Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1996-04-08
2001-05-15
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S327000, C530S387300, C530S868000
Reexamination Certificate
active
06232444
ABSTRACT:
TECHNICAL FIELD
The field of this invention is the diagnosis and treatment of nephritis associated with systemic lupus erythematosus.
BACKGROUND
Antibodies to deoxyribose nucleic acid (DNA) are the hallmark of systemic lupus erythenotesus (SLE) and are associated with the development of kidney disease (nephritis). However, anti-DNA antibodies are heterogeneous and the structural basis for pathogenic anti-DNA antibodies has remained elusive. Idiotypes are structural determinants on or near antibody binding sites recognized by a second (antiidiotype) antibody. Antiidiotype antibodies can be used to identify unique structural features of potentially pathogenic anti-DNA antibodies. Although several anti-DNA antibody antiidiotypes have been produced, an anti-idiotype has not been produced that characterizes unique structural features of anti-DNA antibodies that are conserved in both murine and human lupus associated with nephritis.
Because of the substantial interest in being able to diagnose and treat lupus associated with nephritis, various efforts have been made to determine the causative agent of nephritis associated lupus and to find therapeutic techniques. It is therefore of interest to be able to develop antibodies which are capable of recognizing disease related anti-DNA antibodies.
RELEVANT LITERATURE
References describing the relationship between anti-DNA antibodies, systemio-lupus erythematosus and development of kidney disease are Koffler et al.,
J. Exp. Med.
134, 294 (1971); Miller et al.,
Arthritis Rheum.
24, 602 (1981), Koffler et al.,
J. Exp. Med.
126, 607 (1967); Koffler,
Annu. Rev. Med.
25, 149 (1974); Tron and Bach,
Clin. Exp. Immunol.
28, 426 (1977). Articles describing anti-DNA antibody antiidiotypes are Rauch et al.,
J. Immunol.
129, 236 (1982); Isenberg et. al., Lancet 2, 417 (1984), Hahn and Ebling,
J. Immunol.
138, 2110 (1987); Livneh et al.,
J. Immunol.
138, 123 (1987).
MRL-lpr/lpr mice are known to develop severe nephritis resulting in death (Theofilopoulos and Dixon, Adv. Immunol. 37, 269 (1985); Dixon,
Arthritis Rheum.
28, 1081 (1985).
SUMMARY OF THE INVENTION
Methods and compositions are provided for inducing an immune response for protection against diseases where an idiotype is expressed and related to the disease, e.g., lupus nephritis, and for diagnosis and treatment of such diseases. Particularly, monoclonal antibodies are provided which may serve as vaccines and for diagnosis and treatment of such diseases.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Methods and compositions are provided associated with the treatment and diagnosis of diseases where antibodies are produced where the idiotype is diagnostic of the disease and can be used as an immunogen for inducing immune protection. Diseases of particular interest include lupus nephritis and B-cell lymphomas. For lupus nephritis, the monoclonal antibodies are directed to binding to double stranded DNA (dsDNA) or monoclonal antibodies which specifically bind to an idiotype associated with antibody binding to dsDNA, which is also associated with lupus nephritis. By employing particular variable regions of antibodies which bind to dsDNA as an immunogen, a mammalian host may be immunized for protection against lupus nephritis or may be used to produce antibodies which can provide for diagnosis of the presence of antibodies associated with lupus nephritis or therapeutic treatment of lupus nephritis.
Lupus nephritis may be associated with a wide variety of mammalian hosts, including murine, primate, particularly human, especially women. To obtain monoclonal antibodies specific for binding to dsDNA, one may employ lymphocytes from a host suffering from the disease. The lymphocytes may be splenocytes, lymphocytes from tonsils or lymph nodes, peripheral blood lymphocytes, or the like.
The lymphocytes from the host may be immortalized by any convenient means, e.g., fused with any convenient fusion partner for production of hybridomas in accordance with conventional techniques. Various techniques may be employed for immortalizing B-lymphocytes producing desired antibodies, using myeloid cells, heteromyelomas, Epstein-Barr virus infection, FOX-NY myeloid cells, etc. The resulting cells may then be cloned by limiting dilution and screened for production of the desired antibodies. Conveniently, DNA, e.g., calf thymus DNA may be used as the ligand for screening and any convenient assay technique for detecting complex formation between the DNA and the antibody may be employed. Assays which may be used include ELISA, RIA or the like. Other characteristics of the antibody may be binding to single stranded DNA (ssDNA) and poly dT, but may also include poly dA, poly dC, poly dG or cardiolipin.
An antibody of particular interest was designated mAb 3E10 which is Characterized as an IgG 2a kappa with an isoelectric point of about pH 7.5 to 8.
Antibodies having these characteristics may be used as immunogens for producing antiidiotypes, either as a vaccine, or for the production of antibodies for use in therapy and diagnosis. The entire antibody need not be used, only the variable region is required or even either the light or heavy chain variable region.
Immunization may be achieved in any conventional manner, by immunizing the host, particularly with an adjuvant. Various adjuvants include BCG, aluminum hydroxide, monophosphoryl lipid A, trehalose 6,6′- dimycolate, etc. In addition, the antibody or fragment thereof may be conjugated with various carriers, such as keyhole limpet hemocyanin, tetanus toxoid, gamma globulin, etc.
Antiidiotype monoclonal antibodies may then be produced as described previously. The resulting antiidiotype monoclonal antibodies thus produced should show specific binding to the idiotype of the dsDNA binding antibody. These antibodies may be further screened to demonstrate binding to antibodies present in a host having lupus nephritis. Desirably, the antiidiotype antibody should be at least about 25% inhibited from binding by the presence of the DNA ligand. Furthermore, the antibody will normally remove at least 10%, usually at least 15% of the total antibody binding to dsDNA in a lupus nephritis diseased host.
Depending upon the application, syngeneic, allogeneic, or xenogeneic antibodies may be employed. Chimeric antibodies may be used, where the variable region may be from one host and the constant region from the host to be treated. Antibody fragments may be employed, such as F(ab′)
2
, F(ab′), recombinant F
v
, or the like, where the fragment is free of the constant region. The important factor in the case of therapy is to provide effective binding to endogenous antibodies associated with lupus nephritis.
The antibodies may be administered in any convenient form, particularly parenteral, more particularly intravascularly or subcutaneously. The antibodies or fragments thereof will be administered in an effective amount in a physiologically acceptable medium, such as water, PBS, saline, aqueous ethanol, or the like. The amount administered will generally be in the range of about 10 mg/kg to 50 mg/kg.
One antibody of particular interest is referred to as mAb 1C7. This antibody is a murine IgG 2b antibody and is specific for an idiotype or anti-DNA antibodies. The antibodies may be murine, e.g., rat or mouse, lagomorph, bovine; equine, primate, e.g., monkey, gorilla, human, etc., ovine, porcine, etc. The antibody may be cross reactive with cell membrane determinants and serve as a diagnostic for the presence of such determinants.
The cells producing the desired antibodies may be used as a source of DNA or mRNA for production of cDNA to provide the sequence for the variable region. By employing known techniques, the genes may be isolated. Techniques include sequencing a portion of the protein and preparing redundant probes encoding for the particular sequence. These probes may then be used to screen the genomic or cDNA library from the host cell. Alternatively, one may use subtraction libraries, where a cDNA library from a pre-B cell may be used to subtract from a lib
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Saunders David
The Regents of the University of California
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