WO2002093519A2 - Fragments d'anticorps humains a affinite elevee, opposes aux proteines essentielles du virus de l'hepatite c - Google Patents

Fragments d'anticorps humains a affinite elevee, opposes aux proteines essentielles du virus de l'hepatite c Download PDF

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WO2002093519A2
WO2002093519A2 PCT/EP2002/005227 EP0205227W WO02093519A2 WO 2002093519 A2 WO2002093519 A2 WO 2002093519A2 EP 0205227 W EP0205227 W EP 0205227W WO 02093519 A2 WO02093519 A2 WO 02093519A2
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peptide
seq
gly
ser
antibody
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PCT/EP2002/005227
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WO2002093519A3 (fr
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Tobias Heintges
Kathi Tessmann
Dieter HÄUSSLINGER
Olga Artsaenko
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Tobias Heintges
Kathi Tessmann
Haeusslinger Dieter
Olga Artsaenko
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Priority to AU2002342858A priority Critical patent/AU2002342858A1/en
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Publication of WO2002093519A3 publication Critical patent/WO2002093519A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • 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.
  • hepatitis C The treatment of hepatitis C is currently one of the greatest challenges in pharmaceutical research.
  • HCV hepatitis C virus
  • these antibodies do not have a sufficiently high binding affinity to increase HCV inhibit in vivo.
  • HCV has a (+) stranded RNA genome that encodes a single polyprotein with approximately 3000 amino acids.
  • This single polyprotein is cloned into functional or viral proteins by proteases of the host or the virus, translationally or post-translationally (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. Sei., 90: 10773-7 (1993)).
  • the suspected structural proteins include the core protein (C) and two coat proteins (El and E2), while the non-structural proteins (NS2, NS3, NS4, NS5) are probably components of a complex that is responsible for viral RNA replication (Bartentscher R. et al., J. Virol., 68: 5045-55 (1994); Suzich JA 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 which has 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 likely to be processed by cellular proteases (Shoji I. et al., Virology, 254: 315-23 (1999)).
  • protease and helicase domains of NS3 are active in vitro in the absence of the other domain (Wardell AD et al., J. Gen. Virol., 80: 701-9 (1999); Kim DW et al., J Virol., 71: 9400-9 (1997); Kolykhalov AA et al., J. Virol., 68: 7525-33 (1994)).
  • NS3 protease belongs to the serine protease family and is responsible for the processing of the HCV polyprotein at the connection points NS3 / NS4A (cis cleavage), NS4A / NS4B, NS4B / NS5A and NS5A / NS5B (trans cleavage)
  • NS3 / NS4A cis cleavage
  • NS4A / NS4B NS4B / NS5A
  • NS5A / NS5B trans cleavage
  • the structure of the NS3 protease shows a flat substrate binding pocket, which makes it difficult to design effective protease inhibitors (Kim JL et al., Cell, 87: 343-55 (1996); Yan Y. et al., Protein Sei. 7: 837-47 (1998)).
  • the helicase activity consists of a polynucleotide-stimulated NTPase activity, a 3 '- »5' relaxing activity and a binding activity for single-stranded nucleotides (Wardell AD et al., J. Gen. Virol., 80: 701-9 (1999); Gwack Y. et al., Eur. J.
  • HCV-NS3 helicase is therefore a promising antiviral target that is essential for viral replication.
  • Single-chain variable fragments (sFv) of antibodies contain variable domains of the heavy (V H ) and the light chain (V), 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 the reducing environment found in the cytoplasm. The intracellular sFv can be expressed directly within cells, thereby creating an active molecule for protein-based gene therapy.
  • adenoviral vectors are used to express anti-erB-2 intra-bodies (Deshane J. et al., 3rd Clin. Invest., 96: 2980-9 (1995)).
  • Other groups use a retroviral MuLV vector to express anti-gpl20 intra-bodies in T helper cells in HIV-infected patients (Marasco WA et al., J. Immunol. Methods, 231: 223-38 (1999); Chen SY et al., Proc. Natl. Acad. Sci. USA, 91: 5932-6 (1994)).
