Abstract:
Pooled human plasma is processed by cold ethanol fractionation to produce purified immunoglobulin G antibodies for intravenous administration. Immunoglobulin A is an unwanted by-product since intravenous administration of immunoglobulin A-containing immunoglobulin G can cause life-threatening anaphylaxis in some people. The present invention is the aerosol administration, by metered dose inhaler or nebulizer, of by-product immunoglobulin A for the prevention or treatment of diseases including immunodeficiencies and infections. Antigen-specific monoclonal immunoglobulin A may be used. Immunoglobulin A from any of the aforementioned sources may then be coupled with recombinant J chain, and may then be additionally coupled with recombinant secretory component in order to render the immunoglobulin A more physiologically active. Immunoglobulin A, with or without J chain and secretory component, is then administered by aerosol inhalation.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to human medical treatment by aerosol inhalation of immunoglobulin (Ig) A. More specifically the invention relates to treatment of immunodeficiencies and viral or bacterial infections of the lower respiratory tract by administering doses of IgA or an antibody composition rich in immunoglobulin A.  
         BACKGROUND OF THE INVENTION  
         [0002]    Immunoglobulins (also called antibodies) are a group of structurally related proteins composed of heavy and light chains. These proteins are categorized as IgM, IgG, IgD, IgE, and IgA depending upon the characteristics of the constant regions of their heavy chains (designated μ, γ, δ, ε, and α, respectively). The variable regions of the heavy chains along with the variable regions of the light chains determine the molecular (antibody) specificity of the complete molecule. These molecules are secreted by B lymphocytes in response to signals from other components of the immune system. Their function is to prevent and combat infection by viruses and bacteria.  
           [0003]    Purified IgG from pooled human plasma is administered intravenously in humans to treat a variety of conditions. In the purification, a fraction rich in IgA is considered an unwanted by-product, since intravenous administration of IgA containing immunoglobulin can cause life threatening anaphylaxis in some patents.  
           [0004]    IgA on mucosal surfaces is produced locally and not derived from circulating IgA. IgA is one of the γ globulins on the basis of its electrophoretic mobility. IgA is composed of two α heavy chains and two light chains. It may be monomeric (i.e. a single molecule), dimeric (composed of two molecules) or trimeric (composed of three molecules). IgA monomers are joined together as dimers at the constant regions of their heavy chains by a J chain. IgA is secreted as one of two subclasses, IgA1 and IgA2. IgA1 predominates in the circulating blood wherein most of it occurs as a monomer. Most IgA on mucosal surfaces, such as the surfaces of the trachea, bronchi, and bronchioles in the lungs, occurs as dimers or trimers joined by J chains. IgA dimers and trimers have an increased ability to bind to and agglutinate target molecules (antigens). Agglutinated antigens are more readily phagocytosed and thereby eliminated. In addition, IgA dimers and trimers, because of the presence of their J chains, have the ability to attach to secretory component. Such molecules then have increased resistance to proteolytic enzymatic degradation. Human J chains (Symerski, et al., Mol Immunol 2000; 37:133-140) and murine secretory component (Crottet, et al., Biochem J 1999; 341:299-306) have been produced by genetic recombinant biological techniques. Recombinant expression of polymeric IgA with the incorporation of J chain and secretory component of human origin has been accomplished (Johansen, et al., Eur J Immunol 1999; 29:1701-1708)  
           [0005]    IgA can attach to the cell surface of phagocytic leukocytes and thereby facilitate antibody-dependent cell-mediated killing of microorganisms. It also interacts with lactoperoxidase and lactoferrin which enhances the latter&#39;s antibacterial actions. Monomeric IgA interferes with influenza virus replication (Taylor, et al., J Exp Med 1985;161:198-209) and polymeric IgA interferes with influenza binding to and entry into target cells (Taylor, et al., J Exp Med 1985;161:198-209; Outlaw and Dimmock, J Gen Virol 1990;71:69-76).  
