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WO2008055889A1 - Compositions et procédés permettant de traiter des maladies oncologiques, inflammatoires et autoimmunes par l'intermédiaire de sema4a - Google Patents

Compositions et procédés permettant de traiter des maladies oncologiques, inflammatoires et autoimmunes par l'intermédiaire de sema4a Download PDF

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Publication number
WO2008055889A1
WO2008055889A1 PCT/EP2007/061906 EP2007061906W WO2008055889A1 WO 2008055889 A1 WO2008055889 A1 WO 2008055889A1 EP 2007061906 W EP2007061906 W EP 2007061906W WO 2008055889 A1 WO2008055889 A1 WO 2008055889A1
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Prior art keywords
sema4a
plexin
cells
interaction
cell
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PCT/EP2007/061906
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English (en)
Inventor
Hitoshi Kikutani
Atsushi Kumanogoh
Kenji Sugiyama
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Boehringer Ingelheim International GmbH
Boehringer Ingelheim Pharma GmbH and Co KG
University of Osaka NUC
Boehringer Ingelheim Pharmaceuticals Inc
Original Assignee
Boehringer Ingelheim International GmbH
Boehringer Ingelheim Pharma GmbH and Co KG
Osaka University NUC
Boehringer Ingelheim Pharmaceuticals Inc
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Publication of WO2008055889A1 publication Critical patent/WO2008055889A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8

Definitions

  • This invention relates to the involvement of Sema4A in activation and effector functions of CD8 + T-cells and NK-cells.
  • Sema4A is involved in activation and differentiation of CD4 + T-cells.
  • the present invention demonstrates the role of Sema4A in regulation of CD8 + T-cell and NK-cell functions which up until the present invention has been unclear.
  • Enhancing Sema4A/Plexin-B2 interaction or signaling may be useful in the treatment of diseases where increased T lymphocyte and/or NK cell activation and/or lytic activity is beneficial, for example cancer and viral infections, or for enhancing the immune response to vaccines.
  • Inhibition of Sema4A/Plexin-B2 interaction or signaling may be useful in the treatment of diseases where decreased T lymphocyte and/or NK cell activation and/or lytic activity is beneficial, for example inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, multiple sclerosis, psoriasis, Lupus, and inflammatory bowel diseases.
  • FIG. 1 Binding of Sema4A-Fc to activated T cells are shown.
  • Figure 2 Sema4A promotes T-cell activation.
  • A Sema4A-Fc dose-dependently enhances in vitro T-cell activation of purified CD4 + and CD8 + T-cells that were stimulated with anti-CD3. The effects of various concentrations of Sema4A-Fc are shown.
  • B Sema4A-Fc dose-dependently induces IFN-gamma by anti-CD3-stimulated CD8+ T-cells.
  • NK cells express a receptor for Sema4A other than Tim-2.
  • An analysis of the binding of Sema4A-Fc to NK cells, and expression of Sema4A and TIM molecules on NK cells is shown.
  • FIG. 4 Sema4A treatment induces IFN-gamma from NK cells.
  • Purified NK cells were cultured in presence of IL-2 or IL- 12 or in combination with and without addition of Sema4A fusion proteins.
  • FIG. 5 Sema4A enhances CTL activity of NK cells against YAC-I cells.
  • Purified NK cells were treated with 50 U/ml of recombinant IL-2 for 24 h either with 20 ug/ml of Sema4A-Fc fusion protein or human IgGl and mixed with target YAC-I cells at various effector to target ratios as indicated. Cytotoxicity was measured using an LDH- calorimetric assay.
  • FIG. 6 Generation of P815 mastocytoma cells stably expressing Sema4A.
  • P815 were transfected with full length Sema4A expression vector and selected for 10 days in 0.5 mg/ml neomycin. Sema4A expressing cells were separated by FACS sorting. Stable P815 cells expression was tested using anti-Sema4A antibody. Anti-CD 100 and mouse isotype were used as controls.
  • FIG. 7 Expression of Sema4A in P815 cells results in increased primary CTL generation.
  • DBA/2 syngeneic splenocytes were cultured for 9 days with 20,000 rad irradiated P815 cells that were non-transfected or stably expressing B7.1 or Sema4A.
