WO2002059340A1 - Immunopolypeptides diriges contre le virus de l'hepatite c - Google Patents

Immunopolypeptides diriges contre le virus de l'hepatite c Download PDF

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Publication number
WO2002059340A1
WO2002059340A1 PCT/US2002/002303 US0202303W WO02059340A1 WO 2002059340 A1 WO2002059340 A1 WO 2002059340A1 US 0202303 W US0202303 W US 0202303W WO 02059340 A1 WO02059340 A1 WO 02059340A1
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immunopolypeptide
ofthe
human
cdr
hepatitis
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PCT/US2002/002303
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English (en)
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Toshiaki Maruyama
Ian M. Jones
Dennis R. Burton
Robert I. Fox
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The Scripps Research Institute
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to immunopolypeptides produced by a phage transfected cell library.
  • the immunopolypeptides have binding specificity for certain envelop glycoproteins and nonstructural proteins of hepatitis C virus (HCV).
  • the immunopolypeptides include monoclonal antibodies, fragments thereof and variants thereof.
  • the invention also relates to a method for the detection and treatment of HCV.
  • Viral hepatitis is now known to include a group of viral agents with distinctive viral organization, protein structure and mode of replication, causing hepatitis with different degrees of severity of hepatic damage through different routes of transmission.
  • Acute viral hepatitis is clinically diagnosed by well-defined patient symptoms including jaundice, hepatic tenderness and an elevated level of liver transaminases such as Aspartate Transaminase and Alanine Transaminase.
  • One of these viral agents, hepatitis C virus has recently been recognized as a primary causative agent of NANB hepatitis (Non- A, Non-B).
  • Hepatitis A virus HAV
  • HBV hepatitis B virus
  • HDV delta hepatitis virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • HCV Hepatitis C virus
  • HCV chronically infected
  • 73.7% is genotype I (56.7% with genotype la and 17.0%> with lb) according to a study by CDC (NEngJMed 1999, 341 :556-562).
  • Hepatitis C is responsible for about 90%) ofthe cases of post- transfusion hepatitis. Hollinger et al. in N. R. Rose et al., eds., Manual of Clinical Immunology, American Society for Microbiology, Washington, D. C. , 558-572 (1986).
  • hepatitis C antigens and antibodies are agar-gel diffusion, counter- immunoelectrophoresis, immuno-fluorescence microscopy, immune electron microscopy, radioimmunoassay, and enzyme- linked immunosorbent assay.
  • serologic tests for HCV infection have been developed and are commercially available. Although such serologic tests eliminate 70 to 80% of hepatitis C infected blood from the blood supply system, the antibodies apparently are readily detected during the chronic state ofthe disease, while only 60% ofthe samples from the acute NANBH stage are HCV antibody positive. H. Alter et al., New Eng. J. Med. 321 :1994-1500 (1989).
  • Envelope glycoprotein E2 and nonstructural protein NS3 which is believed to be related to viral replication, are thought to be responsible for the binding of HCV to target cells and contain neutralizing epitopes.
  • CD81 has been found to be the receptor for E2 (Science 1998, 282:939-941).
  • NOB neutralization of binding
  • E2 glycoprotein also may be involved in the pathogenesis of sialadenitis among subset of patients with chronic HCV infection who develop Sj ⁇ gren's syndrome (SS) according to a study using transgenic mice with E1/E2 developing sialadenitis histologically resembling SS (Proc Nad Acad Sci USA 1997, 94:233-236).
  • the immunopolypeptide includes full-length monoclonal antibodies, monoclonal antibody fragments thereof as well as antibody and fragment variations containing designated CDR's and framework variations.
  • the invention is further directed to a phage library displaying such an immunopolypeptide on phage molecule surfaces.
  • Additional aspects ofthe invention include a nucleotide sequence encoding the immunopolypeptide, a vector carrying the nucleotide sequence, a recombinant cell for expression ofthe immunopolypeptide, a diagnostic technique for hepatitis C screening and a method of treatment of hepatitis C or passive immunization using the immunopolypeptide.
  • the immunopolypeptide ofthe invention demonstrates significant immunobinding, preferably high affinity binding, with hepatitis C viral particles.
  • the immunopolypeptide ofthe invention immunoreacts with the E2 envelop glycoprotein or the non-structural protein-2 of hepatitis C.
  • the immunopolypeptide includes monoclonal antibody fragments such as Fab fragments, Fab' fragments, F(ab') 2 fragments, Fd fragments, Fv fragments, single light and heavy chain fragments, full length single light and heavy chains, complete monoclonal antibodies and Variants ofthe fragments, single chains and complete antibodies.
  • the amino acid sequence for the immunopolypeptide ofthe invention includes at least any ofthe 141 CDR amino acid sequences given in Fig's. 12 and 13.
  • the CDR's for the immunopolypeptide are selected as triplets so that the immunopolypeptide will contain at least three CDR sequences.
  • the immunopolypeptide amino acid sequence also includes any ofthe framework regions having the amino acid sequences given in Fig's 12 and 13 .
  • the CDR and framework sequences are matched from one Fab of Fv fragment. Variants of the amino acid units of these framework regions are also included.
  • the immunopolypeptide sequences also include any ofthe CDR triplet sequences in appropriate position with any ofthe framework sequences such that the CDR and framework sequences from one Fab or Fv fragment are mixed with those of another.
  • the immunopolypeptide also includes a match or mix ofthe specified CDR sequences in appropriate position with other known human and other mammalian framework region sequences.
  • the specified CDR sequences of Fig's. 12 and 13 constitute two groups of individual variable region heavy and light chain CDR amino acid sequences that bind to epitopes ofthe E2 envelop protein or the NS3 nonstructural protein of hepatitis C.
  • the single chain sequences incorporating at least a triplet of CDR sequences within framework regions as described above constitute single chain fragments alone or such fragments may be combined with another chain fragment from a whole or partial constant region of human or other mammalian immunoglobulin.
  • These individual sequences may be combined as pairs of light and heavy sequences or a pair of heavy sequences to constitute the Fab, Fab', Fd and Fv antibody fragments respectively.
  • the Fab' pairs may be again combined to form the F(ab') 2 fragments.
  • the combinations are produced either by matching the single chain sequences or by mixing them.
