DE102005028545A1 - New Cherkasky fusion protein comprises antibody binding protein, non-antibody binding domain and/or any domain useful as materials, medicines, coloring- and labelling materials, adhesives and diagnostic systems - Google Patents

Abstract

Fusion proteins, named as Cherkasky fusion proteins (comprises at least one antibody binding protein or its domain or any domain and at least a non antibody binding domain or any domain) are new. Independent claims are included for: (1) a nucleic acids, vectors, expressions and transformation systems for fusion proteins; and (2) a linker or spacer having a formula (Pro(Gly)n)m, where breaks are formed in glycine by proline.

Description

The The invention relates to the fields of molecular biology, immunology and biotechnology.

The state of the art includes works and publications ( DE 102 02 191 A1 . DE 103 501 31.A1 , and WO 2005/040382 ), the fusion proteins containing Fc region binding domains such. Staphylococcal protein A (SPA) or Fc receptor ecto or ligand binding domain and sequence - specific nucleic acid binding domains as well as the fusion proteins containing antibody binding regions such as preferably staphylococcal protein A (SPA), extracellular region of Fc receptor CD 64 or their regions and Microtubule binding regions, preferably gephyrin, tau, MAP or describe their regions. These substances can not be used effectively.

The broad spectrum and uses of the fusion proteins containing Antibody binding regions are not worked out yet. The object of the invention is therein, novel substances with novel properties and uses to create and develop antibodies that bind antibodies and, consequently, those Can bind antigens.

The The object of the invention is obtained by novel fusion proteins Antibody binding regions according to the invention solved.

fusion proteins containing antibody binding Proteins or their regions comprise novel classes of substances, Materials, drugs, diagnostic system, adhesives, color and marking substances and many other substances and systems. Based on a few examples, the width or the range of use and advantages of these substances and their uses being represented. This width or numerous uses and Advantages Are by far not only on these following examples limited.

example 1

Cherkasky Fusion Proteins containing IgE antibody binding regions and hydrophilic regions

Increased values of IgE antibodies Mark asthma, atopic eczema, some allergies and inflammations. The goal of certain therapies is therefore to diminish the IgE values are directed, i. Develop drugs that remove IgE molecules and thereby lower their concentration.

Known are anti-IgE antibodies from Genentech under the trade name Xolair. Because the removal of IgE using antibodies by macrophages or their activation should take place and this even when activating inflammatory factors release, the effect of anti-IgE antibodies is not as effective as expected.

Cherkasky Fusion Proteins containing IgE antibody binding regions and hydrophilic regions IgE antibodies bind and through hydrophilic regions with the circulation of a inflammation and are not on the activation of macrophages reliant. This makes the treatment more effective and less side effects carried out become.

Example 2

Cherkasky Fusion Proteins containing IgE antibody binding regions and Receptors The task has been discussed in Example 1.

The Receptors of the fusion proteins preferentially bind inflammatory factors such as TNF-alpha, C5a, C3a and C4a. Hallway possess fusion proteins thereby a double effect. On the one hand, the binding of IgE antibody and their receptors by binding to fusion proteins according to the invention or the example 2 blocks, on the other hand become inflammatory factors like TNF-alpha, the are bound to the receptor regions of the fusion proteins. The anti-inflammatory Double action can be a more effective treatment with fewer side effects enable.

Example 3

Cherkasky Fusion Proteins containing. IgE or IgG antibody binding regions and enzymes.

enzymes preferably, DNA sen, DNAse 1, S1 endonuclease, catlase or Peroxidase are also anti-inflammatory and soothing in systemic lupus erythematosus (SLE).

The anti-inflammatory Double action of the fusion proteins according to this example is therefore similar to the double action according to Example 2.

Example 4

Cherkasky Fusion Proteins containing IgE or IgG antibody binding regions, Receptors and enzymes

These Fusion proteins possess a triple anti-inflammatory effect Fusion proteins described in Examples 2 and 3, if enzymes are DNA se 1, S1 endonuclease, catalase, peroxidase or other anti-inflammatory Enzymes, and receptors, e.g. TNF alpha receptor, C5a, C 3a, C4a are receptors or other receptors that are inflammatory factors tie.