  • Recombinant IL-2, 10 U/ml or IL-4, 20 U/ml was added separately or in combination to the cultures every alternate day.
  • a CTL assay was performed using non-transfected P815 cells at a 1 :10 ratio of Effector to Target cells.
  • Figure 8 Effect of Sema4A on tumorigenicity of P815 tumor.
  • Figure 9 Na ⁇ ve and activated CD8 + T cells and NK cells do not express Tim-2. FACS analysis was performed with purified CD8 + T-cells before and after activation with anti- CD3 and anti-CD28 antibodies (5 mg/ml each) for 24h and with purified NK cells before and after activation with 200 U/ml of recombinant IL-2 for 24h. Cells were stained with biotinylated anti-TIM-2 or biotinylated Sema4A-Fc and streptavidin-APC. Biotinylated human IgGl and rat IgG2a were used respectively as isotype controls.
  • Figure 10 Purified NK cells were treated with IL-2, IL-12 or both for 16 hours. RT- PCR was performed from cDNA isolated from the treated NK cells for analysis of the expression of various plexin-B family members. An expression library made from NK cells was used as another source of NK cell cDNA.
  • Figure 11 cDNA isolated from NK cells was analyzed for the expression of various TIM family members.
  • Figure 12 Purified NK cells were infected with either control lentivirus or Plexin-B2 silencing shRNA lentivirus particles. NK cells were selected for 6 days using 100 U/ml recombinant IL-2 and 2.5 mg/ml Puromycin followed by 48 h activation with Sema4A or control IgG. IFN-gamma production was measured in culture supernatants by ELISA.
  • FIG. 13 OT-I transgenic purified CD8 + T-cells were infected with either control lentivirus or Plexin-B2 silencing lentivirus particles.
  • CD8 + T-cells were selected for 6 days using 100 U/ml recombinant IL-2 and 2.5 mg/ml Puromycin followed by 48 h activation with Sema4A or control IgG. IFN-gamma production was measured in culture supernatants by ELISA.
  • Figure 14 Sigma shRNA lentiviral Vector used for Plexin-B2 silencing.
  • Plexin-B2 is a ligand for Semaphorin 4A on CD8 + T cells and NK cells.
  • Treatment with Sema4A-Fc augments production of IFN-gamma by NK cells and CD8 + T lymphocytes.
  • Sema4A-Fc treatment also enhances NK cell lytic activity.
  • Expression of Sema4A on tumor cells leads to increased generation of cytotoxic T lymphocytes and resultant tumor regression.
  • Knockdown of Plexin-B2 expression on CD8 + T cells or NK cells abrogates the ability of Sema4A-Fc to enhance IFN-gamma production from these cells.
  • Inhibition of Sema4A/Plexin-B2 interaction or signaling may be useful in the treatment of diseases where decreased T lymphocyte and/or NK cell activation and/or lytic activity is (are) beneficial, for example inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, and inflammatory bowel diseases.
  • inflammatory and autoimmune diseases including sepsis, rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, and inflammatory bowel diseases.
  • Sema4A-Ig fusion protein bound to activated CD8 + T-cells and NK-cells, induced IFN- ⁇ production and enhanced their CTL activities.
  • Adoptive transfer of OT-I CD8 + T-cells into Sema4A "/" mice and subsequent challenge with peptide antigen in CFA revealed that the expansion of CD8 + T-cells was dependent on Sema4A.
  • the stable expression of Sema4A in P815 tumor cells enhanced in vivo tumor immunogenecity in inoculated mice. When syngeneic DBA/2 mice were inoculated with parental P815 cells, mice developed progressively large tumors and died.
  • mice did not develop large tumors due to Sema4A-enhanced CTL activity. Furthermore, the mice that previously experienced regression of Sema4A-P815 tumors were resistant to tumor development when freshly inoculated with parental P815 cells. Boehringer Ingelheim application WO03080673 demonstrates that Sema4A binds to Tim-2 expressed on activated CD4 + T-cells. See also Kumanogoh, A. et al. Nature Vol. 419 October 10, 2002. However, CD8 + T-cells and NK-cells do not express Tim-2.
