB9110 and deposited with the American Type Culture Collection, and CD40CR, described more fully infra . If the second molecule is an - 14 -
  • a portion of CD40 may be produced using nucleic acid sequence that encodes a CD40 protein that is truncated upstream from the transmembrane domain.
  • nucleic acid sequence may be prepared by digesting a plasmid containing a cDNA encoding CD40 antigen, such as that described in Stamenkovic et al . , EMBO J. , 8:1403-1410 (1989), with PstI (P) and Sau 3A (S3) restriction enzymes. The resulting P/S3 fragment may be subcloned into the same plasmid digested with P and Bam HI (B) , to produce a truncated CD40 gene (see Figure 8) .
  • an expression vector used to produce ligands containing at least a portion of CD40 as well as immunoglobulin sequence may preferably comprise a virally-derived origin of replication, a bacterial origin of replication, a bacterial selectable marker, and eukaryotic promoter and enhancer sequences separated from DNA sequences encoding an immunoglobulin constant region by restriction endonuclease sites which allow subcloning of DNA sequences encoding at least a portion of CD40, followed by a polyadenylation signal sequence (see Figure 8.b.) . ⁇ 15 -
  • the truncated CD40 gene may be subcloned into an immunoglobulin fusion plasmid, such as that described in Aruffo et al . , Cell , 61:1303-1313 (1990), using an Mlu I and B digest, to form plasmid pCD40-Ig, which encodes the fusion molecule CD40-Ig (see Figure 8) .
  • CD40-Ig fusion protein may then be produced by transfecting the PCD40-Ig plasmid into COS cells to form a transient expression system.
  • CD40-Ig produced may be collected from the COS cell supernatant and purified by protein A column chromatography as described in Aruffo et al . , Cell , 161:1303-1313 (1990).
  • the soluble ligands of the invention will preferably comprise antibody molecules, monoclonal antibody molecules, or fragments of these antibody molecules which contain an antigen combining site that binds to CD40CR (gp39) preferably human gp39.
  • Such ligands may further comprise a second molecule which may be a protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphokine, antiproliterative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound and may be linked to the antibody molecule or fragment.
  • the ligand is a monoclonal antibody, or a fragment thereof
  • the monoclonal antibody can be prepared against CD40CR (gp39) using any technique which provides for the production of antibody molecules by ⁇ 16 -
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be generated by treating the antibody molecule with pepsin; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment; the F(ab') 2 fragment which can be generated by treating the antibody molecule with papain; and the 2Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.
  • the present invention also provides for chimeric or human antibodies produced by techniques known in the art, such as those set forth in Morrison et al., Proc . Nati . Acad . Sci . U. S . A . , 81:6851-6855 (1984) ⁇ 17 -
  • Immunogen for the production of antibodies may be any source that contains CD40CR.
  • activated T h e.g. activated human T h cells may be used as an immunogen.
  • substantially purified CD40CR prepared as set forth in section 5.3, infra, may be used. If activated Th are used as immunogen, antiserum may be tested for reactivity against activated but not resting T h cells.
  • the immunogen may comprise recombinant gp39 or a fragment thereof.
  • the DNA' s encoding both murine and human gp39 have been cloned and expressed by recombinant methods. . These proteins provide a potential immunogen for producing anti-gp39 antibodies .
  • the soluble ligand is an anti-human gp39 monoclonal antibody, more preferably a humanized or chimeric anti- human gp39 antibody.
  • the following method was used to produce the MRl monoclonal antibody, which specifically binds murine gp39 and may be used to generate other antibodies directed toward CD40CR.
  • Hamsters were immunized intraperitoneally with 5-10 6 activated T h l cells (D1.6) at weekly intervals for six weeks. When the serum titer against murine T h l was greater than about 1:10,000, cell fusions were performed • 18 -
  • MRl was produced in ascites and purified by ion exchange HPLC.
  • antibodies against human gp39 can be prepared according to U.S. Serial No. 08/475,847, incorporated by reference in its entirety herein.
  • the present invention also provides for ligands comprising monoclonal antibodies, and fragments thereof that are capable of competitively inhibiting the binding of MRl to its target antigen or CD40-Ig to its receptor.
