DE10123041A1 - New single-chain human antibody fragment, useful for treating or diagnosing hepatitis C virus infection, has affinity for an essential viral protein - Google Patents

Abstract

Single-chain fragment (I) of a human antibody that inhibits replication of hepatitis C virus (HCV), and comprises the variable regions (Vl and Vh) of the light and heavy chains of an antibody directed against at least one essential viral protein, is new. Independent claims are also included for the following: (1) DNA sequence (II) that encodes (I); (2) (gene transfer) vector containing (II); (3) identifying antibody fragments that inhibit replication of HCV; (4) host cells, preferably prokaryotic, that are transformed with the vector of (2) and/or contain (II); and (5) preparing (I) by growing cells of (4).

Description

The present application relates to human antibody fragments which inhibit the proliferation of hepatitis C virus and which control the variable regions of the light chain (V _L ) and heavy chain (V _H ) of antibodies against one or more essential proteins of hepatitis C virus. Virus include. The invention further relates to the use of the antibody fragments for the treatment and diagnosis of hepatitis C, DNA sequences which code for these antibody fragments and the use of these DNA sequences in vector constructs for gene therapy for hepatitis C.

Background of the Invention

The treatment of hepatitis C is one of the greatest challenges at the moment gene pharmaceutical research. Antibodies are known to attack the Bind hepatitis C virus (HCV) antigens (e.g. from WO 93/04084 and WO 00/05266), but these antibodies do not have a sufficiently large binding affinity to inhibit the proliferation of HCV in vivo. Furthermore is murine, d. H. non-human antibodies and they are complete dige, intact antibodies, which are unstable due to their disulfide bridges in the Cytoplasmic reducing conditions prevail. These antibodies are therefore unsuitable for gene therapy purposes in humans.

HCV has a (+) - stranded RNA genome that is unique to a single polyprotein encoded about 3000 amino acids. This single polyprotein is made by proteases of the host or of the virus co- or post-translationally into functional viral proteins cleaved (Grakoui A. et al., J. Virol., 67: 1385-95 (1993); Bartenschlager R. et al., J. Virol., 68: 5045-55 (1994); Hijikata M. et al. Proc. Natl. Acad. Sci. 90: 10773-7 (1993)). This is one of the suspected structural proteins Core protein (C) and two coat proteins (E1 and E2), while the nonstructured len proteins (NS2, NS3, NS4, NS5) presumably components of a complex are responsible for viral RNA replication (Bartenschlager R. et al., J. Virol., 68: 5045-55 (1994); Suzich J.A. et al., J. Virol., 67: 6152-8 (1993);  

Steinkuhler, C. et al., J. Virol., 70: 6694-700 (1996); Tomei L. et al., J. Virol., 67: 4017-26 (1993)).

The HCV-NS3 protein (see SEQ ID NO: 18) is a bifunctional enzyme that Protease activity in the N-terminal third and RNA helicase activity in the C- terminal position (Bartenschlager R. et al., J. Virol., 68: 5045-55 (1994); Tomei L. et al., J. Virol., 67: 4017-26 (1993)). It has been reported that HCV-NS3 is probably processed by cellular proteases (Shoji I. et al., Virology, 254: 315-23 (1999)). The protease and helicase domains of NS3 are active in vitro in the absence of the other domain (Wardell A. D. et al., J. Gen. Virol., 80: 701-9 (1999); Kim D.W. et al., J. Virol., 71: 9400-9 (1997); Kolykhalov A.A. et al., J. Virol., 68: 7525-33 (1994)). NS3 protease belongs to the serine protease family and is for the processing of the HCV poly proteins at the junctions NS3 / NS4A (cis-cleavage), NS4A / NS4B, NS4B / NS5A and NS5A / NS5B (trans-cleavage) responsible (Tomei L. et al., J. Virol., 67: 4017-26 (1993); Hahm B. et al., J. Virol., 69: 2534-9 (1995); bar tenschlager R. et al., J. Virol., 68: 5045-55 (1994); Bartenschlager R. et al., J. Virol., 67: 3835-44 (1993); Tomei L. et al., J. Virol., 67: 4017-26 (1993)). The Enzymatic activity of the NS3 protease depends in part on complex formation with NS4A (Hahm B. et al., J. Virol., 69: 2534-9 (1995); Failla C. et al., J. Virol., 68: 3753-60 (1994); Bartenschlager R. et al., J. Virol., 69: 7519-28 (1995); Koch J. O. et al., Virology, 221: 54-66 (1996); Lin C., Rice C. M., Proc. Natl. Acad. Sci. 92: 7622-6 (1995)).

The structure of the NS3 protease shows what a flat substrate binding pocket it is makes it difficult to design effective protease inhibitors (Kim J.L. et al., Cell, 87: 343-55 (1996); Yan Y. et al., Protein Sci. 7: 837-47 (1998)). The Helicase activity consists of a polynucleotide-stimulated NTPase activity, a 3 '→ 5' relaxation activity and a binding activity for single stranded nucleotides (Wardell A.D. et al., J. Gen. Virol., 80: 701-9 (1999); Gwack Y. et al., Eur. J. Biochem., 250: 47-54 (1997); Preugschat F. et al., J. Biol. Chem., 271: 24449-57 (1996)). The crystal structure of NS3 helicase was (Yao N. et al., Nat. Struct. Biol., 4: 463-7 (1997); Cho H. S. et al., J. Biol. Chem., 273: 15045-52 (1998); Kim J.L. et al., Structure, 6: 89-100 (1998)).  

Helicase activity appears to be necessary to single-stranded Unwinding RNA during duplication of the viral genome (Wardell A. D. et al., J. Gen. Virol., 80: 701-9 (1999); Gwack Y. et al., Eur. J. Biochem., 250: 47-54 (1997)). HCV-NS3 helicase is therefore a promising antivi ral target that is essential for viral replication.

Single chain variable fragments (sFv) of antibodies contain variable domains of heavy (V _H ) and light chain (V _L ), which are linked by a polypeptide linker, which makes them a fully functional antigen-specific binding unit (Marasco WA, Gene Ther., 4: 11-5 (1997; Chen SY et al., Hum. Gene Ther., 5: 595-601 (1994)). Both variable domains are expressed on a protein chain, which makes the sFv fragments stable compared to the reducing environment found in the cytoplasm. The intracellular sFv can be expressed directly within cells, creating an active molecule for protein-based gene therapy. The expression of human sFv should minimize the immune response against the foreign transgene (Marasco WA, Gene Ther., 4: 11-5 (1997; Chen SY et al., Hum. Gene Ther., 5: 595-601 (1994); Rader C., Barbas Cr., Curr. Opin. Biotechnol., 8: 503-8 (1997)).