  • a method of treating an oncological disease by administering to a patient a composition which enhances Sema4A/Plexin-B2 interaction.
  • One embodiment of the present invention relates to a method to identify a compound that controls interaction of Sema4A with Plexin-B2 activity in a cell, comprising: (1) contacting a cell with a putative regulatory compound, wherein the cell includes a Plexin-B2 protein or a Sema4A protein; and (2) assessing the ability of the putative regulatory compound to inhibit or enhance the interaction of Sema4A with Plexin-B2.
  • the assessment step preferably involves either i) determining the cytokine production as described herein-below, or ii) in vitro assays performed as described previously and methods known in the art.
  • the term “regulate” refers to controlling the activity of a molecule and/or biological function, such as enhancing or diminishing such activity or function.
  • patient includes both human and non-human mammals.
  • treating mean the treatment of a disease-state in a patient, and include: (i) preventing the disease-state from occurring in a patient, in particular, when such patient is genetically or otherwise predisposed to the disease-state but has not yet been diagnosed as having it;
  • Yet another embodiment of the present invention relates to an antibody or antibody binding site which binds Sema4A, Plexin-B2 or fragments thereof.
  • Embodiments of the present invention further include polyclonal and monoclonal antibodies.
  • Preferred embodiments of the present invention include a monoclonal antibody such an anti- Plexin-B2 monoclonal antibody.
  • the above antibody or antibody binding site which binds Plexin-B2 or Sema4a inhibits binding of Sema4A with Plexin-B2.
  • Yet another embodiment of the present invention relates to a biotherapeutic comprising Sema4A or Plexin-B2 or fragments thereof, wherein the biotherapeutic is useful for treating inflammatory, autoimmune or oncological diseases.
  • Another embodiment of the present invention provides a method of enhancing an immunological response to a vaccine comprising administering to a patient a biotherapeutic composition as described immediately above.
  • the composition which enhances immunological response to a vaccine can be administered together or separately with the vaccine.
  • vaccines include any vaccine whose effectiveness is enhanced by stimulation of the cell-mediated lytic immune response including but not limited to HIV vaccine.
  • composition as referred to herein include a putative compound, or a substantially pure protein selected from Sema4A, Plexin-B2 or fragments thereof, an antibody or antibody binding site which binds Sema4A, Plexin-B2 or fragments thereof, to an expression vector encoding Sema4A, Plexin-B2 or fragments thereof, a fusion protein comprising Sema4A, Plexin-B2 or fragments thereof.
  • the antibody binding site may be: specifically immunoreactive with a mature protein selected from the group consisting of the Sema4A and Plexin-B2; raised against a purified or recombinantly produced human or mouse Sema4A or Plexin-B2; in a monoclonal antibody, Fab, or F(ab)2; immunoreactive with denatured antigen; or in a labeled antibody.
  • the antibody binding site is detected in a biological sample by a method of: contacting a binding agent having an affinity for Sema4A or Plexin-B2 with the biological sample; incubating the binding agent with the biological sample to form a binding agent: Sema4A, Plexin-B2 protein complex; and detecting the complex.
  • the biological sample is human, and the binding agent is an antibody.
  • Putative compounds as referred to herein include, for example, compounds that are products of rational drug design, natural products and compounds having partially defined signal transduction regulatory properties.
  • a putative compound can be a protein-based compound, a carbohydrate-based compound, a lipid-based compound, a nucleic acid-based compound, a natural organic compound, a synthetically derived organic compound, an anti-idiotypic antibody and/or catalytic antibody, or fragments thereof.
  • a putative regulatory compound can be obtained, for example, from libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks; see for example, U.S. Pat. Nos. 5,010,175 and 5,266,684 of Rutter and Santi, which are incorporated herein by reference in their entirety) or by rational drug design.
  • the three-dimensional structure of a compound such as a signal transduction molecule can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography.
  • NMR nuclear magnetic resonance
  • This three-dimensional structure can then be used to predict structures of potential compounds, such as putative regulatory compounds by, for example, computer modelling.
  • the predicted compound structure can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi).