  • CD40CR may be characterized by (i) its ability to bind CD40, fusion molecules comprising at least a portion of CD40, and antibodies such as MRl; (ii) its functional characteristic of being able to stimulate B- cell cycle entry, proliferation, and differentiation, and (iii) its cellular distribution.
  • CD40CR may be characterized by its ability to bind to ligands such as CD40, fusion molecules comprising CD40, and antibodies directed toward CD40CR. As discussed in greater detail infra, several techniques were used to characterize CD40CR. For example, CD40-Ig and MRl were shown to recognize the same 39 kD molecule. Both CD40-Ig and MRl were found to - 19 -
  • CD40CR may also be characterized by its ability to stimulate B-cell cycle entry, proliferation, and differentiation.
  • the induction of B-cell activation may be measured by techniques such as [ 3 H] -uridine incorporation into RNA (as B-cells differentiate, RNA synthesis increases) , or by [ 3 H] -thymidine incorporation, which measures DNA synthesis associated with cell proliferation.
  • interleukin-4 IL-4
  • IL-4 may be added to the culture medium at a concentration of about 10 ng/ml.
  • B-cell activation may be measured as a function of immunoglobulin secretion.
  • CD40CR in substantially purified form, or as present in PM, or otherwise, may be added to resting B-cells together with IL-4 (10 ng/ml) and IL-5 (5 ng/ml) . After • 20 -
  • CD40CR may also be characterized by its cellular distribution. For example, CD40-Ig was observed to bind to activated, but not resting T h l, as assessed by flow cytometry ( Figure 3) . Furthermore, CD40-Ig was observed to bind to Jurkat cells, HSB2 cells, and activated T- cells from human peripheral blood, but did not appear to bind significantly to CEM cells, HPBALL cells, or murine thyoma cells.
  • test cells may be evaluated by flow cytometry as follows. Test cells may be tested in parallel with resting (negative control) and activated (positive control) Th cells. All cells may be incubated at a concentration of about 1 x 10 s cells/50 ⁇ l with ligand (e.g. CD40-Ig or MRl) for 20 minutes at 4°C, followed by FITC-conjugated anti-ligand antibody. Propidium iodide may be added to all samples to a final concentration of 2 ⁇ g/ml . Flow cytometric analysis may then be performed, for example on a BD FACSCAN. After positive gating of cells by forward versus side scatter, and by red negativity (for propidium iodide exclusion) , and the log green fluorescense of viable cells may be ascertained. - 21 -
  • CD40CR may be prepared from cells bearing CD40CR, such as activated helper T-cells, Jurkat, and HSB2 cells, by the following method.
  • Plasma membranes may be prepared from appropriate cells, such as activated T h l cells, by discontinuous sucrose gradient sedimentation, as described in Noelle et al., J. Immunol . , 146:1118-1124 (1991).
  • CD40CR may then be isolated by dissociating the crude membrane extract with mild detergent, and then performing size exclusion chromatography followed by either affinity chromatography using appropriate ligands (e.g. MRl or CD40-Ig) bound to a solid support, immunoprecipitation (e.g. by CD40-Ig or MRl), and/or gel electrophoresis .
  • the resulting protein may be expected to have a molecular weight of about 39 kD.
  • the present invention provides for a soluble CD40CR (i.e. cell-free) which may be comprised in pharmaceutical compositions together with a suitable carrier. It further provides for CD40 CR which is linked to a second molecule which may be a peptide, protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphokine, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound.
  • a soluble CD40CR i.e. cell-free
  • CD40 CR which is linked to a second molecule which may be a peptide, protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphokine, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound.
  • the present invention further provides for substantially purified CD40CR which has been prepared by -22 -
  • the gene for CD40CR may be isolated by inserting cDNA prepared from activated helper T-cells into the ⁇ gtlO expression system, and then screening with MRl or CD40-Ig binding to identify CD40CR- expressing clones.
  • cDNA prepared from activated helper T-cells may be transfected into COS cells, the supernatants of which may be screened with MRl or CD40-Ig to identify CD40CR producers.
  • the gene for CD40CR may be then used to express CD40CR using expression systems known in the art.
  • the present invention provides for methods of controlling B-cell activation that utilize ligands that bind to CD40CR.
