DE10238846A1 - Active fusion proteins and processes for their production - Google Patents

Abstract

The invention relates to fusion proteins, comprising an expression protein and a phage coat protein fragment, the phage coat protein fragment being at least one domain D1 or a functionally analogous fragment of a pIII of a filamentous bacteriophage M13.

Description

The invention relates to active fusion proteins, a process for the production of active recombinant proteins and the use of this in medical diagnostics and therapy.

One of the main goals of genetic engineering is the synthesis and purification of proteins from cloned genes and especially the production of therapeutically important proteins. Lots Proteins can are already genetically engineered, such as insulin, Interferons or factor VIII. Crucial for the biological activity of the proteins is their correct folding.

Originally it was assumed that the spatial Arrangement of a protein the state of lowest free energy corresponds and that the protein by chance this State of lowest energy. However, it could be shown that such a process does not take place in the cells.

The folding of a protein is primarily due to its amino acid sequence determined, but it is not possible if the sequence is known, the secondary and / or tertiary structure to predict exactly.

Over the past few years found numerous complex folding mechanisms. So it was discovered that certain proteins - like Chaperones or foldases - the correct one Either support or catalyze protein folding.

Proteins fold in vitro within less seconds. Because proteins are not random according to the lowest fold free energy, there must be different metabolic pathways for folding and it can be assumed that the correct folding of a Protein is not necessarily the most stable fold. The research of the folding kinetics is only possible to a limited extent, since intermediate stages of the Proteins can hardly be considered in isolation when folded.

There were different options proposed to the biotechnological extraction correctly folded Optimize proteins:

• (i) modification of the fermentation technique,
• (ii) Exchange of amino acids and
• (iii) change the intracellular Concentration of folding modulators.

(i) From in vitro experiments known that when refolding denatured proteins a low protein concentration a correct one Folding favors. In vivo it will used so that the overexpression does not is fully induced so that the intracellular concentration of recombinant Protein is moderate.

It is also possible to go through Compilation of the medium, the pH value, the fumigation or the Trunk selection has a significant impact on folding. Also an increase osmotic pressure, e.g. by adding polyols a stabilizing influence on the folding of the protein.

Furthermore, the lowering of the growth temperature leads to a higher one Proportion of correctly folded proteins. That's how the production was of 5-lipoxygenase initially at 37 ° C carried out, by lowering it to 16 ° C the proportion of correctly folded proteins could be increased.

(ii) The change in the amino acid sequence leads to - often dramatic - changes of folding. This has been the case for interferon and interleukin, for example shown.

(iii) Another option consists in the fusion of the target protein with a folding mediator. This includes for example thioredoxin, the gluthation-S-transferase, the Staphylococcus aureus protein A and the maltose binding protein. Furthermore, the Use of heat shock protein systems from E. coli such as the systems DnaK-DnaJ-GrpE and GroEL-GroES known. These chaperones can be used in a co-expression system expressed simultaneously with the target protein become. For TNF, the growth hormone and the dihydrofolate reductase lie as Target protein already results with co-expressing chaperones in front.

A specialty in the manufacture recombinant proteins, especially in connection with their correct Folding is the formation of aggregates (inclusion bodies, IB). The formation of IBs can be beneficial if the target proteins susceptible for proteolytic operations are because IBs are hardly attacked by proteases or when there are the goal is inactive proteins or proteins that have no active folding need, in large To produce quantities. Due to their denatured condition, IBs at higher Temperatures and with a much higher shear stress be isolated. Activities, with which the in vivo folding of the proteins can be improved, also modify the formation of the IBs. IBs are insoluble and not biologically active. In order to obtain active proteins, they have to therefore denatured by drastic methods and then - with the above methods - in the correct folding can be renatured.

However, these methods of correct folding have several disadvantages. The temperature reduction measures reduce productivity and do not guarantee the correct folding of the proteins. Ultimately, production has to be individually optimized for each protein. The change in the amino acid sequence requires a prior determination of the structure and, if possible, the folding process. When using Depending on the target protein, fusion proteins may result in the fusion constructs failing in poorly soluble protein aggregates. The use of coexpressing chaperones is very complex and costly; furthermore, the proteins in these systems need enough space for their folding, especially if disulfide bridges have to be formed. Therefore, the proteins must be diluted very strongly before folding or refolding. In the production of recombinant insulin, this means, for example, that the production reactor has a volume of 35 square meters and the refolding kettle has a volume of 73 square meters.

The object of the invention is therefore provide correctly or actively folded proteins that have a structure have a high biological activity.

The invention solves this technical problem by providing a multimeric fusion protein comprising an expression protein and a phage coat protein fragment, the Phage coat protein fragment one Dömane D1 or a functionally analogous fragment of that of the pIII of the filamentous bacteriophage M13 is.

The filamentous bacteriophage expresses M13 on the surface Proteins made with Roman Numbers are numbered consecutively: for example pI, pII, pIII to pIX. PIII is a coat protein and together with pVI forms the infectious end area of the phage. The coat proteins consist of different domains. The protein comprises pIII three domains, D1, D2 and D3. It was surprising that the invention D1-containing fusion proteins actively folded structures that Expression or target proteins, and that the D1 contain fusion proteins according to the invention Can form multimers i.e. without requiring interaction with D2. D1 in the sense the invention also encompasses proteins or their fragments, the one have sufficient homology to a naturally occurring one D1 domain to be functionally analogous and also such proteins or peptides respectively Fragments caused by deletions, additions, substitutions, translocations, Inversions and / or insertions modified and functionally analogous to D1. It was surprising that D1 itself interacts and binds multimers. You get over the Multimerization of the D1 domains several fusion proteins and in particular an increase in affinity due to the avidity, e.g. when the expression proteins are fragments of antibodies. Advantages of these technologies with regard to multimeric fusion proteins will be described in more detail below. The invention shows the advantage of D1 as a fusion component to the expression protein compared to D1-D2 or all of pIII. The advantages of the fusion protein according to the invention or peptide are still the smaller size and their immunogenicity.

The fusion proteins according to the invention can be homomultimers or hetero multimers. Heterologous multimeric fusion proteins (Hetero-multimers) accordingly comprise two or more different fusion proteins.

The phage coat protein fragment D1 or functionally analogous fragment of the expression protein in the fusion protein by a linker be separated. This can be beneficial for activity and multimerization of the fusion protein. One or more linkers can also occur elsewhere in the fusion protein, for example between individual domains of the expression protein as in the example of scFv or between that Phagenhüllproteinfragment D1 or its functionally analogous fragment or the expression protein and other sequences such as tags (see below). The Characteristically, polypeptide linkers have a length of 1 to 50 amino acids. If there is no linker, one speaks according to the invention also a linker of length 0th

In particular, this enables targeted with the help of fusion proteins with D1 as a partner to the expression protein to produce active multimeric fusion proteins. These have the advantage that when interacting with the binding partner of the expression protein an increased binding strength achieve through the high avidity can. The presence of D1 primarily forms tetramers fusion proteins who achieve particularly high avidity. The high avidity is for example particularly advantageous for receptor-ligand interactions and for antibody binding or also in enzymatic reactions.