  • Potential regulatory compounds can also be identified using SELEX technology as described in, for example, PCT Publication Nos.
  • a naturally-occurring intracellular signal transduction molecule can be modified based on an analysis of its structure and function to form a suitable regulatory compound.
  • a compound capable of regulating Sema4A-Plexin-B2 binding or signaling can comprise a compound having similar structure to the amino acid residues in the intracellular domain of Sema 4A or Plexin-B2.
  • Such a compound can comprise a peptide, a polypeptide or a small organic molecule.
  • a compound capable of regulating Sema4A-Plexin-B2 binding or signaling can comprise a compound that binds to the intracellular domain or Sema4A or Plexin-B2.
  • a compound can comprise a peptide, a polypeptide or a small organic molecule.
  • Putative regulatory compounds can also include molecules designed to interfere with Sema4A and Plexin-B2. For example, mutants of Plexin-B2 can be created that interfere with the coupling of the protein with Sema4A. Putative regulatory compounds can include agonists and antagonists of Sema4A and Plexin-B2 binding. Such agonists and antagonists can be selected based on the structure of a naturally-occurring ligand to these proteins.
  • an immortal cell line typically myeloma cells
  • lymphocytes typically splenocytes
  • a mammal immunized with whole cells expressing a given antigen e.g., Plexin-B2
  • the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen.
  • a given antigen e.g., Plexin-B2
  • Immunization may be accomplished using standard procedures.
  • the unit dose and immunization regimen depend on the species of mammal immunized, its immune status, the body weight of the mammal, etc.
  • the immunized mammals are bled and the serum from each blood sample is assayed for particular antibodies using appropriate screening assays.
  • anti-integrin antibodies may be identified by immunoprecipitation of 1251-labeled cell lysates from integrin-expressing cells.
  • Antibodies, including for example, anti-Plexin-B2 antibodies may also be identified by flow cytometry, e.g., by measuring fluorescent staining of antibody-expressing cells incubated with an antibody believed to recognize Plexin-B2 molecules.
  • the lymphocytes used in the production of hybridoma cells typically are isolated from immunized mammals whose sera have already tested positive for the presence of anti- Plexin-B2 antibodies using such screening assays.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, arninopterin and thymidine ("HAT medium").
  • HAT medium containing hypoxanthine, arninopterin and thymidine
  • HAT medium a culture medium containing hypoxanthine, arninopterin and thymidine
  • HAT medium Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using 1500 molecular weight polyethylene glycol (“PEG 1500").
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants.
  • hybridomas prepared to produce anti- Plexin-B2 antibodies may be screened by testing the hybridoma culture supernatant for secreted antibodies having the ability to bind to a recombinant Plexin-B2-expressing cell line.
  • hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium.
  • Tissue culture techniques and culture media suitable for hybridoma cells are well known.
  • the conditioned hybridoma culture supernatant may be collected and the anti- Plexin-B2 antibodies optionally further purified by well-known methods.
  • the desired antibody may be produced by injecting the hybridoma cells into the peritoneal cavity of an unimmunized mouse.
  • the hybridoma cells proliferate in the peritoneal cavity, secreting the antibody which accumulates as ascites fluid.
  • the antibody may be harvested by withdrawing the ascites fluid from the peritoneal cavity with a syringe.
  • Fully human monoclonal antibody homologs against, for example Plexin-B2 are another preferred binding agent which may block antigens in the method of the invention. In their intact form these may be prepared using in vitro-primed human splenocytes, as described by Boerner et al, 1991, J. Immunol. 147:86-95, "Production of Antigen-specific Human Monoclonal Antibodies from In Vitro-Primed Human Splenocytes".
  • EBNA2 function which is required for immortalization, is subsequently shut off, which results in an increase in antibody production.
  • U.S. Pat. No. 5,789,650 (Aug. 4, 1998, "Transgenic non-human animals for producing heterologous antibodies”) describes transgenic non- human animals capable of producing heterologous antibodies and transgenic non-human animals having inactivated endogenous immunoglobulin genes. Endogenous immunoglobulin genes are suppressed by antisense polynucleotides and/or by antiserum directed against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of that species of non- human animal.