  • the full length complete monoclonal antibodies constitute two Fab fragments and an Fc fragment from a human or other mammalian immunoglobulin including IgG, IgM, IgA, and IgE.
  • the Fab fragments may be mixed or matched.
  • the phage library ofthe invention constitutes an expression vector useful for transforming bacteria so that they will express the immunopolypeptide ofthe invention.
  • the library includes a recombinant phage incorporating DNA encoding the immunopolypeptide ofthe invention.
  • the phage library incorporates nucleotide sequences encoding the individual variable region sequences V H and V L ofthe immunopolypeptide ofthe invention.
  • nucleotide sequences are given in Figs. 14 and 15.
  • the phage embodiments of the phage library display binding with the E2 and NS2 proteins of hepatitis C.
  • the invention also provides nucleotide sequences encoding the individual V H and V L chains of Fig's. 12 and 13. These nucleotide sequences are given in Figs. 14 and 15.
  • Nucleotide sequences encoding the remaining immunopolypeptides ofthe invention can be constructed from segments of these V H and V L nucleotide chains in combination with known human or non-human mammalian consensus constant and framework regions. These consensus nucleotide sequences are known in the art.
  • Vectors encoding any ofthe individual variable region sequences given in Fig's 12 and 13 are also included in the invention. These include plasmids, phages, viruses and nucleotide segments for insertion into prokaryotic and eucaryotic cells.
  • a vector for insertion of DNA encoding the immunopolypeptide into Chinese hamster ovary (CHO) cells is preferred.
  • the vectors may appropriate regulatory sequences for expression, including but not limited to promoter, operator and transcription element intron sequences.
  • the recombinant cells ofthe invention include bacterial host cells, which have been transfected with a phage embodiment ofthe phage library. Also included are eucaryotic host cells and mammalian host cells such as CHO cells, which have, been transformed with an expression vector carrying a DNA sequence for the immunopolypeptide ofthe invention.
  • the process ofthe invention includes any recombinant technique to express the immunopolypeptide ofthe invention.
  • the DNA sequence encoding the immunopolypeptide ofthe invention may be inserted into an expression vector, that vector used to transfect an appropriate host cell and the host cell cultured to provide the immunopolypeptide.
  • the process ofthe invention also includes a screening technique for obtaining fully human monoclonal antibodies to hepatitis C. This screening technique involves obtaining mononuclear cells from a patient infected with hepatitis C. The cDNA derived from those cells is combined with a host phage that will display the protein on its coat.
  • the recombinant phage is then panned against the selected hepatitis C protein to identify those phage particles that bind to the selected protein.
  • the selected hepatitis C protein is obtained by recombinant expression ofthe corresponding hepatitis C DNA sequence using an appropriate expression vector and host cell.
  • the method of diagnosis according to the invention involves conducting an immunoreactive test of a patient's blood or blood serum against an immunopolypeptide ofthe invention. Identification of an antigen- immunopolypeptide (antibody or fragment) complex is made by such assay methods as radioimmunoassay, sandwich assay or ELISA assay.
  • the method of treatment according to the invention involves administration of an effective amount of an immunopolypeptide ofthe invention to a patient infected with hepatitis C.
  • the immunopolypeptide may be combined with a suitable pharmaceutically acceptable carrier as well as appropriate adjuvants and immune reaction enhancers.
  • Figure 1 shows a graph representing the reactivity of patient serum to E2 glycoprotein (genotype la, aa 388-644) (J. Med. Virol. 1995, 45:415-422).
  • Library patient serum Y and a Japanese patient serum J (genotype lb) were tested.
  • Normal healthy serum, NHS as included was a negative control.
  • FIG. 2 shows the scheme for recombinant production of E2.
  • Fragments encoding HCV envelope protein El and E2 were generated by PCR from a full- length cDNA clone of hepatitis C virus type la.
  • El was cloned into the baculovirus transfer vector pAcATMl to fuse to GST preceded by an insect signal peptide.
  • E2 was cloned into the transfer vector pAcATMl to fuse to an insect signal peptide without the GST domain.
  • the regions ofthe HCV genome (accession number: m62321) represented in the final transfer vectors were; nucleotides (nt) 904-1421 (for El), (nt) 1471-2754 (for E2).
  • Figure 3 shows a graph representing the reactivity of human Fab fragments with E2 glycoprotein. Reactivity of anti-E2 Fabs with recombinant E2 (4 ⁇ g/ml) and ovalbumin (4 ⁇ g/ml) determined by ELISA. B IF is a flag- tagged Bl. Ovalbumin (4 ⁇ g/ml) is included as a control antigen. OD405, optical density as 405 nm.
  • Figure 4 show a series of graphs representing the reactivity of human Fab fragments to GST-E1/E2.
  • GST-E1/E2 (8 ⁇ g/ml) was captured by goat anti-GST Ab (10 ⁇ g/ml) to microtiter wells.
  • Ovalbumin (4 ⁇ g/ml) was used as a control antigen.
  • Figure 5 shows a bar graph representing the reactivity of human Fab fragments to a GST-El /E2 complex and E2 alone. GST-El /E2 and E2 alone were coated at 8 and 4 ⁇ g/ml, respectively.
  • Ovalbumin (4 ⁇ g/ml) was used as a control antigen.
  • Figure 6 shows a graph representing inhibition of binding of GST-El /E2 and CD81 by human anti-E2 antibodies.
  • CD81 was coated at 4°C overnight and blocked with 4% nonfat dry milk/PBS.
  • GST-E1/E2 was preincubated with human anti-E2 antibodies and KZ52 IgG (a negative control Ab) for 1 hour at room temperature and added to the wells.
  • Detection of bound GST-E1/E2 was performed with rat anti-E2 antibody and AP conjugated anti-rat IgG (H+L) Ab (1:500). Percentage was calculated by 100-(OD405 (human anti-E2 Abs at each concentration)/OD405 (KZ52 IgG at each concentration)).
  • Cl IgG and Bl Fab effectively blocked the binding of GST-E1/E2 and CD81 (50%) at 0.1 ⁇ g/ml and 2 ⁇ g/ml, respectively).
  • FIG. 7 shows a series of graphs representing competition assay results.