According to the invention, all proteins which have been selected as the target structure can be used as the expression protein or expression peptide. Thus, it can be provided to provide multimeric antibodies, in particular by actively expressing scFv as the expression protein with the D1 as the fusion protein. If the same scFv is used, it is advantageous to obtain a mostly higher affinity, because avid, multimer from antibody fragments, preferably tetramers. These are advantageous for different in vitro and in vivo applications known per se, in which a higher affinity / avidity brings advantages. For example in immunological test systems and therapies. When using different scFv in hetero-multimers, it is easily possible to produce bi- or multispecific antibodies which are used in a wide variety of known in vivo and in vitro applications, for example in biochemical, immunological, immunohistochemical, cell-biological test systems, preferably for example immunohistology, immunocytochemistry, ELISA, RIA, Western blots, FACS analyzes, in vivo radio diagnostics, in vivo radiotherapeutics, immunoprecipitations, for immunization and others. The technological application and the individual methods are known to the person skilled in the art or can be derived. For these purposes it may be advantageous to directly label the fusion molecules according to the invention with, for example, fluorescence molecules, radioisotopes or other molecules suitable for detection, such as, for example, molecules of the streptavidin-biotin system, or with a tag which is detected by secondary detection systems known per se (see below). Spacers or chleators may be necessary or advantageous for this. The technologies required for this are known in principle to the person skilled in the art and can be adapted and / or optimized by him for the corresponding application.

But of course it is too possible, to use a single-chain MHC class molecule as the expression protein. The MHC class molecules the multimerization advantageously results in a higher valence. The purified single chain MHC class fusion proteins comprising molecules can are loaded with peptides according to known methods or in the form of single-chain molecules as MHC class molecules with the corresponding Peptide can be expressed as a fusion protein. Suitable can Linker can be used. Such MHC class multimers with peptides can be used, for example, in a technology analogous to that known Use tetramer technology to detect specific T cells. The MHC class multimers with the peptides can be used with known Methods can be marked with fluorescent dyes, for example.

It is also possible, for example, to use enzyme complexes provide where multiple enzymes or active portions of Enzymes can be stored together in a homo- or hetero-multimer. The advantage in a homo-multimer is the increase in efficiency the concentration of the enzymes. With the help of a hetero multimer, several Enzymes spatially are close together. This is especially beneficial if which correspond to different enzymes from a catalytic series, making it possible Response equilibria efficiently due to the spatial proximity to the side Product and thus efficient catalysis in one Generate multi-step process. In another preferred form in the multimeric complexes, substrates or intermediates, Stabilizers or other catalysts included in order to get as possible to achieve high efficiency.

The multimeric fusion proteins over the D1 give the possibility to construct highly affine and highly active multibodies. The invention also provides the option of described selection system (see below) directly multimers in their function to win. The multimeric fusion proteins can be used for different applications through stabilization for certain applications are improved, where possible stable multimer desired is, for example of multibodies. There are a number of these from the stand of known techniques. In particular, the individual fusion proteins of the multimeric fusion protein stabilized by covalent cross-linking become. Therefor come a number of known chemical, biochemical and physicochemical technologies, such as networking through Formaldehyde or glutaraldehyde, crosslinking via sulfur bridges, carboxy groups, Amide groups or photochemical activation methods. covalent Connections are preferred over so-called spacer molecules produced, which are known per se to the person skilled in the art. In a preferred one Variant will be additional special tags or mutations, for example cysteines or lysines, introduced in the fusion protein that allow or promote stable networking Techniques for the individual applications are optimized according to known methods and adjusted. That explained below The selection process can be used to help by the phages which carry the fusion proteins according to the invention treated according to the appropriate stabilization method and subsequently the selection is made. Only such fusion proteins are placed over it associated with the phage genetic material that identifies its function after the stabilization treatment to keep.

A cleavage site is preferably inserted between the expression protein and the D1 of the fusion protein according to the invention, which makes it possible to cleave the expression protein in the form of an active protein from the portion with the D1. For this, a suitable nucleic acid sequence encoding the cleavage site is inserted into the nucleic acid encoding the fusion protein using methods known per se. The expressed fusion protein is isolated according to the techniques described above and then cleaved by enzymatic, ribozyme or chemical cleavage with cleavage molecules. In connection with a suitable cleavage site, specific enzymes or chemical molecules that do not deactivate the active protein to be obtained serve as cleavage molecules. Table 4 shows a preferred selection of cleavage molecules with a correspondingly suitable amino acid sequence. Of course, it is also possible for the fusion protein to comprise at least one additional day, for example a His day, which remains on the D1 portion after cleavage. The cleavage is preferably carried out with an enzyme which also bears a corresponding day, for example His day. In this way, the cleavage molecules and the D1 portion can be separated from the active protein in one step. This is advantageous, for example, if the active protein to be obtained can preferably be used without the day. An example of this is the production of active antibody fragments which are intended for use in humans and are said to cause as little or as little immune response as possible there. This is a method that is particularly suitable for the production of active antibodies for antibody therapies or antibody diagnostics in humans. Such antibodies can then be used in further, the Steps known to those skilled in the art are loaded, for example, with radioisotopes with or without chelators or are coupled to toxins or other substances with the aid of corresponding coupling steps and molecules. For diagnostics, fluorescence labels are preferred, which are used directly or via secondary binding systems, such as the streptavidin-biotin system.

Some terms are to be defined in connection with the invention:
According to the invention, active proteins are understood to mean those proteins which contain an activity due to a folding or a spatial structure; this activity can be, for example, a binding activity, enzymatic activity, catalytic activity or an activity which is based on interaction. Such proteins can also be post-translationally modified.

Fusion proteins are understood according to the invention two or several polypeptides or proteins that together form a gene construct as one coherent Protein, the fusion protein, can be expressed. The polypeptides or proteins by further sequences, for example linkers or Separate tags from each other and / or to them on one of the two Pages attached become.

An expression protein means the proportion according to the invention of the fusion protein that contains the activity.

According to the invention, the D1 is understood to be a polypeptide or its corresponding coding nuclienic acid sequence, that of the domain D1 des phage coat pIII of the filamentous Corresponds to bacteriophage M13 or their functional analogues. Understands according to the invention one under the D1 but also derivatives of the D1, for example Mutations of D1 or D1 are those with additional amino acid sequences is connected (see also above). D1 can also be part of the Sequences of the domain D2 of the phage coat protein pIII of the filamentous Bacteriophage M13 included as long as the full length of the D2 is not reached. Prerequisite for the derivations of the D1 is that this continues to express active proteins in the senses of the invention and / or a multimerization of the fusion proteins allow.

One understands the D1 portion according to the part of the fusion protein or the nucleic acid encoding the fusion protein, the that contains D1 and is not the expression protein, with additional sequences may be included for example tags, linkers, and / or signal sequences.