           [0006]    Exogenous IgA has been topically applied to the nose in both animals and humans for the purpose of preventing and treating disease. In mice, nasal application of exogenous IgA has been demonstrated to be efficacious in protecting animals from influenza (Tamura, et al., Vaccine 1990;8:479-485, Tamura, et al., Eur J Immunol 1991;21:1337-1344), Sendai virus (Mazanec, et al., J Virol 1987;61:2624-2626, Mazanec, et al., Virus Res 1992;23:1-12) and respiratory syncytial virus (Weltzin, et al., Antimicrob Agents Chemother 1994;38;2785-2791) challenge. Intranasal monoclonal IgA also protects rhesus monkeys against respiratory syncytial virus infection (Weltzin, et al., J Infect Dis 1996;174:256-261). In humans, nasal administration of approximately 70% IgA/30% IgG resulted in decreased frequency of upper respiratory tract infections in elite skiers (Hemmingsson and Hammarstrom, Scand J Infect Dis 1993;25:73-75), and in children (Giraudi, et al., nt J Pediatr Otorhinolarynol 1997;39:103-110, Heikkinen, et al., Pediatr Infect Dis J 1998;17:367-372) but not in elite canoeists (Lindberg and Berglund, Int J Sports Med 1996;17:2335-238).  
           [0007]    Aerosol administration of human γ globulin (Fruchtman, et al., Clin Med 1972 (September);79:17-20), pooled human IgG (Rimensberger and Schaad, Pediatr Infect Dis J 1994;13:328-330) and murine recombinant humanized IgG (Fahy, et al., Am J Respir Crit Care Med 1999; 160:1023-1027) demonstrated that there are no adverse effects from the aerosol inhalation of human  65  globulin or human or humanized IgG.  
           [0008]    Individuals suffering from hypogammaglobulinemia or with bronchial infections from other sources have been treated by a number of means, none of which has proven to be completely satisfactory. On the one hand, such patients have been treated by administration of antibiotics. However, antibiotics treatment is not completely effective in preventing infection in patients with immunoglobulin deficiency or whose immune systems are otherwise compromised. Another method of treating such patients has been intravenous infusion of immunoglobulin. The immunoglobulin administered by intravenous infusion does not contain the secretory piece. As a result, the infused immunoglobulin may not reach the mucosal surface of the bronchial tree. In addition, intravenous infusion of immunoglobulin is usually administered by trained medical personnel and can be associated with systemic reactions. There is thus a need for methods which can be used to deliver IgA to the bronchial mucosal surface. It would be advantageous if such treatment could be administered by the patient without the need for intervention by trained medical personnel. It would further be desirable to make use of unwanted by-products resulting from the preparation of purified immunoglobulin G from pooled human plasma. The present invention provides these advantages and others as will be apparent to one with skill in the art from the disclosure that follows.  
         SUMMARY OF THE INVENTION  
         [0009]    The invention provides a method for medical treatment of humans that involves pulmonary administration by inhalation of an immunoglobulin (Ig) A composition. In one embodiment, the IgA is prepared as a by-product from pooled human plasma and is derived from a Cohn fraction component enriched in IgA. In another embodiment, the IgA composition contains a monoclonal antigen-specific IgA. In a preferred embodiment, the IgA component is further combined with recombinant human J chains and recombinant secretory component to produce a more physiologically effective composition. Conditions treatable by pulmonary administration of such compositions include immunodeficient diseases, immune suppression, bacterial infections, and viral infections.  
         DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
         [0010]    The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.  
           [0011]    In one embodiment, the invention provides a method for medical treatment of humans comprising the step of administering by inhalation an aerosol composition. The aerosol composition contains an IgA component which can be derived from a number of sources. The aerosol composition contains an IgA component which can be derived from a number of sources. The by-product is obtained from pooled human plasma following Cohn cold ethanol fractionation to produce fraction III precipitate as performed by those of skill in the art of protein separation. IgA by-product is further purified by adsorption onto a ion exchange medium in neutral or slightly acidic conditions as performed by those of skill in the art of protein purification.  
           [0012]    A more detailed description of isolation of an IgA component as a by-product from pooled human plasma or hyperimmune pooled human plasma is as follows. Ethanol fractionation of pooled human plasma is a well known process to prepare immunoglobulin G. Pooled human plasma is first obtained from licensed plasmapheresis centers in the United States and tested for various pathogens including the HIV virus. The first manufacturing step of most commercial immunoglobulin G preparations involves a modified cold ethanol fractionation according to Cohn to produce Cohn fraction II. In the fractionation process, many infectious viruses are eliminated from the pooled human plasma. Following fractionation, the Cohn fraction II is subjected to adsorption onto an ion exchange medium. This step may selectively reduce the IgA concentration to less than 0.1%. Such a step is important for producing immunoglobulin G for intravenous infusion into humans. This is because some individuals undergo an anaphylactic-like reaction if treated with intravenous IgG that contains IgA as an impurity.  