It is known in the art that the intracellular expression of sFv- Fragments act as "intra-bodies" of various intracellular proteins can inhibit. Thus shows the intracellular expression of anti-tat intra-bodies inhibition of HIV replication after retroviral transduction (Marasco W.A. et al., J. Immunol. Methods, 231: 223-38 (1999); Mhashilkar A.M. et al., Hum. Gene Ther., 10: 1453-67 (1999); Poznansky M.C. et al., Hum Gene Ther., 9: 487-96 (1998)). Inhibition of HIV integrase by sFv intra-bodies resulted on the resistance of cultured cells to HIV infection (Kitamura Y. et al., 20: 105- 14 (1999)). This was also possible after the retroviral transduction of cells an sFv-expressing vector can be observed (Bouhamdan M. et al., Gene Ther., 6: 660-6 (1999)). The phenotypic knockout of interleukin-2 Receptors were developed by transducing primary T cells with a lentiviral Reached vector expressing IL-2 receptor binding sFv fragments in the ER (Richardson J.H. et al., Gene Ther., 5: 635-44 (1998)). Inhibition of CREB and the activating transcription factor 1 (ATF-1) reduced the malignancy  potential of melanoma cells significantly (Jean D. et al., Oncogene, 19: 2721-30 (2000)). Inhibition of the oncogene erB-2 by adenoviral Vectors expressing anti-erB-2 sFv led to the regression of the malignant Phenotype of ovarian tumor cells (Deshane J. et al., J. Clin. Invest., 96: 2980- 9 (1995)).

In addition, clinical trials will be conducted with patients who are under ovary cancer suffered. Adenoviral vectors are used to Express anti-erB-2 intrabodies (Deshane J. et al., J. Clin. Invest., 96: 2980-9 (1995)). Other groups use a retroviral MuLV vector, anti-gp120 intra-bodies in helper T cells in HIV-infected patients express (Marasco W.A. et al., J. Immunol. Methods, 231: 223-38 (1999); Chen S.Y. et al., Proc. Natl. Acad. Sci. USA, 91: 5932-6 (1994)).

The object of the present invention was to develop special antibody derivatives to provide vate that effectively inhibit the proliferation of hepatitis C and which can be used intracellularly, making them suitable for gene therapy Applications are suitable. It has been found that special single chain Antibody fragments against hepatitis C virus (HCV) essential proteins, especially high affinity human single chain (sFv) antibody fragments against HCV-NS3, which is from a large library of recombinant antibodies elements have been selected that meet these requirements.

Summary of the invention

The present invention thus relates

• 1. a single-chain fragment of a human antibody that inhibits the multiplication of hepatitis C virus (hereinafter also referred to as "HCV") and which the variable regions of the light chain (V _L ) and the heavy chain (V _H ) one Antibody to one or more HCV essential proteins;
• 2. in a preferred embodiment of (1) inhibits the single-chain Fragment of an HCV NS3 protein, in particular NS3 protease and / or NS3 Helicase, particularly preferably NS3 helicase;
• 3. a DNA sequence which is suitable for an antibody fragment according to (1) or (2) encoding;  
• 4. a vector or gene transfer vector comprising a DNA sequence according to (3);
• 5. A method for identifying antibody fragments which inhibit the multiplication of HCV according to (1) or (2)
  • a) Cloning of a DNA library encoding antibody fragments against one or more essential proteins of HCV;
  • b) Expression of said antibody fragments on the surface of bacteri phages with subsequent enrichment of the phages which carry antibody fragments with high affinity for the essential protein by means of several cycles of selection and reamplification (panning); and continue if necessary
  • c) multiplication of said antibody fragments with high affinity by soluble expression in bacterial cells and isolation of the antibody fragments from the periplasm of these cells;
• 6. a host cell, in particular a prokaryotic host cell, with a Vector is transformed according to (4) and / or a DNA sequence according to (3) contains;
• 7. a method for producing an antibody fragment according to (1) or (2), comprising culturing a host cell according to (6);
• 8. a pharmaceutical or diagnostic composition containing one or contains several antibody fragments according to (1) or (2);
• 9. the use of the antibody fragments according to (1) or (2) as a vaccine in particular for passive immunization;
• 10. a pharmaceutical composition containing one or more Gene transfer vectors according to (4);
• 11. the use of the gene transfer vector according to (4) for gene therapy, especially for gene therapy of HCV infections in humans; and
• 12. a gene therapy method, in particular a method for the treatment of HCV infections comprising administering the gene transfer vector according to (4) to a patient.

Brief description of the figures

Fig. 1 shows the serum antibody titers of 4 patients with chronic HCV infection against recombinant NS3 protein, which were determined by EIA.

Figure 2 shows the results of gel electrophoresis of sFv cDNA fragments generated by immunoglobulin specific PCR.

Figure 3 shows the enrichment of phages with sFv fragments that show binding affinity for recombinant NS3 during various passes of affinity selection (panning).

Figure 4 shows the identification of monoclonal phages carrying sFv with binding activity against NS3 protein by phage EIA.

Figure 5 shows EIA of purified soluble sFv fragments against NS3.

Fig. 6 shows the immunoblot analysis of bacterially expressed recombinant sFv fragments.

Figure 7 shows the amino acid sequence of the variable regions of the H and L chain of selected human sFv.

Fig. 8 shows the activity of bacterially expressed sFv, which were purified to homogeneity by Ni-NTA columns, in the competition EIA.

Detailed description of the invention

The single-chain fragment of a human antibody according to embodiment (1) of the invention (hereinafter also referred to as "antibody fragment") preferably inhibits at least one essential protein of HCV, but can also inhibit several. The antibody fragment preferably has an affinity K _D ≦ 10 ^-6 M and particularly preferably ≦ 10 ^-7 M (determined by a competition ELISA as described, for example, under "Materials and Methods") for at least one essential virus protein. "Essential virus proteins" in the sense of the present invention include surface proteins (envelope), structural proteins (core) and non-structural proteins (NS) and are preferably selected from envelope 1 (E1), envelope 2 (E2), core, NS3 protease, NS3- Helicase, NS4A cofactor, NS5B RNA polymerase, etc.

