WO2005025650A1

WO2005025650A1 - Apheresis device

Info

Publication number: WO2005025650A1
Application number: PCT/AT2004/000312
Authority: WO
Inventors: Frank Mattner; Walter Schmidt
Original assignee: Frank Mattner; Walter Schmidt
Priority date: 2003-09-12
Filing date: 2004-09-13
Publication date: 2005-03-24

Abstract

The invention relates to an apheresis device for use in the treatment of lupus patients. Said device comprises a solid support with which a flow of blood or plasma can be contacted, said support comprising a specific receptor that inhibits DNA autoantibodies.

Description

Apheresis device

The invention relates to an apheresis device comprising a solid support that can be contacted with the blood or plasma flow.

Apheresis is understood to mean treatment methods whose therapeutic effects are based on the extracorporeal elimination of pathogenic proteins, protein-bound pathogenic substances, free pathogenic substances or pathogenic cells of the blood. If the pathogenic protein can only be eliminated from the cell-free plasma, the plasma is separated from the blood cells beforehand using a membrane plasma separator (plasma separation) or using a hemocentrifuge. In the case of unselective plasma exchange (plasmapheresis), the patient plasma exchanged is separated as a whole, with all other vital proteins being eliminated in addition to the pathogens. It is therefore necessary to substitute the removed plasma with electrolytes, human albumin or fresh plasma. In selective plasmasheresis methods, pathogenic proteins can be removed from the separated plasma very specifically with the aid of adsorption, precipitation or filtration, the plasma being able to be reinfused after the removal without significant loss of volume. These selective processes have the advantage that a substitution solution can be dispensed with. In selective whole blood apheresis methods, the pathogenic proteins are specifically adsorbed directly from the non-pretreated blood without prior plasma separation, which means that - in contrast to the plasma separation methods - both the plasma separation and the addition of a substitution solution can be omitted. Another sub-form of apheresis is zytapheresis, in which cells are removed from the blood. Leukocytes, erythrocytes, thrombocytes, granulocytes or even stem cells can be obtained selectively.

Although apheresis (eg as plasmapheresis or zytapheresis) is currently mainly used to obtain donor plasma (as a plasma preserve, to isolate various plasma fractions or to obtain blood products), apheresis methods are becoming increasingly popular in the therapeutic field Importance. A whole range of metabolic diseases (eg (familial) hypercholesterolemia, progressive coronary heart disease with isolated Lp (a) elevation, chylomicronemia syndrome, liver failure, ...), kidney diseases (Goodpasture syndrome, systemic lupus erythematosus with Lu - pusnephritis, Wegner's granulomatosis, haemolytic-uraemic syndrome, idiopathic focal-sclerosing glomerulonephritis, paraprotein-associated syndromes, cryoglobulinemic purpura, HLA sensitization in kidney transplantation, ...), diseases of the nervous system (myasthenia barri syndrome, guillainia syndrome, Guillasthenia gravis , Chronic demyelinating polyradiculoneuritis, para-protein polyneuropathy, Lambert-Eaton syndrome, Refsum syndrome, ...), diseases of the immune system (rheumatoid arthritis, inhibitor hemophilia, pemphigus, ...), diseases of the blood circulation and microcirculation ( Hyperviscosity syndrome, antiphospholipid antibody syndrome, Thrombotic microangiopathy after bone marrow transplantation, age-related macular degeneration, sudden hearing loss, peripheral disorders of the microcirculation, idiopathic dilated cardioopathy, transplant vasculopathy after cardiac transplantation, homozygous familial hypercholesterolemia, focal segmental glomeruloskeleton, hematolytic disorders, hemolytic insufficiency, hemolytic insufficiency , Neoplasms, hyperhydration, thyrotoxicosis, etc., treated with apheresis procedures (see Pschyrembel (257. Edition) Keyword "plasmapheresis"; www.nephrologie.de/172Apha- rese.htm).

Lupus is a chronic inflammatory autoimmune disease with complex clinical manifestations. The lupus in humans known as "systemic lupus erythematosus" (SLE) affects between 40 and 250 out of 100,000 people, mostly women. Lupus, as the most important immunological abnormalities, includes the presence of pathogenic autoantibodies, the lack of B and T lymphocyte regulation and disturbed removal of autoantigens and immune complexes. Almost all autoantibodies in lupus cases are directed against intracellular nucleoprotein particles. Antibodies against (double-stranded) DNA (dsDNA) are found in 50 to 80% of SLE patients, these antibodies only being linked to SLE The etiology is largely unknown, although genetic, hormonal and environmental factors Vulnerability can affect, as with all autoimmune diseases. The course of the disease is very variable and can range from the (rare) acute fatal to chronic decades.

All lupus patients have joint and / or skin disorders. Furthermore, kidneys, heart, lungs and the central nervous system (CNS) are affected in 30 to 70% of the cases, whereby the prognosis is always poor when several organs are affected. Around 90% of patients experience arthralgia, but are less inflammatory than in rheumatoid arthritis. Lymphocytopenia is also common in SLE patients.

Lupus patients have a high level of autoantibodies. Anti-DNA antibodies bind to nucleosomes, laminin, collagen type IV and heparan sulfate, whereby these antibodies can induce nephritis. The anti-DNA antibodies probably lead directly to tissue damage. The most important immunological abnormalities also include abnormalities in T lymphocyte functions, complement deficiencies and disorders in the cytokine network (in particular production of and response to IL-2).

