Classifications

• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype

Definitions

• the present invention relates generally to IgM autoantibodies and, more particularly, to a method of inhibiting disease progression. Discussion of the Background
• Chemokines or chemotactic cytokines, are a class of cytokine molecules capable of chemotactically attracting migratory cells. Chemokines are essential in
• Chemokines generally have small molecular weights in the range of about 8-10 kD.
• chemokines can be divided into three major families, CC, CXC and
• CXXXC based on the number of amino acids (referred to as "X") separating the two cysteines (referred to as "C") in the chemokine molecule.
• X the number of amino acids
• C cysteines
• chemokines are further grouped into related sub-families based on amino acid sequence similarity between them.
• CC chemokine sub-families include the monocyte chemoattractant protein ("MCP") sub-family and the sub-family
• MlP-l ⁇ macrophage inhibitory protein- l ⁇
• MlP-l ⁇ macrophage inhibitory protein- l ⁇
• MlP-l ⁇ protein- l ⁇
• CXC chemokine sub-families include the IP-10 and Mig sub- family; the interleukin-8 (“IL-8”) sub-family; and the PF4 sub-family.
• IL-8 interleukin-8
• SDF-l ⁇ form a chemokine family that is approximately equally
• RANTES attracts T lymphocytes to
• IL-8 typically attracts neutrophils to inflammatory sites.
• Chemokines exert their effect by binding to chemokine receptors.
• CC chemokines typically bind to members of the CCR class of receptors, while CXC
• chemokines generally bind to members of the CXCR class of receptors.
• Chemokine receptors are involved in certain functions such as, for example
• the HIV-1 virus is known to bind to certain proteins on the surface of cells, i.e. , the CD4 antigen on
• lymphocytes In order to gain entrance into these cells and replicate,
• HIV-1 virus must bind to another receptor, i.e., predominantly, CXCR4 and CCR5 chemokine receptors.
• CXCR4 and CCR5 chemokine receptors i.e., predominantly, CXCR4 and CCR5 chemokine receptors.
• Different HIV-1 viral strains use specific chemokine
• the X4 virus uses CXCR4 receptors
• the R5 virus uses CCR5
• Viral entry through chemokine receptors is of prime importance in influencing viral replication and disease progression after HIV-1 infection.
• HIV-1 viral replication especially because fresh human sera and their antibodies
• Immunoglobulin G (including Immunoglobulin G (“IgG”) anti-HIV-1) have no direct lytic or neutralizing activity on the HIV-1 virus.
• IgM Immunoglobulin M
• leukocytes such as, for example, B and T lymphocytes, without causing cell lysis
• lymphocyte autoantibodies These antibodies may also be referred to herein as
• IgM anti-leukocyte antibodies or "IgM anti-leukocyte autoantibodies" because
• leukocytes very little is known about the leukocyte or lymphocyte antigens or receptors that bind to IgM autoantibodies. Levels of such anti-leukocyte
• autoantibodies increase during inflammatory states, including autoimmune diseases
• infectious diseases i.e. , virus-mediated diseases
• infectious diseases i.e. , virus-mediated diseases
• SLE systemic lupus erythematosus
• Sarcoidosis HIV-1
• malaria Epstein-Barr virus
• CMV cytomegalovirus
• HIV-1 therefore, have high levels of IgM anti-leukocyte autoantibodies.
• the inventor's studies show, however, that chemokine receptors are one of the cell membrane receptors that bind to these IgM autoantibodies and that, through this
• IgM autoantibodies inhibit HIV-1 from infecting cells.
• the inventor's studies also show that IgM autoantibodies that bind to chemokine
• receptors are heterogeneous and that only some of these antibodies have the ability to inhibit HIV-1 from infecting cells.
• Levels of IgM antibodies that inhibit HIV-1 are heterogeneous and that only some of these antibodies have the ability to inhibit HIV-1 from infecting cells.