According to embodiment (2), the fragment of the invention inhibits NS3 protease or helicase, in particular NS3 helicase. Antibody fragments are particularly preferred in which the variable region of the light chain (V _L ) contains one or more of the sequences of the complementarity-determining regions (CDR) shown in FIG. 7A, namely one or more of the sequences CDR-L1, CDR-L2 or CDR-L3, and particularly preferably comprises one of the peptides shown in SEQ ID NOs: 1 to 8 or a derivative thereof, and / or in which the variable region of the heavy chain (V _H ) one or more of those in FIG. 7B CDR sequences shown, namely one or more of the sequences CDR-H1, CDR-H2 or CDR-H3, and particularly preferably comprises one of the peptides shown in SEQ ID NOs: 9 to 16 or a derivative thereof. For the purposes of the present invention, include truncated forms of the starting peptide (C- and / or N-terminally truncated), deleted forms of the starting peptide (individual amino acid residues or sequence segments in the starting peptide are deleted), substituted forms of the starting peptide (individual amino acid residues or sequence segments in the starting peptide) are replaced by other amino acid residues or sequence sections) and combinations thereof.

In the antibody fragment according to embodiment (1) and (2) of the invention, it is preferred that the fragments V _L and V _H are linked to one another by a covalent bond or by a linker molecule, in particular by a linker peptide. In addition, a linkage by means of non-proteinogenic polymeric compounds (such as, for example, polycarbonates, polyamides, polyethylene glycol derivatives, polyamine derivatives, etc.) and low molecular weight compounds (such as diamines, etc.) is also possible. For some applications of the antibody fragment according to the invention in which the stability under reductive conditions is not critical (for example in diagnostics), V _L and V _{H can} also be linked by a disulfide break. For the purposes of the present invention, however, it is particularly preferred that V _L and V _{H are} via a linker peptide, preferably via a hydrophilic and flexible linker peptide, particularly preferably via a peptide with the amino acid sequence (Gly _a Ser _b ) _x (where a is an integer from 1 to 7, preferably from 2 to 5, b is an integer from 1 to 4, preferably 1 or 2 and x is an integer from 1 to 10, preferably from 2 to 7) which are linked to one another. In particular, there is a linker peptide with the amino acid sequence (Gly ₄ Ser) _x , where x can have the values given above.

The most preferred antibody fragments for the purposes of the present invention have one of the following peptide sequences or a derivative thereof (where "derivatives" has the meaning given above):
Peptide (SEQ ID NO: 1) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 9)
Peptide (SEQ ID NO: 2) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 10)
Peptide (SEQ ID NO: 3) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 11)
Peptide (SEQ ID NO: 4) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 12)
Peptide (SEQ ID NO: 5) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 13)
Peptide (SEQ ID NO: 6) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 14)
Peptide (SEQ ID NO: 7) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 15)
Peptide (SEQ ID NO: 8) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 16),
where x is 3, 4 or 5 and in particular is 4.

The antibody fragments defined above are in different ways accessible. Because a classic chemical synthesis due to the size of the Antibody fragments is not the method of choice, it is preferred that recombinant techniques (e.g. expression in prokaryotes, in particular Expression in E. coli) used to produce these antibody fragments become. This ensures a high degree of homogeneity of the antibody product. For the synthesis of compounds that are linked by means of non-proteino have generally polymeric compounds or low molecular weight compounds, however, additional chemical reaction steps are unavoidable.

The production process of the fragments according to the invention is as follows based on the fragments against NS3 helicase and protease explained.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) (see SEQ ID NO: 18) shows protease and helicase activity which are essential for the life cycle of HCV. For the isolation of antibody fragments that inhibit NS3 at the protein level, cloning of antibody fragments that bind to NS3 was first carried out for stable intracellular expression. Antibody fragments of human origin were chosen to prevent an immune reaction after intracellular expression (intracellular immunization). A phage display system was used to isolate human sFv fragments against NS3, which was based on the expression of sFv fragments on the surface of filamentous phages. A large phagemid library with 2 × 10 ⁶ representatives was cloned from plasma cells from 5 patients who were infected with HCV. Phages expressing human sFv fragments with binding activity against NS3 were highly enriched during affinity selection. Selected soluble sFv antibody fragments that were expressed in E. coli showed a high affinity for NS3 protein, which was detected by immunoblot and measured in an EIA. K _D values describing the affinity of sFv for NS3 were measured by competitive EIA and estimated to be between 10 ^-6 and 10 ^-7 M. Antibody fragments could be cloned as complete human IgG antibodies, which means that 5 coli can be used as a safe and inexpensive source of recombinant human high-affinity antibodies for diagnostic and therapeutic purposes.

K _D values between 10 ^-6 and 10 ^-7 M (ie ≦ 10 ^-6 M) or less than 10 ^-7 M with respect to the affinity of sFv for NS3 in the competitive EIA mean that the antibody fragments inhibitor activity against NS3 protease / helicase exhibit. The use of vectors which contain a cDNA coding for these inhibiting antibody fragments for transduction into infected cells opens up a new gene therapy strategy for intracellular immunization against chronic HCV infection.

The phage display technique enables an affinity selection of proteins in several orders of magnitude from combinatorial libraries (Rader C., Barbas Cr., Curr. Opin. Biotechnol., 8: 503-8 (1997); Gram H. et al., Proc. Natl. Acad. Sci. USA, 89: 3576-80 (1992); Hoogenboom H.R., Winter G.J. Mol. Biol., 227: 381-8 (1992); Winter G. et al., Annu. Rev. Immunol., 12: 433-55 (1994)). A library of human antibody fragments was generated by immunoglobulin specific PCR cloned from plasma cells from HCV infected patients. Antibody fragments are generated by fusion with a smaller coat protein (gene III protein; gIIIp) at the top of the phage on the surface of filamentous Bacteriophage expressed (phage display) (Marks J.D. et al., J. Mol. Biol., 222: 581-97 (1991); Pope A.R. et al., New York: IRL Press, 1-40 (1996); McCafferty J. et al., Nature, 348: 552-4 (1990)).

Then phages, the antibodies with high affinity against the target Carrying antigen in several cycles of selection and reamplification (panning) be dramatically enriched.