Today lupus is mostly treated with glucocorticoids, non-steroidal anti-inflammatory drugs and immunosuppressants. Plasmapheresis is also used to treat lupus (see above), but so far all immunoglobulins have always been removed non-specifically (for example by immunoadsorption on protein A, peptide GAM, tryptophan or on dextran sulfate (JP 54/090896 A, Kutsuki et al., Therapeutic Apheresis 2 (1) (1998), 18-24; Zemlin et al., Journal of Obstetrics and Neomatology 206 (2002), 22-25; Suzuki et al., J.Clin.Aph. II (1996), 16 -22; Autoim unity 19 (1994), 105-112; Arter. & Rheum. 34 (1991), 1546-1552)), but not only immunoglobulins of a certain specificity (see, for example, US 6,030,614, 5,817,528 and 6,551,266). A method for the selective adsorption of anti-DNA antibodies by therapeutic plasmapheresis over a column with monoclonal anti-idio-typical antibodies is described by Harata et al. (J. Clin. Apheresis 6 (1991), 34-39). Antibodies used in apheresis procedures, however, have several disadvantages: they can due to their nature only be applied gently to the apheresis column and they can easily diffuse from the column. In addition, because of their size, antibodies can only be provided on the column in low densities (binding site per unit area) and they are complex and expensive, especially industrial production (GMP guidelines). For this reason, the one described in Harata et al. The approach described has not yet been implemented in product development.

To date, specific biological therapies have focused on the use of recombinant cytokines (e.g. TGF-ßl), blocking antibodies and soluble receptors. Gene therapy approaches (e.g. with cytokine inhibitors) have already been proposed (Mageed et al., Gene Therapy 10 (2003), 861-874).

The object of the present invention was therefore to provide a new treatment and prevention strategy for lupus.

Accordingly, the present invention provides an apheresis device comprising a solid support which can be contacted with the blood or plasma flow and which has specific low-molecular receptors for a DNA-binding autoantibody. With the present apheresis device, apheresis can be used to clear DNA autoantibodies in a targeted manner in lupus patients or people at risk of lupus. It is not critical here whether the low molecular weight receptors in the apheresis device, which are contacted with the patient's blood or plasma, are specific for a specific antibody, it is only important that with this specific adsorption the harmful autoantibodies that target the cell - and cause tissue damage in lupus, from which blood is eliminated, so that they cannot have their fatal effect. The present invention is therefore based on a completely different application approach for apheresis than the lupus treatment that is proposed in US Pat. No. 6,030,614, 5,817,528 and 6,551,266, namely on the elimination of the specific DNA autoantibodies and not on the (unspecific) elimination of all antibodies , which also removes the autoantibodies, but also the other immunoglobulins that are otherwise required, which of course leads to decreased immune defense in the patient.

According to the invention, “low molecular weight” means those affinity molecules for DNA-binding autoantibodies which have a molecular weight of less than 10 kDa, preferably less than 5 kDa, in particular less than 2 kDa. Preferred receptors according to the invention therefore have a molecular weight of 0.1 to 10 kDa , preferably between 0.2 and 8 kDa, in particular between 0.3 and 7 kDa. The affinity molecules are preferably biocompatible, in particular peptides or short nucleic acids. The low molecular weight receptor according to the present invention can either be directly or indirectly (for example via a suitable linker Suitable linkers include organic at least bifunctional compounds as well as peptide or carbohydrate linkers, etc. (the linkers are of course not to be regarded as part of the low-molecular CETP affinity molecule according to the invention (with a correspondingly low molecular weight).

In a preferred embodiment, the affinity or receptor molecule according to the invention is a DNA autoantibody affinity peptide. This DNA autoantibody affinity peptide according to the present invention is an oligo or polypeptide which, immobilized on an apheresis column, is able to bind DNA-binding autoantibodies from the plasma stream as part of an apheresis treatment. Such affinity peptides can be easily produced, for example by chemical synthesis. In contrast to antibodies, the peptides according to the present invention are, on account of their size alone, much more stable and resistant to denaturing effects (such as can occur specifically when binding to the solid phase and influence affinity). These stability advantages can be further enhanced by also providing one or more non-natural amino acids (ie other than the 20 "classic" amino acids) in the peptide, or by providing one or more D-amino acids Antibody fragments - many times smaller size of the peptides, according to the invention, much higher densities (binding sites per unit area) can be provided on the apheresis column: in comparison with anti bodies enables the use of a CETP affinity peptide according to the invention (with a length of about 20 amino acids) a packing of binding activities on the column which is 200 times larger. However, higher densities enable the column to be more efficient. As a result, higher flow rates and thus shorter apheresis treatment times can be achieved, an important acceptance factor with this type of therapy, whereby the advantages compared to that of Harata et al. proposed variant with anti-idiotypic antibodies clearly stand out.

For practical reasons, the affinity peptide used according to the invention has a length of 1 to 50 amino acid residues. With longer peptides, the production becomes increasingly complex and complex. The low-molecular DNA autoantibody affinity peptide preferably has a length of 10 to 40, in particular 15 to 20, amino acid residues. This size is easy to handle chemically and synthetically and can also be used in large-scale production, even when non-natural amino acids are used.