  • transgenes containing sequences of unrearranged heterologous human immunoglobulin heavy chains are introduced into a non- human animal thereby forming a transgenic animal capable of functionally rearranging transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes.
  • Such heterologous human antibodies are produced in B-cells which are thereafter immortalized, e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line capable of producing a monoclonal heterologous, fully human antibody homo log.
  • the conditions under which the cell of the present invention is contacted with a putative regulatory compound, such as by mixing, are conditions in which the cell can exhibit Plexin-B2, Sema4A activity if essentially no other regulatory compounds are present that would interfere with such activity. Achieving such conditions is within the skill in the art, and includes an effective medium in which the cell can be cultured such that the cell can exhibit Sema4A with Plexin-B2 activity.
  • effective media are typically aqueous media comprising RPMI 1640 medium containing 10% fetal calf serum.
  • Cells of the present invention can be cultured in a variety of containers including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and carbon dioxide content appropriate for the cell. Such culturing conditions are also within the skill in the art. For example, for Ramos cells, culturing can be carried out at 37° C, in a 5% CO 2 environment.
  • Acceptable protocols to contact a cell with a putative regulatory compound in an effective manner include the number of cells per container contacted, the concentration of putative regulatory compound(s) administered to a cell, the incubation time of the putative regulatory compound with the cell, the concentration of ligand and/or intracellular initiator molecules administered to a cell, and the incubation time of the ligand and/or intracellular initiator molecule with the cell. Determination of such protocols can be accomplished by those skilled in the art based on variables such as the size of the container, the volume of liquid in the container, the type of cell being tested and the chemical composition of the putative regulatory compound (i.e., size, charge etc.) being tested.
  • a suitable number of cells are added to a 96-well tissue culture dish in culture medium.
  • a preferred number of cells includes a number of cells that enables one to detect a change in Sema4A with
  • Plexin-B2 binding activity using a detection method of the present invention (described in detail below).
  • a more preferred number of cells includes between about 1 and 1 x 10 6 cells per well of a 96-well tissue culture dish. Following addition of the cells to the tissue culture dish, the cells can be preincubated at 37° C, 5% CO 2 for between about 0 to about 24 hours.
  • a suitable amount of putative regulatory compound(s) suspended in culture medium is added to the cells that is sufficient to regulate the activity of a Sema4A, Plexin-B2 protein in a cell such that the regulation is detectable using a detection method of the present invention.
  • a preferred amount of putative regulatory compound(s) comprises between about 1 nM to about 10 mM of putative regulatory compound(s) per well of a 96-well plate.
  • the cells are allowed to incubate for a suitable length of time to allow the putative regulatory compound to enter a cell and interact with Sema4A or Plexin-B2 protein.
  • a preferred incubation time is between about 1 minute to about 48 hours.
  • cells suitable for use in the present invention are stimulated with a stimulatory molecules capable of binding to Sema4A or Plexin-B2 protein of the present invention to initiate a signal transduction pathway and create a cellular response.
  • a stimulatory molecule capable of binding to Sema4A or Plexin-B2 protein of the present invention to initiate a signal transduction pathway and create a cellular response.
  • Suitable stimulatory molecules can include, for example, antibodies that bind specifically to Sema4A or Plexin-B2 protein.
  • a suitable amount of stimulatory molecule to add to a cell depends upon factors such as the type of ligand used (e.g., monomeric or multimeric; permeability, etc.) and the abundance of Sema4A or Plexin-B2 protein.
  • the method of the present invention include determining if a composition is capable of regulating Sema4A and Plexin-B2 protein activation. Such methods include assays described in detail in the Examples section.
  • the method of the present invention can further include the step of performing a toxicity test to determine the toxicity of the composition.
  • kits to identify compositions capable of regulating Sema4A and Plexin-B2 protein activity in a cell includes: (1) a cell comprising Sema4A and Plexin-B2 protein; and (2) a means for detecting regulation of either the Sema4A and/or Plexin-B2 protein.
  • a means for detecting the regulation of Sema4A or Plexin-B2 protein include methods and reagents known to those of skill in the art, for example, Plexin-B2 protein activity can be detected using, for example, activation assays.