An intracellular expression of human monoclonal antibody fragments in patients should not be accompanied by an immune response to the transgene his. Therefore human monoclonal antibodies against the essential HCV-  Protein NS3 (see SEQ ID NO: 18) for possible use as Cloned intra-body for intracellular immunization. bone marrow aspirates of HCV infected patients were used to encode Amplify regions of variable immunoglobulin domains. This PCR Products were used to build a large human sFv phagemid library clone. An EIA against recombinant NS3 was used to measure high serum To ensure antibody titers against NS3. Using phage Display and several cycles of affinity selection, phages were selected, who carried high affinity sFv fragments against NS3. Monoclonal phages were isolated and tested for binding activity against NS3. Most antibodies were directed against NS3 helicase and not against NS3 protease as it was has already been described (Chen M. et al., Hepatology, 28: 219-24 (1998)). Soluble recombinant sFv fragments were expressed, purified and again to be tested for their ability to bind to NS3 protein. The coding cDNA binding sFv was isolated and sequenced. The variable domains of six sFv fragments were characterized. The sequence showed κ-light- Chains and λ light chains. VDJ germline segments were identified. The Affinity of sFv fragments was determined by competitive EIA. The sFv Fragments showed the high binding affinities described above that suggest inhibition of NS3 protease / helicase.

After cloning the sFv fragments, cloning is also complete against human IgG, IgA or IgM antibodies possible (Boel E. et al., J. Immu nol. Methods, 239: 153-66 (2000)). The recombinant expression of complete gene antibodies is simple and cheap compared to the isolation of Antibodies from the serum of HCV infected patients. Moreover, there is at the use of recombinant high affinity antibodies no risk of Infection with HCV or other human pathogens. These antibodies are for diagnostic and therapeutic purposes can be used.

Thus, the antibody fragments according to embodiment (8) of the invention Part of pharmaceutical compositions (such as vaccines, etc.) and of diagnostic compositions (also "diagnostic kits" ge called). In addition to the antibody fragments, these contain compositions  general additives, auxiliaries, etc. In the diagnosti The antibody fragments of the invention can be used, for example, in kits. B. in one Cell culture system for evaluating the NS3 protease function (Heintges T. et al., J. Med. Virol., (2000)), in assays for testing for NS3 helicase function (Hsu C. C. et al., Biocem. Biophys. Res. Commun., 253: 594-9 (1998)) in a replicon system for evaluating the function of non-structural HCV proteins (Lohmann V. et al., Science, 285: 110-3 (1999)) etc. can be used.

According to embodiment (4), the present invention relates to a gene trans fervektor, which is a DNA sequence for an antibody according to the invention fragment encoded, includes. This gene transfer vector can in addition to the above DNA sequence and other functional DNA sequences (such as promoters sequences, recognition sequences, viral sequences, splice donor and acceptor sequences, etc.) included. So, in order to have antiviral effects in hepatocytes achieve the coding cDNA for antibody fragments that replicate the HCV inhibit, are transduced by vectors. The most promising at the moment Vectors for gene therapy of the liver are lentiviruses or "boned" adeno viruses (Richardson J.H. et al., Gene Ther., 5: 635-44 (1998); Naldini L. et al., Science, 272: 263-7 (1996); Zufferey R. et al., Nat. Biotechnol., 15: 871-5 (1997); Ferry N., Heard J.M., Hum. Gene Ther., 9: 1975-81 (1998)). A efficient expression of human antibody fragments in hepatocytes Prerequisite for intracellular immunization with intra-bodies, what as a promising approach to treating chronic HCV Infection is considered.

The sequence and structure of the antibody fragments according to the invention are still helpful for the design of inhibitors of the NS3 protease or - Helicase in the form of small molecules. Based on the structure of the Antibody fragments are derived from low molecular weight compounds that inhibit these essential viral proteins of HCV.

The present invention is based on the Experi described below ment and the detailed description of the figures.  

experiments Materials and methods patients

Bone marrow was taken from five patients with chronic hepatitis C. won aspirate. All patients had serologically documented HCV Infection (HCV antibody positive in the enzyme-linked immunoassay of the third Generation and serum HCV-RNA detectable by RT-PCR). Of all patients Bone marrow aspirate was obtained during procedures based on ver were indicated for various medical reasons. Sign all patients a written declaration of consent for the removal of another 5 ml Aspirate and 10 ml serum according to an assessment by the local ethics expert Commit- tee. Serum was available from 4 of the 5 patients.

HCV NS3 ELISA

An ELISA plate (Nung, Gibco BRL, Karlsruhe) was coated overnight at 4 ° C. with 0.5 μg NS3 in PBS and then blocked for 1 h at RT with 3% BSA / PBS. Patient sera were diluted 1: 1000, 1: 2000, 1: 4000, 1: 8000 and 1: 16000 and then added to the wells. After 1 h of incubation at RT, the plate was washed ten times with TBST / 0.1% Tween®-20. Subsequently, a conjugate of alkaline phosphatase (AP) and goat anti-human (Fab) ₂ IgG (Boehringer, Mannheim) was added to each well at a dilution of 1: 5000 and incubation was carried out for 1 hour. After washing ten times, p-nitrophenyl phosphate (Sigma, Aldrich, Taufkirchen) was added and incubated for 20 minutes. The optical density at 405 nm was then measured using an EIA reader (Anthos Labtec, Salzburg, Austria). A signal-to-noise ratio of over 2.4 was seen as positive.

Cloning of a combinatorial human sFv library

Bone marrow aspirate was used to prepare total RNA. RNA was extracted using Tri-reagent (Gibco BRL, Karlsruhe) and transcribed into cDNA using oligo (dT) primers (Gibco BRL, Karlsruhe). The V _H and V _L genes were amplified by PCR separately with V _H and V _L -specific primers. Furthermore, the reverse primer of V _{L was} modified by incorporating a flag tag at the 5 'end (Knappik, Plückthun, 1994). The V _H antisense primer and the V _L sense primer contain sequences which encode a flexible amino acid linker (Gly ₄ Ser) ₄ which is inserted between the variable domains of the heavy and the light chain. This enables a recombinant hybridization between V _H and V _L products in the following overlap PCR reaction. The cycle parameters were as follows: 30 cycles, 94 ° C 1 min, 57 ° C 2 min, 72 ° C 1 min. For extension overlap PCR, gel-purified V _H and V _L PCR fragments (approximately 300 bp in size) were mixed together in equimolar ratios and used to form a template in the absence of primers (2 ×: 92 ° C. for 1 min , 63 ° C 30 s, 58 ° C 50 s, 72 ° C 1 min). Then five cycles (92 ° C 1 min, 63 ° C 30 s, 58 ° C 50 s, 72 ° C 1 min) and 23 cycles (92 ° C 1 min, 63 ° C min, 72 ° C 1 min) in the presence of specific external sFv primers containing SfiI restriction sites at the 5 'end. The primers used were:

Lambda (λ) -Sense primer

Lambda (λ) antisense primer

Kappa (κ) -Sense primer

Kappa (κ) antisense primer

VH sense primer

VH antisense primer

outer sFv primer (pull through)

Underlined are flag-tag sequences within each V _L back primer (kappa; κ and lambda; λ).