According to the invention, the existing and known apheresis devices in all embodiments can be easily adapted to the present invention. In particular, when choosing the solid support (and the apheresis device), consideration should be given to its medical technology suitability. Such carriers, methods or devices are described, inter alia, in US Pat. Nos. 5,476,715, 6,036,614, 5,817,528 or 6,551,266. Corresponding commercial apheresis devices are also marketed by companies such as Fresenius, Affina, Plasmaselect, ASAHI, Kaneka, Braun, etc., such as the LDL-Therasorb ^R , the Immunosorba ^R , the Prosorba ^R , the Globaffin ¹ *, the Ig-Therasorb ^R , the Immusorba ^R , the Liposorba ^R , the HELP ^R , the DALI ^R , the bilirubin-bile acid absorber BR-350, the Prometheus ^R detoxification, the MARS ^R , the ADAsorb system from Medicap or the plasma FLO -System. All of these systems - although in the commercial form are not always primarily aimed at the specific elimination of a single protein - can be readily adapted to the present invention by an expert in apheresis, for example as immunapheresis and / or with the incorporation of the solid carrier according to the invention (for example as a column) in the apheresis device.

According to the invention, “DNA autoantibodies” are understood to mean all DNA-associated autoantibodies occurring in lupus patients, that is to say antibodies which are specific for the nucleosome, dsDNA (dsDNA antibody) and for dsDNA: protein complexes (ie complexes of dsDNA with nucleic acid associated proteins), both complexes of a structural nature (for example histones or dsDNA with histones) and those of a functional nature (for example with repressors or transcription factors), for example DNA: histone complexes, nucleoprotein particles, nucleosomes, laminin , Collagen type IV and heparan sulfate; complexes of nucleic acids with nucleic acid-associated proteins or peptides, such as polymerases, telomerases and the aforementioned proteins, insofar as they occur in lupus patients. All of these antibodies are, according to the invention, as anti-DNA autoantibodies or lupus View autoantibodies (in contrast to non-specific materials such as protein A, peptide GA M, tryptophan, dextran sulfate, etc.). The low molecular weight receptors for these DNA autoantibodies which can be used according to the invention must be able to specifically remove these autoantibodies from the blood or plasma of lupus patients or persons at risk of lupus. The present invention accordingly also relates to all apheresis devices which contain a carrier with any low-molecular receptor specific for these lupus antibodies. The only important thing is that the carriers with the low molecular weight receptors are able to specifically bind the lupus antibodies from the blood or plasma flow in the aphe- rese device and thus selectively separate them (from the other immunoglobulins). These DNA antibody receptors can preferably be peptides or nucleic acids.

Particularly preferred receptors are dsDNA molecules, dsDNA mimicks, e.g. dsPNA molecules, peptide mimicks for (ds) DNA, or mixtures of these substances, and molecules of the same binding specificity.

Peptides as DNA autoantibody-binding low-molecular receptors can consist of D- or L-amino acids or combinations be composed of D- and L-amino acids and, if necessary, have been changed by further modifications, ring closings or derivatizations. suitable

Peptide receptors for the DNA antibodies can be provided from peptide libraries that are commercially available. These peptides are preferably at least 5, preferably 6, amino acids long, in particular at least 8 amino acids, preferred lengths being up to 11, preferably up to 14 or 20, amino acids. According to the invention, however, longer peptides can also be readily used as receptors which bind DNA autoantibodies, but, as mentioned above, the advantages of the present invention described for molecules which are larger than 10kDA are no longer so clear and therefore are not preferred. Oligomers (such as polyethyleneimine and polylysine) are also suitable as receptors.

For the production of such DNA autoantibody-binding low molecular weight receptors, phage libraries, peptide libraries, e.g. generated by combinatorial chemistry or by high throughput screening techniques for various structures, suitable (see e.g. in Phage Display: A Laboratory Manual by Carlos F. Barbas (Editor), et al; Willats WG, Phage display: practicalities and prospects. Plant Mol Biol. 2002 Dec; 50 (6): 837-54; http: //www.microcollections .de / showpublications .php #). In addition to phage libraries based on randomization, libraries that use the ribosomal display or the bacterial display are also suitable. Corresponding libraries and methods for their generation are known to the person skilled in the art and, inter alia, in US 2004/0110281 A, WO 00/72880 and WO 02/059148 A.

Examples of such peptides, which represent mimicks for antigenic and immunogenic epitopes of dsDNA i-lupus, are described in Sun et al. (Int. Immun. 13 (2) (2001), 223-232) (“positive clones” in Table 1, in particular RLTSSLRYNP; found by “rando peptide libraries”), in US Pat. No. 6,001,964 (in particular the sequence XGWXRV) , Gaynor et al. (PNAS 94 (1997), 1955-1960; in particular DWEYSVWLSN as consensus sequence), etc. Furthermore, according to the invention, in addition to dsDNA (plasmids, circular dsDNA, linear dsDNA, dsRNA, cDNAs, PCR fragments), complementary oligonucleotides, “decoy” -01igodeoxynuleotides (ds oligonucleotides, which in terms of sequence represent binding sites for transcription factors), etc., can also be used low-molecular-weight DNA autoantibody-binding receptors based on nucleic acids, even as "aptamers", are used, and these can also be found using a wide variety of (oligonucleotide) libraries (for example with 2-180 nucleic acid residues) (for example Burgstaller et al., Curr. Opin. Drug Discov. Dev. 5 (5) (2002), 690-700; Famulok et al., Acc. Chem. Res. 33 (2000), 591-599; Mayer et al., PNAS 98 (2001 ), 4961-4965, etc.). Plasmid DNAs to be used according to the invention can be found, for example, in corresponding databases (http: //www.belspo.be/bccm/db/plasmid search. Asp) from commercial providers (http: // www.strataqene. Com /) or in the scientific literature (aniatis T, et al. Molecular Cloning A Laboratory Manual; http: //www.ncbi. nlm. nih. qov / entrez / query. fcqi? db = pubmed & cmd = search & term = plasmid). Biological organisms from the group of prokaryotes and eukaryotes as well as viruses and bacteriophages are suitable sources of genomic DNA.