  • the object of the present invention was to provide special antibody derivatives which effectively inhibit the multiplication of hepatitis C and which can be used intracellularly, so that they are suitable for gene therapy applications. It has been found that special single chain antibody fragments against essential hepatitis C virus (HCV) proteins, especially high affinity human single chain (sFv) antibody fragments against HCV-NS3, selected from a large library of recombinant antibody fragments, meet these requirements. It could be shown that some of the antibody fragments according to the invention bring about an almost 100% inhibition of the HCV helicase activity.
  • HCV essential hepatitis C virus
  • sFv high affinity human single chain
  • the present invention thus relates
  • a single-chain fragment of a human antibody which inhibits the proliferation of hepatitis C virus (hereinafter also referred to as "HCV") and which contains the variable regions of the light chain (V) and the heavy chain (V H ) Antibody to one or more HCV essential proteins;
  • the single-chain fragment inhibits an HCV-NS3 protein, in particular NS3 protease and / or NS3 helicase, particularly preferably NS3 helicase;
  • a method of identifying antibody fragments that inhibit HCV proliferation comprising a) cloning a DNA library encoding antibody fragments against one or more essential HCV proteins; b) expression of said antibody fragments on the surface of bacteriophages 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 optionally further 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;
  • a host cell in particular a prokaryotic host cell, which is transformed with a vector according to (4) and / or contains a DNA sequence according to (3);
  • HCV infections comprising administering the gene transfer vector according to (4) to a patient;
  • a vaccination method comprising administering an antibody fragment according to one or more of claims 1 to 9 to the patient to be vaccinated.
  • Figure 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.
  • FIG. 3 shows the enrichment of phages with sFv fragments which show binding affinity for recombinant NS3 during various passes of the 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.
  • Figure 6 shows the immunoblot analysis of bacterially expressed recombinant sFv fragments.
  • FIG. 7 shows the amino acid sequence of the variable regions of the H and L chain of the human sFv selected in experiment 1.
  • FIG. 8 shows the activity of bacterially expressed sFv (Experiment 1), which were purified to homogeneity by Ni-NTA columns, in the competition EIA.
  • FIG. 9 shows the amino acid sequence of the variable regions of the H and L chain of the human sFv selected in experiment 2.
  • FIG. 10 shows the result of the production of recombinant HCV helicase, namely the detection of helicase expression by Western blot (A) and the result of the purification of His-tag helicase by means of Ni-NTA agarose.
  • FIG. 11 shows the influence of the binding of the antibody sFvl-2 on the HCV helicase activity (A), the inhibition of the HCV helicase activity by this antibody (B) and the concentration dependence of this inhibition (C).
  • FIG. 12 shows the influence of the binding of the antibodies from experiment 2 on the HCV helicase activity.
  • the single-chain fragment of a human antibody according to embodiment (1) of the invention preferably inhibits at least one essential protein of the 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.
  • 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) contains one or more of the sequences of the complementarity-determining regions (CDR) shown in FIG.
  • V H variable region of the heavy chain
  • “Derivatives” for the purposes of the present invention include truncated forms of the respective 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 segments replaced) and combinations thereof, the derivatives having essentially (ie at least 75%, preferably at least 90%) the activity (inhibition, inhibition) of the parent compound.
  • 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.
  • a linkage by means of non-proteinogenic polymeric compounds such as polycarbonates, polyamides, polyethylene glycol derivatives, polyamine derivatives, etc.
  • low molecular weight compounds such as diamines, etc.
  • V and V H can also be linked by disulfide breaks.
  • 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 t> ) x (where a is a whole Number 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.
  • a linker peptide with the amino acid sequence (Gly 4 Ser) x where x can have the values given above.
  • Particularly preferred DNA sequences of embodiment (3) of the invention are those shown in SEQ ID NOs: 59 to 65.
  • the antibody fragments defined above are accessible in various ways. Since classic chemical synthesis is not the method of choice due to the size of the antibody fragments, it is preferred that recombinant techniques (for example expression in prokaryotes, in particular expression in E. coli) are used to produce these antibody fragments. This ensures a high degree of homogeneity of the antibody product. For the synthesis of compounds that are linked by means of non-protein generally polymeric compounds or low molecular weight compounds, additional chemical reaction steps are unavoidable.
  • the non-structural protein 3 (NS3) of hepatitis C virus (HCV) shows protease and helicase activity which are essential for the life cycle of HCV.