  • Competition assays were performed to see the inhibition of bindings of human monoclonal Fabs to E2 by mouse monoclonal conformational Ab, H53 (gift of Dr. Jean Dubuisson, J. Virol., 72:2183-2191, 1998).
  • GST-E1/E2 was captured with goat anti-GST Ab (10 ⁇ g/ml) to microtiter wells and preincubated with H53 (0.032, 016, 0.8, 4, 20 and 100 ⁇ g/ml).
  • Detection of human Fabs was performed with alkaline-phosphatase (AP) conjugated goat anti-human IgG F(ab') 2 Ab (1:500) in 1% BSA/PBS.
  • AP alkaline-phosphatase
  • Figure 8 shows a graph representing the inhibition of GST-El /E2 complex binding of Cl IgG by a human Fab fragments. Detection of Cl IgG was done with AP conjugated anti-human IgG Fc (1 : 1000). Binding of Cl IgG was completely blocked by Fabs A, Cl, H2, 1, J3, L4, and M. Fab Bl and Cl were used as a negative and a positive control, respectively.
  • Figure 9 shows the titration of patient sera on NS-3 antigen.
  • Library patient serum (Y) (genotype la) and a Japanese patient serum (J) (genotype lb) with chronic HCV infection were titrated on recombinant NS-3.
  • Normal human serum (NHS) was included as a negative control.
  • Ovalbumin (4 ⁇ g/ml) was included as a control antigen.
  • Figure 10 shows a bar graph representing isotyping of patient serum IgG reactivity to NS-3. Detection of human IgGl, 2, 3, and 4 was performed with mouse IgG anti-human IgG isotype specific antibodies (PharMingen). Reactivity of sera of both library patient (Y) and another patient (J) sera was restricted to IgGl .
  • Figure 11 shows a bar graph representing the reactivity of phage clones against NS3 after 4 rounds of panning. The clones showed specific reactivity to NS-3. Ovalbumin (4 ⁇ g/ml) was included as a control antigen.
  • Figures 12 and 13 show the amino acid sequences ofthe framework and CDR segments used for the immunopolypeptides ofthe invention.
  • Figures 14 and 15 show the nucleotide sequences corresponding to the amino acid sequences of Figures 12 and 13.
  • Figure 16 shows the scheme for production ofthe recombinant cells producing preferred immunopolypeptides ofthe invention.
  • Figures 17 and 18 show the amino acid sequences for CDR and framework sequences ofthe invention.
  • the present invention provides an immunopolypeptide that is immunoreactive with hepatitis C virus.
  • This immunopolypeptide includes a group of complete monoclonal antibodies, antibody fragments, complete and partial single antibody chains and variations thereof that are immunoreactive with hepatitis C.
  • the immunopolypeptide may incorporate amino acid sequences from other mammalian antibody classes, the immunopolypeptide preferably is fully human so that its immunogenicity as a foreign protein is minimal or negligible.
  • the immunopolypeptide is obtained by recombinant methods involving phage amplification and selection. Other methods such as hybridoma preparation may also be used.
  • the immunopolypeptide can be formulated as a pharmaceutical composition and administered as a treatment of acute and chronic hepatitis C. In its fully human form, it will not cause development of immunosensitivity or anaphylactic sensitivity upon repeated administration. It can also form the basis of diagnostic tests to determine whether a patient is infected with hepatitis C. At least three conformational epitope(s) on E2 are recognized by the preferred Fab species of immunopolypeptide ofthe invention.
  • the immunopolypeptide ofthe invention may be not only therapeutically effective but also useful for the design of effective vaccine development or for passive immunization.
  • An immunopolypeptide the invention immunoreacts with epitopal sites ofthe E2 envelop glycoprotein and with the NS3 protein.
  • the E2 glycoprotein is believed to be responsible for target cell binding and contains neutralizing epitopes.
  • Development of antibodies that are immunoreactive with the E2 glycoprotein is believed to provide an especially effective regimen of treatment for chronic hepatitis C infection. This regimen is especially useful in situations where the sera of a patient does not exhibit an immunoresponse to a viral challenge but the patient nevertheless carries the virus.
  • the immunopolypeptide has an amino acid sequence that incorporates any ofthe CDR amino acid sequences set forth in Figures 12 and 13. These CDR sequences have SEQ ID NO's 78-308and in its most basic form is a single amino acid chain.
  • the immunopolypeptide contains a triplet of these CDR sequences wherein each CDR is individually chosen from either or both ofthe light and heavy CDR groups.
  • the triplet of CDR sequences is chosen from one ofthe light and heavy CDR groups. More preferably, the triplet is chosen so that it matches the CDR's of a single chain of an Fab fragment of Figure 12 or 13.
  • the CDR's chosen for the immunopolypeptide are selected so as to bind to the antigenic E2 glycoprotein or the NS3 protein of hepatitis C.
  • the triplet of CDR's is appropriately spaced so as to provide a trifunctional binding site.
  • the spaced triplet binds to the E2 glycoprotein or the NS3 protein.
  • the trifunctional binding site has spacer amino acid sequences between the CDR sequences that mimic the consensus number of amino acid units between CDR sequence of a human or other mammalian antibody.
  • a preferred spacer peptide sequence is a mammalian antibody variable region framework sequence as is well known in the art, such as those in the National Center for Biotechnology Information (NCBI) genebank database.
  • the immunopolypeptide mimics the variable region of a single chain of an antibody.
  • the selected CDR's ofthe immunopolypeptide are spaced with a human framework.
  • the human framework is a consensus human framework of a human immunoglobulin, especially an IgG.
  • the framework has a sequence as given in Figures 12 and 13. These framework sequences have SEQ ID NO's 309-401, 433-537, 573-587 and 593-610.
  • the immunopolypeptide incorporates a matched CDR and framework region of a single chain of an Fab fragment provided in Figures 12 and 13. Immunopolypeptide sequences having non-CRD amino acid segments substantially identical to those these Fab fragments and having CRD sequences identical to those of these Fab fragments are also preferred.
  • the most basic structure ofthe immunopolypeptide is a single amino acid chain having the CDR selections as described above.
  • the immunopolypeptide may also have a structure that combines this single chain with a single chain of a constant region of a human or other mammalian immunoglobulin.
  • the immunopolypeptide may be a combination of single chains. In particular, it may be a combination of any pair of single chains having CDR triplets.