  • a method of inhibiting B-cell activation comprising exposing a mixture of B-cells and Th cells to an effective concentration of ligand that binds to CD40CR.
  • Ligands that may be used are described supra in section 5.1.
  • the method of the invention may be practiced in vi tro or in vivo .
  • An effective concentration refers to a concentration of a ligand that inhibits B-cell activation, measured by any technique known in the art (including those set forth in section 5.2, supra) by at least about 30 percent, and preferably by about 75 percent.
  • CD40-Ig may be used as ligand, in which case an effective concentration -23 -
  • an effective concentration of ligand may refer to plasma concentration of ligand or to a local concentration. For example, it may be desirable to inhibit B-cell activation in a localized area in order to limit the effects on the immune system as whole.
  • the invention provides for a method of treating a subject suffering from a disorder associated with B-cell activation, comprising administering to the subject a therapeutic amount of ligand that binds to CD40CR.
  • a subject may be a non- human or, preferably, a human animal.
  • Disorders associated with B-cell activation include, but are not limited to, allergy (including anaphylaxis) ; autoimmune conditions including drug induced lupus, systemic lupus erythematosus, adult rheumatoid arthritis, juvenile rheumatoid arthritis, scleroderma, Sjogren's Syndrome, etc.; and viral diseases that involve B-cells, including Epstein-Barr infection, and retroviral infection including infection with a human immunodeficiency virus.
  • allergy including anaphylaxis
  • autoimmune conditions including drug induced lupus, systemic lupus erythematosus, adult rheumatoid arthritis, juvenile rheumatoid arthritis, scleroderma, Sjogren's Syndrome, etc.
  • viral diseases that involve B-cells including Epstein-Barr infection, and retroviral infection including infection with a human immunodeficiency virus.
  • B-cell activation is associated with the induction of human immunodeficiency virus replication from latency, it may -24 -
  • anti-gp39 antibodies As discussed above, and in the examples which follow, a particularly preferred application of anti- gp39 antibodies, or fragments thereof, comprises their use for the treatment of drug-induced lupus or systemic lupus erythematosus. It has been discovered, as substantiated by the underlying data and experiments in the examples which follow, that anti-gp39 antibody therapy reduces autoantibody production, and renal disease, and results in prolonged survival in NZB/NZW, an accepted animal model for human SLE.
  • mice allowed to develop 2-3+ proteinuria active lupus disease condition
  • anti-gp39 antibody actually reversed disease (as evidenced by prolonged survival and absence of proteinuria after antibody administration) .
  • treatment with anti- gp39 antibodies was shown, after development of renal disease in an accepted animal model of human lupus, to reverse the lupus disease process.
  • This substantiates the potential of anti-gp39 antibodies for human therapeutic applications in the treatment of lupus and other autoimmune diseases.
  • such antibodies may be used to treat persons having an active ongoing disease, even at advanced stages of the disease. Such treatment will -25 -
  • Ligands may be administered, in a suitable pharmaceutical carrier, by any method known in the art, including intravenous, intraperitoneal, subcutaneous, intrathecal, intraarticular or intramuscular injection, and oral, intranasal, intraocular and rectal administration, and may be comprised in microspheres, liposomes, and/or sustained release implants.
  • a therapeutic amount of ligand is defined as an amount which significantly diminishes the deleterious clinical effects of B-cell activation or T cell activation, and may vary among ligands used and conditions treated. If CD40-Ig is used, therapeutic concentration may be about 10 ⁇ g/ml either systemically (plasma concentration) or locally. If MRl or another anti-gp39 antibody, e.g., a humanized or chimeric anti- human gp39 antibody or fragment thereof, is used, a therapeutic concentration may be about 10 ⁇ g/ml either systemically (plasma concentration) or locally.
  • the above methods may utilize a ligand comprising a toxin or antimetabolite such that Th cells are killed or damaged and B-cell activation is increased as a result of Th cell destruction.
  • the ligands of the invention may also be used to label activated T cells, a technique which may be useful - 26 -
  • ligand comprising an enzyme, radioisotope, fluorescent compound or other detectable label may be exposed to T cells in vi tro or in vivo and the amount of binding may be quantitated.
  • the ligands of the invention may also be used to deliver substances, e.g. growth factors, to activated T- cells .