According to the invention, a linker is understood to be an amino acid sequence or the nucleic acid encoding the sequence, the two polypeptides or Combines proteins with each other in a fusion protein. A leftist can also be one between two or more polypeptides Expression protein occur, for example between the variable domains the light and heavy chain of antibodies in single chain antibody fragments or for example between the alpha unit and the beta unit of an MHC Class I molecule and the MHC class I peptide in the form of a single chain MHC I / peptide in the Fusiosnprotein are included .. The linkers can have a length of 0 to 50 amino acids (see also above).

According to the invention, a day means longer or shorter amino acid Sequences or the nucleic acid encoding the sequence, the for example, the identification or affinity purification by one against the Tad specifically targeted antibody or binding molecule, cleaning through complex binding to metals (His-Tag), the fluorescent label (e.g. green fluorescence protein) or labeling or coupling of molecules serve, for example by radioactive or fluorescent labeling or coupling to biotin, enzymes or other structures. This and other tags are known to the person skilled in the art, as well as their insertion in corresponding nucleic acid or protein constructs and linkage with the molecules, as well as advanced Cleaning and marking techniques.

A host cell is understood to mean according to the invention a prokaryotic or eukaryotic cell expressing the fusion protein allowed in the periplasm or in a secreted form. In a preferred variant, the cell is a bacterial cell. The host cell can also be part of an organism, for example plant cells.

Cleavage molecules are understood enzymes according to the invention, for example proteases, ribozymes or other chemical molecules, for example Bromocyanine, which specifically recognize the cleavage site in the fusion protein and specifically cleave them, thereby releasing the active protein comes

Under a multimeric fusion protein one understands according to the invention the non-covalent Storage of two or more fusion proteins by the domain D1 or its functional analogue of the phage coat protein pIII of the bacteriophage M13 is conveyed. Homologous multimeric fusion proteins exist thereby from fusion proteins with the same expression proteins. heterologous Fusion proteins consist of fusion proteins with different Fusion proteins.

According to the invention, multibodies are understood to mean multimers Antibody fragments, for example slide, tria or tetrabodies. These are, for example by shortening it or omit the linker between the variable domains VH (variable domain heavy chain) and VL (light chain variable domain). Multibodies have binding sites of the same specificity, for example Dia-, Tria-, Tetrabodies, or different specificity, for example bispecific diabodies.

According to the invention, a vaccine is understood to be a substance for the immunization of humans or Animals. According to the invention, a vaccine contains a fusion protein according to the invention or a multimeric fusion protein.

Under a drug one according to the invention a pharmaceutical Product containing a vaccine according to the invention in a suitable pharmaceutical dosage form and for the Application to humans.

According to the invention, an abbot means one Antibody, that against the binding region of a primary antibody that contains the desired antigen recognizes against which to immunize, specifically directed is and immunologically imitates the antigen, and in an immunization causes an immune response against the antigen.

Under a bacteriophage a virus according to the invention, that can infect a bacterium.

Taking a mimicry peptide against Conformation epitopes are understood according to the invention as a peptide that is a conformation epitope or a non-protein structure, for example a carbohydrate structure, imitated immunologically and in an immunization an immune response against the conformation epitope.

A nucleic acid means one according to the invention DNA or RNA molecules, which is an amino acid sequence encode the fusion proteins of the invention includes. The DNA and / or RNA is made according to methods known per se synthetic or natural Sources, for example eukaryotic or prokaryotic cells, for example bacteria, or viruses, for example bacteriophages, won. Appropriate methods are known to the person skilled in the art. Nucleic acids that used as a vaccine have additional sequences that one Enable use as a vaccine, for example as a DNA vaccine or improve. Corresponding sequences are known to the person skilled in the art. It is also advantageous to use the corresponding sequences CpG forms either synthetically or from bacteria. Appropriate methods are known to the person skilled in the art. Nucleic acids that for the Expression used in host cells include additional ones Sequences localizing the expressed active fusion proteins into the periplasm or in the form of secreted active fusion proteins cause. Corresponding sequences are known to the person skilled in the art, for example preferred sequences are listed in Tables 5 and 6.

In a preferred embodiment of the invention, the fusion peptide comprises a polypeptide linker, a secretory localization sequence and / or a periplasmic one Localization sequence. The polypeptide linker can comprise zero to 50 amino acids (see also above). The localization sequences enable in particular, that the fusion peptide or fusion protein at its Obtained in the periplasm or in the host cell supernatant. It goes without saying it possible in a preferred embodiment the localization sequences and / or the polypeptide linker (s) and / or to maintain the tag or tags on the fusion peptide. In a further preferred embodiment the localization sequences and / or the polypeptide linker (s) and / or the tag or tags after the fusion protein has been obtained cleaved.

In a further preferred embodiment the fusion peptide has at least one cleavage site between the Target protein and the domain D1 on. It is possible with advantage to provide several cleavage sites already in the coding vector, to find a suitable one for to contain the expression protein (see above).

In another embodiment In the invention, the expression protein is selected from the group consisting of from an antibody fragment, a single chain antibody, a multibody, a Fab fragment, a single chain MHC molecule, one MHC peptide fusion peptide and / or Dept. The expression peptides can also Expression proteins related to immune response can stand such as ligands, receptors, enzymes or other structural proteins.

In another embodiment In the invention, the multimeric fusion peptide comprises the same expression peptides or at least two different expression peptides.

The invention also relates to a nucleic acid molecule which encodes the fusion peptide according to the invention. The nucleic acid molecule in particular comprises a nucleic acid sequence which encodes one or more signal sequences for the transport into the periplasm or into the surrounding medium. The production of the nucleic acids and their introduction into bacteria is known to the person skilled in the art. Advantageously, additional expression vectors can be provided with additional tags and / or linkers. The technologies required for this are known in principle to the person skilled in the art and, with the aid of the disclosed invention, allow the production of such expression vectors. The nucleic acid molecule or a vector comprising the nucleic acid molecule can be introduced into suitable liposomes or host cells, it being possible for the enzymes required for the expression of the fusion peptide to be introduced into the liposomes. An expression vector system is obtained in this way. The expression vector system accordingly comprises fragments of the bacteriophage coat protein pIII, such as the domain D1 of the pIII of the filamentous bacteriophage M13. D1 is linked to an expression protein in the form of a fusion protein and is expressed either in the periplasm or soluble in the culture supernatant of bacteria. But host cells can also be used which make it possible to express soluble proteins, for example yeast cells or higher eukaryotic ones Cells, such as insect cells, preferably vertebrate cells, preferably mammalian cells, here preferably human cells and cell lines. For this purpose, the nucleic acids which encode the fusion protein additionally contain corresponding signal sequences which are known per se and which allow the protein to be expressed in a soluble form. Such signal sequences encode signal peptides, for example periplasmic localization signals or export signals, which are responsible for localization of the fusion protein in the periplasm or in secreted form outside the host cell, as are exemplified in Tables 5 and 6.