In the case of receptors based on nucleic acids, the nucleic acid backbone can be provided, for example, by the natural phosphoric diester compounds, but also by phosphorothioates or combinations or chemical variations (for example as PNA), the bases being U, T, A, C according to the invention , G, H and mC can be used. The 2 'residues of the nucleotides which can be used according to the present invention are preferably H, OH, F, Cl, NH ₂ , O-methyl, O-ethyl, O-propyl or O-butyl, the nucleic acids also can be modified, for example provided with protective groups, as are usually used in oligonucleotide synthesis. “Protecting group” is understood to mean etherification of the oxygen atom, whereas in the 2 modification the —OH group is replaced by something else. For the two variants, there are very diverse possibilities in the prior art, particularly preferred protecting groups being methyl and allyl -, Propyl, and the like (e.g. 2 ^Λ -0CH ₃ , 2 -0-CH = CH ₂ , etc.); particularly preferred modifications are 2 -deoxy, 2 ^Λ -amino, 2 -Fluoro, 2 ^λ -Bromo, 2 -azido, but also metals, such as selenium, etc. In addition, according to the invention, the oligonucleotide stabilization techniques used for the antisense technology (ribozymes, RNAi, etc., for example with Phosphorothioates) were developed to provide the nucleic acids (eg leading companies ISIS and Ribozyme Pharmaceuticals, especially their patent documents and homepage).

The aptamers for the DNA autoantibodies can be found, for example, using the method described below. Immobilized DNA autoantibodies are contacted with a mixture of nucleic acids, high-affinity binding nucleic acids being separated from the less affine or non-binding nucleic acids. The mixtures with nucleic acids are typically nucleic acid libraries which have been produced, for example, by combinatorial chemistry. A nucleic acid library contains a multiplicity of nucleic acids that differ from one another, with randomization (with natural and / or non-natural nucleotides) being set up at least in a partial sequence region. A conserved sequence area can, but need not, be provided. Randomization in n positions with m different nucleotides leads to a library with n ^m elements.

The following steps are carried out to find the DNA autoantibody-specific affinity nucleic acids. a) a column is loaded (inside) with DNA autoantibodies and DNA autoantibodies are immobilized in the column, b) the mixture of nucleic acids is applied to a first end of the column, with a defined volume flow of carrier substance running through the column, running from the first End to the second end of the column, c) the nucleic acids are bound and immobilized with decreasing affinity of the nucleic acids for DNA autoantibodies at an increasing distance from the first end of the column, d) the volume flow of carrier substance through the column is after a defined runtime terminated, e) the column is separated by a plurality of divisions into column segments, each segment being assigned a path coordinate, f) the immobilized nucleic acids are unspecifically desorbed from at least one segment and the segment is assigned assigned route coordinate. The expression inside the column means in general within a lumen. The printout of the column should include all types of solid support systems, for example, support systems that are not completely enclosed are also possible.

The immobilization of DNA autoantibodies can be carried out according to the usual methods of column chromatography. Any mechanical construct that has a lumen with two ends is called a column. All materials customary for columns, such as metals, glass and / or plastics, are suitable as the structural material. The inside of the column can be provided with a matrix that binds DNA autoantibodies and / or the structural material can be suitable or prepared for the direct binding of the target molecules. A mixture of nucleic acids denotes nucleic acid libraries with a number of typically 10 ⁶ to 10 ²² / mol, in particular 10 ¹⁰ to 10 ²¹ / mol, of different nucleic acid species. In the library applied to the column, each nucleic acid species is represented statistically, for example with 10 to 10 ¹⁷ , in particular 100 to 10 ¹³ , molecules. The carrier substance is usually a liquid in which the nucleic acid library is soluble and stable. All buffers and the like customary for nucleic acid libraries can be used for this. The volume flow of carrier substance can be adjusted before the nucleic acid library is applied. The nucleic acid library is then added to the carrier substance stream on the column inlet side. The nucleic acid library can also be given up immediately. After a running time, which is determined by the structure of the column and the volume flow set, the "grafting", which was given by the nucleic acid library, emerges again on the column exit side (widened by folding with diffusion), bound nucleic acids from the "grafting" separated and immobilized in the column. The volume flow through the column is expediently set to be low or non-turbulent, preferably laminar (sum of the acceleration vectors of the carrier substance over the column volume, in particular over the column cross section, minimally, ideally 0). The total number of DNA autoantibody molecules in the column is typically 10 ² to 10 ¹⁶ times, in particular 10 ³ to 10 ¹⁵ times, the amount Number of nucleic acid molecules of a single species in the applied nucleic acid library. The binding of the nucleic acids to the DNA autoantibody molecules is preferably carried out under conditions which correspond to a later use of the nucleic acids in apheresis, for example in a suitable buffer which is appropriately matched with regard to temperature, ionic strength, pH and buffer conditions. The carrier substance and the solvent of the nucleic acid library are then to be selected accordingly with regard to their components. The column can be divided into a plurality of column segments, for example by cutting the column, the cuts preferably being carried out orthogonally to the volume flow vector. However, the column may also have previously been composed of column segments, with one column segment preferably being closely aligned with the next column segment in the direction of the volume flow vector (joining cross section orthogonal to the volume flow vector). Then the division can take place by dissolving the previously formed composite of column segments. The non-specific desorption can take place by elution with a sufficiently strong ligand by displacement, complexation, modification and / or destruction of DNA autoantibodies, physico-chemically or thermally. Mechanical methods, for example ultrasound, can also be used for desorption or for increasing desorption. Combinations of the above desorption methods can also be used. It goes without saying that the nucleic acids must not be decomposed by the desorption process used.

