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U.S. Patent Jan. 14,1997 Sheet 2 of 5 5,593,897
7 9 11 13 15 17 19 Days after Tumor Implantation
Antigen + Antibody = Antigen-Antibody Complexes
BINDING OF IMMUNE COMPLEXES BY
MODIFIED FORMS OF C-REACTIVE
This application is a continuation of application Sen No. 5 07/582,884, filed Oct. 3, 1990 now abandoned, which is the national phase under 35 USC 371 of PCT/US 89/01247 filed on Mar. 31,1989 which is a CIP of Ser. No. 07/176,923 filed on Apr. 4, 1988, now abandoned.
FIELD OF INVENTION
This invention relates to a method for binding immunoglobulin, aggregated immunoglobulin or immune complexes, including all immunoglobulin isotypes of mammals, 15 particularly IgG, IgA, and IgM, using a modified form of C-reactive protein, a naturally-occuring protein. In particular, this invention relates to the use of modified C-reactive protein to bind immune complexes in the treatment of a variety of diseases and as part of assays for the detection and quantitation of immune complexes. The modified form of C-reactive protein may be used as a reagent that can bind immune complexes either when coated onto a solid surface or when added to a solution containing immune complexes. Preferred is modified C-reactive protein expressing the neo-CRP antigen.
The invention further relates to a method of preparing modified C-reactive protein, and to a method of binding aggregated immunoglobulin, immune complexes and immu- 3Q noglobulin comprising contacting them with antibody to neo-CRP. Finally, the invention also relates to test kits for detecting and quantitating immune complexes and to devices for removing immune complexes and aggregated immunoglobulin from fluids. 35
BACKGROUND OF THE INVENTION
Immune complexes are formed by the binding of antibodies with antigens, sometimes in conjunction with other 40 proteins. The antigens which form immune complexes include components of infectious organisms, other molecules foreign to the host organism, tumor-associated molecules and, in many diseases, normal tissue molecular components. Antibodies produced to an antigen are specific 45 for the particular antigenic substance. Antibodies bind to antigens and essentially neutralize them by, e.g., altering the biologic activity of a toxin, neutralizing the infectivity of microorganisms, or by providing the recognition signal whereby the antibody plus bound antigen are removed from 50 the circulation or tissues. When an antibody binds to an antigen, the antibody-antigen complex is termed an immune complex.
foreign substance + immune system = immune complexes
Immune complexes are removed from the circulation and 60 tissues by a variety of normal mechanisms such as by fixed macrophages found in the liver, spleen and lymph nodes, and by circulating macrophages. The formation of antibodies and immune complexes is part of the natural response of the individual to combat diseases, such as infections and 65 cancer. The binding of antibodies to antigens and the removal of the immune complexes are the mechanisms by
which antigens are neutralized, taken out of tissues and blood and degraded.
The continued presence of immune complexes in the circulation and their deposition in tissues, contributes to compromised immune system function and inflammatory pathology. Immune complexes can deposit in tissues such as the lung, kidney, heart and joints causing both transient and permanent damage to those organs. In cancer, it is thought that the immune complexes may block the proper function of immune mechanisms which would otherwise destroy the cancer cells and prevent the growth and spread of the cancer within the body.
There may be one or several reasons why circulating immune complexes are found in high levels in diseases. The antigens and the immune system responses may be near maximal, and the immune complexes formed may simply overwhelm the capacity of the systems for their removal. Alternatively, some mechanism may have compromised the efficiency of the immune complex removal system, or the nature of the antigens and antibodies involved may result in inefficient removal. Immune complexes are found to persist in many individuals with cancer, autoimmune, arthritic, and infectious diseases. Therefore, the binding and removal of immune complexes, by an extracorporeal device, may result in a clinical improvement of individuals with cancer and improve the effectiveness of other treatments for cancer, may result in the prevention of lesions in organs associated with immune complexes that occur in infectious diseases, and may prevent lesions in organ systems in arthritic and autoimmune diseases, as well as result in the clinical improvement of these latter diseases. Indeed, many published studies indicate that the removal of circulating immune complexes, or enhancing their clearance from the circulation, may constitute an effective therapeutic treatment. [See Theofilopoulos, A. M. and Dixon, F. J., Adv. Immunol, 28:90-220 (1979); Theofilopolous, A. N. and Dixon, F. J., Immunodiagnostics of Cancer, p. 896 (M. Decker Inc., New York 1979)].
There can be a wide variety in the number of anti-body molecules which bind to antigen, and antigens are of various sizes and shapes, thus leading to wide variations in the sizes of the immune complexes. Some immune complexes can also affix complement proteins such as Clq and C3 which may result in larger, more heterogeneous structures. Some immune complexes stimulate leukocytes while others stimulate lymphocytes or platelets [See Ritzmann, et al., Clin. Chem., 28:1259-71 (1982); Make, et al., Clin. Exp. Immunol, 51:215-224 (1983), and Schifferli, et al., New Eng. J. Mad., 315:448-495 (1986)]. Immune complexes may remain in circulation for long periods of time and deposit in various tissues contributing to the inflammatory and erosive manifestations of autoimmune and other diseases [See Emancipator, et al., Lab. Invest, 54:475^178 (1986)].
Circulating immune complexes may block or reduce the efficiency of the natural effector mechanisms of the immune system, as has been postulated for malignant transformations [See Feldman, et al., J. Exp. Med., 131:247 (1970); Mingari, et al., J. Immunol, 121:767 (1978); Theofilopoulos, etal.,7. Immunol, 119:657-663 (1977); Theofilopoulos, et al., Immunodiagnostics of Cancer, p. 896, M. Decker, New York (1979), and Levinsky, et al., Lancet, 1:564 (1977)]. The removal of immune complexes from the circulation may reduce many of the clinical problems associated with autoimmune and infectious diseases and cancer. For example, it is beneficial to monitor circulating immune complex levels in blood and to specifically bind and remove circulating immune complexes which may otherwise com