As further components, the Nucleic acids according to the invention for linkers encode that separate the D1 and the expression protein, and / or which are inserted between parts of the expression protein, such as scFv. The production of nucleic acids Encode variances of scFv or individual scFv or the extraction the nucleic acid sequences or relevant parts of other antibodies or antibody fragments from cells are known to the person skilled in the art.

Whether linker in the specific places are necessary or advantageous, or whether it is cheaper to link to certain Omission is dependent on the fusion protein and the expression protein expressed there and can be according to known Methods are determined. For example, it can be beneficial be the linker between the two domains of the variable chains of shorten scFv or omit, which results in multimerization of the antibody, which is known per se to the person skilled in the art. This multimerization is different from the multimerization via D1 domains described below.

As a further component, the the nucleic acids encoding the fusion protein also contain tags, for example for cleaning, detection, stabilization or Mark can serve. This Tags can both before, after or between individual components of the fusion protein, including are within the expression protein portion.

The examples explain in detail that the expression of expression proteins in the form of fusion proteins leads to the expression of active protein. In doing so, certain Proteins previously found in conventional Expression systems could not be actively expressed, actively expressed and others have increased activity. The examples also show that this is based on activation of the fusion protein and not on a raise expression alone. Moreover it also becomes clear that the effect is not due to multimerization comes about on its own because it also contains monomers that previously had no fusion were inactive on D1, in the form of the monomeric fusion protein according to the invention became active. Monomeric fusion proteins can be determined by chromatographic Methods that are explained in detail in the examples can be obtained.

The invention also relates to a Vector comprising the nucleic acid molecule especially pKJB3.

The invention also relates to a Phages comprising the vector, the part of the vector, the phage coat protein portion, for example the D1 domain or their functional analogue, does not encode the part of the phage coat protein coded, which serves to anchor it in the phage shell. anchoring proteins are all proteins that are used for integration into the phage shell.

The invention also relates to recognition molecules that directed against a fusion protein, the nucleic acid molecule and / or the host cell are.

In a preferred embodiment of the invention, the recognition molecule is an antibody, an antibody fragment and / or an antisense construct, in particular an RNA interference molecule. Prefers is the antibody a monoclonal or polyclonal antibody against the active protein, namely the expression protein, the fusion protein. With the help of the phages Display technology using active protein or active fusion protein as antigen, antibody phage display libraries can be obtained become. Methods for generating the recognition substances are known to the person skilled in the art known.

The invention also relates to a Vaccine comprising the fusion peptide, the nucleic acid molecule, the host cell and / or the recognition molecule optionally with a pharmaceutically acceptable carrier. The vaccine can in particular Include expression proteins that correspond to an abbot or one Mimic crypeptide that mimics a conformational epitope. Preferably include the vaccine is a monomeric fusion protein or multimeric fusion protein spanning the domains D1 or functionally analogous domains like certain structural parts of D2, or fragments thereof and a or several antibody fragments and / or at least one additional Adjuvant or other suitable substance to enhance the effect in a suitable solution contains.

The invention also relates to a method for the production of an active fusion protein comprising the steps:

• - provision of a nucleic acid molecule a fusion peptide comprising a domain D1 of a pIII or its Functional analogs and an expression protein,
• - bring in of the nucleic acid molecule into a Host cell
• - extraction of the one encoded by the nucleic acid molecule Fusion peptides from a periplasm and / or a host cell supernatant.

In the process, in addition to D1 homologs / analogs can also be used which have a D1 activity. For example this is a different phage coat protein of a filamentous Phages, for example pVI or pVIII, preferably a phage coat protein of filamentous Phage M13, particularly preferably domain 2 of pIII of the bacteriophage M13. The preferred form is the D1 or its Fumktionsanalog (see above). The provided procedure enables to be active efficiently To express fusion peptides or proteins. The with the inventive method Proteins or peptides obtained can be monomeric or multimeric available. The extraction of multimeric structures can be advantageous if, for example, good avidity should be achieved. Of course is it also possible win active monomers. Advantageously, the Proteins produced cannot be renatured such fusion peptides or proteins can be obtained by conventional Method or the use of known expression systems in one inactive form or in a reduced active form in bacteria were expressed. The method allows fusion peptides or proteins in an active one or a higher one active form can be expressed. The fusion protein can so selected be that it includes: at least one polypeptide linker length of 0 to 50 amino acids, one or more tags and / or one or more additional Cleavage sites between the phage coat protein and the expression protein. The expression protein can in particular be: an antibody fragment, a single chain antibody, a multibody, a Fab fragment or an MHC molecule / MHC peptide Fusion peptide. The invention thus provides a method that allows To express proteins in an active form in the periplasm of bacteria, which were previously not or less active when expressed in the periplama were. This is the protein that is to be actively expressed in the form of a fusion protein with a phage coat protein, preferably the pIII of filamentous B13 bacteriophages or homologues that are functionally analogous are expressed and through a periplasmic localization signal into the periplasm brought. There are a number of proteins that are also used in expression cannot be folded into an active form in the periplasm or the activity is relatively small. The method according to the invention provides the possibility by fusion of the proteins with a phage coat protein, preferably the pIII of filamentous Bacteriophage M13, or fragments thereof, when expressed to produce active proteins in the periplasm of such proteins, that could not previously be actively expressed in the periplama, or the activity of the proteins compared to expression in the periplasm without the phage coat protein portion increase. The proteins can can be used as active fusion proteins or via inserted proteolytic Cleavage sites by cleavage with cleavage molecules without the phage coat protein be used.

The expression protein portion preferably carries one day with the active protein after cleavage of the D1 portion can be cleaned from the other parts with the process. A preferred method is binding on a nickel column via a His day and the cleavage of the D1 portion with the help of the cleavage molecule. To followed by Washing, the active protein is eluted with imidazole. The corresponding Individual technologies are known to the person skilled in the art and can be used for the method be adapted and optimized.

The skilled worker is aware that he a method of obtaining a biological structure with others Can combine procedures. For example, the method according to the invention be combined with a purification process of the fusion protein, with a cleavage of the phage coat protein or its fragment from the expression protein using an or several specific cleavage molecules and / or with one cleaning of the active protein thus obtained. Furthermore, it is of course possible inventive method with a method of selecting an active fusion protein with the help of phage display technology phage coat in the expression of the fusion protein in the host cell as a fragment can be present. The combination with other display systems is also preferred, such as the ribosome display or its separate use.

With the method according to the invention can especially antibody fragments getting produced. About that can out Expression proteins can also be all other proteins contained in one active form must be expressed and / or which have a specific conformation which is based on a folding or interaction of amino acids of the expression protein or fusion protein. among them fall, for example, enzymes, antibody fragments, receptors, Ligands, structural proteins and others, but also peptides certain conformation or those that have a certain conformation Imitating the conformation immunologically.

The invention also relates to the use the fusion protein, the nucleic acid molecule, the vector, the phage, the host cell, the recognition molecule, the vaccine and / or the Drug in diagnostics and therapy.

In a preferred embodiment relates to the use of the fusion protein p, the nucleic acid molecule, the Vector, phage, host cell, recognition molecule, Vaccine and / or the drug initiate an immune response in a cell, a cell suspension, a tissue and / or a Organism.