  • NS3 hepatitis C virus
  • a large phagemid library with 2 x 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 E. 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 for the affinity of sFv for NS3 in 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 opens up for transduction into infected cells a new gene therapy strategy for intracellular immunization against chronic HCV infection.
  • the phage display technique enables affinity selection of proteins of 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 HR, Winter GJ Mol. Biol., 227: 381-8 (1992); Winter G. et al., Annu. Rev. Immunol., 12: 433-55 (1994)).
  • a library of human antibody fragments was cloned by immunoglobulin-specific PCR from plasma cells from HCV-infected patients.
  • Antibody fragments are expressed by fusion with a smaller coat protein (Gen III protein; glllp) at the tip of the phages on the surface of filamentous bacteriophages (phage display) (Marks JD et al., J. Mol. Biol., 222: 581-97 (1991); Pope AR et al., New York: IRL Press, 1-40 (1996); McCafferty J. et al., Nature, 348: 552-4 (1990)).
  • Gene III protein Gene III protein
  • phages carrying antibodies with high affinity for the target antigen can be dramatically enriched in several cycles of selection and reamplification (panning).
  • HCV protein NS3 human monoclonal antibodies against the essential HCV protein NS3 (see SEQ ID NO: 18) were cloned for possible use as an intra-body for intracellular immunization. Bone marrow aspirates from HCV infected patients were used to amplify the coding regions of immunoglobulin variable domains. These PCR products were used to clone a large human sFv phagemid library. An EIA against recombinant NS3 was used to ensure high serum antibody titers against NS3.
  • Phages carrying high affinity sFv fragments against NS3 were selected using phage display and several cycles of affinity selection. Monoclonal phages were isolated and tested for binding activity against NS3. Most of the antibodies were directed against NS3 helicase and not against NS3 protease, as already described (Chen M. et al., Hepatology, 28: 219-24 (1998)). Soluble recombinant sFv fragments were expressed, purified and in turn tested for their ability to
  • binding to NS3 protein tested.
  • the coding cDNA from binding sFv was isolated and sequenced.
  • the variable domains of six sFv fragments were characterized.
  • the sequence showed ⁇ light chains and ⁇ 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, which indicate an inhibition of
  • the antibody fragments according to embodiment (8) of the invention can be part of pharmaceutical compositions (such as vaccines, etc.) and of diagnostic compositions (also called “diagnostic kits").
  • these compositions also contain conventional additives, auxiliaries, etc.
  • the antibody fragments according to the invention can e.g. in a 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 CC et al., Biocem. Biophys. Res. Commun., 253: 594-9 (1998)) can be used in a replicon system for evaluating the function of nonstructural HCV proteins (Lohmann V. et al., Science, 285: 110-3 (1999)) etc.
  • the present invention relates to a gene transfer vector which comprises a DNA sequence which codes for an antibody fragment according to the invention.
  • This gene transfer vector can in addition to the above DNA sequence still contain further functional DNA sequences (such as promoter sequences, recognition sequences, viral sequences, translocalization sequences, splice donor and acceptor sequences, etc.).
  • the coding cDNA for antibody fragments which inhibit HCV replication must be transduced by vectors.
  • the currently most promising vectors for liver gene therapy are lentiviruses or "boned" adenoviruses (Richardson JH et al., Gene Ther., 5: 635-44 (1998); Naldini L.
  • compositions, vaccines and medicaments according to embodiments (8), (9), (10) and (11) can contain, in addition to the antibody fragments or gene transfer vectors according to the invention, further pharmacologically suitable auxiliaries and carriers and solvents.
  • the exact composition and the content of the antibody fragments and gene transfer vectors depends both on the chosen form of application and on the type and severity of the disease etc. and is set in a suitable manner by the person skilled in the art.
  • embodiments (12) and (13) the treating doctor will determine the exact dosage and the treatment regimen.
  • the sequence and structure of the antibody fragments according to the invention are also 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, low-molecular compounds are derived that inhibit these essential viral proteins of HCV.
  • Bone marrow aspirate was obtained from five patients with chronic hepatitis C. All patients had a serologically documented HCV infection (HCV antibodies positive in the third generation enzyme-linked immunoassay and serum HCV-RNA detectable by RT-PCR). Bone marrow aspirate was obtained from all patients during procedures that were indicated for various medical reasons. All patients signed a written consent for the removal of an additional 5 ml of aspirate and 10 ml of serum following an assessment by the local ethics committee. Serum was available from 4 of the 5 patients.