  • Means for detecting the regulation of Sema4A with Plexin-B2 protein also include methods and reagents known to those of skill in the art.
  • Suitable cells for use with a kit of the present invention include cells described in detail herein.
  • a preferred cell for use with a kit includes a human cell.
  • Plexin-B2 is a ligand for Semaphorin 4A on CD8 + T cells and NK cells.
  • Enhancing Sema4A/Plexin-B2 interaction or signaling may be useful in the treatment of diseases where increased T lymphocyte and/or NK cell activation and/or lytic activity is (are) beneficial, for example cancer and viral infections, or for enhancing the immune response to vaccines.
  • a composition according to the invention will also be useful for treating oncological diseases.
  • diseases include but are not limited to solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases.
  • Those disorders also include lymphomas, sarcomas, and leukemias.
  • breast cancer examples include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
  • cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma and mesothelioma .
  • brain cancers include, but are not limited to brain stem, optic and hypophtalmic glioma, cerebella and cerebral astrocytoma, medulloblastoma, ependymoma, as well as pituitary,neuroectodermal and pineal tumor.
  • peripheral nervous system tumors include, but are not limited to neuroblastoma, ganglioneuroblastoma, and peripheral nerve sheath tumors.
  • tumors of the endocrine and exocrine system include, but are not limited to thyroid carcinoma, adrenocortical carcinoma, pheochromocytoma, and carcinoid tumors.
  • Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer.
  • Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.
  • Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallblader, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
  • Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, and urethral cancers.
  • Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
  • liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without f ⁇ bro lamellar variant), hepatoblastoma, cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cho langiocarcinoma.
  • Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Head-and-neck cancers include, but are not limited to laryngeal/ hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and lip and oral cavity cancer.
  • Lymphomas include, but are not limited to AIDS-related lymphoma, non- Hodgkin's lymphoma, Hodgkins lymphoma, cutaneous T-cell lymphoma, and lymphoma of the central nervous system.
  • Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, Ewings sarcoma, malignant fibrous histiocytoma, lymphosarcoma, angiosarcoma, and rhabdomyosarcoma.
  • Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
  • Plasma cell dyscrasias include, but are not limited to multiple myeloma, and Waldenstrom's macroglobulinemia.
  • a composition that would block the interaction of Plexin-B2 with Sema4a would block inflammatory cytokine production from cells.
  • the inhibition of cytokine production is an attractive means for preventing and treating a variety of cytokine mediated diseases or conditions associated with excess cytokine production, e.g., diseases and pathological conditions involving inflammation, autoimmune responses or bone resorption.
  • the compositions are useful for the treatment of diseases and conditions including the following:
  • osteoarthritis atherosclerosis, contact dermatitis, bone resorption diseases including osteoporosis, reperfusion injury, asthma, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host disease, systemic lupus erythematosus and insulin-dependent diabetes mellitus, rheumatoid arthritis, toxic shock syndrome, Alzheimer's disease, diabetes, inflammatory bowel diseases, acute and chronic pain as well as symptoms of inflammation and cardiovascular disease, stroke, myocardial infarction, alone or following thrombolytic therapy, thermal injury, adult respiratory distress syndrome (ARDS), multiple organ injury secondary to trauma, acute glomerulonephritis, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system disorders, syndromes associated with hemodialysis, leukopherisis, granulocyte transfusion associated syndromes, and necrotizing enterocolitis, complications including restenosis following percutaneous trans
  • Anti-cytokine activity can be demonstrated by using methods known in the art. See for example Branger et al., (2002) J Immunol. 168: 4070-4077, and the 46 references cited therein, each incorporated herein by reference in their entirety.
  • compositions may be administered in any conventional dosage form in any conventional manner.
  • Routes of administration include, but are not limited to, intravenously, intramuscularly, subcutaneously, intrasynovially, by infusion, sublingually, transdermally, orally, topically or by inhalation.
  • the preferred modes of administration are oral and intravenous.
  • compositions may be administered alone or in combination with adjuvants that enhance stability of the inhibitors, facilitate administration of pharmaceutic compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients.
  • combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.