• gpl20 glycoprotein or is designed for a specific purpose, i.e. , to function as
• IgM autoantibodies that inhibit HIV-1 from infecting cells are depleted in patients with AIDS but not in asymptomatic HIV-1 infected individuals or in normal
• one object of the present invention is to provide a method of
• Another object of the present invention is to provide a method of inhibiting
• FIG. 1A is a graph depicting binding of Normal IgM, HIV IgM and AIDS
• FIG. IB is a graph depicting binding of Normal IgM, HIV IgM and AIDS
• FIG. 1C is a flow cytometry (FASCAN) dot plot showing lymphocytes
• neutrophils separated by size and derived from human blood.
• FIG. ID is a graph depicting binding of Normal IgM to human T
• FIG. IE is a graph depicting binding of Normal IgM to human neutrophils derived from peripheral blood cells.
• FIGS. 1F-1G are Western blot assays depicting IgM binding to cell
• FIG. 2 A is a graph depicting affinity purified Accurate IgM inhibition of I 125 RANTES binding to CCR5 present in denatured U373-MAGI-CCR5E
• FIG. 2B is a Western blot assay depicting affinity purified Accurate IgM
• FIG. 2C is a dot plot FASCAN display depicting CK15 IgM inhibition of
• FIG. 2D is a graph depicting affinity purified Accurate IgM inhibition of
• FIG. 3A is a graph depicting AIDS and HIV-1 IgM inhibition of SDF-l ⁇
• FIG. 3B is another graph depicting IgM inhibition of SDF-l ⁇ binding to
• FIG. 4A is a FACSCAN exemplifying a dot plot quantitating SDF-l ⁇
• the bottom panel depicts
• FIG. 4B is a graph depicting the effect of AIDS IgM, HIV IgM, Normal
• FIG. 4C is a graph measuring the effect of IgM on SDF-l ⁇ induced change
• tracing B depicts the effect of SDF-l ⁇ only; and tracing C depicts
• FIG. 5 A depicts the inhibitory effect of IgM from normal, HIV and AIDS
• FIG. 5B depicts the inhibitory effect of IgM from normal, HIV and AIDS
• FIG. 5C is a flow cytometry dot plot exemplifying the inhibitory effect of Normal IgM on infection of ghost CCR5 cells by the R5 virus 8658.
• the middle dot plot depicts that about 17.2% of ghost CCR5 cells were infected, and infected
• present invention relates to the expression, stimulation and administration of IgM
• the IgM autoantibodies in the blood of normal individuals have been found to bind to various extracellular receptors present on lymphocytes. Such receptors
• chemokine receptors include, but are not limited to, chemokine receptors and other lymphocyte-surface receptors.
• Representative chemokine receptors are, for example, CXCR4, CCR5, CCR3 and CCR2b.
• Representative lymphocyte-surface receptors include, for example, glycolipid receptors.
• IgM anti-lymphocyte autoantibodies According to the present invention, IgM anti-lymphocyte autoantibodies
• IgM autoantibodies prevent lymphocytes from being infected with HIV-1.
• anti-leukocyte antibodies increase after HIV-1 infection and may
• IgM anti-chemokine receptor IgM anti-chemokine receptor
• antibodies function as "blocking" antibodies as they are not cytolytic at 37° C.
• IgM autoantibodies do
• IgM autoantibodies are heterogeneous and function normally to regulate receptor
• anti-chemokine receptor autoantibody inhibits HIV-1 from infecting cells. It is
• autoantibodies may be important in slowing down disease progression in the event of viral infection or other virus-mediated disease.
• Sup T-l and Jurkat cells are lymphoma T cell lines expressing the CXCR4
• An HOS osteosarcoma cell line is co-transfected with CD4 and either
• Ghost CCR5 and ghost CXCR4 are HOS-CD4 cells co-transfected
• the cell line and the transfectants are obtained from the AIDS Reagent Program at NIH.