In a further preferred embodiment, the phage according to the invention is in a pha gene bacterial system used. Here, a selection technology via the D1 domain is used in a phage bacterial system with an expression plasmid in combination with a helper phage rescue. This use within a technology differs from the known phage display systems. It does not include a phagemid or phage vector system. The use takes advantage of the surprising properties of the multimerization of the D1. The core of the use is an expression vector which comprises an expression protein and a D1 in the fusion protein according to the invention and the expression vector does not encode the part of the phage coat protein which is responsible for the anchoring in the phage coat, ie no anchor proteins are encoded which encode the invention Fusion proteins encoded, which comprises an expression protein and a D1 or its functional analogue, with the property that such expressed fusion proteins cannot integrate into the phage envelope, but only adhere to them via interactions. Bacteria which contain the expression plasmid or the vector are infected according to methods known per se with the filamentous helpferphage M13 known per se or with helper phages derived therefrom, for example VCS-M13 or KM13. A preferred variant of the helper phage is a deletion mutant in which DII or parts of DII deleted parts of the DII are mutated in order to avoid or reduce an interaction between D1 and D2. Such helper phages retain their infectiousness, which in principle can be reduced. It comes according to known methods for the production of phages. In addition to the pIII or modified pIII of the helper phage, these phages do not carry the fusion protein covalently. (The multimerization via D1 domain is preferably used here. However, D1-D2 domains can also be used in a special form. The phages, preferably the pIII, are preferably changed such that multimerization with the fusion proteins is enhanced suitable induction systems are used, such as, for example, the IPTG Lac promoter system or constitutive expression vectors (in order to increase the efficiency of the system). These phages according to the invention, which were produced by methods known per se, carry the genetic material of the expression vector contain the nucleic acid encoding the fusion protein, the genetic and thus the sequence information of the expression protein is coupled with the expressed active fusion protein on the surface of the phage.This serves for the identification and the multiplication of the fusion protein.Thus this system can be used as a selection system for Bibliot expression proteins can be used. The selection is carried out according to known methods of biopanning, in which the phages with the fusion protein are selected for their binding to a target structure, for example a protein, and / or unwanted phages are removed by depletion. Selected phages are applied to correspondingly suitable bacteria according to methods known per se for infection, and the bacteria to which the expression vector has been transmitted by infection are selected by antibiotic-resistant or other markers and can be cloned. The sequence of the selected expression proteins and / or fusion proteins can be determined from the clones by methods known per se, for example with the aid of PCR. A great advantage of the system is that no recloning into an expression vector is necessary to generate appropriate amounts of active protein or fusion protein. The system therefore does not require an amber-stop codon or a system similar to that in the case of the phagemid system. The expression rate of the active fusion proteins for obtaining them as a multimeric fusion protein or in connection with the phage is in most cases significantly better than with the conventional phagemid system. Another advantage of the system is that due to the smaller size of the expression vector compared to phagemid or phage vector systems, a larger variance or simply a large variance in different nucleic acids encoding different expression protein portions of the fusion proteins in connection with the infection of the bacteria in the Manufacturing libraries can be obtained. The variances of the library are usually produced synthetically by methods known per se by PCR methods known per se. It is also advantageous that the disclosed system allows simple and rapid expression of the multimeric fusion protein and it enables active proteins or active fusion proteins to be obtained directly. Many of these fusion proteins are active, which are not or only slightly active compared to conventional phagemid systems (see above).

The production of Antibody libraries and the selection of antibodies with the help of technology. Due to the multimerization, that expression proteins can also be selected that a comparably low affinity of a single fusion protein to the binding partner. The latter are replaced by conventional ones Selection systems often not identified. This problem can be caused by a multiple display due to binding to several pIII derived proteins (D1 and Functional analogues) of the phage.

The invention also relates to the use the fusion protein, the nucleic acid molecule, the vector, the phage, the host cell, the recognition molecule, the vaccine and / or the Drug in research, diagnostics and therapy.

Use is preferred research, diagnosis and therapy of tumors, inflammation, Autoimmune diseases and / or infectious diseases.

Use is particularly preferred to modify or prevent angiogenesis.

The invention accordingly also relates to Use of multimers according to the invention Fusion peptides or proteins for obtaining monomeric forms as well as the monomeric forms themselves.

In a further preferred variant The invention uses multimeric fusion proteins for immunization used. As stated in the examples, immunization is achieved a strong immune response with the multimers. The D1 component acts as an adjuvant, with the unexpectedly high immune responses likely come about because technology has an advantage causing slow release of each fusion protein comes from the multimer, which is underpinned by the fact that it is a Giving balance of multimers and individual fusion proteins can. Such slowed-down release can have a depot effect or have a functionally similar effect to the depot effect. Both is otherwise relative to other adjuvants, for example, by large bad to characterize and standardizable and therefore for In clinical use, GMP only has limited suitability for giant molecules such as reaches the KLH (Keyhole limpet hemocyanin) or water-oil mixtures. This Technology can take the form of a vaccine with appropriate antigens as expression proteins for immunization against different Diseases, for example tumor or infectious diseases serve or for the production of antibodies by immunization of Animals, for example mice, be used. In a preferred embodiment, Ab2 scFv in the form of homo- or hetero-multimeric fusion proteins as vaccines or administered for immunization to an immune response against the original antigen to achieve. This is advantageous for tumor therapies, for example to break anergies. Ab2 that mimic a tumor epitope in the form of a multimeric fusion protein in suitable pharmacological Formulations, doses and vaccination intervals are administered to provide an immune response to achieve against tumor cells that carry the tumor antigen (s). Analogously, over this system also mimicry peptides from antigens that are active Have conformation or which are not proteins in active multimers Fusion proteins advantageous for vaccination against diseases used.

Another preferred embodiment is the use of multimeric fusion proteins with ligands or receptor-inhibiting molecules, for example in the form of antibody fragments or peptides, as an expression protein portion. This serves for example as an activator or inhibitor of receptor-mediated processes. So that can secondary processes be controlled in the cells or the interaction between cells be modulated. For example, the interactions inhibited by tumor cells with endothelial cells in the blood, whereby it reduces or prevents an inflammatory process or metastasis is coming. These processes are ongoing thereby, for example, via selectin-mediated Adhesion of cells to the endothelium by antibodies or Mimikrymoleküle, Ab2 or mimicry peptides, can be prevented. The application in the multimeric form according to the invention is included advantageous as it is comparatively due to the increased avidity strong inhibition comes. Another example is interaction of the scFv L36 with laminin, which can prevent angiogenesis. The application technologies and forms of administration are beyond Technologies described in the examples are also known per se.