  • HCV-NS3 ELISA An ELISA plate (Nunc, Gibco BRL, Düsseldorf) was coated overnight at 4 ° C. with 0.5 ⁇ g NS3 in PBS and then blocked with 3% BSA / PBS at RT for 1 h. Patient sera were diluted 1: 1000, 1: 2000, 1: 4000, 1: 8000 and 1:16 000, respectively, 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.
  • the V H and V L genes were amplified by PCR separately with V H and V L -specific primers.
  • V's reverse primer was further enhanced by incorporating a flag modified at the 5 'end (Knappik, Plückthun, 1994).
  • the V H antisense primer and the V sense primer contain sequences which encode a flexible amino acid linker (Gly Ser) 4 which is inserted between the variable domains of the heavy and the light chain.
  • the cycle parameters were as follows: 30 cycles, 94 ° C 1 min, 57 ° C 2 min, 72 ° C 1 min.
  • gel-purified V H and V L PCR fragments (size approximately 300 bp) were mixed with one another in equimolar ratios and used to form a template in the absence of primers (2x: 92 ° C. 1 min, 63 ° C 30 s, 58 ° C 50 s, 72 ° C 1 min).
  • HuV ⁇ 2back 5 'GCC ATG GCG GAC TAC AAA GAC CAG TCT GCC CTG ACT CAG
  • HuV ⁇ 3back 5 'GCC ATG GCG GAC TAC AAA GAC TCT TCT GAG CTG ACT CAG
  • HuV ⁇ 5back 5 'GCC ATG GCG GAC TAC AAA GAC CAG GCT GTG CTC ACT CAG
  • HuV ⁇ öback 5 'GCC ATG GCG GAC TAC AAA GAC AAT TTT ATG CTG ACT CAG CCC
  • Lambda ( ⁇ ) antisense primer HuJ ⁇ lfor 5 'GGA GCC GCC GCC GCC AGA ACC ACC ACC ACC AGA ACC ACC ACC
  • VH sense primer HuJHla back 5 'GGC GGC GGC GGC TCC GGT GGT GGT GGA TCC CAG GTG CAG
  • 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 tag.
  • the resulting plasmids of all 5 patients were used to transform electro-competent E. coli XLi-Blue (Stratagene, Amsterdam Zuidoost, The Netherlands) and thus to create a large library of recombinant human sFv fragments in the pAKlOO expression vector (Krebber A. et al., J. Immunol. Methods, 201: 35-55 (1997)). Transformants were raised in 2 x YT medium containing 25 ⁇ g / ⁇ l 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 library size of all 5 patients was 2 x 10 6 .
  • the library was then infected with 10 11 "12 VCSM13 helper phages (Amersham Pharmacia, Freiburg) to achieve sFv-bearing phages. Expression was induced by the addition of 0.5 mM IPTG (Roth, Düsseldorf) and for 2 hours at 24 ° C. After 3-4 hours of incubation, kanamycin (30 ⁇ g / ⁇ l; Fluka, Neu-Ulm) was added, and the recombinant phage culture grew overnight at 24 ° C. The phage particles were removed by precipitation with 20% PEG / 15% NaCI (Sambrook et al., 1989) harvested, dissolved in PBS and stored at 4 ° C. or used directly for further experiments.
  • Affinity Selection Recombinant phages carrying human sFv fragments were selected by panning based on their affinity for binding to NS3.
  • a microtiter plate (Nunc, Gibco BRL, Düsseldorf) was coated overnight at 4 ° C. with 1 ⁇ g recombinant NS3 protein / well (Mikrogen, Kunststoff). After blocking with 3% BSA for 1 h at RT, a phage suspension (approximately 10 9 , diluted in 3% BSA) was added and it was incubated for 2 h at RT. The microtiter plate was washed thoroughly (20x) 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 XLl-Blue, which was in exponential growth. This panning process was repeated 4 times selected phage population was diluted and streaked to obtain single monoclonal colonies.
  • Phage EIA Selected single colonies were grown separately in 8 ml of 2 x YT medium containing chloramphenicol (25 ⁇ g / ⁇ l) and glucose (1%) at 37 ° C. with shaking and then infected with 10 ′′ VCSM13 helper phages.