After digestion with the restriction enzyme SfiI, the sFv fragments (approx. 750 bp) either gel cleaned (Qiaquik; Gel Extraction Kit, Qiagen, Hilden) or electroeluted (Schleicher & Schüll, Dassel) and then into the phagemid vector pAK100 (Krebber, A. et al., J. Immunol. Methods, 201: 35-55 (1997)) ligated. This vector enables expression of sFv as a gene III fusion protein the surface of bacteriophages (phage display) with the introduction of a C- terminal c-myc tags.  

The resulting plasmids of all 5 patients were used to transform electrocompetent E. coli XL ₁ blue (Stratagene, Amsterdam Zuidoost, Netherlands) and thus to create a large library of recombinant human sFv fragments in the pAK100 expression vector (Krebber A. et al., J. Immunol. Methods, 201: 35-55 (1997)). Transformants were raised in 2 × YT medium containing 25 μg / μl chloramphenicol and 1% glucose to an OD ₅₅₀ of 0.5. At this point, the built-up phage mid library was titrated as colony forming units (cfu). The size of the library of all 5 patients was 2 × 10 ⁶ . The library was then infected with 10 ^11-12 VCSM13 helper phages (Amersham Pharmacia, Freiburg) in order to obtain phages carrying sFv. Expression was induced by the addition of 0.5 mM IPTG (Roth, Karlsruhe) and carried out at 24 ° C. for 2 hours. After 3-4 hours of incubation, kanamycin (30 µg / µl; Fluka, Neu-Ulm) was added, and the recombinant phage culture grew overnight at 24 ° C. The phage particles were harvested by precipitation with 20% PEG / 15% NaCl (Sambrook et al., 1989), dissolved in PBS and stored at 4 ° C. or used directly for further experiments.

Affinity selection (panning)

Recombinant phages carrying human sFv fragments were selected by panning based on their affinity for binding to NS3. A microtiter plate (Nung, Gibco BRL, Karlsruhe) was coated overnight at 4 ° C. with 1 μg recombinant NS3 protein / well (Mikrogen, Munich). After blocking with 3% BSA for 1 h at RT, a phage suspension (approximately 10% diluted in 3% BSA) was added and it was incubated for 2 h at RT. The microtiter plate was washed thoroughly (20 ×) with PBS / 0.1% Tween-20 to eliminate non-specifically bound phages. The phages bound to NS3 with high affinity were then eluted with 0.1 M glycine / HCl, pH 2.2. The phage solution was neutralized with 2 M Tris, and the phages (typically 10 ^3-4 ) were used to reinfect E. coli XL1-Blue, which was in exponential growth. This panning process was repeated 4 times. The selected phage population was diluted and streaked to obtain individual monoclonal colonies.

Phage EIA

Selected single colonies were grown separately in 8 ml of 2 × YT medium containing chloramphenicol (25 μg / μl) and glucose (1%) at 37 ° C. while shaking and then infected with 10 ¹¹ VCSM13 helper phages. Human sFv expression was induced with 0.5 mM IPTG. After overnight incubation, monoclonal phages were precipitated with 20% PEG / 15% NaCl and dissolved in 200 µl PBS. The phage titer was estimated as cfu / ml and the phage solution was used in an EIA assay as follows. An EIA plate was first coated overnight at 4.degree. C. with 0.3 μg NS3 (Mikrogen) and then blocked for 1 h at RT with 3% BSA. Then 50 µl of a phage solution (approximately 109 phages) was added. After 1 h incubation at RT and washing three times with PBS / 0.05% Tween-20, HRP-conjugated anti-M13 antibody (Amersham Pharmacia, Freiburg) diluted in 3% BSA was added and incubated for 1 h. For detection, an incubation with ABTS substrate was carried out according to the manufacturer's instructions (Boehringer, Mannheim). After 15 min incubation, the absorbance at 405 nm was measured with an EIA reader (Anthos Labtec, Salzburg, Austria). All assays were carried out four times in 3 independent experiments.

Sequencing

Sequencing reactions were carried out using an automatic ABI-310 sequencer (Perkin Elmer, Rodgau-Jügesheim) performed where Standard sequencing protocols and specific vector oligonucleotides were used. Then the deduced amino acid sequences were carried out Computer research with BLAST (http: / / www.ncbi.nlm.nih.gov/igblast) and DNAPLOT (http: / / www.genetik.uni-koeln.de/dnaplot) and the Kabat database (http: // immuno.bme.nwu.edu) (Kabat E.A. et al., 5th ed. Bethesda, MD (1991); Martin A.C.R., Proteins: Structure, Function and Genetics, 25: 130-133 (1996)) analyzed.

Soluble expression of human sFv fragments in E. coli

For soluble expression, sFv fragments were first cloned into pQE-70 (Quiagen, Hilden). This vector enables strong inducible expression in E. coli and introduces a His tag that is useful for protein purification. Individual colonies were incubated overnight at 37 ° C. in 30 ml of 2 × YT medium (Chlorampheni col, 25 μg / μl, and glucose, 1%). With an OD ₅₅₀ = 0.8, expression of the human sFv fragments was induced with 1 mM IPTG and the bacteria were grown for 4 hours at 24 ° C. with shaking. After sedimentation, bacterial cells were incubated for 0.5 h at 4 ° C in spheroblast buffer (200 mM Tris / HCl, pH 8.0, 0.5 M sucrose). Soluble sFv fragments secreted into the periplasm were obtained by lysozyme treatment (0.8 mg / ml, only the outer membrane of E. coli being destroyed) and mild osmotic shock, with the spheroblasts remaining intact (Skerra, 1989). The cell sediment from 10 ml expression culture was resuspended in 200 ul ice-cold spheroblast buffer, and 790 ul ice-cold 50% (v / v) spheroblast buffer was immediately added. The lysis was completed on ice for 30 minutes. A standard procedure for the purification of His-tagged sFv was chosen, which consisted of a Ni-NTA affinity purification step (HisTrap columns, Amersham Pharmacia, Freiburg) with an elution step under native conditions using imidazole (100-150 mM) existed. This process was carried out using an FPLC system according to the manufacturer's instructions (Amersham Pharmacia, Freiburg).