  • the present invention provides for methods of controlling B-cell activation that utilize CD40CR or a molecule comprising CD40CR, prepared as described supra .
  • it provides for a method of promoting B- cell activation comprising exposing B-cells to an effective concentration of CD40CR.
  • the method may be practiced in vivo or in vi tro .
  • An effective concentration refers to a concentration of receptor that induces B-cell activation, measured by any technique known in the art by at least about 30 percent.
  • the concentration of CD40CR may be about 10 ⁇ g/ml locally or systemically.
  • the invention provides for a method of treating a subject suffering from an immunodeficiency disorder associated with diminished humoral immunity, comprising administering to the subject a therapeutic amount of CD40CR.
  • a subject may be a non-human or, preferably, a human animal. -27 -
  • Immunodeficiency disorders associated with diminished humoral immunity include acquired immunodeficiency caused, for example, by chemotherapy or radiation therapy, as well as genetic disorders involving humoral immunity.
  • CD40CR may be administered, in a suitable pharmaceutical carrier, by any method known in the art, including intravenous, intraperitoneal , subcutaneous, intrathecal, intraarticular, or intramuscular injection, and oral, intranasal, intraocular, and rectal administration and may be comprised in microspheres, liposomes, and/or sustained release implants.
  • a therapeutic amount of CD40CR for CD40 is defined as that amount which increases immunoglobulin production by at least about 30 percent.
  • a CD40CR may be conjugated to a toxin, and then administered to a subject under circumstances in which it would be preferable to destroy B-cells that express CD40. Examples of such circumstances include patients receiving organ transplants or suffering from multiple myeloma or another B-cell malignancy, or from autoimmune disease.
  • CD40CR may also be used to label B-cells expressing CD40, a technique which may be useful in the diagnosis of B-cell disorders.
  • receptor linked to an enzyme, radioisotope, fluorescent compound or other detectable label may be exposed to B-cells in vivo or in vitro and the amount of binding may be quantitated.
  • CD40CR may also be used to deliver molecules that are linked to it to B-cells.
  • mice Female DBA/2J mice (Jackson Laboratories, Bar Harbor, ME) were used for the preparation of filler cells to support the growth of Th clones and in the preparation of resting B-cells.
  • T h l a I-A d -restricted, rabbit Ig-specific T h l clone (Kurt-Jones et al . , J. Exp . Med . , 166:1774-1787 (1987)) was obtained from Dr. David Parker, University of Massachusetts at Worcester. Dl .6 will be referred to herein as T h l .
  • T h l were cultured (8 x 10 6 /well) in cluster wells (6 well, Corning, NY) coated with 40 ⁇ g/4 ml of PBS/well with anti-CD3 for 16 hours, as described in Noelle et al. (J. Immunol . , 146:1118-1124 (1991)).
  • Plasma membranes were prepared by discontinuous sucrose gradient sedimentation, as described in Noelle et al. ( Id. ) . Resting splenic B-cells were prepared by sedimentation on discontinuous Percoll gradients, as -29-
  • anti-CD3 145- 2C11 (Leo et al . , Proc . Nati . Acad . Sci . USA, 84:1374- 1378 (1987); anti- ⁇ , ⁇ :H57-597 ; anti-CD4 :GK1.5 (Wilde et al., J. Immunol . , 131:2178-2183 (1983); anti- ICAM:YN1/1.7.4 (Prieto et al . , Eur. J. Immunol .
  • the CD40 fusion protein was prepared by digesting a plasmid containing a cDNA encoding the CD40 antigen (Stamenkovic and Seed, EMBO J. , 8:1403-1410 (1989)) with the restriction enzyme Pst I (P) and Sau 3A (S3) . This P/S3 fragment was subcloned into the same plasmid digested with P and Bam HI (B) . This allowed the preparation of the CD40 ⁇ which encoded a CD40 protein truncated upstream from the transmembrane domain. The DNA fragment encoding a CD40 ⁇ was then subcloned into the immunoglobulin fusion plasmid (Aruffo et al . , Cell , 61:1303-1313 (1990)) using a Mlul and B digest. The CD40-Ig fusion protein was produced by transient -30-
  • Interleukin 4 Recombinant mouse IL4 was generously provided by Drs . C. Maliszewski and K. Grabstein, Immunex Corporation, Seattle, WA.