The steps described above are based on the knowledge that a nucleic acid library can be separated spatially according to the affinity for the target molecule, in the case described here, for DNA autoantibodies by means of affinity chromatography analogously to a protein mixture. The invention is further based on the knowledge that a blending of desorbing nucleic acids of different affinity which occurs by means of non-specific desorption can be avoided by dividing the column with the nucleic acids bound therein, as it were, into affinity sections and that in the affinity sections or column segments thus obtained bound nucleic acids easily and without disruptive ligand Desorb couplings, for example in the context of a PCR or RT-PCR, and have them amplified non-specifically as well. Subsequent selection artifacts are avoided. Ligands, especially high concentrations of ligands, are also not required for desorption. Finally, practically all bound and then desorbed nucleic acid molecules are available for amplification. This makes it possible to work with low nucleic acid concentrations. In principle, it is sufficient if every species is represented on average in the nucleic acid library with one molecule. If the number of DNA autoantibody molecules in a column segment is statistically 1, then even individual nucleic acid species can be separated according to their affinity for the target molecule.

Basically, it is sufficient if a segment to which a desired affinity is assigned is (isolated) processed for desorption. However, this requires - except for maximum affinity, where the first column segment, based on the volume flow vector, is processed further - an idea of the affinity distribution in the applied nucleic acid library. It is therefore preferred, as a rule, that the immobilized nucleic acids are desorbed and obtained separately from each segment with the respective assignment of the travel coordinates of each segment to the nucleic acids obtained therefrom.

In principle, any type of desorption is possible. The non-specific desorption is preferably carried out by means of conventional physico-chemical or thermal processes. Thermal desorption takes place by heating the column segment or the solution contained therein. The heating can be carried out, for example, by electrical heating or irradiation with microwaves or IR. In particular, the heating techniques from the PCR technology are suitable. In addition to the amplification of nucleic acids or aptamers using polymerase, other amplification methods, such as using ligase, can of course also be used. The non-specific desorption can be supported by chemical modification of the DNA autoantibodies, eg oxidation by sodium perjo- dat or the like, or by unspecific complex formation, for example by means of borate or the like for blocking cis-trans-diol bonds in carbohydrates.

It is therefore preferred if the non-specific desorption is carried out by thermal desorption in a, preferably extended, high-temperature phase of a PCR or RT-PCR. A synergy effect is achieved here, since amplification is generally required anyway, especially when working with nucleic acid libraries with low concentrations of the nucleic acid species. To increase the yield, for example, 5 to 60, preferably 20 to 60, most preferably 45 to 55 cycles are used. Within the scope of the amplification, at least one labeled primer can be used. The primer can have at least one endonuclease interface. Such an interface serves, for example, to free the amplificate from larger areas of the primer sequence. Nucleotide building blocks, whether in the primer or in the nucleic acids to be selected, can be labeled, for example, using fluorescent dyes. Examples of fluorescent dyes are: Alexa ™ Fluor 488, Fluor 532, Fluor 546, Fluor 568, Fluor 594, Oregon Green 488, Fluorescein, Rhodamine 6G, Tetramethylrhodamine, Rhodamine B and Texas Red. The amplificate can also be obtained through two different chemical modifications Different ends can be marked, provided that the groups introduced during the modification are suitable for being able to be bound as ligands to a different affinity matrix.

For safe separation of non-affine or low-affine nucleic acids, it is advisable to insert washing process steps between suitable process steps. In particular, it is preferred if at least one washing process step is carried out between process steps d) and e). The solvent or the medium of the nucleic acid library or the carrier substance, for example, is suitable for washing.

It is preferred if the inside of the column is coated with DNA autoantibodies and its immobilization by means of covalent binding, preferably after activation with chemically highly reactive groups (for example tresyl chloride, cyanogen bromide and / or periodate) or via bifunctional spacer compounds after modification with chemically less reactive groups (for example amine, hydroxy, keto and / or carboxyl). Examples of spacer structures of suitable spacer compounds are: substituted and unsubstituted C2-C10 alkyl groups, substituted and unsubstituted C2-C10 alkenyl groups, substituted and unsubstituted C2-C10 alkynyl groups, substituted and unsubstituted C4-C7 carbocylo alkyl groups, substituted and unsubstituted C4-C7 carbocylo, carbocylo and unsubstituted C7-C14 aralkyl groups, a heterocyclic molecule with heteroatoms selected from nitrogen, oxygen, sulfur, where said substitutions can consist of alkyl, alkenyl, alkynyl, alkoxy, thiol, thioalkoxy, hydroxyl, aryl, benzyl, phenyl, Nitro, halogen, ether groups with 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, polyalkyl glycol, halogen, hydroxyl, thiol, keto, carboxyl, amides, ether compounds, thioethers, arnidine derivatives, guanidine derivatives, glutamyl derivatives, nitrate (0N0 ₂ ), Nitro (N0 ₂ ), nitrile, trifluoromethyl (-CF3), trifluorome - thoxy (-OCF3), O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N ₃ ), hydrazino (NHNH ₂ ), Hy - Droxylamino (ONH ₂ ), sulfoxides (SO), sulfones (S0 ₂ ), sulfides (S-), disulfides (SS), silyl can exist. Spacer compounds are typically bifunctional, the functionalities being the same or different and being selected, for example, from the group consisting of "N-hydroxysuccinimide and hydrazides".