Immunoblot analysis

1/100 purified sFv fraction was used for a further immunoblot analysis. Specifically, a 20 µl aliquot of each sample was loaded onto 0.1% SDS / 12% PAGE. After transfer to a nitrocellulose membrane (Protran, Schleicher & Schüll, Dassel) using standard instructions, the sFv fragments were detected by immunoblot. The membrane was first blocked at RT with 3% BSA in TBS for 1 h to prevent non-specific adsorption, and then washed twice with TBS. This was followed by incubation either with anti-flag mAB M1 (Sigma Aldrich, Taufkirchen) or with anti-tetra-His antibodies (1: 2000 in 3% BSA / TBS) (Qiagen, Hilden) for 30 min using 10 µg / ml in TBS / 1 mM CaCl ₂ . After washing with TBST (twice) for anti-His detection or with TBS + 1 mM CaCl ₂ for anti-flag detection, conjugate of horseradish peroxidase (HRP) and rabbit anti Mouse down (Biorad, Munich) added. The detection was carried out by chemiluminescence via the ECL ™ system (Amersham Pharmacia, Freiburg).

sFv EIA

The binding activity of soluble sFv fragments was tested by EIA. The wells were coated overnight at 4.degree. C. with 0.5 μg of recombinant NS3 protein in PBS and then blocked for 1 h at RT with 3% BSA. Then periplasmic extract containing soluble monoclonal sFv fragments was added (1, 0.1 or 0.01 µg in PBS / well) and incubated for 1 h at room temperature. After 5 washing steps with TBST / 0.5% Tween®-20, the anti-His-Ab (1: 2000 in 3% BSA; Qiagen, Hilden) was added and it was incubated for 1 h at RT. The wells were then washed and incubated for 1 hour with HRP-conjugated rabbit anti-mouse IgG-Ab (1: 5000 in 10% milk powder). ABTS substrate was added for detection and A ₄₀₅ was measured as described above.

affinity measurements

Determination of the affinity of the antibody fragments was carried out with the help of a competitive EIA, which was first developed by Friguet et al. beschrie (Friguet B. et al., J. Immunol. Methods, 77: 305-19 (1985)). The too testing antibody was used with various concentrations of the antigen incubated until an association-dissociation equilibrium was reached. Then a solid phase EIA with immobilized antigen was used to control the Determine concentration of free antibody that is not soluble Antigen bound (Friguet B. et al., J. Immunol. Methods, 77: 305-19 (1985)). The EIA conditions were chosen so that only a small proportion of the unsaturated antibody in equilibrium by the immobilized Antigen is captured. This ensures that the balance in Solution is not significantly affected by the EIA conditions (Goldberg M.E., Djavadi-Ohaniance L., Curr. Opin. Immunol., 5: 278-81 (1993)).

Different concentrations of the human sFv fragments were tested to to determine the linear range of the saturation curve. For this purpose, serial Dilution series of the antibodies with subsequent determination of the OD carried out. Dilution of the sFv fragments resulted in a decrease in OD by at least 40% was used for further competitive EIA experiments selected. sFv fragments in this concentration were mixed at 4 ° C for 2 h soluble recombinant NS3 protein incubated to balance to reach. Then the concentration of the free sFv fragments in sFv-EIA determines as described above, coating with 0.3 µg NS3 plates were used that were previously blocked with 10% milk powder  were. The affinity was determined for each individual antibody perfragment in correlation to the linear part of the competition curve.

results

Sera from 4 patients were analyzed in the immunoassay for the presence of antibodies against NS3 protein. Data obtained from this antibody screening showed that 3 out of 4 patients had high titers of 1: 16,000 against NS3. The titer of one patient (patient 3) was low and similar to the negative controls (no first antibody or irrelevant recombinant sFv fragments). The individual titers of the patients are shown in Fig. 1.

In order to create a recombinant human phage display library, we used the sFv-expressing phagemid vector pAK100. Initially, overlap extension PCR gave rise to single-chain fragments which consisted of recombined variable domains of the heavy (V _H ) and light chain (V _L ) with an inserted flexible polypeptide (Gly ₄ Ser) ₄ linker ( FIG. 2) ,

Subsequently, sFv fragments were included in the pAK100 expression vector niert (Krebber A. et al., J. Immunol. Methods, 201: 35-55 (1997)), the TH- Terminator, lacI gene and lac promoter region included, making optimal Growth conditions for transformed E. coli XL1-Blue were created, which is incubated in 2 × TY medium to which 1% glucose has been added (see Section "Materials and Methods"). Under these conditions, a undesirable toxic effects as well as a background expression completely be excluded before further induction with IPTG takes place (Krebber A. et al., J. Immunol. Methods, 201: 35-55 (1997)). Optimal production of gIII-fused sFv was obtained by incubation at RT for 4 h.

The recombinant sFv expressed on the surface of filamentous phages were selected in a panning reaction with NS3 bound to a solid phase. Polyclonal phage were fixed in 4 cycles of phage panning used functional binding phage to enrich and to eliminate non-binding phage (see the following Table 1 and Fig. 3).

Table 1

Table 1 shows the relative enrichment of phages carrying monoclonal sFv against NS3 protein (SEQ ID NO: 18) before use in panning and after the first, second, third and fourth round of affinity selection. The phage titer was measured as cfu / ml. The number of sFv-carrying phages during panning was determined as cfu / ml by series of dilutions. Before each round of panning, the phage population was amplified to 10 ^10-12 and then subjected to an affinity selection against recombinant HCV-NS3. After washing, phages bound to NS3 were eluted with 0.1 M glycine / HCl, pH 2.2, as described in the "Materials and Methods" section. The percentage of phages eluted after panning increases steadily, indicating that phages binding against HCV-NS3 are being enriched.