  • Interleukin 5 IL5 Recombinant mouse IL5 was purchased from R&D Research, Sarrento, CA.
  • T h l or T h 2 membrane protein was added. From 42-48 hrs, wells were pulsed with 2.5 ⁇ Ci of 3 H-uridine (New England Nuclear, Boston, MA) , harvested, and the radioactivity determined by liquid scintillation spectroscopy. The results were expressed as cpm/culture +/- s.d.
  • Resting B-cells were cultured as described above. To culture wells, 0.5 ⁇ g of T h l membrane protein, IL4 (10 ng/ml) and IL5 (5 ng/ml) were added. On day three of culture, an additional 50 ⁇ l of cRPMI was added. On day six of culture, SN from individual wells were harvested and quantitated for IgM and IgG 17 as described in Noelle et al. (J. Immunol . , 146:1118-1124 (1991)).
  • T h l were rested or activated with insolubilized anti-CD3 for 16 hrs .
  • Proteins from resting and activated T h (20 x 10 6 /ml) were labelled with 1 mCi of [ 35 S] -methionine/cysteine for one hour, at which time they were washed twice in RPMI/10%FCS and the cell pellet was lysed in extraction buffer, as described (Noelle et al . , (1986) J. Immunol . 137 : 1718-1726 ) .
  • Purified antibodies or fusion proteins (1-10 ⁇ g) were added to 500 ⁇ l of lysate (5xl0 6 cell equivalents) at 4°C for 16 hours.
  • the lysates were transferred to tubes containing 50 ⁇ l of packed Protein A-sepharose.
  • the pelleted Protein A-Sepharose was resuspended and tubes were incubated at 4°C for 1 hr with agitation. The samples were then washed 3x with high stringency wash buffer.
  • the pelleted protein A-Sepharose was resuspended and tubes were incubated at 4°C for 1 hr with agitation. The samples were then washed 3x with high stringency wash buffer. The pelleted protein A-
  • Sepharose was resuspended in 30 ⁇ l of SDS sample buffer and run on a 10% polyacrylamide gel. After running the gel, the gel was fixed and fluorography performed.
  • CD40-Ig a soluble fusion protein of the extracellular domains of human CD40 and the F c domain of human IgG x
  • Ig fusion protein (Damle and Aruffo, (1991) Proc . Nati .
  • B- cells were cultured in the presence of LPS and CD40-Ig.
  • RNA synthesis was assessed (Fig. lc) .
  • CD40-Ig was ineffective at inhibiting B-cell activation by LPS, yet inhibited the response of B-cells to PM ACT .
  • T h l were activated for 16 hours with insolubilized anti-CD3, harvested and irradiated.
  • the irradiated T h l were cultured with B-cells in the presence of IL4 and B-cell proliferation was determined on day 3 of culture.
  • An exogenous source of IL4 was required to achieve B-cell proliferation with T h l, because T h l do not produce IL4 (Noelle et al . , (1989) J. Immunol .
  • CD40-Ig inhibited the induction of B-cell proliferation by irradiated T h in a dose-dependent manner, similar to that observed with PM ACT (Fig. 2b) .
  • T h l express a binding protein for CD40
  • resting and activated (16 hours) T h l were stained with CD40-Ig or CD7E-Ig, followed. by FITC-anti-HigG. Binding of CD40-Ig was assessed by flow cytometry (Fig. 3) .
  • T h l proteins were biosynthetically labelled with [ 35 S] - methionine/cysteine and proteins immunoprecipitated with CD40-Ig or CD7E-Ig.
  • the immunoprecipitated proteins were resolved by SDS-PAGE and fluorography ( Figure 4) .
  • a 39 kd band was also immunoprecipitated from activated T h that were vectorially labelled with 15 I, confirming that the 39kD protein was a membrane protein.
  • T h molecule responsible for the T h effector phase activity.
  • One such mab, MRl recognized an antigen that was selectively expressed on activated T h l .
  • flow cytometry and blocking studies were performed.
  • CD40-Ig and MRl stained approximately 56% and 61%, respectively, of activated, but not resting Th (Fig. 5a) .