• a glioblastoma cell line, U373-MAGI is co-transfected with CD4 and either CXCR4 or CCR5 to produce U373-MAGI-CXCR4 and U373-MAGI-CCR5,
• the cell line and the transfectants are obtained from the AIDS
• PBL Human peripheral blood lymphocytes
• PBL (2xl0 6 cells in 1 ml RPMI culture media containing 10% fetal calf serum are activated by initially pre-treating ficol/hypaque separated PBL with IL-2 (40 units/ml) and phytohemagglutinin ("PHA-P", 5 mcg/ml) and then washing the PBL after the cells are cultured at 37° C in about
• the R5 HIV-1 virus used to infect ghost CCR5 is obtained from Dr.
• Human IgM is obtained from the following sources: affinity purified,
• Normal IgM asymptomatic patients with HIV- 1 infection
• HIV IgM HIV- 1 infection
• AIDS IgM culture supernatants of EBV transformed human
• the HIV IgM is pooled from seven asymptomatic HIV-1 infected patients
• the HIV-1 infected patients all have greater than 500 CD4 positive cells per ml and a DNA viral load of less than 2,000.
• the AIDS IgM is pooled from nine AIDS patients having an opportunistic infection and less than 150 CD4 positive cells per ml and a viral load of greater than 10,000 despite
• the culture supernatants of EBV transformed human B cell clones are:
• the human B cell clones are derived from B lymphocytes
• the B cell clones are developed by
• non-T cells capable of secreting a specific antibody, i.e., IgM. More particularly, non-T cells
• the culture medium is replaced about every 4 to 5 days. After about 3 to 4
• B cell lines appear as "clumps" in the wells. Feeder cells die during this period. When the "clumps” appear, these clumped cells are transferred to a 24-
• well plate i.e., cells from one well are transferred into a single larger well.
• Culture media is changed when the media changes to a yellowish color, usually
• K6H6/B5 plasmacytoma cell line to develop
• the clones are screened to identify and obtain those clones that react with CCR5 and CXCR4 chemokine receptors present on the transfected cells.
• transfectants i.e., HOS-CD4-CXCR4 and HOS-CD4-CCR5 when compared to the HOS-CD4 control.
• IgM is also obtained using Sephacryl S-300 HR column chromoatography
• RANTES RANTES
• Radio-labeled RANTES (referred to as "I 125 RANTES" or “I 125 ”) is obtained from NEN Life Science of Boston, Massachusetts. RANTES
• CXCR4 and CCR5, respectively, are used.
• the 12G5 is obtained from Becton
• Flow cytometry is used to quantify IgM binding to the cells. Prior to flow
• FITC fluorescein-isothiocyanate
• IgM (Fc specific). Binding of IgM to human peripheral blood T lymphocytes is
• controls are made by combining about 50 ⁇ g of crude membrane proteins obtained
• the first approach is to determine if affinity purified Accurate IgM and/or
• CK15 IgM inhibit binding of I 125 RANTES to non-denatured, crude membrane
• RANTES in varying molar concentrations ranging from about 10 "6 to about 10 "10
• RANTES available from NEN Life Science is added to each mixture, and each
• I 125 RANTES unbound I 125 RANTES.
• Specific I 125 RANTES binding is calculated by subtracting the counts of radioactivity per minute ("c.p.m.") of I 125 RANTES when used with
• CCR5 membrane proteins are incubated, under non-reducing conditions, with each of about 0.1 nM Normal IgM, 350 nM IgG, 0.4 mcM unlabeled RANTES and a
• CCR5 membrane proteins is used. Each mixture is then electrophoresed onto 12%
• a procedure is used to determine if IgM inhibits binding of RANTES to
• CCR5 receptors present on intact cells, e.g., U373-MAGI-CCR5E and IL-2-
• calf serum or with about 150 nM of affinity purified Accurate IgM or with about 400 nM purified human IgG or with about 5 nM, aboutl.25 nM or about 0.45 nM
• CK15 IgM prior to adding about 1 microgram of RANTES to each mixture.