Example 5

Cherkasky Fusion Proteins containing antibody binding regions and fluorescent proteins or their regions

With Help these fusion proteins can antibody e.g. Specific IgG or IgE antibodies be marked. The main advantage of labeling the antibodies by these fusion proteins are the genugkeit. An example of a fluorestent Protein is e.g. GFP (Green fluorescent protein) or its region.

Example 6

Cherkasky Fusion Proteins containing antibody binding regions and metal binding proteins or their regions

These Fusion proteins complexed with metal atoms or ions can pass through Magnetic field- be directed or oriented. This will be the task solved the molecular orientation. Metal binding proteins are e.g. Astacin (from Astecus fish), Ferritin, Ceruloplasmin, a cysteine rich Region u.v.a. Protein or its regions.

Example 7

Cherkasky Fusion Proteins containing antibody bandage, Metal bandage - and fluorescent regions

The Fusion proteins can be marked and oriented, which e.g. in the insulation and purifying the corresponding antigens of fusion proteins bound antibody is beneficial.

Example 8

Cherkasky Fusion Proteins containing antibody bandage, Polymer binding and metal binding region

These Fusion proteins can to a polymer, preferably biopolymer, e.g. a DNA sequence or a polysaccharide such as cellulose, chitin, pectin, starch Polymer binding region, through antibody binding region, e.g. IgG or monoclonal antibodies bound and directed by metal ions and atoms complexed metal binding domain or to be oriented. Such complexes of fusion proteins and Polysaccharides can e.g. for the infiltration of certain substances or substances, preferably Antigens e.g. viral antigens.

Example 9

Cherkasky fusion proteins containing antibody binding, polymer binding and fluorescent proteins or their regions

The Advantages and uses of these fusion proteins are similar to those of Example 8, where the fluorescent regions are their color, Brightness or intensity dependent on of concentrations of bound or infiltrated antigens.

Example 10

Cherkasky Fusion Proteins containing antibody binding, Metal binding, polymer bandage and fluorescent proteins or their regions

The Uses of these fusion proteins are similar to those of the examples 8 and 9, being bound by metals bound to metal binding regions or complexed, using magnetic fields better molecular orientation is reached.

Example 11

Cherkasky Fusion Proteins containing antibody binding region, and at least one receptor or its region

The Antibody binding region is preferably an IgG binding region, e.g. SPA, being receptor e.g. an inflammatory factor how TNF binds Alpha, C5a, C4a or C3a.

The Effect is SLE-alleviative and similar, as in Examples 2-4 is described.

Example 12

Cherkasky Fusion Proteins containing antibody binding regions and ligands or their regions

ligands are e.g. C3b or C4b proteins of the immune system or their regions, which bind to erythrocyte CR 1 receptors and to opsonization and the elimination of immune complexes are involved. By such Fusion proteins can antibody like IgE in inflammation or IgG autoantibodies, e.g. bound to Epidermolysis bullosa acquesita (EBA) and to alternative Routes dissipated and disposed of, which leads to relief.

Example 13

Cherkasky Fusion Proteins containing antibody binding regions, Receptors and ligands.

receptors are e.g. Receptors of inflammatory factors such as TNF alpha, ligands are e.g. C3b and C4b or their regions. These fusions have the double anti-inflammatory effect as the proteins described in Examples 2-4, 11 and 12.

Example 14

Cherkasky Fusion Proteins containing antibody binding regions, Receptors and enzymes

receptors are e.g. Inflammatory factor receptors and enzymes such as DNAse1, S1 endonuclease, calalase or peroxidase and have an anti-inflammatory effect. The additional Binding of antibodies IgE or IgG increased the anti-inflammatory Effect of the fusion proteins according to this example.

Example 15

Cherkasky Fusion Proteins containing antibody binding regions or their regions and autoantigens or their regions

These fusion proteins can be used to diagnose autoimmune diseases. The Fitsionsproteine are thereby added in serum. If autoantibodies are found in low or high concentrations in the serum, they bind to the autoantigens or auto-antigenic regions of the fusion proteins. However, the Fc regions are nonspecifically bound antibody. This creates the in mul timolecular complex that precipitates.

Should there are no or only a few or in low concentration, the autoantibodies are so the autoantigens become regions of the fusion proteins accordingly unbound and the multimolecular complex arises according to that Not. The precipitation to the extent is not observed.