  • MRl, but not another hamster anti-T cell mab, anti- ⁇ / ⁇ TCR blocked the staining of activated T h l with CD40-Ig, in a dose-dependent manner.
  • the antigen that bound MRl was identified by immunoprecipitation of proteins from radiolabelled Th lysates. Both CD40-Ig and MRl immunoprecipitated a 39kD protein (Fig 1. 5b). Finally, immunoprecipitation of the 39kD protein with CD40-Ig removed the antigen recognized by MRl from radiolabelled lysates of activated T h supporting the tenet that the MRl antigen and the CD40 binding protein were identical.
  • T h surface molecules LFA-1, CD4, ICAM-1, CD3 , ⁇ , ⁇ TCR
  • CD40-Ig or a maB specific to the CD40 binding protein blocked T h - dependent B-cell activation in a dose-dependent manner.
  • the CD40 binding protein was identified as a 39 kD protein that is selectively expressed on the membranes of activated, but not resting T h . Both CD40-Ig and a mab specific to the 39kD CD40 binding protein blocked B-cell activation by PM AT .
  • CD40-Ig and a mab specific to the CD40 binding protein inhibits T h - dependent B-cell activation.
  • the ligand for CD40 is a 39Kd protein that is expressed on activated, but not resting T h .
  • Biochemical studies indicate that the 39kD protein is a single chain molecule since electrophoretic migration was not influenced by reducing agents.
  • both activated T h l and T h 2 express the 39 kD CD40 binding protein. This is consistent with the functional studies that show both T h l and T h 2 induce B-cell cycle entry.
  • cDNA encoding CD proteins in the MW range of 39kD Cd 53, CD27 and CD69
  • None of the transfected COS cells expressed proteins that bound CD40-Ig. It is therefore suspected that the 39 kD protein is not one of these CD proteins.
  • CD40 is a member of the nerve growth factor ⁇ 3 8 -
  • NGFR neuropeptide-like protein kinases
  • NGFR nerve growth factor receptor
  • Signalling through CD40 by mab has been shown ( Uckun et al . , J. Biol . Che . 266. : 174 8 -1 485 (1991 ) ) to involve the activation of tyrosine kinases resulting in the increased production of inositol triphosphate and the activation of at least four distinct serine/threonine kinases.
  • T h and B Based on information obtained from signaling through other members of the NGF receptor family, it is anticipated that interaction between activated T h and B will result in many of the same biochemical processes.
  • CD40 Ig fusion protein was conjugated with biotin using biotin- succinimide (Sigma) .
  • Flow cytometry analysis was then performed by two-step staining using phycoerythrin (PE) - streptavidin (Becton-Dickinson) with a Coulter Epics C instrument. Representative results of screening multiple T cell lines is presented below. The Jurkat and HSB2 cell lines were found to bind specifically, whereas other T cell lines including CEM, HPBALL, and murine thyoma did not bind the CD40 Ig fusion protein (Fig. 7) .
  • SLE Systemic lupus erythematosus
  • One example is the second signal delivered by the interaction between CD28/CTLA4 on the Th cell and B7.1/B7.2 on the B cell. Blocking antibodies to either participant can render the T cell hyporesponsive in vi tro .
  • a fusion protein consisting of the extracellular domain of murine CTLA-4 linked to a murine Ig Cg2a chain was shown to block autoantibody production and prolong life when given to NZB/NZW mice, even with advanced stages of disease, presumably by competitively inhibiting the binding of B7.2 to endogenous CTLA-4 in treated mice.
  • anti-CD40L antibody therapy may circumvent two major concerns with the use of anti-lymphocyte cell-surface molecule antibodies for immunotherapy : the development of an anti-antibody response and prolonged post-treatment immunosuppression.
  • mice who responded to anti-CD40L antibody treatment in terms of survival at 11 months did not develop an anti-antibody response, an almost invariable phenomenon in animals receiving antibodies derived in another species.
  • MR-1 no anti-MR-1 responses were seen suggesting that the responses we have observed in the
  • NZB/NZW mice may represent a particular hyperreactivity limited to autoimmune mice.
  • this observation has important implications for the therapeutic application of an anti-human CD40L antibody, since this serious complication of antibody therapy may be obviated with anti-CD40L antibody therapy, particularly with a "humanized” version of the antibody.