  • the above described compositions may be physically combined with the conventional therapeutics or other adjuvants into a single pharmaceutical composition.
  • the compositions may then be administered together in a single dosage form.
  • the pharmaceutical compositions comprising such combinations of compositions contain at least about 5%, but more preferably at least about 20%, of a composition (w/w) or a combination thereof.
  • the optimum percentage (w/w) of a composition of the invention may vary and is within the purview of those skilled in the art.
  • the compositions may be administered separately (either serially or in parallel). Separate dosing allows for greater flexibility in the dosing regime.
  • dosage forms of the compositions described herein include pharmaceutically acceptable carriers and adjuvants known to those of ordinary skill in the art.
  • These carriers and adjuvants include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, buffer substances, water, salts or electrolytes and cellulose-based substances.
  • Preferred dosage forms include, tablet, capsule, caplet, liquid, solution, suspension, emulsion, lozenges, syrup, reconstitutable powder, granule, suppository and transdermal patch. Methods for preparing such dosage forms are known (see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., Lea and Febiger (1990)).
  • Dosage levels and requirements are well-recognized in the art and may be selected by those of ordinary skill in the art from available methods and techniques suitable for a particular patient. In some embodiments, dosage levels range from about 1-1000 mg/dose for a 70 kg patient. Although one dose per day may be sufficient, up to 5 doses per day may be given. For oral doses, up to 2000 mg/day may be required. As the skilled artisan will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific dosage and treatment regimens will depend on factors such as the patient's general health profile, the severity and course of the patient's disorder or disposition thereto, and the judgment of the treating physician.
  • Splenic CD4 + T cells and CD8 + T cells were stained with biotinylated Sema4A-Fc (blue) or biotinylated human IgGl (red) and streptavidin-APC before and after 24 h of 10 mg/ml of anti-CD3 and ConA activation as shown in Figure 1: Binding of Sema4A- Fc to activated T cells.
  • Figure 2 shows that Sema4A promotes T-cell activation.
  • Sema4A-Fc enhances in vitro T-cell activation of purified CD4 + and CD8 + T cells that were stimulated with anti-CD3, with or without various concentrations of Sema4A-Fc.
  • B Dose dependent induction of IFN-gamma by Sema4A-Fc treated CD8 + T-cells, culture supernatants were estimated by ELISA.
  • FIG. 3 shows Sema4A treatment induces IFN-gamma from NK cells.
  • Purified NK cells were cultured in presence of IL-2 and IL- 12 or in combination with and without addition of Sema4A fusion proteins. 48h later culture supernatants were estimated by ELISA.
  • NK cells were treated with 50 U/ml of recombinant IL-2 for 24 h either with 20 mg/ml of Sema4A-Fc fusion protein or human IgGl and mixed with target YAC-I cells at various effector to target ratio as indicated and cytotoxicity was measured using LDH-calorimetric assay.
  • P815 were transfected with full length Sema4A expression vector and selected for 10 days in 0.5 mg/ml neomycin. Sema4A expressing cells were separated by FACS sorting. Stable P815 cells expression was tested using anti-Sema4A antibody. Anti-CD 100 and mouse isotype were used as controls.
  • DBA/2 syngeneic spleenocytes were cultured for 9 days with 20,000 rad irradiated parental or P815 stably expressing B7.1 or Sema4A.
  • Recombinant IL-2, 10 U/ml or IL- 4, 20 U/ml was added separately or in combination to the cultures every alternate day.
  • CTL assay was performed using parental P815 cells using 1 :10 ratio of Effector to Target cells as shown in Figure 7.
  • the effect of Sema4A on tumorigenicity of P815 tumor demonstrated in Figure 8.
  • (A) 10 6 non-transfected or Sema4A expressing cells were injected subcutaneously and tumor size was measured after 4 weeks.
  • Figure 10 shows purified NK cells were treated with either IL-2 and IL- 12 or in combination for 16 hours.
  • RT-PCR was performed from cDNA isolated from treated NK cells for analysis of various plexin-B family members.
  • Expression library made from NK cells was used as another source of NK cell cDNA.
  • cDNA was isolated from NK cells was analyzed for expression of various TIM family members.