Example 16

Cherkasky Fusion Proteins containing antibody binding, autoantigenic and fluorescent regions

The Use of these fusion proteins as diagnostic tools or agents is similar as in Example 15, wherein the complex formation by fluorescent Proteins or their regions clarified and better visualized becomes.

Example 17

Cherkasky Fusion Proteins containing antibody binding, and T cell response triggering regions

The Modification of monoclonal IgG antibodies with these fusion proteins leads to improved use of the antibodies in use against viral, fungal bacterial and protozoan pathogens because of these T cells are attacked.

Example 18

Cherkasky Fusion Proteins containing antibody binding proteins or their regions and storage proteins or their regions

When Storage proteins can e.g. those of animal origin such as caseins or the herbal ones Origin like Viciline, Legumine, Gliadin, Glutenin, Lupine, Avenine, Secaline, Hordeine, Trifoliin can be used. The use of this Fusion proteins or fusion protein-antibody complexes consists of novel substances, which are amorphous or elastic, and certain antigens preferably on the surface recognize and bind. Such substances can preferably used as novel antigen-specific filters or applied to certain surfaces as security features become.

Example 19

Cherkasky Fusion Proteins containing antibody binding proteins or their regions and structural proteins or their regions

structural proteins can preferably selected from the following proteins: Lamprin, a cell protein, Resilin, abducin, crystalline alpha, beta, gamma, collagen or Kerstin.

The Uses are similar as in Example 18, wherein the fabric is elastic or solid can.

Example 20

Cherkasky Fusion Proteins containing antibody binding proteins or their regions, storage proteins or their regions, and structural proteins or their regions

The Substances consisting of these fusion proteins or fusion protein-antibody complexes have similar Applications and properties like those described in Examples 18 and 19 have been described.

Example 21

Cherkasky Fusion Proteins containing antibody binding proteins or their regions, metalbinding regions and storage proteins or their regions The properties and uses are similar to those in Examples 18-20 are described.

Example 22

Cherkasky Fusion Proteins containing antibody binding proteins or their regions, metalbinding regions and structural proteins or their regions

The Properties and uses are similar to those in the examples 18 and 19 are described.

Example 23

Cherkasky Fusion Proteins containing antibody binding proteins or their regions, metal-binding proteins or their regions, structural proteins or their regions and storage proteins or their regions

The Properties and uses are similar to those in the examples 18 and 19 are described.

Further Examples include fusion proteins containing antibody binding proteins or their regions and vegetable proteins such. B. phloem protein 1 (PP1) from cucurbitas, fish proteins, e.g. Astacin from Astacus, Neuron proteins such as e.g. GFAP, MBP, (Myelin Basic Protein), Polymer binding protein-enzyme fusion, the polymer binder region preferably being a cellulose binder region such as. Cip A is; rubredoxin; which is a tick stable protein is, antifreeze protein or. Glycoprotein, a serine or serine rich Region, a laminin, actin, myosin, a cytokine, an interleukin, a blood protein, e.g. Serum albumine, a transprotein, a basic Protein or its region, an acidic protein or its region, a spacer, a hydrophobic protein or its region, a hydrophilic one Protein or its region, a cell protein, a nuclear protein or their regions, a neurofilament or its region, a viral Protein, e.g. phage T4, Tobacco mosaic virus TMV, an opsonin, CRP or C-reactive protein, MBP (mannose binding protein), nucleic acid binding proteins or their regions, LH, FSH, Vicilin like protein or another Protein or its regions. The fusion proteins or fusion protein Antibody complexes can as novel substances for different purposes are used. Everyone has elastic, amorphous or solid mechanical properties and specific binding ability the antibody.

At surfaces bound antibodies can detect certain antigens and be used as a basis for sensors. Once these fusion protein-antibody complexes conduct electricity while keeping antibodies specific to their antigens recognize, the conductivity is or the resistance changed slightly, and while allowing this change easily registered and measured. This is the basis for the Construction of novel diagnostic, test; Control sensor and alarm or warning systems that are greatly reduced or miniaturized can be and work much more efficiently as electronic systems with immobilized antibodies, because for large concentrations of fusion proteins or fusion protein-antibody complexes already a few Antigens, such as dangerous Toxins such as anthrax, botulinus, or tetanus toxins, viruses such as e.g. HIV or Ebola virus, bacteria such as Bacillus anthracis, for registration in the interaction with the bound antibodies and measurement off or suffice.