  • this combination includes the spacer amino acid units as discussed above. More preferably, this combination includes a triplet selected from the light CDR group and a triplet selected form the heavy CDR group. Especially more preferably, this combination includes the matched triplets and framework regions discussed above. Most preferably, this combination includes a light chain sequence and a heavy chain sequence with matched triplets and framework regions as discussed above.
  • variable region Fab or Fv fragment as provided by Figures 12 and 13.
  • This preferred version may also be combined with the constant regions of an Fab or Fab' fragment of a human or other mammalian immunoglobulin to provide the complete Fab or Fab' fragment.
  • the heavy chains of such complete Fab or Fab' fragments may be combined with a single heavy chain of an Fc fragment of an human immunoglobulin to provide at least one side of a complete antibody. Any of these pairs may also be combined to provide a double pair combination, which will have a structure mimicking the "Y" form of a truncated or complete antibody.
  • the immunopolypeptide When the immunopolypeptide has a structure like the variable region of an antibody (i.e. a CDR triplet spaced with an antibody framework sequence), it mimics, or in certain versions is, the variable region single chain of an Fab monoclonal antibody fragment. If the appropriate constant region sequence of an Fab fragment is added, the immunopolypeptide has a structure mimicking, or is, a complete single chain of an Fab or Fab' antibody fragment. If the CDR triplets are chosen from the light and heavy groups as discussed above, immunopolypeptide is a variable region single chain of an Fab monoclonal antibody fragment.
  • the immunopolypeptide is a full length heavy or light single chain of a monoclonal antibody.
  • the immunopolypeptide may also be a combination of two such single chains of any ofthe foregoing descriptions. This combination may be two light chains, two heavy chains, two mixed CDR chains, or preferably a light and heavy chain combination.
  • the last combination includes the variable region and the option constant region, it has a construction like that of an Fab or Fab' fragment.
  • this combination provides an Fab' fragment and two of such fragments are combined, the resulting immunopolypeptide is an F(ab') 2 monoclonal antibody fragment.
  • the immunopolypeptide is a F(ab) x fragment plus a constant region Fc of a human or other mammalian immunoglobulin, it is a complete human or other mammalian monoclonal antibody.
  • Preferred species ofthe immunopolypeptide ofthe invention include the Fab variable region amino acid sequences provided in Figures 12 and 13. These sequences have SEQ ID NO's 1-77. Also preferred are the variants of these Fab fragments as well as amino acid sequences that are substantially identical in their non-CDR segments and identical in their CDR segments. Additional preferred species include these Fab variable region sequences to which have been added human consensus constant region sequences as provided within the genebank of the National Center for Biotechnology Information.
  • Preferred CDR sequences for the immunopolypeptide ofthe invention include the amino acid sequences designated in the CDR columns of Figures 12 and 13. These CDR's have SEQ ID NO's 78-308.
  • Preferred framework sequences for the immunopolypeptide of the invention include the amino acid sequences designated in the framework columns of Figures 12 and 13. These framework sequences have SEQ ID NO's 309-401, 433-537, 573-587 and 593-610.
  • Preferred Fab' human consensus constant region sequences for the immunopolypeptide ofthe invention, which provide the heavy chain constant region include those provided within the genebank ofthe National Center for Biotechnology Information.
  • Further preferred embodiments ofthe invention include the Fabs to E2 glycoprotein and to NS3 presented in Figures 12 and 13. These Fab fragments have SEQ ID NO's 1-77. These Fabs were isolated from a phage display library made from the bone marrow of a patient with chronic HCV infection and SS. These Fabs showed good neutralizing ability by NOB assay (50% at 0.5-1.0 ⁇ g/ml). They may be divided into three groups.
  • Group I does not bind to the same epitope as the known H53 antibody and binds better to E1/E2 complex than it does to E2 alone. This group also binds to the same E2 epitope as Cl IgG.
  • Group II does bind to the same epitope with H53 and binds better to E2 alone than to the E1/E2 complex.
  • Group III does not bind to the same epitope of E2 as H53 does and binds better to E2 alone than to the E1/E2 complex.
  • the nucleotides ofthe invention are produced by manipulation ofthe
  • DNA sequences obtained from the phage library as discussed below.
  • the recombinant techniques for obtaining the DNA encoding the source antibodies that immunoreact with HCV provide the DNA sequences encoding the matched
  • nucleotide sequences encoding the CDR's and framework amino acid sequences may be produced. Religation of these CDR and framework nucleotide segments using techniques known in the art will produce the nucleotide sequences encoding the other variations ofthe immunopolypeptide.
  • nucleotide sequences for the consensus constant regions may be obtained from the gene bank and used in known ligation procedures to engineer additive DNA sequences for still other forms ofthe immunopolypeptide such as but not limited to the complete antibody, Fab' fragments, Fd fragments, complete single chains, as well as Fab and Fv fragments containing consensus constant regions. See the Cold Spring Harbor Laboratory Manuals cited below for the details involved in DNA sequence engineering. Amino acid sequences of the invention may also be produced through synthetic methods well-known in the art (Merrifield, Science, 85:2149 (1963)). Process For Preparation ofthe Immunopolypeptide
  • the CDR sequences for the immunopolypeptide ofthe invention preferably are derived from the mononuclear cells of a human patient chronically infected with hepatitis C using known techniques.
  • Alternative CDR sequences may be developed by known techniques through non-human mammal challenge with the E2 envelop glycoprotein or NS2 protein of hepatitis C virus as an antigen. These techniques and development of CDR sequences from antibodies is described, for example, in Andbodies, A Laboratory Manual by Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988, and in Molecular Cloning, A Laboratory Manual by Sambrook, et al., Cold
  • the CDR development technique using a human source was the procedure used to provide the CDR sequences of Figs. 12-15.
  • the CDR development technique first involves a collection of immune cells sensitized to the specified antigen.
  • mononuclear cells from a patient infected with hepatitis C are collected.
  • these cells are from the bone marrow ofthe infected human patient.
  • Speen cells may also be harvested from a non-human mammalian host.
  • the mononuclear cells are processed according to the phage display technology described by Barber et al. Proc. Natl.
  • the mRNA or the cDNA may be amplified by PCR techniques to provide full length genes or through the use of selected primers to provide antibody fragments such as Fab, F(ab') , V H , V L , scFv, the complete partial constant region for the Fab, and the like.