• the cells are then re-incubated for about 90 minutes at 4° C and then washed at 4°
• FITC-labeled RANTES binding to CCR5 on these cells is analyzed by flow cytometry.
• chemokine receptors i.e. , CXCR4
• CXCR4 chemokine receptors
• Sup T-l cells are initially incubated at room temperature for about 45 minutes with
• RPM1 media containing about 10% fetal calf serum or with about 150 nM of each
• FITC avidin is added to the cells, and the cells are
• chemokines binding to CCR5 or CXCR4 is indeed specific for chemokines. More
• these procedures are used to determine if IgM, through some nonspecific mechanism, also inhibits binding of radio-labeled IL-2 to the IL-2R
• phytohemagglutinin-activated PBL (lxlO 6 ) is incubated with I 125 labeled IL-2
• a chemotaxis assay is performed with each of Normal IgM, HIV IgM,
• AIDS IgM and Waldenstrom IgM at concentrations of about 20 nM, about 40 nM, about 100 nM and about 200 nM IgM using 24-well Costar transwell tissue culture
• CI chemotaxic index
• chemotactic index of SDF-l ⁇ alone is about 3.1.
• Assays are performed to determine intracytosolic Ca +2 flux using known methods, for example, as described in Haverstick, G., MD, Molecular Biol. of
• second assay is done using about 45 nM of AIDS IgM in place of HIV IgM.
• IgM antibody is evaluated by a complement dependent microlymphocytotoxicity assay. Various dilutions of IgM antibody are reacted for 1 hour with either 2xl0 5 PBL or
• IL-2-activated PBL 7 days) before adding fresh rabbit serum as a source of complement. After about 2 hours, the cells are washed twice before adding trypan blue and enumerating dead cells that stain blue. Experiments are performed at 15°
• HIV-1 R5 virus utilizes CCR5 receptors for
• the ghost cells are derived from HOS cells transfected with HIV-1.
• the hGFP construct enables cells infected with HIV-1 virus to emit a green fluorescence so that the number of infected cells can be quantified using flow cytometry. These cell lines are particularly suited for these studies because single-cycle viral replication can be detected in less than 48 hours.
• Infected cells emitting green fluorescence are enumerated with flow cytometry.
• Normal IgM contains IgM antibodies that bind to Sup T-l (FIG. 1A) and ghost CD4-CXCR4 cells (FIG. IB).
• FIGS. ID and IE Normal IgM contains antibodies that bind to T-lymphocytes
• FIG. IE The negative control in each figure indicates that no IgM was incubated with the various cells.
• IgM autoantibodies bind to several cell membrane proteins. However, IgM bound to an additional 48 kD protein present in cell lines transfected with cDNA of CCR5 ("CCR5") and
• CXCR4 (“CXCR4").
• Membrane proteins of 48 kD are similar to the molecular
• IgM can bind to several membrane proteins because the IgM antibody is known to be
• IgM autoantibodies inhibit binding of radio-labeled RANTES to CCR5
• IL-2R radio-labeled IL-2 receptors
• the CK15 IgM inhibit binding of I 125 RANTES to CCR5 in a dose-dependent
• FIG. 2 and FIG. 3B clearly indicate that inhibition of chemokines to their
• anti-leukocyte autoantibodies are heterogeneous and recognize different epitopes on the chemokine receptor. It is, therefore, possible that AIDS IgM lacks the
• FIG. 4A shows flow cytometry data from another experiment to visually
• tracing C represents Jurkat cells with HIV IgM
• tracing B represents
• Tracing A indicates that AIDS IgM enhances the rise in the intracellular
• HIV IgM HIV IgM.
• IgM anti-lymphocyte autoantibodies with AIDS IgM containing IgM that predominantly enhances chemotaxis and Ca +2 flux after AIDS IgM containing IgM that predominantly enhances chemotaxis and Ca +2 flux after AIDS IgM containing IgM that predominantly enhances chemotaxis and Ca +2 flux after AIDS IgM containing IgM that predominantly enhances chemotaxis and Ca +2 flux after
• CK15 lysed cells at concentrations of about 5 nanograms or more.