  • Sequencing reactions were carried out using an automatic ABI-310 sequencer (Perkin Elmer, Rodgau-Jügesheim), using standard sequencing instructions and specific vector oligonucleotides. Then the derived amino acid sequences were determined by 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 EA et al., 5th ed.Bethesda, MD (1991); Martin ACR, 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.
  • Immunoblot analysis 1/100 purified sFv fraction was used for a further immunoblot analysis. Specifically, a 20 ul 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.
  • 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 ⁇ g 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 ⁇ g in PBS / well) and incubated for 1 h at room temperature. After 5 washes with TBST / 0.5% Tween ® - 20 was the anti-His Ab (1: 3% BSA in 2000; 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 The affinity of the antibody fragments was determined using a competitive EIA, which was first described by Friguet et al. (Friguet B. et al., J. Immunol. Methods, 77: 305-19 (1985)). The antibody to be tested was incubated with various concentrations of the antigen until an association-dissociation equilibrium was reached. A solid phase EIA with immobilized antigen was then used to determine the concentration of the free antibody that is not bound to soluble antigen (Friguet B. et al., J. Immunol.
  • sFv fragments Different concentrations of the human sFv fragments were tested to determine the linear region of the saturation curve. For this purpose, serial dilution series of the antibodies were carried out with subsequent determination of the OD. The dilution of the sFv fragments, which resulted in a drop in OD by at least 40%, was used for further competitive EIA experiments selected. sFv fragments at this concentration were incubated with soluble recombinant NS3 protein at 4 ° C for 2 hours to achieve equilibrium. The concentration of the free sFv fragments in sFv-EIA was then determined as described above, using plates coated with 0.3 ⁇ g NS3, which had previously been blocked with 10% milk powder. The affinity was determined for each individual antibody fragment in correlation to the linear part of the competition curve.
  • Optimal production of glll-fused sFv was achieved by 4 h incubation at RT.
  • the recombinant sFv expressed on the surface of filamentous phages were selected in a panning reaction with NS3 bound to a solid phase.
  • Polyclonal phages were used in 4 cycles of phage panning to enrich functional binding phages and to eliminate non-binding phages (see Table 1 and Figure 3 below).
  • Table 1 shows the relative enrichment of phage carrying monoclonal sFv against NS3 protein (SEQ ID NO: 18) before use in panning and after the first, second, third and fourth rounds of affinity selection.
  • the phage titer was measured as cfu / ml.
  • the number of sFv-carrying phages during the panning was determined as cfu / ml by dilution series.
  • the phage population was amplified to 10 10 "12 and then subjected to affinity selection against recombinant HCV-NS3.
  • phages bound to NS3 were eluted with 0.1 M glycine / HCl, pH 2.2, as as described in the "Materials and Methods" section.
  • the percentage of phage eluted after panning is increasing steadily, indicating that phages binding against HCV-NS3 are being enriched.
  • the recombinant sFv was further purified using the Ni-NTA column cleaning system and FPLC as described in the section "Materials and Methods". After cleaning, no non-specific bands were seen when sFv was loaded on 12% PAGE, transferred to a nylon membrane and stained with Ponceau's Red or Coomassie Brilliant Blue.
  • the coding sequence of phagemid DNA was determined and the amino acid sequence of the variable domains of the antibody fragments was derived (see Figure 7).
  • the positions of scaffold regions (FR) and complementarity-determining regions (CDR) were determined according to the numbering system by Kabat et al. (Kabat E. A. et al., 5th ed. Bethesda, MD (1991)).
  • Figure 7 shows a comparison of the sFv sequences.
  • Tables 2A and B summarize the classification and germline segments of variable domains of cloned antibody fragments.
  • Recombinant NS3 helicase, expression and purification Recombinant NS3 helicase was produced in insect cells using the Bac-to-Bac baculovirus expression system (Gibco BRL). The helicase coding sequence was cloned into pFastBacHTa and transformed into MAX Efficiency DHlOBac. Recombinant bacmid was isolated, treated with CellFECTIN reagent (Gibco BRL) according to the manufacturer's instructions and transfected into SF21 insect cells.