  • the cDNA or PCR products may then be inserted into a vector, such as a bacteriophage or a phagemid through use of recombinant DNA techniques well-known in the art. Sambrook et al. (1989).
  • the vectors containing the cDNA or PCR products may then be transformed into a bacteria to produce a library.
  • This procedures will provide a vector for transfection of bacteria and allow expression ofthe desired cDNA or PCR product, such as an antibody, single variable chains, or Fab fragments.
  • the procedure also allows for the production of a polypeptide which is fused to a coat protein.
  • the library of recombinant phage may be panned as described in the foregoing references and patents to select those phage carrying antibody chains that will bind with the E2 glycoprotein or the NS2 protein.
  • the panning may be accomplished by combining the phage library with immobilized glycoprotein or protein, removing the phage not bound, followed by removing the bound phage.
  • the mRNA or cDNA stock material may be amplified using selected primers to provide antibody variable regions.
  • the DNA encoding constant regions may be recombined in appropriate orientation once the desired expression vector is obtained.
  • the host bacterial cells such as E. coli or other suitable bacteria are transfected with panned phage library to provide a library of transfected cells.
  • the cells are separated to colonies carrying only single antibody genes by plating onto culture medium.
  • the phage may also carry a selection marker such as an antibiotic resistance gene. Selection with culture medium carrying the selection marker provides cultures of bacteria that have been transfected. Examination of single cell cultures from single colonies by a binding assay using the E2 or NS3 protein identifies those cultures exhibiting specific immunoreactivity.
  • nucleotide sequences encoding CDR's, framework, single chain variable regions or single chain variable and constant regions of Fab, Fab', Fd fragments may be conveniently identified by known procedures for nucleotide sequence identification.
  • nucleotide sequences encoding the Fab sequences provided in Figures 12 and 13 are determined by this technique. These nucleotide sequences are provided in Figures 14 and 15.
  • the cultures providing expression ofthe desired polypeptides may also be manipulated by known recombinant techniques to insert into a vector (e.g. the recombinant phage) the nucleotide sequences for remainder ofthe desired immunopolypeptide amino acid sequence.
  • Such sequences include, for example, the constant antibody regions of light and heavy chains as well as the Fc chain.
  • the CDR DNA sequences obtained through sequencing ofthe phage or phagemid DNA may be cloned into a vector carrying the DNA sequences encoding the spacers, framework and constant regions ofthe immunopolypeptide ofthe invention. Those DNA sequences are consensus sequences, are known and are available from gene bank sources as described above. If the phage library is designed to carry the nucleotide sequences for the antibody constant regions as well as the variable regions, those constant region DNA sequences may be used instead.
  • the framework DNA sequences obtained by sequence identification ofthe phage DNA from the immunoreactive bacterial cultures may be used as the nucleotide sequences encoding the framework amino acid sequences ofthe immunopolypeptides ofthe invention.
  • mammalian host cells such as Chinese hamster ovary cells may also be used.
  • the nucleotide sequence encoding the desired immunopolypeptide obtained as described above may be inserted into an expression cassette for mammalian host cells. Transfection and expression of the nucleotide sequence in the mammalian host cells will produce the immunopolypeptide.
  • These recombinant cells are capable of expressing the appropriately folded, complete monoclonal antibody.
  • Combinations of chains such as chains for an Fab fragment or light and heavy variable region chains can also be expressed by a single cell following the techniques given in the foregoing references and patents.
  • the techniques described above may be followed to provide antibody fragments or full length antibodies. Alternatively, single chain expression products may be mixed at appropriate ratios and coupled by disulfide ligation to provide two chain combinations.
  • the immune cells from a source such as an experimental non-human mammal or a patient infected by hepatitis C may also be fused with immortalized cells to provide hybridomas expressing the library of antibodies derived from the patient.
  • the techniques described above and in the Cold Spring Harbor Laboratory Manuals cited above provide the protocols for obtaining monoclonal antibodies from hybridomas.
  • Single chains typically are produced by the bacterial cell culture techniques described above.
  • the three dimensional structure of a typical antibody is known to be highly stable and reconstitutable. Consequently, under appropriate conditions known in the art, these single chains may be ligated and folded to provide active antibody configurations. Ligation may be achieved by conducting in vitro disulfide bond formation. Proper folding may be accomplished by dilute constitution in aqueous physiological media. Folding and disulfide ligation techniques are well known in the art. The following detailed procedure provides further explanation for production ofthe immunopolypeptide ofthe invention.
  • PCR amplification of Fd and K regions from the mRNA ofthe source mononuclear cells a may be performed as described by Sastry et al., Proc. Natl. Acad. Sci U.S.A., 86, 5728 (1989).
  • the PCR amplification is performed with cDNA obtained by the reverse transcription of the mRNA with primer specific for amplification of heavy chain sequences or light chain sequences.
  • the PCR amplification of messenger RNA (mRNA) isolated from the mononuclear cells with oligonucleotides that inco ⁇ orate restriction sites into the ends ofthe amplified product may be used to clone and express heavy chain sequences (e.g., the amplification ofthe Fd fragment) and K light chain sequences from mouse spleen cells.
  • the oligonucleotide primers which are analogous to those that have been successfully used for amplification of V H and V L sequences (see Sastry et al., Proc. Natl. Acad. Sci U.S.A., 86, 5728 (1989)), may be used for these amplifications.
  • Restriction endonuclease recognition sequences are typically inco ⁇ orated into these primers to allow for the cloning ofthe amplified fragment into a suitable vector (i.e. a phagemid or a ⁇ phage) in a predetermined reading frame for expression.
  • Phage assembly proceeds via an extrusion-like process through the bacterial membrane.
  • filamentous phage Ml 3 may be used for this process.
  • This phage has a 406-residue minor phage coat protein (cpIII) which is expressed before extrusion and which accumulates on the inner membrane facing into the periplasm of E. coli.
  • cpIII The two functional properties of cpIII, infectivity and no ⁇ nal (nonpolyphage) mo ⁇ hogenesis have been assigned to roughly the first and second half of the gene.
  • the N-terminal domain of cpIII binds to the F' pili, allowing for infection of E. coli, whereas the membrane- bound C-terminal domain, P198-S406, serves the mo ⁇ hogenic role of capping the trailing end ofthe filament according to the vectorial polymerization model.