• IgM anti-lymphocyte autoantibodies are lytic at colder temperatures but not at 37° C.
• IgM anti-chemokine receptor autoantibodies contribute to the resistance
• FIGS. 5A-5C show the effect of various IgM antibodies
• FIG. 5A which shows the
• IgM isolated from five individual normal sera and one HIV sera.
• IgM from pooled normal sera partially inhibits (about 40 to 50% inhibition) HIV-1 infectivity with the R5 virus 8442 and the X4 virus RF,
• the IgG antibody 2D7 a murine anti-CCR5 antibody, inhibited infectivity
• serum is calculated to have about 168 nM IgM. HIV-1 infected sera and AIDS
• infectivity is mediated via reactivity of IgM to
• IgM IgM receptors important for HIV-1 entry into cells.
• receptors include, but are not limited to, CXCR4 and
• IgM from normal sera has no direct anti-viral neutralizing effect and yet has the most inhibitory effect on HIV-1 infectivity.
• IgM binds to Ghost cells and T cell lines and also enhances chemotaxis and cytosolic Ca +2 induced by SDF-1. With respect to IgM binding and inhibition of
• Normal IgM and AIDS IgM contain a heterogeneous group of IgM anti- lymphocyte antibodies with different binding epitopes that give rise to different
• AIDS IgM appears to lack this subset of IgM anti-lymphocyte antibody.
• anti-chemokine receptor antibody may be more important in influencing
• IgM anti-lymphocyte autoantibodies limit the entry of the HIV-1 virus into cells and prolong the latency period because
• lymphocyte autoantibodies especially the subset of antibodies that inhibit HIV-1 entry into cells.
• subset could be exhausted or could be infected with HIV-1, thereby leading to a
• lymphocyte antibody may only partially prevent entry of certain HIV-1 viral isolates, as indicated by some of the studies herein. This latter mechanism may provide another explanation for disease progression despite the presence of IgM
• infectivity i.e. , through receptor blockade
• lymphocytes present on the lymphocytes may be particularly useful in situations where the HIV-1 virus switches its receptor usage, e.g. , from CCR5 to CXCR4.
• Maintaining increased levels of such protective antibodies could also increase the latency period after HIV-1 infection.
• the source of IgM antibodies may be heterologous, autologous or
• the leukocytes may be raised in vivo (i.e., in mice or other animals or in humans) or in vitro using cell culture techniques.
• IgM antibodies may be produced either in vivo or in vitro by genetic engineering whereby genes specific for IgM anti-lymphocyte antibodies are
• antibody-producing cells may be introduced into antibody-producing cells. These antibody -producing cells may be introduced into antibody-producing cells. These antibody -producing cells may be introduced into antibody-producing cells. These antibody -producing cells.
• these antibody-producing cells may be grown in vitro using hybridoma or other technology.
• IgM antibodies with specificity for chemokine receptors may also be produced by isolating human antibody-producing cells specific for IgM antibodies
• human lymphocytes may be transplanted into immunodeficient mice, and the lymphocytes may then be stimulated with an agent that will activate B cells
• LPS lipopolysaccharide
• Another method of producing IgM antibodies is by isolating human
• antibody -producing cells capable of generating human IgM from animals such as,
• IgM antibody production by such cells may then be enhanced in vitro employing hybridoma or other technology such as, for example, the XenoMouseTM.
• IgM antibody production by such cells may then be enhanced in vitro employing hybridoma or other technology such as, for example, the XenoMouseTM.
• hybridoma or other technology such as, for example, the XenoMouseTM.
• the cells e.g., the EBV virus.
• IgM antibodies may also be produced in vitro by isolating, from an
• viruses, bacteria and other antigens may be used.
• viruses, bacteria and other antigens e.g. , mitogens
• IgM antibodies produced outside an infected individual may be delivered to the individual by one of several routes of administration including, but not limited to, intravenous and intramuscular delivery.

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