To reduce the variety within the nucleic acids obtainable from a column segment, it is preferred if each column segment contains 0.1 to 10 ³ , preferably 1 to 10 ² , most preferably 1 to 10, DNA autoantibody molecules on average. Matched to this, the nucleic acid library, as applied, can contain on average 0.1 to 10 ³ , preferably 1 to 10 ² , most preferably 1 to 10, nucleic acid molecules of a species.

For the structural material of the column, basically all the work known, for example, from affinity chromatography substances in question. These include columns made of silica gel or polymers, such as polyethylene after activation by chemical derivatization or plasma activation. The length of the column segments is expediently in the range from 0.1 μm to 1 mm, preferably 0.1 to 100 μm, most preferably 0.5 to 10 μm. Such cuts can easily be made using a microtome, for example. The inside diameter of the column is expediently in the range from 0.05 to 1 mm, preferably from 0.1 to 0.5 mm, most preferably from 0.2 to 0.4 mm.

It is expedient if, before the actual separation (binding or mechanical separation) of the nucleic acids, undesired nucleic acids are eliminated. Undesirable nucleic acids are, for example, nucleic acids which bind to inner surfaces of the column that are free of DNA autoantibodies. A DNA autoantibody-free column can then be connected upstream and the nucleic acid library can be guided through it beforehand.

In the above procedures, i. e. for example by cascading columns, or discontinuously, for example by temporarily collecting the eluent from the precolumn.

In principle, there are various ways of achieving a further improvement in the affinity separation. For example, the nucleic acids desorbed from one or more segments can be subjected repeatedly to the method according to the invention, if appropriate after amplification, and / or the elutropy of the conditions during the desorption can be increased (temperature, ionic strength, pH, buffer conditions). Alternatively, the spatial density of DNA autoantibodies and consequently the bound nucleic acids, i. e. the number of DNA autoantibody molecules in a column segment can be reduced.

DNA autoantibody binding aptamers are therefore also preferred DNA autoantibody binding receptors in the context of the present invention.

According to the invention, the DNA autoantibody-binding low-molecular receptors, which are preferably composed of peptides, antibodies or nucleic acids are used on a suitable carrier material for the extra-corporal elimination of DNA autoantibodies in lupus (risk) patients.

When the present invention is used in routine medical practice, it is necessary for the carrier in the apharea apparatus to be sterile and pyrogen-free, so that any carrier substance or each receptor / carrier combination which fulfills these features is preferred according to the invention (see e.g. US 6 030 614 or US 5 476 715). Suitable examples include porous homopolymers, copolymers or terpolymers of vinyl-containing monomers (for example acrylic acid, such as TSK Toyopearl, Fractogel TSK), supports with modifications (activations) with oxirane-containing compounds (for example epichlorohydrin) and, if appropriate, further reactions with Compounds containing NH ₃ , amino or carboxyl, or CNBr or CNCl adsorbents, as described in EP 110 409 A and DE 36 17 672 A. Particularly preferred adsorption materials for therapeutic purposes are suitable for avoiding a loss of blood cells, do not activate the complement system or only activate it slightly and keep aggregate formation in the extra-corporeal circulation as far as possible. Furthermore, the carrier materials used should preferably also be sufficiently stable in the form of a receptor coupled to sterilization measures, in particular to ethylene oxide saturation, glutaraldehyde saturation, gamma radiation, steam treatment, UV treatment, solvent treatment and / or detergent treatment, etc. For example, also Products based on Sepharose, Agarose, Acrylic, Vinyl, Dextran etc. are used, which preferably have suitable functional groups for binding the DNA autoantibody-binding receptors already commercially available. Other suitable supports also include monoliths (supports based on cross-linked glycidyl methacrylate-co-ethylene glycol dimethacrylate polymer) and Subpol (Poschalko et al., J Am Chem Soc. 2003 Nov 5; 125 (44): 13415-26.).

The chemistry known to the person skilled in the art can be used to couple the receptors to the suitable supports (for example Bioconjugate Technologies, Greg T Herson, Ed., Academic Press, Ine, San Diego, CA, 1995, 785pp). According to a further aspect, the present invention relates to the use of the device according to the invention for providing a treatment or treatment device for lupus diseases or for preventing such a disease, in that the device is suitably prepared for the treatment of the respective patient. In performing the treatment, a patient is attached to the apheresis apparatus for a period of time sufficient to effectively eliminate DNA autoantibodies, contacting the patient's blood or plasma flow with the solid support comprising the DNA autoantibody binding receptor, followed by the DNA autoantibodies are bound. In the course of apheresis treatment, peripheral or central venous vein access or arterial venous fistula must of course be ensured, as well as sufficient anticoagulation and the necessary quantification and measurement data recorded. Furthermore, most apheresis procedures will require a primary separation of plasma and blood cells before the actual plasma treatment. Special persons who require preventive measures are family members, persons at risk from special exogenous / endogenous factors (sunlight, hormones, gender, diagnosis (e.g. detection of DNA autoantibodies in the blood), ...) or persons with a different risk factor for lupus, especially due to genetic factors.