The invention is based on the following an example closer explained are limited to this without to be:

example 1

manufacturing of the pKBJ construct

The vector pUC119His6MycXbaI was used as the starting vector for the production of the pKBJ vectors. An scFv antibody was cloned between the HindIII and NotI sites of the vector. Gen III (Nucleotide Accession Code: V00604) from pHEN2 (www.mrc-cpe.cam.ac.uk/~phage/g1p.html) was amplified by PCR using specific primers for each vector. pKBJ1 was produced by PCR amplification with the primers gIII N-term NotI and DII-DIII EagI and by subsequent cloning of the EagI-cut PCR product into the NotI site of the scFv-containing pUC119His6MycXbaI. For pKBJ2, gene III was amplified with the primers VL-Link and DII-III OpaI-EcoRI, digested with NotI and EcoRI and cloned into the corresponding sites in the starting vector. The QuikChange mutagenesis kit (Stratagene) and the primers gIII N-term QC Amber-back and gIII N-term QC Amber-fw were used to remove the amber stop codon between the scFv and GenIII in pHEN2. The pKBJ3 construct was prepared analogously to pKBJ1 with the primers gIII N-term NotI and DI-DII EagI. The three constructs are all in 1 shown schematically. Three different scFv antibody fragments L36, D4 and XR5 were subcloned from the phagemid pHEN2 into three pKBJ vectors and the vector pUC119 using the restriction enzymes Nco1 and Not1. The DNA encoding the D1 fusion protein of the XR5 was subcloned from pKBJ3 into the pROOSTER vector encoding a kanamycin resistance using the restriction enzymes HindIII and EcoRI. All constructs were then primed with M13-Rev, VL-link and M13-20 using the 373A sequencer (Applied Biosystems) and the ABI Prism Dye Terminator sequencing kit (Per kin-Elmer) sequenced. All enzymes were ordered from New England Biolabs unless otherwise specified and used according to the manufacturer's instructions. The primers used are listed in Table 1.

Table. 1: List of primer sequences

Expression of aritic bodies

The clones were picked from TYE plates and overnight at 37 ° C. in 2xTY [J. Sambrook, Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, New York, (1989)] cultured with 100μg / ml ampicillin and 1% glucose, then diluted 1: 100 in 2xTY with 100μg / ml ampicillin and 0.1% Glucose and shaken for 4 hours at 37 ° C. The cultures were induced by adding 1mM IPTG and incubated overnight at room temperature. The cells were pelleted at 6000xg, resuspended in 50mM Na _x H _y PO ₄ pH 8.0 and lysed in a French press (American Instruments Co., Inc. Silver Spring, MD, USA). Before the immobilized metal affinity chromatography (IMAC) the suspension was centrifuged at 26000xg and the supernatant was mixed with 30mM imidazole and 300mM NaCl. Ni-NTA was incubated with the supernatant for 2 hours at 4 ° C. and then with at least 100 ml wash buffer (50 mM Na _x H _y PO ₄ pH 8.0; 30 mM Imidazole and 300 mM NaCl) and then 50 ml high salt wash buffer (50 mM Na _x H _y PO ₄ pH8.0; 30mM imidazole and 750mM NaCl). The protein was eluted with 300mM imidazole in water buffer. The concentrations were determined according to Bradford [MM Bradford Anal Biochem 72 (1976) 248-254] and are given in Table 2 for the various antibodies and their pKBJ constructs and the purity by means of SDS-PAGE [UKLaemmli, Nature 227 (1970) 680- 685] analyzed ( 2 ).

Table 2

Affinity determination using ELISA

For ELISAs with the pKBJ derivatives of the scFv antibodies L36 and D4, the plates (Maxi-sorp ^TM , NUNC, Roskilde, Denmark) with laminin-1 (Sigma) or fibronectin (Sigma) in a concentration of 0.5 μg per well coated in 50mM NaHCO ₃ pH 9.6 overnight at 4 ° C. The remaining binding sites on the plastic surface were then blocked with 2% MPBS (2% w / v skim milk powder in PBS) for 2 h, the antibody derivatives were added in various concentrations and incubated for 1 h. The plates were then washed 6 times with PBS and the bound antibody with the murine antibody 9E10 (ECACC), which recognizes the c-myc tag of the antibodies, was detected in a concentration of 0.5 μg / ml. After 6 further washes in PBS, the plates were incubated with 1: 1000 diluted HRP-conjugated rabbit anti-mouse antibody (DAKO) in 2% MPBS. To 6 Washing steps, the reaction with o-phenylenediamine tablets (DAKO, Denmark) was developed according to the manufacturer's instructions ( 3.a . b ).

In order to test the activity of the scFv XR5 and its derivatives, the MUC1 antibody A76-A / C7 was coated with a concentration of 0.1 μg / well overnight in PBS. The remaining binding sites were blocked with 2% BSA (Sigma) in PBS for 2 h and the plates were then incubated for 2 h with the antibody derivatives. The XR5 antibody was detected with a polyclonal rabbit antibody against c-myc (A-14; Santa Cruz Biotechnology) and then with an HRP-conjugated pig anti-rabbit antibody (DAKO, Denmark). Between the incubation steps, the plates were washed 5 times with PBS ( 3.c ).

stability measurements

To see the effect of the merger on the stability of the antibody fragments Activity determinations were to be examined after incubation of the fusion proteins at different temperatures carried out for different periods. All L36 and XR5 constructs showed no change in activity after incubation up to 4 days at room temperature, 30 ° C and 37 ° C.

The stability was exemplified under denaturing conditions based on the constructs of L36 examined by using the constructs with the help of guanidinium chloride (GdmCl) induced folding removal using "Steady State Fluorescence Spectroscopy "was determined.

Purified L36 fusion scFv was diluted to a final concentration of 1μM in various concentrations of guanidine chloride (GdmCl) in 10mM Tris-HCl pH8 and transferred to quartz cuvettes with a path length of 3mm. The cuvette was placed in an RTC2000 fluorimeter with a 75 W xenon arc lamp (Photon Technology International, Lawrenceville, NJ) and an emission spectrum of wavelengths from 300 to 400 nm was recorded (⎕ _ex 285 nm, excitation slot width 2 nm, emission slot width 6 nm). Folded scFV had an emission maximum at 330nm, while the maximum for unfolded scFv was 355nm. The ratio between the fluorescence of these two wavelengths was therefore determined as the signal to be determined [GdmCl] _50% [MM Santoro, Biochemistry 27 (1988) 8063-8068 gel filtration analyzes].

[GdmCl] _50% , the concentration required to denature half of scFv-L36, pKBJl-L36, pKBJ2-L36 and pKBJ3-L36 was 2.73 ± 0.11 M, 2.70 ± 0.07 M, 2.67 ± 0.03 M and 2.41 ± 0.09 M .. It follows that the observed increase in activity is not due to increased stability.

gelfiltration

Gel filtration analyzes were performed to determine whether the antibody fusion proteins mutimerize. Gel filtration was carried out on a TSK-Gel G3000 SW column with a pre-column (ToSoHaas) using HPLC (Biotek instruments). To correlate the retention times with the globular molar mass, bovine plasma fibronectin, murine IgG, BSA and GST were applied to the column and the protein was detected using a diode array detector 540+ (Biotek Instruments). At least 1 mg of scFv fusion protein from each construct was applied to the column and the fractions were collected for subsequent analysis in the ELISA ( 4 ) Table 3 shows the percentages of monomers, dimers and multimers for the different antibodies and their constructs.