           [0013]    The modified cold ethanol fractionation process according to Cohn is a series of fractionations using various levels of ethanol, pH, and temperature to produce a fraction II which is further treated to produce immunoglobulins as described above. In the fractionation method, pooled human plasma is first treated to produce a cryoprecipitate and cryo-supernatant. The cryo-supernatant is subjected to a first ethanol fractionation to yield a supernatant I. Supernatant I is subjected to a second ethanol fractionation to yield fraction II+III. Fraction II+III is subjected to a third ethanol fractionation procedure to yield a supernatant III and Fraction III precipitate.  
           [0014]    The fraction III precipitate enriched in IgA is generally discarded as an unwanted by-product. According to the invention, this unwanted IgA following ion exchange adsorption purification, is further treated by incubation with immobilized hydrolases to inactivate viruses and vasoactive substances. Such treatment has been proven to eliminate many viruses tested including HIV, Sindbis, and vaccinia. Following incubation to remove viruses, the concentration of the active material is adjusted with sterile saline or buffered solutions to ensure a constant amount of active material per milliliter of reconstituted product. Finally, the solution with a constant amount of reconstituted product is sterilized by filtration before use.  
           [0015]    The ethanol fractionation process according to Cohn is well known in the art and is described in Cohn, et al., J Am Chem Soc 1946;68:459-475, Oncley, et al., J Am Chem Soc 1949;71:541-550, and in most detail in pages 576-602, Kirk-Othmer Encyclopedia of Chemical Technology, Vol 3, second edition (1963), the disclosure of which is hereby expressly incorporated by reference.  
           [0016]    In a preferred embodiment, the compositions of the invention contain, in addition to the IgA component, one or more further components selected from the group consisting of recombinant human J chains, recombinant secretory component, and combinations thereof. The production of human J chains by genetically recombinant biological techniques is disclosed in Symerski, et al., Mol Immunol 2000; 37:133-140, the disclosure of which is hereby incorporated by reference. Human secretory component can be produced by recombinant techniques as described in Crottet, et al., Biochem J 1999; 341:299-306, disclosure of which is hereby incorporated by reference. In a preferred embodiment the IgA may be coupled to recombinant J chains by disulfide bonding which is accomplished in mildly oxidizing conditions. The resulting IgA-J chain conjugates are purified. IgA-J chain conjugates may then be further coupled to recombinant secretory component. In a preferred embodiment, the coupling is accomplished by forming disulfide bonds under mildly oxidizing conditions. IgA containing both J chain and secretory component is again purified by ion-exchange and size exclusion chromatography and/or ultrafiltration as described in Lullau, et al., J. Biol Chem 1996; 271:16300-16309, Corthesy, Biochem Soc Trans 1997; 25:471-475, and Crottet, et al., Biochem J 1999; 341:299-306, as performed by those of skill in the art of protein purification, the disclosures of which are hereby incorporated by reference. While recombinant expression of IgA with the incorporation of J chain and secretory component has been accomplished, hybridoma production of IgA may not include incorporated J chains and secretory component. According to the invention, the recombinant J chains, recombinant secretory component, or mixtures of them may be combined with the monoclonal IgA after production of the IgA by hybridoma techniques. Such IgA may be coupled to recombinant J chains and secretory component as described above. Purified IgA containing J chain and secretory components can be stabilized for example by the addition of human serum albumin to a final concentration of 5%. The presence of the human J chains and secretory component in the compositions of the invention leads to inhaled doses of immunoglobulin which are more physiologically effective than compositions without such components.  
           [0017]    In another embodiment, an IgA containing component is isolated as a by-product from hyperimmune pooled human plasma for coupling with J chain and secretory component. Hyperimmune pooled human plasma is obtained from donors who have been immunized against a specific disease.  
           [0018]    In another embodiment, the IgA component can be prepared by hybridoma techniques to provide antigen-specific IgA. Hybridoma techniques are described originally in Kohler and Milstein, Nature 1975;256:495-497 with more recent advances summarized in Berzofsky et al., Fundamental Immunology, Third Edition, 1993, pp 455-462, the disclosures of which are hereby incorporated by reference. Hybridoma production involves the fusion of an immortalized immunoglobulin-producing myeloma cell with an antibody-producing cell from an immunized individual. The product is an immortalized cell culture which produces the specific antibody against the antigen that the donor individual is immune to. For example, a mouse monoclonal IgA antibody has been prepared against respiratory syncytial virus F glycoprotein as described in Weltzin, et al., J Infect Dis 1996;174:256-261 and Weltzin, et al., Antimicrob Agents Chemother 1994;38:2785-2791.  