The bound antibodies or the fusion protein-antibody complexes form an amorphous or elastic mass, which also contain salts can and the sensor component of the registration devices according to this Invention represents. Electricity is passed through this mass and any interaction of the antigens with the bound antibodies of the Fusion protein-antibody complexes change the amperage and the resistance.

These changes are registered and evaluated, which leads to a meaningful result.

In the 1a is a system for making diagnoses, controls and assays for detecting or registering certain antigens such as HIV viruses, bacteria, proteins, protozoa, or other molecules, microorganisms, or cells or substances, shown schematically.

The system contains the registration body 7 which is a mass consisting of antibody-fusion protein complexes, which are preferably applied to a substrate.

The registration body 7 contains antibodies 1 , _That the fusion proteins consisting of antibody binding domains 14 and storage proteins 10 , or structure or other proteins are bound. The registrar body 7 is through the line 22 with the current amplifier 6 and the evaluation system or with the computer 5 connected to the evaluation of changes in electrical signals.

In the 1b is the interaction of the antigen 2 shown schematically, which is eg HIV virus, with the antibodies 1 attached to the fusion proteins consisting of the antibody binding regions 14 and the storage proteins 10 giving.

In the 2a is the fusion protein-antibody complex which binds to CR 1 receptor 21 , the erythrocyte 19 binds, presented. The fusion protein-antibody complex consists of the fusion protein containing antibody binding domain 14 and the specific ligand 23 C3b or C4b, and antibodies 1 , By means of this fusion protein, an antigen can be removed and disposed of in an inflammatory manner by alternative means.

The fusion protein antibody 1 - Ligand 20 - Complex, in the 2 B which is involved in the erythrocytes 19 binds, contains additional middle receptor domain 13 which, for example, binds inflammatory factors such as TNF-alpha and dissipates or mitgesorgt. In this case, an additional anti-inflammatory effect is achieved.

In the 3 is a possible embodiment of the test, diagnosis or control system according to the invention, and the corresponding molecular process shown schematically.

The antibody eg a monoclonal antibody in the 3a 1.1 is linked to a polymer such as a nucleic acid sequence or a polysaccharide by the fusion protein containing an antibody binding region and a polymer binding region. Bin Antigen eg a virus becomes of this antibody in the 3b 1.1 bound. The optical visualization ( 3c 1.1 ) is carried out by fluorescent fusion protein containing an antibody binding domain - eg SPA and a fluorescent domain such as GFP. The fusion proteins in the 3a 1.2 , b 1.2 and c 1.2 contain longer spacer regions such as polyglycine to increase the range of the Reichsweite.

In the 3a 2 b 2 and c 2 and the corresponding test procedure manual or automatic execution shown. The pencil 25 with the handle 24 contains the molecular surface structure corresponding to the figures a 1.1 or a 1.2. This pen in the 3 a 3 gets into the vessel 26 done with a blood sample or serum, held for some time eg 2 minutes and taken out. Thereafter, this pin (b2) is placed in the solution containing fusion protein-antibody complexes. The fusion proteins consist of antibody binding regions and fluorescent regions.

Should in the blood or serum sample antigens e.g. Viruses like HIV, or SARS or hepatitis, these are by appropriate antibody the fusion protein-antibody complexes recognized and bound. After the pen is removed from the sample this shines. To prove if this is the case either high or medium affinity specific antigen - antibody interactions or non-specific or low-affinity interactions the pen is done in water or in a buffer solution and stirred gently, and then from the solution taken (c2). If the light stays on, the intensity is measured. The light is interpreted as follows: The antigens have specific to the antibodies bound to the fusion proteins bound to polymers and these are on the lateral surface tied to the pen. The fusion protein-antibody complexes are linked to these antigens specifically bound to the other free sides of the antigen.

The Fusion proteins are linked to the antibody binding domains to antibodies and shine through fluorescent domains. So that means persistent Fluorescence is the fact of being in the blood sample or serum sample Containing antigens. If the lighting disappears after stirring, this means that there are no antigens in the serum or in the blood sample were.

The fusion proteins in the 4a . 4b and 4c contain fluorine-resistant regions and can be used as constituents of the sensors, because the light intensity is changed by antigen-antibody interactions. This change will be registered.