The D1 fusion proteins show the highest activity on multimers and the multimerization ability was therefore further investigated. In order to test whether the purified monomers, dimers and multimers are stable with regard to multimerization, they were incubated for 36 h at 37 ° C and then examined on a TSK-Gel G3000 SW column ( 5 ). The results show that the multimerization is conditionally reversible and equilibria are established.

GX4000 column separation analysis shows that tetramerization is by far the most common form of multimerization of D1 fusion proteins ( 6 ).

The possibility of generating hetero-multimeric fusion proteins was examined by double transformation of the D4 antibody fragment as D1 fusion protein in the pKBJ3 vector and the XR5 D1 fusion protein in the pROOSTER vector in E. coli with subsequent expression and purification. A sandwich ELISA of the purified fusion protein shows the formation of hetero multimers ( 7 ).

Table 3

Each scFv construct was created using HPLC gel filtration analyzed. This method differentiates between Monomers and dimers as single peaks, whereas multimers be eluted in the exclusion volume. The proportion of monomer, dimer and Multimer varies with the different constructs (see table). The numerical values give the area below the gel filtration curve. There were three different ones for each scFv Constructs compared - pUC119 (unfused scFv), pKBJ1 (scFv fused to domains I-II) and pKBJ3 (scFv merges with domain I).

Immunization of Balb / c mice

The mice were injected with purified protein expressed in either the FuncFAb format or the scFv format, with or without emulsion in Freund's Incomplete Adjuvant (FIA). After a boost immunization on day 14, the mice were sacrificed on day 28 and blood was taken to test the serum for scFv-specific antibodies of the IgM and IgG type. Compared to day -1, scFv-specific antibodies were generated in all mice tested, although the titers and isotypes varied. Injection of the scFv-L36 alone induced a limited IgG response in one of the mice ( 8.a ), possibly analogous to the HAMA reaction observed in humans after administration of murine antibodies, in which case the mouse produces a mouse anti-human antibody response. The addition of FIA significantly increased the IgG response, but the IgM response was not changed (a third mouse died during immunization) ( 8.b ). The mice immunized with the scFv-L36 in pKBJ1 format showed a stronger immune response than the mice injected with scFv-L36 alone, but this response was not as strong as when immunized with FIA ( 8.c ). The effect of the protein III domain I fusions of the scFv-L36, however, differed from the other immunizations. In addition to a positive effect on the IgG response compared to the immunizations with scFv-L36 alone and pKBJ1-L36, an IgM response was generated which did not occur with the other immunizations ( 8.d ).

The fusion proteins with domains I-II and domain I of Protein III produced an immune response against the fusion partner (scFv-L36) and can therefore used as an adjuvant. The observed effect on however, the IgG response is not as pronounced as when using FIA. In addition, the effect of Domain I alone appears to be better than the combination with domain I-II, which is partly due to the composition of the protein contained in the mouse was injected can be.

It has been reported that proteins as components of complexes are better immunogens (Claassen et al., 1998; Morein et al., 1984). The antibodies are in the complex that through the domain I-Fusion is created, better presented, since it is more active than the complex created by the merger with domain I-II is formed. It can also be discussed whether the observed Adjuvant effect of the protein III fusion products Chaperone properties are imparted because of such properties recently have been associated with potent adjuvants (Brenner and Wainberg, 1999).

The anti-idiotypic answer

L36 is an antibody obtained by phage display, which is directed against human, purified laminin-1 and is able to inhibit tumor neovascularization (Sanz et al. 2002; Sanz et al. 2001). Preliminary results indicate the presence of anti-idiotypic antibodies in the serum, since the sera of the mice immunized with scFv-L36 in FIA, L36-pKBJ1 and L36-pKBJ3 have a dose-dependent effect on the binding of scFv-L36 to laminin -1 own ( 9 ). In addition, the sera of the mice immunized with scFv-L36 in FIA and L36-pKBJ3 and to a lesser extent also the mice immunized with L36-pKBJ1 recognize a polyclonal rabbit anti-laminin-1 antibody ( 10 ). This indicates that a certain epitope of laminin-1 is present in the serum of the immunized mice.

Legend for the figures:

1 Schematic structure of the fusion proteins

Schematic representation of the domain structure of the pIII protein of the filamentous bacteriophage. The amino acid sequence is marked in color in accordance with the schematic domain structure. The pelB signal sequence is violet, the three pIII domains are blue and the connecting linkers are shown in red. The transmembrane domain, on the other hand, is marked in gray. The three pKBJ constructs 1-3 are marked in the schematic representation with the same colors for the corresponding pIII domains. The positions for the tags are shown in green and for the scFv portions in brown. The pKBJ2 has the same tag structure as in the phage display system, while the pKBJ1 and pKBJ3 have the tags arranged at the C-terminal. The tag sequence includes a c-myc tag (AAEQKLISEEDLNGAA) and a hexa-histidine tag.

2 : SDS gel of the various antibodies

SDS-PAGE of four different constructs for each of the three scFvs- D4 (lanes 1-4 ), L36 (lane 5-8 ) and XR5 (lane 9- 12 ). The four constructs are pUC119 unfused scFv (lane 1 . 5 and 9 ), pKBJ3 (lane 2 . 6 and 10 ), pKBJ1 (lane 3 . 7 and 11 ) and pKBJ2 (lane 4 . 8th and 12 ).

3.ac : ELISA of the seFv, and the scFv constructs in pKHJ1, pKBJ2 and pKBJ3 on laminin, fibronectin and A76-A / C7.

und pKBJ2

welche wiederum eine höhere Aktivität als der nicht-fusionierte scFv, pUC119 (⧫) besitzen. In den Verdünnungsreihen sind Mittelwerte aus Zweifachbestimmungen dargestellt.ELISA of the three different scFv and the pKBJ constructs of these scFvs, showing the binding of D4 to fibronectin from L36 to laminin, and of XR5 to the mouse monoclonal antibody A / C7. The domain I fusion construct pKBJ3 (•) is generally more active than the domain I-II fusion construct pKB1

and pKBJ2

which in turn have a higher activity than the unfused scFv, pUC119 (⧫). Average values from double determinations are shown in the dilution series.

4 , Activity in the preparative HPLC fractions

The activity of the various stages of multimerization The pKBJ1 and pKBJ3 constructs were used for the different constructs of the individual scFv when using equal amounts of monomer (empty Bars), dimer (hatched bars) and multimer (filled bars) compared. The pKBJ3 multimers are generally active, but not the pKBJ1 multimers from L36 and D4. L36 appears to be the most active as a dimer to be indicative of a tendency of this scFv to form active dimers suggesting.