  • mice who did develop an anti-anti-CD40L antibody response fared no better than control mice in their anti-DNA antibody production or survival, suggesting that, for whatever reason, antibody production by this subset of mice was not prevented by anti-CD40L antibody.
  • the NZB/NZW Fl mice were treated with 200 ug of MR-1 intraperitoneally twice a week from age 4-10 months. Also, treatment has also been effected by a treatment protocol, increasing the dosage to 250 mg of MR-1 three times a week intraperitoneally from 4 to 10 months of age. This dose had been used in the collagen-induced model of rheumatoid arthritis and resulted in 100% survival in -43 -
  • mice were treated mice (Durie, Science, 1993) . Using this protocol, the NZB/NZW mice now have 100% survival at month 11 (Fig. 10) , whereas two control groups of mice had all expired by age 10 months.
  • mice with anti-gp39 antibody While the prevention of SLE in the NZB/NXW F 1 mice with anti-gp39 antibody is of interest, it is not predictive of the utility of this antibody to be able to therapeutically intervene in active progressed disease states.
  • a cohort of mice were allowed to develop 2 - 3+ proteinuria (equivalent to 100 mg/day to 500 g/day) , determined by urine dipstick. At that point, mice were randomly assigned to continue no treatment or to receive MR-1 at 100 ⁇ g/day. The ten untreated mice were all dead by 10 months of age. By contrast, 5 of 10 MR-1 treated mice were alive at 11 months of age. Three of these mice were healthy and without proteinuria. Intriguingly, one of these mice nevertheless has high-titer anti-DNA antibody (Table 1) . Two of the 5 survivors appear sick and have high titer anti-DNA antibodies and 4+ proteinuria.
  • NZB/NZW mice tumor necrosis factor (TNF- ⁇ ) significantly delayed the development of nephritis (Jacob, Science, 1988; Gordon, 1989) .
  • TNF- ⁇ tumor necrosis factor
  • the NZW mice have reduced levels of TNF- ⁇ that correlates with a polymorphism in the TNF- ⁇ gene which is located within the H-2 complex.
  • the NZB/NZW Fl mice also have significantly reduced levels of TNF- ⁇ .
  • these results substantiate the utility of anti-gp39 antibodies and active fragments thereof for treating human lupus, i.e., systemic lupus erythematosus or drug-induced lupus.
  • these antibodies can be used for the therapeutic intervention of active, ongoing lupus which is often characterized by nephritis. Such antibodies should inhibit the progression of the disease and potentially even reverse the disease process .
  • TNF- ⁇ ELISA kit from Genzyme, Cambridge, MA.

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Abstract

L'invention concerne une méthode de traitement de lupus utilisant des anticorps anti-gp39 ou des fragments de ceux-ci. Un tel traitement permet de faire régresser la maladie, et en particulier une maladie rénale associée au lupus, qui constitue la cause principale de mortalité chez des sujets atteints de lupus.