  • CellFECTIN reagent Gibco BRL
  • SF21 cells were grown at 27 ° C in Grace's supplemental insect medium (Invitrogen) containing 10% heat inactivated fetal bovine serum (Biochrom KG) and 10 ⁇ g / ml gentamycin. Recombinant baculovirus was collected 96 hours after transfection, propagated in SF21 cells and used to infect High Five TM insect cells (Invitrogen) for recombinant protein production. High Five TM insect cells were grown in Ultimate Insect TM serum-free medium (Invitrogen) as an adherent cell culture and harvested four days after infection.
  • Invitrogen Grace's supplemental insect medium
  • fetal bovine serum Biochrom KG
  • Recombinant baculovirus was collected 96 hours after transfection, propagated in SF21 cells and used to infect High Five TM insect cells (Invitrogen) for recombinant protein production.
  • High Five TM insect cells were grown in Ultimate Insect TM serum-free medium (Invitrog
  • the lysate-Ni-NTA mixture was applied to a column and washed with 20 ml of lysis buffer containing 20 mM imidazole.
  • the fusion protein attached to Helikase-His (6) was eluted with 200 mM imidazole in lysis buffer without protease inhibitors and ⁇ -mercaptoethanol and overnight against 50 mM NaPO 4 , 0.3 M NaCl, 20% glycerol, 0.5% Triton ® -100, dialyzed at pH 8.0.
  • the results of the helica production and cleaning are summarized in FIG. 10.
  • Helicase activity assay The helicase activity assay was performed as an ELISA as described by Hsu, C. et al. (Biochemical and Biophysical Research Communications, 253: 594-599 (1998)) with the following modifications.
  • the substrate consisted of two annealed complementary DNA strands, the 5'-biotin-labeled template strand: 5'-GGTTTAAAAA ATAGGAGGGA CAACGTCGTG ACTGGGAAAA CTCCCCGGGT ACCGAGCTCG-3 '(SEQ ID NO: 66) and the 5'-digoxigenangin-labeled '-GTTTTCCCAG TCACGACGTT GT-3' (SEQ ID NO: 67; MWG Biotech).
  • the annealing of the two oligonucleotides in a molar ratio of 1: 3 was carried out in 10 mM Tris / HCl, 25 mM NaCl, 1 mM EDTA at pH 7.6 by heating to 100 ° C. for 10 minutes and slowly cooling Room temperature.
  • High binding polystyrene Vi area microtiter plates (Costar) were coated with 10 ⁇ g / ml streptavidin (Fluka) and blocked with 2% BSA in PBS. The substrate was transferred to the blocked plate at a concentration of the template strand of 0.2 pM per well (50 ⁇ L) and incubated for 1 hour at room temperature.
  • HCV helicase in buffer M was supplemented with 2.5 mM ATP and 20 pM per well of trapping oligo, complementary to the release strand: 5'-ACAACGTCGT GACT- GGGAAA AC-3 '(SEQ ID NO: 68) added.
  • the plate was incubated with shaking at room temperature for 15 minutes and stationary at 37 ° C. for one hour, then washed three times with buffer M and three times with PBS. Release train, which was not wrung out by helicase was treated with anti-digoxigenin-alkaline phosphatase, Fab-fr.
  • FIG. 1 Serum antibody titers of 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 ) detected. The 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 against HCV-NS3.
  • AP alkaline phosphatase
  • Fab goat anti-human
  • 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 ⁇ ⁇ ⁇ 6 (picture A), the lambda chains ⁇ - ⁇ 7 (picture B) and the heavy chains H1-H6 (picture C) are shown, loaded on a 1.5% agarose -Gel.
  • FIG. D shows sFv-PCR products including the light chains V L ⁇ (K + H, lane 2) or V ⁇ ( ⁇ + H, lane 3), which were amplified separately. Different subgroups of V or V H are for individual
  • FIG. 3 Enrichment of phages with sFv fragments which 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 phage.
  • FIG. 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.
  • FIG. 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 antibodies and then HRP-conjugated rabbit anti-mouse antibodies 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.
  • 50 ⁇ l whole cell fraction of E. coli was loaded onto 12% SDS-PAGE gel and treated with anti-flag ( ⁇ -flag) and anti-tetra-His ( ⁇ -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 from experiment 1.
  • the coding cDNA of monoclonal sFv, which is positive in phage EIA and sFv-EIA against NS3 has been 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.