The lupus diseases which can be treated according to the invention include all types which are associated with the presence of DNA autoantibodies within the meaning of the present invention. This includes, in particular, lupus erythematosus, preferably chronic discoid lupus erythematosus (antinuclear antibodies, especially against histones), pseudo-lupus erythematosus (antimitochondrial antibodies), chillblain lupus, anticardiolipin syndrome (cardiolipin antibodies), drug-induced lupus erythematosus, neonatal lupus erythematosus (diaplacental transmission of maternal antibodies), lupus erythematosus profundus, subacute cutaneous lupus erythematosus (antinuclear antibodies), systemic lupus erythematosus (antinuclear antibodies, dsDNA antibodies), especially systemic lupus erythematosus. In the specific treatment of A specific set of DNA antibody receptors can also be used for each lupus sub-form and a disease- or even patient-specific carrier can be used, depending on the type and titer of the specific DNA autoantibodies in the patient.

The invention is illustrated by the following examples, to which it is of course not restricted.

1. Preparation of the DNA Autoantibody Receptor Carrier

1.1 Monolithic column

A ^CIM® epoxy monolithic column (BIA Separations, SI) is equilibrated according to the manufacturer's instructions with 0.5 M Na phosphate buffer at pH 8.0 and a plasmid DNA is likewise activated according to the manufacturer's instructions and coupled to the CIM column , The column is washed several times with phosphate buffer (+ 1 M NaCl) and excess epoxy groups are optionally blocked.

Quality assurance is carried out by means of a control in the washing and equilibration eluate; only columns without active epoxy groups and without DNA leakage in the eluate are used further and installed in an apharesis apparatus.

1.2 Sepharose column

An agarose bulk material (Sepharose CL4B) is aseptically filled into a sterile and pyrogen-free container and the material is washed aseptically, the gel material being dried completely under vacuum between each washing step. The Sepharose is then steam sterilized in an autoclave for 30 minutes at 115 ° C.

After sterilization, the Sepharose is taken up in a sterile container in 60% acetone / water and activated with CNBr and triethylamine (14 g CNBr per 96 ml acton; 30 ml triethylamine in 66.2 ml 87% acetone). Then an acetone / HCl solution was added (392 ml sterile, pyrogen-free water; 16.3 ml 5N HCl, 408 ml acetone). The activated Sepharose will washed and fed to the coupling reaction within 2 hours to prevent the hydrolysis of activated groups.

A sterile-filtered, couplable plasmid DNA solution is introduced into the reaction vessel and stirred for at least 90 min. Finally, the reaction solution is washed thoroughly

(with isotonic phosphate buffer) until no reaction products are detectable in the eluate, and the plasmid-coupled Sepharose is filled into sterile and depyrogenized glass columns with glass sinters and subjected to a final quality control (eluate analysis for reaction products, heavy metals etc.; particle analysis, pyrogenicity; sterility) ,

2. Animal model for apheresis treatment of SLE patients

At the Institute for Diabetes "Gerhardt Katsch" a miniaturized extracorporeal system for the application of apheresis therapy to the rat small animal model has been developed. Such an apheresis test system is only available to a very limited extent worldwide. The repeated apheresis treatment on the same test animal and subsequent examinations in the follow-up phase to assess the therapeutic success have a high novelty value and have not previously been described in international literature.

Apheresis is an alternative therapeutic method in which blood outside the body is taken from substances that cause disease. So far, apheresis has been used in human medicine to treat more than 100 diseases. With the help of apheresis, disease-relevant substances in autoimmune diseases such as rheumatoid arthritis, myasthenia gravis, endocrine ophthalmopathy, multiple sclerosis, systemic lupus erythematosus, Stiff-Man syndrome and type 1 diabetes were completely or at least partially removed from the blood. So far, however, apheresis has only been recognized as an alternative method for the treatment of therapy-resistant chronic diseases, which are associated with severe stress and a significant reduction in the quality of life of the affected patients. A key reason for this is that no detailed knowledge of the mechanisms of action successful apheresis therapy.

By using recognized animal models, such as For type 1 diabetes and rheumatoid arthritis, both the mechanisms of action of the apheresis procedures can be further elucidated and novel indications for apheresis treatment can be tested preclinically. To carry out apheresis treatments, the test animals are first provided with chronic vascular catheters. In the extracorporeal circuit, the plasma and cellular components of the blood are then separated using plasma filters. While the cellular components return immediately to the animal, the plasma can be cleaned by various adsorption processes (immunoadsorption or the like) before being returned. The good tolerability of such repeated apheresis treatments has already been demonstrated for various rat strains (body mass, hematocrit, general condition). The apheresis test system has been tested in animals with a minimum weight of 250 g and has been used on rat models for autoimmune diseases (type 1 diabetes, collagen type II-induced arthritis).

The extracorporeal system for plasmapheresis on a small animal model can be used for various types of adsorption. The application of this test system to models for chronic diseases enables (A) to test new treatment and prevention strategies, (B) to decode the mechanisms of action of various apheresis technologies and (C) to determine the indication for individual apheresis therapy methods. The IDK is your competent partner for the preclinical testing of newly developed apheresis procedures and can make a significant contribution to the transfer and market launch of new therapeutic procedures from the development phase to animal experiments and clinical use in patients (http: //www.praeklinik. De /).