           [0019]    The compositions of the invention for pulmonary delivery of aerosol compositions generally contain in addition to the IgA component and optional J chains and secretory component known pharmaceutical excipients and buffering agents. Non-limiting examples of such excipients include proteins as for example, human serum albumin and recombinant human albumin. Other pharmaceutical excipients include carbohydrates, sugars, and alditols. Non-limiting examples of suitable carbohydrates include sucrose, lactose, raffinose, and trehalose. Suitable alditols include mannitol, and pyranosyl sorbitol. Polymeric excipients include polyvinylpyrolidone, Ficolls, soluble hydroxyethyl starch, and the like of suitable molecular weight. Non-limiting examples of suitable buffering agents include salts prepared from organic acids such as citric acid, glycine, tartaric acid, lactic acid, and the like. Other useful excipients include surfactants and chelating agents. The compositions of the invention are readily aerosolized and rapidly deposited in the lungs of a host. Doses are formulated from the compositions of the invention by combining the IgA component with or without human J chain and secretory component, and pharmaceutical excipients so as to contain an effective dose of the active ingredient. A typical dose would include about 5 milligrams of active material. The dose amount may be adjusted up or down as required to meet the treatment needs of an individual, or to provide for ease and convenience in administering the dose.  
           [0020]    The compositions of the invention can be administered by nebulization or by metered dose inhalers. Nebulizers and metered dose inhalers are well know in the art and are described for example, in Wolff and Niven, J Aerosol Med 1994;7:89-106.  
           [0021]    Diseases and conditions for which aerosol pulmonary administration of the compositions of the invention is to be used therapeutically or prophylactically include, but are not limited to: common variable immunodeficiency, IgA deficiency, human immunodeficiency virus (HIV) infection, lower respiratory tract infection with influenza, lower respiratory tract infection with respiratory syncytial virus, lower respiratory tract infection with rhinovirus, lower respiratory tract infection with adenovirus, chronic lymphocytic leukemia, multiple myeloma, macroglobulinemia, chronic bronchitis, bronchiectasis, asthma, immune suppression associated with bone marrow transplantation, immune suppression associated with cyclophosphamide administration, immune suppression associated with azathiaprine administration, immune suppression associated with methotrexate administration, immune suppression associated with chlorambucil administration, immune suppression associated with nitrogen mustard administration, immune suppression associated with 6-mercaptopurine administration, immune suppression associated with thioguanine administration, severe combined immunodeficiency, adenosine deaminase deficiency, major histocompatibility class I (Bare leukocyte syndrome) and class II deficiencies, purine nucleoside phosphorylase deficiency, DiGeorge Syndrome, transient hypogammaglobulinemia of infancy, X-linked agammaglobulinemia, X-linked agammaglobulinemia with growth hormone deficiency, transcobalamin II deficiency, immunodeficiency with thymoma, immunodeficiency with hereditary defective response to Epstein Barr virus, immunoglobulin deficiency with increased IgM, κ chain deficiency, ataxiatelangiectasia, and immunodeficiency with partial albinism.  
           [0022]    As used here, the term therapeutic treatment means that the patient being administered a dose of a composition of the invention has been diagnosed as having the condition to be treated. Prophylactic treatment means that the patient is being treated to prevent infection. Such treatment is often indicated where a patient is at risk for lower respiratory tract infection. 
       
    
    
     EXAMPLE  
       [0023]    Polyclonal IgA is obtained from pooled human plasma following Cohn cold ethanol fractionation to produce fraction III precipitate. IgA is further purified by adsorption onto an ion exchange medium in neutral or slightly acidic conditions. Alternatively, monoclonal IgA is obtained from an IgA-producing hybridoma. The IgA is then coupled to recombinant J chains by disulfide bonding which is accomplished in mildly oxidizing conditions. The molar ratio of IgA to J chain is 2:1 or 3:1. IgA-J chain conjugates are purified. IgA-J chain conjugates may then be further coupled to recombinant secretory component again by disulfide bonding in mildly oxidizing conditions, preferably at a molar ratio of secretory component to IgA-J chain conjugates of 1:1. IgA containing both J chain and secretory component is again purified. Purified IgA containing J chain and secretory component is stabilized by the addition of human serum albumin to a final concentration of 5%. The final solution, adjusted to a therapeutic dose of 5 mg IgA, is then placed in a nebulizer for self-administration.  
         [0024]    The description of the invention is merely exemplary in nature and, thus, variations that do not depart from the gist of the invention are intended to be within the scope of the invention. Such variations are not to be regarded as a departure from the spirit and scope of the invention.