  • Figure 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, sFv 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 ensure a stable association To achieve 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 in the
  • FIG. 9 Amino acid sequence of the variable regions of the L chain (FIG. 7A) and H chain (FIG. 7B) of selected human sFv from experiment 2.
  • FIG. 10 Production and purification of the recombinant HCV-Helikaseenzyms HCV helicase was produced in insect cells (Bac-to-Bac ® baculovirus expression system, Gibco BRL) and purified by Ni-NTA (Qiagen).
  • A Detection of helicase expression by Western blot in High Five TM insect cells with Tetra-His TM antibodies (Qiagen).
  • Lane 1 total cell extract of the Baculovurs-infected insect cells.
  • Lane 2 control, total cell extract of uninfected insect cells.
  • Lane 3 molecular weight markers.
  • Lane 1 cell extract
  • Lane 2 pass
  • Lane 3 wash fraction
  • Lanes 4, 5, 6 elution fractions
  • Lane 7 molecular weight markers.
  • the recombinant sFvl-2 antibody was preincubated together with HCV helicase, then the helicase activity was measured (PBS control).
  • the sFvl-2 antibody showed a clear inhibitory effect on the
  • Figures 11B and 11C Inhibition of HCV helicase activity by the sFvl-2 antibody.
  • 11B Affinity-reduced recombinant proteins for helica assay (SDS gel electrophoresis, Coomassie staining). Lane 1: molecular weight marker; Lane 2: recombinant HCV helicase; Lane 3: sFvl-2 protein.
  • Figure 12 Inhibition of helicase activity by the recombinant sFv antibodies of Experiment 2.

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Abstract

La présente invention concerne des fragments d'anticorps humains qui inhibent la multiplication du virus de l'hépatite C et qui comprennent les domaines variables de la chaîne légère (VL) et de la chaîne lourde (VH) d'anticorps opposés à une ou plusieurs protéines essentielles du virus de l'hépatite C. Cette invention concerne également l'utilisation des fragments d'anticorps pour le traitement et le diagnostic de l'hépatite C, des séquences d'ADN qui codent pour ces fragments d'anticorps, ainsi que l'utilisation de ces séquences d'ADN dans des constructions de vecteurs servant à mettre en oeuvre une thérapie génique de l'hépatite C.
PCT/EP2002/005227 2001-05-11 2002-05-13 Fragments d'anticorps humains a affinite elevee, opposes aux proteines essentielles du virus de l'hepatite c WO2002093519A2 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7378093B2 (en) 2001-10-16 2008-05-27 The United States Of America As Represented By The Department Of Health And Human Services Broadly cross-reactive neutralizing antibodies against Human Immunodeficiency Virus selected by Env-CD4-co-receptor complexes
US7566451B2 (en) 2002-05-06 2009-07-28 The United States Of America As Represented By The Department Of Health And Human Services Human immunodeficiency virus-neutralizing human antibodies with improved breadth and potency
US7803913B2 (en) 2002-05-06 2010-09-28 The United States Of America As Represented By The Department Of Health And Human Services Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries
US8110192B2 (en) 2002-05-06 2012-02-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Human immunodeficiency virus type 1 (HIV-1)-neutralizing human single-chain antibodies with improved breadth and potency
WO2006071206A2 (fr) * 2003-09-29 2006-07-06 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunoglobulines a activite antivirale potentielle et large
WO2006071206A3 (fr) * 2003-09-29 2006-08-31 Us Gov Health & Human Serv Immunoglobulines a activite antivirale potentielle et large
EP2322624A3 (fr) * 2005-03-25 2012-03-14 National Research Council Of Canada Procédé d'isolation de polypeptides solubles
EP2330196A3 (fr) * 2005-03-25 2012-04-18 National Research Council of Canada Procédé d'isolation de polypeptides solubles
US8293233B2 (en) 2005-03-25 2012-10-23 National Research Council Of Canada Method for isolation of soluble polypeptides
US10150807B2 (en) 2005-03-25 2018-12-11 National Research Council Of Canada Method for isolation of soluble polypeptides
US11091536B2 (en) 2005-03-25 2021-08-17 National Research Council Of Canada Method for isolation of soluble polypeptides
US11993643B2 (en) 2005-03-25 2024-05-28 National Research Council Of Canada Method for isolation of soluble polypeptides

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