  • NK cells were infected with either controls lentivirus or Plexin-B2 silencing ShRNA lentivirus particles.
  • NK cells were selected for 6 days using 100 U/ml recombinant IL-2 and 2.5 mg/ml Puromycin followed by 48 h activation with Sema4A or con-Ig.
  • IFN-g was estimated from culture supernatants by ELISA, see Figure 12.
  • OT-I transgenic purified CD8 + T-cells were infected with either controls lentivirus or Plexin-B2 silencing lentivirus particles shown in Figure 13.
  • CD8 + T-cells were selected for 6 days using 100 U/ml recombinant IL-2 and 2.5 mg/ml Puromycin followed by 48 h activation with Sema4A or con-Ig.
  • IFN-g was estimated from culture supernatants by ELISA.

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Abstract

L'invention porte sur des compositions et des procédés permettant de traiter des maladies en modulant l'interaction de Sema4A avec la Plexine-B2, et sur des procédés permettant d'identifier des composés qui modulent l'interaction de Sema4A avec la Plexine-B2.
PCT/EP2007/061906 2006-11-10 2007-11-06 Compositions et procédés permettant de traiter des maladies oncologiques, inflammatoires et autoimmunes par l'intermédiaire de sema4a Ceased WO2008055889A1 (fr)

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WO2012135332A1 (fr) * 2011-03-28 2012-10-04 President And Fellows Of Harvard College Modulateurs de l'activité de la plexine b2
CN104105706A (zh) * 2011-10-06 2014-10-15 德克萨斯州立大学董事会 用于治疗疾病的抗人sema4A抗体
WO2015105806A3 (fr) * 2014-01-07 2015-09-24 Oklahoma Medical Research Foundation Anticorps contre des biomarqueurs de gliome
EP2903692A4 (fr) * 2012-10-08 2016-06-08 St Judes Children S Res Hospital Thérapies fondées sur la régulation de la stabilité et de la fonction des lymphocytes t régulateurs par l'intermédiaire d'un axe neuropiline-1:sémaphorine
WO2019030461A1 (fr) * 2017-08-11 2019-02-14 Universite Claude Bernard Lyon 1 Biomarqueurs du neuroblastome

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012135332A1 (fr) * 2011-03-28 2012-10-04 President And Fellows Of Harvard College Modulateurs de l'activité de la plexine b2
US9353188B2 (en) 2011-03-28 2016-05-31 President And Fellows Of Harvard College Modulators of Plexin B2 activity
CN104105706A (zh) * 2011-10-06 2014-10-15 德克萨斯州立大学董事会 用于治疗疾病的抗人sema4A抗体
EP2764023A4 (fr) * 2011-10-06 2015-06-10 Univ Texas Anticorps sema4a antihumains utiles dans le traitement de maladies
CN104829716A (zh) * 2011-10-06 2015-08-12 德克萨斯州立大学董事会 用于治疗疾病的抗人sema4A抗体
EP2903692A4 (fr) * 2012-10-08 2016-06-08 St Judes Children S Res Hospital Thérapies fondées sur la régulation de la stabilité et de la fonction des lymphocytes t régulateurs par l'intermédiaire d'un axe neuropiline-1:sémaphorine
US9540439B2 (en) 2012-10-08 2017-01-10 St. Jude Children's Research Hospital Therapies based on control of regulatory T cell stability and function via a neuropilin-1:semaphorin axis
EP3677310A1 (fr) * 2012-10-08 2020-07-08 St. Jude Children's Research Hospital Thérapies fondées sur la régulation de la stabilité et de la fonction des lymphocytes t régulateurs par l'intermédiaire d'un axe neuropiline-1:sémaphorine
WO2015105806A3 (fr) * 2014-01-07 2015-09-24 Oklahoma Medical Research Foundation Anticorps contre des biomarqueurs de gliome
US10030076B2 (en) 2014-01-07 2018-07-24 Oklahoma Medical Research Foundation Antibodies against glioma biomarkers
WO2019030461A1 (fr) * 2017-08-11 2019-02-14 Universite Claude Bernard Lyon 1 Biomarqueurs du neuroblastome

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