The fusion protein in the 4a contains a fluorescent region 18 , a polymeric binder region 16 , and the antibody binding region 14 which is associated with an antibody.

The fusion protein is through the polymer binding region 16 to a polymer 9 bound.

The fusion protein in the 4b additionally contains a longer spacer region 8th , such as a polyglycine domain, which increases the range.

The fusion protein in the 4c additionally contains a domain of a storage protein, structure or metal binding protein to increase stability.

In the 5 some possible embodiments of the fusion protein-antibody-polymer complexes are shown schematically.

The fusion protein in the 5 .1 contains the polymer binder region 16 , and the antibody binding region 14 that with the antibody 1 connected is. The fusion protein in the 5.2 additionally contains a spacer region 8th , The spacer region of the fusion protein in the 5.3 contains Kniks originating from Proline in the polyglycine region.

The spacer region of the fusion protein in the 5.4 contains a helix. The fusion protein in the 5.5 additionally contains a region of a structural protein, storage protein or a metal binding protein to increase stability.

This fusion protein can either be a linear ( 5.6 ), cracked ( 5.7 ) or helical ( 5.8 ) Spacer region.

The fusion protein in the 5.9 additionally contains two regions, eg one of one structure - and the other of a storage protein or a metal binding protein.

This fusion protein can either be a linear ( 5.10 ), cracked ( 5.11 ) or a helical ( 5.12 ) Spacer region.

The fusion protein in the 5.13 contains a terminal domain such as GFP or a metal binding domain, where the middle domain is the polymer binding domain.

This fusion protein can either be a linear ( 5.14 ), cracked ( 5.15 ) or helical ( 5.16 ) Spacer domain included.

The fusion protein in the 5.17 contains two terminal domains before and after the polymer binding domain.

This fusion protein can either be a linear ( 5.18 ), a cracked ( 5.19 ) or a helical ( 5.20 ) Spacer region.

The fusion protein in the 5 .21 contains a middle polymer binding region (terms "region" and "domain" are to be used as synonyms) and two side domains, respectively, where the one N- or C-terminal domain is the antibody binding domain which is linked to an antibody. The adjacent domains are, for example, a metal binding domain, the polymer binding domain, a structural protein domain and a fluorescent domain and / or domain of a storage protein to achieve stability, fluorescence and molecular orientation by directing the complexed zones. The geknikte domain may, for example, the following formula (Pro (Gly) n) m, where n and m may be arbitrary. For example, this fusion protein can be either a linear ( 5.22 ), a cracked ( 5.23 ) or a helical ( 5.24 ) Domain.

The fusion protein in the 6 contains antibody binding domain 14 and metal binding regions 11 , and is with the antibody 1 connected. The antibody can be oriented in solution through the fusion protein and electromagnetic fields.

In the 7 is the complex consisting of antibodies and fusion proteins containing autoantigens or autoantigen regions 15 and antibody binding regions, shown schematically. These fusion proteins serve to diagnostically determine the presence of certain autoantibodies in blood or serum. If autoantibodies are present, the Fab fragments bind the autoantigen domains of the fusion proteins and the complex is formed.

If no antibodies are present, they will not bind to the autoantigen domains of the Fu sion proteins and the complexes do not arise. In the 8th a polymer-antibody-fusion protein complex is shown schematically. Fusion proteins contain antibody binding domains 14 , Polymer binder regions 16 , and receptors 17 , and are due to polymer binding regions 16 on polymer 9 and by antibody binding regions 14 to antibodies 1 bound.

The receptors 17 bind ligands 20 , The receptors bind eg inflammatory factors such as TNF-alpha, these correspond to the ligands. In the 9 is the steering of a T cell 4 against a target cell 3 represented by fusion protein-antibody complexes. antibody 1 binds specifically to a target cell-specific antigen and the ligand domain 20 binds to the receptor 17 the T cell. For example, the ligand domain is a region of HLA-B7.1 or B7.2 and elicits T-cell activation, which leads to targeting the target cell.

The fusion protein in the 10 contains a terminal hydrophilic domain consisting of hydrophilic amino acids such as serine to increase the solubility of the polymer-fusion protein-antibody complexes. The fusion proteins in the 11a , b, c, d and e are characterized by an enzyme, eg protease, nuclease, lipase, catalase, peroxidase or another enzyme.