Before the experimental apheresis therapy begins, the animals are provided with arterial and venous catheters, or chronically catheterized rats (vascular catheters, which are used to apply test substances and to take blood samples, as well as to carry out apheresis therapy and Clamp studies in animal models are suitable). In a first step, apheresis first separates blood cells and plasma using a plasma filter. While the blood cells are being refunded directly into the animal (via the venous catheter), the separated plasma is guided past the adsorbent prepared in Example 1 (the ligands being separated from the plasma by binding to the immobilized affinity peptides) before it is transferred to the animal is fed again.

3. DNA autoantibody aptamers

3.1 Activation of a silica gel column with tresyl chloride

The column is flushed with acetone. For activation, an anhydrous solution (2 ml acetone, 1 ml tresyl chloride, a few drops of pyridine) is passed through the column (10-fold column volume) and incubated overnight on ice. The column is then rinsed with 20 times the column volume of 100% acetone (anhydrous). The activated column can be stored in 1 mM HC1.

3.2 Activation of a polyethylene column

A polyethylene tube is rinsed with 20 times the column volume at room temperature with a solution (2% potassium permanganate (KMn0 ₄ ) (w / v) in concentrated sulfuric acid (H ₂ S0)) and then with distilled water. For further coupling of the column surface, bi- or polyvalent molecules can be used for crosslinking which have at least one reactive aldehyde group (eg 1% glutaraldehyde). These are passed through the column at 4 ° C. for 1 h. The reaction is then stabilized by reducing conditions (for example sodium cyanoborohydride (0.00025% w / v in 0.15 M NaCl, pH 3.9).

3.3 Coupling of DNA autoantibodies to the activated silica column

The tresyl chloride-activated column is flushed with 0.1 M Na ₂ CO ₃ (pH 8.5). A peptide or protein (2 mg / ml 0.1 M Na ₂ CO ₃ , pH 8.5) passed through the column several times for 2 h at 37 ° C. and then for 4 h on ice. To block free binding sites on the column, an excess of 0.2 M glycine, pH 8 is then passed through the column.

3.4 Coupling a glycoprotein to the activated polyethylene column

The activated column is flushed with 0.1 M Na ₂ CO ₃ (pH 8.5). For coupling, DNA autoantibodies (2 mg / ml 0.1 M Na ₂ CO ₃ , pH 8.5) are passed through the column several times for 2 h at 37 ° C. and then for 4 h on ice. To block free binding sites on the column, an excess of 0.2 M glycine, pH 8 is then passed through the column. To improve the reaction, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 5% w / v can be added).

3.5 Production of a column for the elimination of undesired molecules

The tresyl chloride-activated column is flushed with 0.1 M Na ₂ CO ₃ (pH 8.5). If elimination is to take place against a molecule with one or more primary or secondary amino acids, this molecule or the mixture (2 mg / ml 0.1 M Na ₂ CO ₃ , pH 8.5) is passed through the column several times overnight at RT , To block free binding sites on the column, an excess of 0.2 M glycine, pH 8 is then passed through the column. If no elimination against certain molecules with one or more primary or secondary amines is desired, all binding sites of the column are blocked with glycine.

Further derivatization methods can be found, for example, in the following references: Patterson, WJ, National Aeronautics and Space Administration, Technical Memorandum, NASA TMX-73311, US Government Printing Office, Washington, DC, 1976, Ma, SM, Gregonis, DE , by Wagenen, RA, and Andrade, JD, in "Hydrogels for Medical and Related Applications" (JD Andrade, Ed.), Amer. Chem. Soc. Symp. Series, vol. 31, p. 241, 1976, Harris, JM, Struck, E.C., Case, MG, Paley, MS, Van Alstine, JM, and Brooks, DE, J. Polymer Sci., Polymer Che. Ed., 22, 341 (1984), Regnier and Noel, Regnier, FE, and Noel, RJ, J. Chromatog. Sci., 14, (1976), Yalpani, M. and Brooks, DE, J. Polymer Sci., Polymer Chem. Ed., 23, 395 (1985).

3.6 Carrying out the contact of the nucleic acids with DNA autoantibodies and separating the column

The coated columns are leaked freely one after the other, first the columns for the elimination of unwanted molecules, then the DNA autoantibody column. A suitable buffer, e.g. B. a buffer solution (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ and gelatin 0.001% (w / v), pH 8.3), for 1 h on ice over the column. The nucleic acids of the combinatorial nucleic acid library are taken up in 1 ml of the same buffer and heated to 95 ° C. for 10 min to melt double strands and then passed over the column several times (4-30 times). The columns are then separated and washed overnight on ice with the chosen buffer (see above). The column is separated in the direction of flow using a suitable cutting tool.

Claims

Claims:

1. Apheresis device with a solid carrier that can be contacted with the blood or plasma flow, characterized in that the solid carrier has a specific low-molecular DNA autoantibody-binding receptor.

2. Device according to claim 1, characterized in that the DNA autoantibody-binding receptor is selected from dsDNA, dsPNA, DNA autoantibody aptamers, dsDNA-Mimick peptides, dsDNA: protein complexes or mixtures of these substances.

3. Device according to claim 1 or 2, characterized in that the carrier is a sterile and pyrogen-free column.

4. Use of a device according to one of claims 1 to 3, for providing a treatment for lupus diseases or for the prevention thereof.

5. Use according to claim 4, characterized in that the lupus disease is selected from lupus erythematosus, preferably chronic discoid lupus erythematosus, pseudo-lupus erythematosus, chillblain lupus, anticardiolipin syndrome, drug-induced lupus erythematosus, neonatal lupus erythematosus, lupus erythematosus profound, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, especially systemic lupus erythematosus.