  • a phagemid vector may be constructed to fuse the antibody fragment chain such as an Fab, Fab' or preferably an Fd chain with the C-terminal domain of cpIII (see Barbas et al., Proc. Natl. Acad. Sci.
  • a flexible five-amino acid tether (GGGGS), which lacks an ordered secondary structure, may be juxtaposed between the expressed fragment chain and cpIII domains to minimize interaction.
  • the phagemid vector may also be constructed to include a nucleotide coding for the light chain of a Fab fragment.
  • the cpIII/Fd fragment fusion protein and the light chain protein may be placed under control of separate lac promoter/operator sequences and directed to the periplasmic space by pelB leader sequences for functional assembly on the membrane.
  • phage FI intergenic region in the vector allows for packaging of single-stranded phagemid with the aid of helper phage.
  • helper phage superinfection may result in expression of two forms of cpIII. Consequently, normal phage mo ⁇ hogenesis may be perturbed by competition between the cpIII/Fd fragment fusion protein and the native cpIII ofthe helper phage for inco ⁇ oration into the virion.
  • the resulting packaged phagemid may cany native cpIII, which is necessary for infection, and the fusion protein including the Fab fragment, which may be displayed for interaction with an antigen and used for selection.
  • Fusion at the C-terminal domain of cpIII is necessitated by the phagemid approach because fusion with the infective N-terminal domain would render the host cell resistant to infection.
  • the result is a phage displaying antibody combining sites ("Phabs").
  • the antibody combining sites, such as Fab fragments are displayed on the phage coat.
  • This technique may be used to produce Phabs which display recombinantly produced Fab fragments, such as recombinantly produced Fab fragments that immunoreact with a antigen, on the phage coat of a filamentous phage such as Ml 3.
  • a phagemid vector i.e. pComb 3 or pComb3H which allows the display of antibody Fab fragments on the surface of filamentous phage, has been described (see Barbas et al., Proc. Natl. Acad. Sci. USA, 88, 7978 (1991).
  • Xho I and Spe I sites for cloning PCR-amplified heavy-chain Fd sequences are included in pComb 3 and pComb3H. Sac I and Xba I sites are also provided for cloning PCR-amplified antibody light chains.
  • the nucleotide sequences ofthe pelB leader sequences are recruited from the ⁇ HC2 and ⁇ LC2 constructs described in Huse et al, ibid, with reading frames maintained. Digestion of pComb 3 and pComb3H, encoding a selected Fab, with Spe I and Nhe I permit the removal ofthe gene III fragment, which includes the nucleotide sequences coding for the antibody Fab fragments. Because Spe I and Nhe I produce compatible cohesive ends, the digested vector may also be religated to yield a phagemid that produces soluble Fab.
  • Phabs may be produced by overnight infection of phagemid containing cells (e.g., infected E. coli XL-1 Blue) yielding typical titers of 10 11 cfu/ml.
  • phagemid containing cells e.g., infected E. coli XL-1 Blue
  • ratios of clonally distinct phage may easily be determined by titering on selective plates.
  • clonally mixed phage may be incubated with an antigen-coated plate. Nonspecific phage will be removed by washing, and bound phage may then be eluted with acid and isolated.
  • the immunopolypeptides are generally be formulated with a pharmaceutically acceptable carrier and may be administered by any desired route. More particularly, the immunopolypeptides may be formulated with a buffered aqueous, oil or organic medium containing optional stabilizing agents and adjuvants for stimulation of immune binding.
  • a preferred formulation involves lyophilized immunopolypeptide and separate pharmaceutical carrier. Immediately prior to administration, the formulation is constituted by combining the lyophilized immunopolypeptide and pharmaceutical carrier. Administration by a parenteral or oral regimen will deliver the immunopolypeptide to the desired site of action. The dosage and route of administration will generally follow the judgment ofthe patient's attending physician. In particular, intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration may be used.
  • the amount of immunopolypeptide useful to establish treatment of hepatitis C can be determined by diagnostic and therapeutic techniques well known to those of ordinary skill in the art.
  • the dosage may be determined by titrating a sample ofthe patient's blood sera with the immunopolypeptide to determine the end point beyond which no further immunocomplex is formed. Such titrations may be accomplished by the diagnostic techniques discussed below.
  • Available dosages include administration of from about 1 to about 1 million effective units of antibody per day, wherein a unit is that amount of immunopolypeptide, which will provide at least 1 microgram of antigen- immunopolypeptide complex.
  • from about 100 to about 100,000 units of antibody per day can be administered.
  • the immunopolypeptide ofthe invention may be administered in a range of about 0.05 to about 100, preferably 0.5 to about 50 mg per kg of patient body weight per day.
  • compositions ofthe immunopolypeptide ofthe invention can prepared as liquids, gels and suspensions.
  • the formulations are preferably suitable for injection, insertion or inhalation. Injection may be accomplished by needle, cannula catheter and the like. Insertion may be accomplished by lavage, trochar, spiking, surgical placement and the like. Inhalation may be accomplished by aerosol, spray or mist formulation.
  • the immunopolypeptide of the invention may also be administered topically such as to the epidermis, the buccal cavity and instillation into the ear, eye and nose.
  • the immunopolypeptide may be present in the pharmaceutical formulation at concentrations ranging from about 1 percent to about 50 percent, preferably about 1 percent to about 20 percent, more preferably about 2 percent to about 10 percent by weight relative to the total weight ofthe formulation.
  • the carrier for the pharmaceutical formulations includes any pharmaceutically acceptable agent suitable for delivery by any one ofthe foregoing routes and techniques of administration. Diluants, stabilizers, buffers, adjuvants, surfactants, fungicides, bactericides, and the like may also optionally be included. Such additives will be pharmaceutically acceptable and compatible with the immunopolypeptide.
  • Carriers include aqueous media, buffers such as bicarbonate, phosphate and the like; ringers solution, Ficol solution, BSA solution, EDTA solution, glycerols, oils of natural origin such as almond, corn, arachnis, caster or olive oil; wool fat or its derivatives, propylene glycol, ethylene glycol, ethanol, macrogols, sorbitan esters, polyoxyethylene derivatives, natural gums, and the like.
