Drug – bio-affecting and body treating compositions – Lymphokine – Interferon
Reexamination Certificate
1999-10-18
2001-03-13
Fitzgerald, David L. (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interferon
C424S085500, C424S085600, C424S143100, C424S145100, C530S388200, C435S007100, C435S007800, C435S007920
Reexamination Certificate
active
06200559
ABSTRACT:
The invention concerns antibodies and a procedure for indirect detection of interferon production by means of antibodies against Mx proteins, especially MxA protein and/or MxB protein, as well as the utilization of interferon for increasing a lowered Mx protein level and the utilization of anti-interferon medications for lowering an elevated MxA and/or MxB level.
Interferons, especially interferon &agr;, &bgr; and &ohgr;, are a class of naturally occurring proteins, which are known to have a favorable effect in viral infections and in the prevention of tumor growth. Furthermore, interferons stimulate the activity of natural killer cells and modulate the activity of macrophages, B- and T-cells.
Interferons are therefore important factors of the immune system, yet, too high levels of interferon have unfavorable side effects, as for example bone marrow toxicity, CNS-toxicity or fever.
A number of proteins are induced by interferons that take part in the antiviral, anti-prolific and immune-modulatory activity that is ascribed to interferons. Two of these interferon-induced proteins are the MxA and the MxB protein (Markus Aebi et al., Mol. Cell. Biol. 1989, 9: 5062-5072).
Because one can make an assesment, by means of measurement of the endogenous interferon production, of the activity of the immune system, and since with various illnesses one has to know whether a too high or too low interferon production is taking place, it remains the task of the present invention to provide a simple procedure for measurement of the interferon production. Further it remains the task of the present invention to provide an option to adjust a too high or a too low measured interferon production to normal levels. Furthermore, it is a task of the invention to provide the corresponding antibodies.
The inventors have found that type I interferon production in vivo can be measured through the utilization of antibodies against Mx proteins, particularly MxA and/or MxB protein. There exists a correlation between the production of interferons in a patient and the expression of interferon-induced proteins MxA and/or MxB, i.e., at a too high interferon production in the body the concentration of MxA and/or MxB is also elevated, and correspondingly with a too low production of interferon the concentration is lowered.
According to the invention, the determination of MxA and/or MxB contents is done immunologically by the utilization of antibodies against these proteins. According to the invention, it is possible to use polyclonal or monoclonal antibodies, correspondingly fragments or conjugates thereof.
For the production of antibodies one first obtains MxA and/or MxB protein. This is done preferably by cultivating murine or human cells, e.g. lymphocytes, macrophages, monocytes or lymphoblastoid cells in a common growth medium (i.e. RPMI 1640 medium), possibly enriched with vitamins and/or hormones. At the end of the exponential growth-phase, the cells are incubated with natural or recombinant interferon, in concentration of 5×10
4
to 10
8
cells/ml and 3,000 to 12,000 international units of interferon/ml. Following, the cells are harvested and lysed according to standard methods. The protein, respectively the proteins are purified, i.e. by means of column chromatography, HPLC or electrophoresis, and are used as antigenes for the generation of antibodies. Also, a recombinant production is possible.
Polyclonal antibodies are obtained by standard methods of injecting the purified MxA and/or MxB protein into mice or rabbits, and isolating the antibodies from the blood serum of the immunized animal, i.e., by affinity chromatography.
Monoclonal antibodies are obtained by means of standard methods. Preferably, an Mx negative mouse is injected with purified Mx1 and/or MxB protein. Antibody-producing cells (i.e. from the spleen) of the immunized animal are extracted and fused with myeloma cells, whereby the fused cells are cloned afterwards. The thereby obtained hybridoma cells are cultivated in vitro (i.e. in Dulbecco's Modified Eagle Medium, or RPMI 1640 Medium), and the monoclonal antibodies are obtained from the culture supernatant. Suitable methods for that are known to specialists, like precipitation with ammonium sulfate, followed by purification of the immune globulins through standard chromatographic procedures.
The inventors have deposited monoclonal antibodies against MxB protein at the DSM, Braunschweig, Mascheroder Weg 1b, D-38124 Germany on Jun. 16, 1997, listed as number DSM-ACC 2309 (internal label 8-271) and as number DSM-ACC 2309 (internal label 7-88). Monoclonal antibodies against MxA+MxB protein were deposited under the number DSM-ACC 2289 (internal label 2-95) and DSM-ACC 2290 (internal label 5-237) on Dec. 6, 1996.
Objects of the invention are also the corresponding antibodies.
Fragments of the Antibodies, i.e. Fab, Fab′ or F(ab′)
2
fragments can be generated if the above purified antibodies are digested with enzymes (i.e. pepsin or papain), or the disulfide bonds are reduced chemically.
Also, conjugates of the antibodies or fragments thereof can be generated, i.e. if the antibodies or fragments thereof are coupled with glutaraldehyd, biotin or avidin.
According to the invention, the term antibody should be understood in a way that it comprises either monoclonal or polyclonal antibodies as well as fragments or conjugates thereof.
The inventors have uncovered the following new contexts:
1) The Mx proteins indicate activity of illness with systemic
Lupus erythemotodes
(SLE) and with mixed collagenosis;
2) The Mx-proteins indicate the progress of the HIV infection and are hereby a prognostic marker for the survival of the patients;
3) Through staining of histological sections Mx antibodies indicate SLE activity in kidney sections;
4) Through staining of histological sections of transplant tissue Mx antibodies indicate rejection:
5) In in vitro cultures of human HIV infected mononuclear cells high Mx levels are measured. In the presence of anti-retroviral active medications the Mx-levels decrease.
All applications have in common, that killer cells, especially T cell-mediated attacks on human cells, stimulate the latter to express Mx proteins.
The inventors have furthermore found that AIDS patients produce phase-dependent increasing interferon. These patients produce partially more interferon than patients who are on interferon-&agr; medication. Patients on therapeutic interferon-&agr; medication were often suffering of side effects like bone marrow toxicity, CNS toxicity or fever. AIDS-patients who produce too much interferon also have exactly these symptoms.
Similarly is the situation with patients who suffer an autoimmune disease or who are receiving a steroid therapy. Already a short-time administration of steroids, i.e. of cortinsones (i.e. prednisolon), is sufficient to decrease the Mx content below a certain level.
The inventors now found that the increase or the decrease of the endogenous interferon production (interferon type I) can easily be determined by detecting elevated or lowered MxA and/or MxB protein levels in cell-containing body fluids (i.e. blood, urine, cerebrospinal fluid ). This is accomplished by employing suitable immunological detection systems, i.e. ELISA or RIA, and using antibodies or fragments or conjugates thereof against MxA and/or MxB protein, to determine the content of MxA and/or MxB protein. From an elevated or lowered content of MxA protein and/or MxB protein in the examined body fluid of the patient one can infer an elevated or a lowered interferon production of the patient, respectively. It was found that an Mx concentration of 0.5 to 2.0 mU/1000 leukocytes can be regarded as normal and reflects a normal production of interferon in the patient.
If, according to the invention, elevated MxA or MxB concentrations are detected, anti-interferon medications (i.e. steroids, anti-interferon antibodies) are administered in a suitable amount, so that the above mentioned normal concentrations of MxA and/or MxB are reached. Such a treatment has
Fitzgerald David L.
Locke Reynolds LLP
LandOfFree
Use of antibodies against MxA or MxB to determine levels of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of antibodies against MxA or MxB to determine levels of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of antibodies against MxA or MxB to determine levels of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2509608