After re-infection of E. coli XL ₁ -blue with sFv-bearing phages against NS3, cells were streaked out to form individual colonies. Only in the case of an EIA reaction (signal-to-noise ratio) of at least 2.4 or above in comparison to the negative control (neg. Ctrl) were the sFv clones regarded as specifically binding to HCV-NS3, as is shown in FIG. 4 is shown. Monoclonal colonies were selected and sequenced. During the sequencing, some of the selected clones proved to be multiple represented.

For high-level expression of soluble sFv in E. coli and introduction of a hexa-His tag, all sFv fragments were recloned into pQE-70 (Qiagen, Hilden). After induction with IPTG and incubation at RT for 4 h, soluble sFv molecules were harvested from the periplasmic space using the cold osmotic shock method. An immunoblot procedure was performed to determine the correct expression and size of the cloned sFv fragments expressed in E. coli against NS3. A strong and clear band of 32 kDa was made visible with anti-flag and with anti-tetra-His antibodies ( FIG. 6).

The recombinant sFv was further purified using the Ni NTA column cleaning system and FPLC as described in the "Materials and Methods ". After cleaning, there were no non-specific ones Bands seen when sFv loaded on 12% PAGE, on a nylon membrane transferred and stained with Ponceau's Red or Coomassie Brilliant Blue.

The reactivity of soluble purified recombinant sFv fragments against NS3 was tested by EIA ( Fig. 6). Strong and specific signals were obtained from six different clones compared to different controls.

The coding sequence of phagemid DNA was determined and the amino acid sequence of the variable domains of the antibody fragments was derived (see Fig. 7). The positions of scaffold regions (FR) and complementarity-determining regions (CDR) were determined according to the numbering system of Kabat et al. (Kabat EA et al., 5th ed. Bethesda, MD (1991)). Fig. 7 shows a comparison of sFv sequences.

Five antibodies have λ light chains and one antibody has κ light chains Chains on (see Table 2A (light chain) and 2B (heavy chain)). A further analysis by DNA plot (http: / / www.genetik.uni-koeln.de/dnaplot) or Blast (http: / / www.ncbi.nlm.nih.gov/igblast) revealed the germ line-V (D) J- Segments (Table 2). Further characteristics of the antibodies are in Table 2 shown. V (D) J germline segments that most closely resemble the sequence of the antibody showed are called.

Antibody fragment affinity measurements were made using competitive EIA. All antibodies showed high affinities against NS3. The affinity constants K _D were of the order of 10 ^-6 to 10 ^-7 M ( Fig. 8).

Table 2A (light chain (V _L ))

Table 2B (heavy chain (V _H ))

Tables 2A and B summarize the classification and germline segments of variable domains of cloned antibody fragments. V _λ , J _λ , V _κ , J _κ , V _H , D and J _H segments are named according to the nomenclature of the authors named in the methods. The family and the subgroup were determined according to Kabat et al. (Kabat EA et al., 5th ed. Bethesda, MD (1991); Martin A. et al., Proteins: Structure, Function and Genetics, 25: 130-133 (1996)) (nk = not classified).

Detailed description of the figures

Fig. 1 Serum antibody titers from 4 patients with chronic HCV infection against recombinant NS3 protein were determined by EIA. A dilution series of sera was incubated in wells coated with recombinant NS3-O / N and with conjugates of alkaline phosphatase (AP) and goat anti-human (Fab) ₂ IgG (diluted 1: 5000, Boehringer Mannheim) was detected. EIA reactivity was measured in four parallel experiments as OD at 405 nm after p-nitrophenyl phosphate was added as a chromogen. Three patients (patient Nos. 1, 2 and 4) show a very high Ab titer of 1: 16,000. One patient shows no significant titer compared to HCV-NS3.

Fig. 2 gel electrophoresis of sFv cDNA fragments, which were generated by immunoglobulin-specific PCR. Initially, VL and VH were amplified separately by 6 to 7 different primer pairs (see "Materials and Methods" for details). PCR products of the kappa chains κ ₁ -κ ₆ (image A), the lambda chains λ ₁ -λ ₇ (image B) and the heavy chains H1-H6 (image C) are shown, loaded on a 1.5 % Agarose gel. An overlap extension PCR was then used using pull-through primers (outer primers) containing SfiI restriction sites, so that sFv fragments formed, which consisted of VL and VH domains, which were linked by a flexible amino acid linker were connected. Figure D shows sFv-PCR products including the light chains V _{L κ} (K + H, lane 2) or V _{L λ} (λ + H, lane 3), which were amplified separately. Different subgroups of V _L or V _H are more strongly represented than others in individual patients. For example, the patient shown here shows stronger signals for λ ₂ and λ ₆ than for λ ₄ and λ ₅ . Other patients showed different patterns of subgroup representation (data not shown in detail).
St = standard kb marker (Gibco BRL, Karlsruhe).

Fig. 3 Enrichment of phages with sFv fragments that show binding affinity for recombinant NS3 during various passes of affinity selection (panning). HRP-conjugated anti-M13 antibody was used at a 1: 5000 dilution and then ABTS substrate was added as described in the "Materials and Methods" section. The OD was measured at 405 nm. The OD increases steadily with further rounds of panning, indicating that the percentage of phages with binding affinity for NS3 increases. BSA: wells coated with 3% BSA; neg. Ctrl: wells coated with NS3 protein and incubated with non-binding helper phages.

Figure 4 Identification of monoclonal phages carrying sFv with binding activity against NS3 protein by phage EIA. The phage population after 3 panning runs was diluted and monoclonal phages were amplified. Approximately 10 ⁸ monoclonal phages were used in a phage EIA against NS3 to select positive binding to NS3. After incubation with 1: 5000 diluted HRP-conjugated anti-M13 antibody and addition of ABTS substrate, the OD was measured at 405 nm. Clones No. 3, 6, 7, 10, 11, 12, 13, 15, 16 were identified on the basis of their binding properties against NS3 and selected for further analysis.

Figure 5 EIA of purified soluble sFv fragments against NS3. sFv fragments were purified from periplasmic extracts of E. coli through Ni-NTA columns to homogeneity. After coating wells with NS3-O / N, cleaned sFv are incubated. Then anti-tetra-His antibody and then HRP-conjugated rabbit anti-mouse antibody were used, and chemiluminescence was performed using the ECL substrate system. All clones show strong affinity for NS3 compared to BSA (negative control).