  • buffers such as bicarbonate, phosphate and the like
  • ringers solution Ficol solution, BSA solution, EDTA solution, glycerols, oils of natural origin such as almond, corn, arachnis, caster or olive oil
  • wool fat or its derivatives propylene glycol, ethylene glycol, ethanol, macrogols, sorbitan esters, polyoxyethylene derivatives, natural gums, and the like.
  • Diagnostic and screening techniques useful for identification of patients afflicted with hepatitis C include any that identify antibody-antigen binding.
  • An immunopolypeptide ofthe invention can be combined with an appropriate sample from the patient to produce a complex.
  • the complex in turn can be detected with a marker reagent for binding with such a complex.
  • Typical marker reagents include antibodies selective for the complex, antibodies selective for certain epitopes ofthe immunopolypeptide or a label attached to the immunopolypeptide itself.
  • radioimmunoassay RIA
  • radioallergosorbent test RAST
  • radioimmunosorbent test RIST
  • immunradiometric assay IRMA
  • Fair assay fluorescence immunoassay (FIA)
  • sandwich assay ELISA assay
  • northern or southern blot analysis e.g., Western blot analysis
  • color activation assay may be used following protocols well known for these assays. See for example fmmunology, An fllustrated Outline by David Male, CN. Mosby Company, St Louis, MO, 1986 and the Cold Spring Harbor Laboratory Manuals cited above.
  • Labels including radioactive labels, chemical labels, fluorescent labels, luciferase and the like may also be directly attached to the immunopolypeptide according to the techniques described in U.S. Patent No. (BN patent cite), the disclosure of which is inco ⁇ orated herein by reference. Definitions
  • immunopolypeptide refers to a chain of two (2) or more amino acids which are linked together with peptide or amide bonds, regardless of post-translational modification (e.g., glycosylation or phosphorylation). Antibodies are specifically intended to be within the scope of this definition.
  • the immunopolypeptides of this invention may include more than one subunit, where each subunit is encoded by a separate DNA sequence.
  • substantially identical with respect to an antibody or immunopolypeptide sequence means an antibody or immunopolypeptide sequence exhibiting at least 70%>, preferably 80%>, more preferably 90% and most preferably 95% sequence identity to the reference antibody or immunopolypeptide sequence.
  • the term with respect to a nucleic acid sequence means a sequence of nucleotides exhibiting at least about 85%, preferably 90%), more preferably 95% and most preferably 97% sequence identity to the reference nucleic acid sequence.
  • the length ofthe comparison sequences will generally be at least 25 amino acids.
  • nucleic acids the length will generally be at least 75 nucleotides.
  • identity means the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C- terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705).
  • antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, F(ab') 2; Fd and Fv) so long as they exhibit the desired biological activity.
  • Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable region (N H ) followed by a number of constant regions. Each light chain has a variable region at one end (V L ) and a constant region at its other end.
  • the constant region ofthe light chain is aligned with the first constant region of the heavy chain, and the light chain variable region is aligned with the variable region ofthe heavy chain.
  • the variable region of either chain has a triplet of hypervariable or complementarity determining regions (CDR's) spaced within a framework sequence as explained below.
  • CDR's complementarity determining regions
  • the framework and constant regions ofthe antibody have highly conserved amino acid sequences such that a species consensus sequence may typically be available for the framework and constant regions. Particular amino acid residues are believed to form an interface between the light and heavy chain variable regions (Chothia et al., J. Mol. Biol. 186, 651-63, 1985); ⁇ ovotny and Haber, Proc. ⁇ atl. Acad. Sci.
  • variable in the context of variable region of antibodies, refers to the fact that certain portions ofthe variable regions differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variability is concentrated in three segments (a triplet) called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable regions.
  • CDRs complementarity determining regions
  • variable regions are called the framework (FR).
  • the variable domains of native heavy and light chains each comprise three FR regions, largely adopting a ⁇ -Sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation ofthe antigen binding site of antibodies (see Kabat et al.)
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector function, such as participation ofthe antibody in antibody-dependent cellular toxicity.
  • a "species-dependent antibody,” e.g., a mammalian anti-human IgE antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species.
  • the species-dependent antibody "bind specifically" to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1 X IO "7 M, preferably no more than about 1 X 10 "8 and most preferably no more than about 1 X IO "9 M) but has a binding affinity for a homologue ofthe antigen from a second non-human mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen.
  • the species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
  • antibody variation refers to an amino acid sequence variant of an antibody wherein one or more ofthe amino acid residues have been modified. Such mutant necessarily have less than 100%> sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain ofthe antibody, more preferably at least 80%>, more preferably at least 85%, more preferably at least 90%>, and most preferably at least 95%>.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 , Fd and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') fragment that has two antigen binding fragments which are capable of crosslinking antigen, and a residual other fragment (which is termed pFc').
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab') 2 and Fd fragments.
  • an “Fv” fragment is the minimum antibody fragment which contains a complete antigen recognition and binding site.
  • This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V dimer). It is in this configuration that the three CDRs of each variable region interact to define an antigen binding site on the surface of the V H -V L dimer.
  • V H -V dimer dimer of one heavy and one light chain variable domain in a tight, non-covalent association
  • the Fab fragment also designated as F(ab) also contains the constant region ofthe light chain and the first constant region (CHI) ofthe heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) ofthe constant regions have a free thiol group.
  • F(ab') fragments are produced by cleavage ofthe disulfide bond at the hinge cysteines ofthe F(ab') 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • the light chains of antibodies (immunoglobulin) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino sequences of their constant domain.
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG- 1, lgG-2, IgG-3 and IgG4; IgA-1 and IgA-2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called .alpha., .delta., .epsilon., .gamma, and .mu., respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the prefe ⁇ ed immunoglobulin for use with the present invention is immunoglobulin IgG.
  • the term "monoclonal antibody” as used herein as a subclass ofthe immunopolypeptide ofthe invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies composed ofthe population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the a hybridoma or phage infected bacterial culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal" indicates the character ofthe antibody indicates the character ofthe antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al., J. Mol. Biol. 222: 581-597 (1991).