PCT/US1999/007321 1998-04-03 1999-04-02 Utilisation d'anticorps anti-gp39 pour traiter et/ou faire regresser un lupus et une maladie renale associee WO1999051258A1 (fr)

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HU0101689A HUP0101689A3 (en) 1998-04-03 1999-04-02 Use of anti-gp39 antibodies for treatment and/or reversal of lupus and associated kidney disease
EP99915237A EP1067954A4 (fr) 1998-04-03 1999-04-02 Utilisation d'anticorps anti-gp39 pour traiter et/ou faire regresser un lupus et une maladie renale associee
KR1020007011045A KR20010072564A (ko) 1998-04-03 1999-04-02 루푸스 및 관련된 신장 질환의 치료 및/또는 반전에사용되는 안티 ｇｐ39 항체의 용도
JP2000542029A JP2002510643A (ja) 1998-04-03 1999-04-02 狼瘡およびその関連腎臓疾患の治療および／または後退のための抗−ｇｐ３９抗体の使用
AU33796/99A AU743824B2 (en) 1998-04-03 1999-04-02 Use of anti-gp39 antibodies for treatment and/or reversal of lupus and associated kidney disease
NO20004966A NO20004966L (no) 1998-04-03 2000-10-02 Anvendelse av anti-gp-39-antistoff ved behandling og/eller reversering av lupus og assosiert nyresykdom

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017024146A1 (fr) 2015-08-05 2017-02-09 Janssen Biotech, Inc. Anticorps anti-cd154 et procédés d'utilisation correspondant
US10683356B2 (en) 2015-02-03 2020-06-16 Als Therapy Development Institute Methods of treating a CD40L associated disease or disorder by administering anti-CD40L antibodies
US11384152B2 (en) 2017-05-24 2022-07-12 Als Therapy Development Institute Therapeutic anti-CD40 ligand antibodies

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WO1996023071A2 (fr) * 1995-01-26 1996-08-01 Bristol-Myers Squibb Company ANTICORPS MONOCLONAUX SPECIFIQUES DE DIFFERENTS EPITOPES DE gp39 HUMAINE ET PROCEDES CONCERNANT LEUR UTILISATION A DES FINS DE DIAGNOSTIC ET DE THERAPIE
US5672347A (en) * 1984-07-05 1997-09-30 Genentech, Inc. Tumor necrosis factor antagonists and their use

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US5833987A (en) * 1995-06-07 1998-11-10 Trustees Of Dartmouth College Treatment of T cell mediated autoimmune disorders
US6001358A (en) * 1995-11-07 1999-12-14 Idec Pharmaceuticals Corporation Humanized antibodies to human gp39, compositions containing thereof
TR200001249T2 (tr) * 1997-01-10 2000-11-21 Biogen, Inc. Anti-CD40L bileşikleri ile lupus nefritis tedavisi
WO1998039026A2 (fr) * 1997-03-07 1998-09-11 Biogen, Inc. Procedes d'administration therapeutiques de composes anti-cd40l
WO1999000143A1 (fr) * 1997-06-27 1999-01-07 Biogen, Inc. Therapie de blocage par cd154 pour maladies auto-immunes

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US5672347A (en) * 1984-07-05 1997-09-30 Genentech, Inc. Tumor necrosis factor antagonists and their use
US5474771A (en) * 1991-11-15 1995-12-12 The Trustees Of Columbia University In The City Of New York Murine monoclonal antibody (5c8) recognizes a human glycoprotein on the surface of T-lymphocytes, compositions containing same
WO1996023071A2 (fr) * 1995-01-26 1996-08-01 Bristol-Myers Squibb Company ANTICORPS MONOCLONAUX SPECIFIQUES DE DIFFERENTS EPITOPES DE gp39 HUMAINE ET PROCEDES CONCERNANT LEUR UTILISATION A DES FINS DE DIAGNOSTIC ET DE THERAPIE

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10683356B2 (en) 2015-02-03 2020-06-16 Als Therapy Development Institute Methods of treating a CD40L associated disease or disorder by administering anti-CD40L antibodies
US11014990B2 (en) 2015-02-03 2021-05-25 Als Therapy Development Institute Anti-CD40L antibodies
US11692040B2 (en) 2015-02-03 2023-07-04 Als Therapy Development Institute Anti-CD40L antibodies and methods for treating CD40L-related diseases or disorders
WO2017024146A1 (fr) 2015-08-05 2017-02-09 Janssen Biotech, Inc. Anticorps anti-cd154 et procédés d'utilisation correspondant
US10669343B2 (en) 2015-08-05 2020-06-02 Janssen Biotech, Inc. Anti-CD154 antibodies and methods of using them
US11421037B2 (en) 2015-08-05 2022-08-23 Janssen Biotech, Inc. Nucleic acids encoding anti-CD154 antibodies
US11384152B2 (en) 2017-05-24 2022-07-12 Als Therapy Development Institute Therapeutic anti-CD40 ligand antibodies

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NO20004966L (no) 2000-12-04
AU3379699A (en) 1999-10-25
CN1757413A (zh) 2006-04-12
CN1173735C (zh) 2004-11-03
JP2002510643A (ja) 2002-04-09
AU743824B2 (en) 2002-02-07
TWI224969B (en) 2004-12-11
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KR20010072564A (ko) 2001-07-31
CN100352497C (zh) 2007-12-05
NO20004966D0 (no) 2000-10-02

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