5

HPLC gel filtration was carried out with a GX3000 gel filtration column for R5 pKBJ3 fusion protein performed. A) shows the purified monomer after 36 h at 37 ° C, b) shows under the same conditions the data for cleaned Dimereundc) shows the data for cleaned under the same conditions Multimers (trimers, tetramers, pentamers ...). d) shows a gel filtration diagram shows of 10 times concentrated monomer. The retention times correspond in all cases from 21, 18 and 15 minutes to the monomers, dimers and multimers. Gel filtration analysis using the GX3000 column separation allows only that Separation of monomers, dimers and higher multimers.

6

HPLC gel filtration with a GX4000 Pillar enables with a size exclusion of 7MDa the separation of larger multimers The data show that tetramers the most common occurring multimers.

7

und anschließend mit R5/D4 pKBJ3 Fusionsprotein inkubiert und die Bindung mit A/C7 und antimouse HRP Antikörper nachgewiesen.. Beide Tests zeigen eine Konzentrationsabhängige Bindung.Analysis of the hetero-multimerization of pKBJ3 fusion proteins (D1 fusion proteins) using a sandwich ELISA. The ELISA was performed by immobilizing A / C7 in the wells (⧫) and detection of R5 / D4 pKBJ3 fusion protein binding using fibronectin, fibrinogen and anti-Rabbit-HRP antibody binding. Fibronectin was immobilized in a mirror-image ELISA

and then incubated with R5 / D4 pKBJ3 fusion protein and the binding with A / C7 and antimouse HRP antibody was detected. Both tests show a concentration-dependent binding.

8.ad : Immunizations of half / c mice with the antibody L36

3 mice were treated on day 0 and day 14 with 50 μg purified L36 either as a) scFv-L36 alone, b) scFv-L36 in combination with incomplete Freund's adjuvant, c) scFv-L36 as a fusion protein with domains I and II immunized by protein III and d) scFv-L36 as a fusion protein with domain I of protein III. Blood was drawn from the mice on the day before immunization (-1) and after day 28 (+28) and the sera were examined for scFv-L36-specific IgM and IgG antibodies in two dilutions 1:50 and 1: 250. The error bars show the standard deviation of 2 independent measurements.

9 : Dose-dependent effect of the sera on L36 binding to laminin.

• A) L36 detection with 1:50 or 1: 250 diluted sera of mice, those with L36-scFv (A1, A2 and A3), L36-scFv in FIA (B1 and B2), L36 merges with domain I-II (C1, C2 and C3) and L36-scFv fused to domain I (D1, D2 and D3) were immunized prior to immunization (day -1) and after immunization (day 28).
• B) Competition of L36 binding to immobilized laminin-1 with sera (dilution 1:15, 1:45 and 1: 135) from mice, which merges with L36-scFv (A2 and A3), L36-scFv in FIA (B1 and B2), L36 to domain I-II (C2 and C3) and L36-scFv merges with domain I (D1 and D2) were immunized before immunization (day -1) and after immunization (day 28).

10 : Ligand analysis using anti-idiotypic antibodies.

Detection of rabbit anti-EHS laminin polyclonal antibodies using 1: 25-diluted Sera of mice, which merges with L36-scFv (A2 and A3), L36-scFv in FIA (B1 and B2), L36 to domain I-II (C2 and C3) and L36-scFv fused to domain I (D1 and D2) immunized were.

Table 4: Cleavage site for proteolytic enzymes.

Table 5: Periplasmic localization signals

Table 6: Secretory localization signals

Claims (26)

Fusion protein comprising an expression protein and a phage coat protein fragment, characterized in that the phage coat protein fragment is at least one domain D1 or a functionally analogous fragment of a pIII of a filamentous bacteriophage M13. Fusion protein according to claim 1, characterized in that there is at least one secretory localization sequence, one periplasmic localization sequence, a polypeptide linker and / or spanned a day. Fusion protein according to claim 1 or 2, characterized in that that there is at least one cleavage site between the expression protein and the domain D1 has. Fusion protein according to one of Claims 1 to 3, characterized in that that the expression protein is selected from the group consisting of from an antibody fragment, a single chain antibody, a multibody, a Fab fragment, an MHC molecule, an MHC molecule as a single chain, an MHC peptide fusion peptide and / or Ab2. Fusion protein according to one of the preceding claims, characterized characterized that it is a homomultimer or heteromultimer Is fusion protein. Nucleic acid molecule comprising a nucleic acid sequence, which encodes a fusion protein according to any one of claims 1 to 5. Vector comprising a nucleic acid molecule according to claim 6, in particular pKBJ3. Phage comprising a nucleic acid molecule according to claim 6 and / or a vector according to claim 7. Host cell comprising a vector according to claim 7 and / or one Phage according to claim 8. Recognition molecule against a fusion protein according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a phage according to claim 8 and / or a host cell is directed according to claim 9. recognition molecule according to the preceding claim, characterized in that it an antibody, an antibody fragment and / or an antisense construct, in particular an RNA interference molecule. Vaccine comprising a fusion protein according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a phage according to claim 8 and / or a host cell according to claim 9 optionally with a pharmaceutical acceptable Carrier. Medicament comprising a vaccine according to claim 12. A method for producing a fusion protein comprising the Steps: - provision of a nucleic acid molecule a fusion peptide comprising a domain D1 of pIII and a target protein, - bring in of the nucleic acid molecule into a Host cell - extraction of the nucleic acid molecule Fusion peptide from a periplasm and / or a culture supernatant of the host cells. Method according to the preceding claim, characterized in that that the fusion protein using a combinatorial technique is selected, in particular a phage display technique. A method according to claim 14 or 15, characterized in that that the expression protein with the help of cleavage molecules from D1 is separated. Method according to one of claims 14 to 16, characterized in that that as the expression protein an antibody, a single chain fragment, Multibody, a Fab fragment, an MHC molecule, an MHC peptide fusion peptide and / or an Ab2 can be used. Use of a fusion protein according to any one of claims 1 to 5, a nucleic acid molecule Claim 6, a vector according to claim 7, a phage according to claim 8, a host cell according to claim 9, a recognition molecule according to claim 10 or 11, a vaccine according to claim 12 and / or a medicament according to claim 13 in research, diagnostics and therapy. Use according to claim 18 for research, diagnosis and therapy of tumors, inflammation, autoimmune diseases and / or infectious diseases. Use according to claim 18 or 19 for modification or prevention angiogenesis. Use according to one of claims 18 to 20 for initiation an immune reaction in vivo or in vitro of a cell, a cell suspension, a tissue and / or an organism. Use according to one of claims 18 to 21 for control of secondary processes in a cell and / or to modulate an interaction between Cells. Use of a nucleic acid molecule according to Claim 6, a vector according to claim 7 and / or a phage according to claim 8 in a phage bacterial system. Use according to claim 23, characterized in that in a phage display system a nucleic acid molecule according to claim 6 and / or a vector according to claim 7 in combination with a helper phage used for the selection of expression proteins from libraries become. Use according to claim 24, characterized in that the Helper phage in a domain D2 has a mutation. Use according to one of claims 18 to 25 for the production of antibody libraries and for the selection of antibodies.