In the 12 the test pen with 10 sectors or segments as well as the procedure of the preparation and the test execution are shown schematically (see 3 ). Excess of the fusion proteins in solution densifies the fusion proteins bound to polymers.

The Sequences for Proteins are public accessible and on the internet e.g. at the National Center for Biotechnology Information, NIH, Bethesda MP, 20894, USA (NCBI http://www.ncbi.nlm.nih.gov) available.

embodiment 1

cloning and expression of the fusion construct N-keratin SPA C c DNA sequences for keratin and Staphylococcal protein A are cloned by PCR or RT-PCR. The Fusion protein is composed of PCR products. The PCR primers are designed to contain restriction sites on 5 'and 3' ends, for later Ligationsschtitte perform. The 5 'and 3' ends of the keratin PCR Products contain Bam HI and Hind III restriction sites.

The 5 'and 3' ends of the SPA-PCR Products contain EcoRI and KpnI restriction sites.

By Treatment with Bam HI and Hind III will be keratin PCR product in the ligated according to preparatory vector. The vector becomes treated with KpnI and EcoRI to inoculate SPA-PCR product. The ligation product is transformed into E. coli, expressed and finally cleaned.

In the Seq. 1 is the amino acid sequence of the fusion protein containing human low affinity receptor for Fc Fragment of IgG and human keratin 7, shown.

In the Seq. 2 is the amino acid sequence of the fusion protein containing human low affinity receptor for Fc Fragment of IgG and Vicilin-like Protein represented by Lupinus albus. The receptor domain is 290 amino acid long and the length of the vicilin-like Protein is 182 amino acids, so that's the length of the fusion protein 472 amino acids.

Novel Cherkasky fusion proteins containing antibody binding proteins or their regions

Claims (7)

Novel fusion proteins, called Cherkasky fusion proteins, characterized in that they contain at least one antibody binding protein or its region or domain and at least one non-antibody binding region or region. Fusion proteins, called Cherkasky fusion proteins, characterized in that they contain at least one antibody binding protein or its region preferably staphylococcal protein (SPA), another IgG or an IgE binding region and at least one region selected partially or wholly from the following proteins, fusion proteins or their regions HLA-B7.1, HLA-B7.2, a domain encoding it, a T-cell activating domain, an enzyme, DNAse 1, Si endonuclease, a receptor, preferably for inflammatory factors TNF-alpha, C5a, C3a, C4a , a fluorescent protein preferably GFP, a ligand preferably C3b, C4b, an opsonin, CRP or C-reactive protein, MBP or mannose-binding protein, and My elin basic protein, a neural protein, an acidic, a basic, a hydrophilic, a hydrophobic protein, a cysteine-rich region, antifreeze protein or glycoprotein, rubredoxin, an autoantigen or its region, a structural protein preferably, resilin, abducin, lamprin, Collagen, keratin, a storage protein, casein a cell protein, a neurofilament, a muscle protein preferably myosin or its region, active, laminin, a cytokine, an interleukin, a blood protein, preferably serum albumin, a viral protein, a metalloprotein or metal binding protein, a transport protein, or its region, a polysaccharide binding protein (PBD), CipA cellulose binding region, a nucleic acid binding protein or its region, crystallin or crystallin alpha, beta or gamma, an ishtyoprotein, preferably astacin, phloem protein 1 (PP1), phloem protein 2 (PP2 ) from Cucuribitas, a spacer or a linker, a serine or serine-rich region, vicilins, legumes, gliadin, glutenin , Hordeins, Trifolin, Vicilin-like or similar protein from Lupinus albus, a hormone, LH, FSH or PBD-enzyme fusion protein. Fusion proteins according to one of claims 1 and 2, characterized in that it comprises at least one His-tag or another day for cleaning, a linker or a joint region or at least one other region. nucleic acids, Vectors, Expressions Lind transformation systems for the fusion proteins according to the claims 1-3. Uses of the substances according to claims 1 to 4 preferably as materials, medicines, dyes and markers, Adhesives, and diagnostic systems. Linker or spacer, characterized by the formula (Pro (Gly) n) m, whereby in Glycinen by Proline Knicks are formed. Use of the polymers for densification of the fusion proteins in particular that according to claims 1-5.