Fig. 6 Immunoblot analysis of bacterially expressed recombinant sFv fragments. To demonstrate the size and presence of bacterially expressed sFv fragments, 50 µl whole cell fraction of E. coli was loaded onto 12% SDS-PAGE gel and treated with anti-flag (α-flag) and anti-tetra-His ( α-His) - antibodies detected. The detection was carried out using HRP-conjugated rabbit anti-mouse antibodies and the ECL substrate system. As expected, a significant band was detected at around 31 kDa, which was consistent with soluble sFv fragments.

FIG. 7 amino acid sequence of the variable regions of the L chain ( FIG. 7A) and H chain ( FIG. 7B) of selected human sFv. The coding cDNA of monoclonal sFv, which is positive for NS3 in phage EIA and sFv-EIA, was isolated. Automatic two-way sequencing was performed twice to determine the nucleotide and amino acid sequence of V _L and V _H. Framework regions (FR) and complementarity-determining regions (CDR) according to the nomenclature of Kabat et al. (Kabat EA et al., 5th ed. Bethesda, MD (1991); Martin A. et al., Proteins: Structure, Function and Genetics, 25: 130-133 (1996)) are given. Two cysteine residues in the variable regions of V _L and V _H , which are involved in structurally conserved disulfide bridges between the chains, are printed in bold. Sequences that are influenced by the primers used are printed in italics.

Fig. 8 bacterially expressed sFv were purified to homogeneity by Ni-NTA columns. Non-specific bands were not detected when staining with Ponceau red and Coomassie Brilliant Blue after cleaning (data not shown). Purified sFv fragments were used for a competitive EIA to determine antibody affinity. Initially, sEv concentrations of the linear region of the dose-binding curve were chosen (data not shown in detail). sFv were incubated for 2 hours at 4 ° C with different concentrations of soluble NS3 to achieve a stable association-dissociation equilibrium. The concentration of free sFv in solution was then determined using NS3-EIA. The figure shows a decreasing OD with increasing concentrations of soluble antigen on a logarithmic scale. The affinities of the sFv shown are of the order of 10 ^-6 to 10 ^-7 M.

SEQUENCE LISTING

Claims (19)

1. Single chain fragment of a human antibody that inhibits the proliferation of hepatitis C virus (HCV) and that the variable regions of the light chain (V _L ) and heavy chain (V _H ) of an antibody against one or more essential Proteins covered by HCV. 2. Antibody fragment according to claim 1, which inhibits one or more essential proteins of HCV, preferably an affinity K _D ≦ 10 ^-6 M, particularly preferably K _D ≦ 10 ^-7 M, for at least one essential virus protein. 3. Antibody fragment according to claim 1 or 2, wherein the essential Viruspro teins are selected from surface proteins (envelope), structural proteins (Core) and non-structural proteins (NS) and is preferably selected from Envelop 1 (E1), Envelope 2 (E2), Core, NS3 protease, NS3 helicase, NS4A- Cofactor and NS5B RNA polymerase. 4. Antibody fragment according to claim 3, the NS3 protease and / or NS3 Helicase, preferably NS3 helicase inhibits. 5. Antibody fragment according to claim 4, wherein the variable region of the light chain (V _L ) comprises the peptide shown in SEQ ID NOs: 1 to 8 or a derivative thereof, and / or wherein the variable region of the heavy chain (V _H ) comprises peptide or a derivative thereof shown in SEQ ID NOs: 9 to 16. 6. Antibody fragment according to one or more of claims 1 to 5, wherein the fragments V _L and V _H are linked to one another by a covalent bond or by a linker molecule, in particular a linker peptide. 7. Antibody fragment according to claim 6, wherein V _L and V _H via a linker peptide, preferably via a hydrophilic and / or flexible linker peptide, particularly preferably via a peptide with the amino acid sequence (Gly _a Ser _b ) _x (where a is an integer from 1 to 7, preferably from 2 to 5, b is an integer from 1 to 4, preferably 1 or 2 and x is an integer from 1 to 10, preferably from 2 to 7) are linked to one another. 8. The antibody fragment according to claim 7, which has one of the following peptide sequences or a derivative thereof:
Peptide (SEQ ID NO: 1) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 9)
Peptide (SEQ ID NO: 2) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 10)
Peptide (SEQ ID NO: 3) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 11)
Peptide (SEQ ID NO: 4) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 12)
Peptide (SEQ ID NO: 5) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 13)
Peptide (SEQ ID NO: 6) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 14)
Peptide (SEQ ID NO: 7) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 15)
Peptide (SEQ ID NO: 8) - (Gly ₄ Ser) _x peptide (SEQ ID NO: 16),
where x is 3, 4 or 5, preferably 4. 9. Antibody fragment according to one or more of claims 1 to 8, the by expression in prokaryotes, in particular by expression in E. coli are available. 10. DNA sequence required for an antibody fragment according to one or more of claims 1 to 8 coded. 11. Vector or gene transfer vector comprising a DNA sequence according to Claim 10. 12. A method for identifying antibody fragments that inhibit the proliferation of HCV according to claims 1 to 8, comprising

• a) Cloning of a DNA library encoding antibody fragments against one or more essential proteins of HCV;
• b) Expression of said antibody fragments on the surface of bacteri phages with subsequent enrichment of the phages which carry antibody fragments with high affinity for the essential protein by means of several cycles of selection and reamplification (panning); and continue if necessary
• c) Multiplication of said antibody fragments with high affinity by soluble expression in bacterial cells and isolation of the antibody fragments from the periplasm of these cells.

13. Host cell, in particular prokaryotic host cell, with a Vector is transformed according to claim 11 and / or a DNA sequence according to Claim 10 contains. 14. A method for producing an antibody fragment according to one or several of claims 1 to 9, comprising culturing a host cell according to Claim 13. 15. Pharmaceutical or diagnostic composition containing one or contains several antibody fragments according to claims 1 to 9. 16. Use of the antibody fragment according to one or more of the Claims 1 to 9 as a vaccine, in particular for passive immunization. 17. Pharmaceutical composition containing one or more Gene transfer vectors as defined in claim 11. 18. Use of the gene transfer vector according to claim 11 for gene therapy, especially for gene therapy of HCV infections. 19. Gene therapy methods, in particular methods for the treatment of HCV Infections comprising administering the gene transfer vector according to claim 11 to the patient.