  • the immunopolypeptide subclasses including monoclonal antibodies, fragments and single chains thereof include "chimeric" forms in which a portion ofthe heavy and or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder ofthe chain(s) is identical with or homologous to co ⁇ esponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567); Morrison et al. Proc. Natl. Acad. Sci. 81, 6851-6855 (1984).
  • the immunopolypeptide subclasses also include fully human forms in which the entire sequence is derived from human immunoglobulins (recipient antibody) including the complementary determining region (CDR) ofthe immunopolypeptide
  • CDR complementary determining region
  • Fv framework residues ofthe human immunoglobulin are replaced by co ⁇ esponding non-human residues.
  • an immunopolypeptide include residues which are found neither in a human immunoglobulin nor in a non-human mammalian sequence.
  • Single-chain Fv” or “sFv” antibody fragments include the V H and V regions of an antibody, wherein these regions are present in a single immunopolypeptide chain.
  • the Fvimmunopolypeptide further includes an immunopolypeptide linker between the V H and V L regions which enables the sFv to form the desired structure for antigen binding.
  • an immunopolypeptide linker between the V H and V L regions which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to a small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable region (V H ) connected to a light chain variable domain (V L ) in the same immunopolypeptide chain (V H -V L ).
  • V H heavy chain variable region
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161, and Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
  • a functional fragment or analog of an antibody is a compound having qualitative biological activity in common with a full-length antibody.
  • a functional fragment or analog of an anti-IgG antibody is one which can bind to an IgG immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, Fc gamma receptor.
  • amino acid and “amino acids” refer to all naturally occurring L- ⁇ -amino acids.
  • substitutional variants refers to substitutional, insertional and/or deletional variants.
  • “Substitutional” variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule as been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • “Insertional” variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to either the .alpha. -carboxyl or .alpha.-amino functional group ofthe amino acid.
  • deletional variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region ofthe molecule.
  • cell cell line and cell culture are used interchangeably, and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.
  • the "host cells” used in the present invention generally are prokaryotic or eukaryotic hosts.
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed.
  • transfected host cell and “transformed” refer to the introduction of DNA into a cell.
  • the cell is termed "host cell” and it may be either prokaryotic or eukaryotic.
  • Typical prokaryotic host cells include various strains of E. coli.
  • Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary or cells of human origin.
  • the introduced DNA sequence may be from the same species as the host cell of a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA.
  • replicable expression vector and "expression vector” refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA.
  • Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell.
  • the vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently ofthe host chromosomal DNA and several copies ofthe vector and its inserted (foreign) DNA may be generated.
  • vector means a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression ofthe DNA in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genome itself.
  • phage and vector are sometimes used interchangeably, as the phage is the form of vector used in the present invention.
  • vector is intended to include such other form of vectors which serve equivalent function as and which are, or become, known in the art.
  • Typical expression vectors for bacterial expression and mammalian cell culture expression are based on pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen).
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • an "isolated" nucleotide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguishable from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • a nucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • This can be a gene and a regulatory sequence(s) which are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences(s).
  • DNA for a presequence or secretory leader is operably linked to DNA for an immunopolypeptide if it is expressed as a preprotein that participates in the secretion ofthe immunopolypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ofthe sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription ofthe sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a “disorder” is any condition that would benefit from treatment with the immunopolypeptide. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • “Mammal” for pu ⁇ oses of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • solid phase means a non-aqueous matrix to which the antibody ofthe present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • affinity maturation using phage display refers to a process described in Lowman et al., Biochemistry 30(45): 10832-10838 (1991), see also Hawkins et al., J. Mol Biol. 226, 889-896 (1992). While not strictly limited to the following description, this process can be described briefly as: several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle.
  • the phage expressing the various mutants can be cycled through rounds of binding selection, followed by isolation and sequencing of those mutants which display specific immuno-binding, preferably high affinity binding.
  • the method is also described in WO 92/09690, issued Jun. 11, 1992.
  • a modified procedure involving pooled affinity display is described in Cunningham, B. C. et al., EMBO J. 13(11), 2508-2515 (1994).
  • phage library refers to the phage library used in the affinity maturation process described above and in Hawkins et al., J. Mol Biol. 226: 889-896 (1992), and in Lowman et al., Biochemistry 30(45): 10832- 10838 (1991).
  • Each library includes a variable region (e.g. 6-7 sites) for which all possible amino acid substitutions are generated.
  • the antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle and expressed on the exterior ofthe phage.
  • high affinity means an affinity constant (Kd) of at leastlO “5 M and preferably at least IO “7 M, and especially preferably at least 10 "10 M under physiological conditions.
  • Example 1 Human recombinant antibodies to HCV E2 glycoprotein and nonstructural protein-3
  • the scheme for recombinant construction ofthe phage and subsequent expression ofthe immunopolypeptides as Fab antibody fragments according to the invention is presented in Fig. 16.
  • the light chains and immunoglobulin (Ig) GI heavy chain fragments were amplified by PCR using gene specific primers and following restriction enzyme digestion they were sequentially cloned into a phagemid vector, pComb3H using solid phase amplification method.
  • Final library size was 1.3xl0 7 for IgGl, kappa library and 2.1xl0 6 for IgGl, lambda library.
  • the wells were washed 3 times with phosphate-buffered saline (PBS) and blocked with 4% non-fat dry milk in PBS.
  • the milk was discarded and the wells were incubated with library phage at 37°C for 1-2 hours.
  • the phage was discarded and the wells were washed with PBS.
  • the remaining phage was eluted and freshly grown E. coli (XL-1 Blue) was infected. Phage was titrated on LB agar plates with carbenicillin and propagated overnight with VCS Ml 3 helper phage over night for the next round.
  • the libraries were panned for four to five consecutive rounds with increasing washing stringency.
  • Fabs were analyzed for DNA sequence with a 373A or 377 A automated DNA sequencer (ABI, Foster City, CA) using a Taq fluorescent dideoxy terminator cycle sequencing kit (ABI).
  • Neutralization of binding (NOB) assay Four of five Fabs initially isolated by panning on recombinant E2 protein were purified and tested on E2 glycoprotein and GST-El /E2. They were tested for neutralizing ability by neutralization of binding (NOB) assay at Chiron Italy and showed good neutralizing ability (50% neutralization at 0.5-1.0 ⁇ g/ml) (TABLE 1).
  • FIG 3 shows the test results for reactivity of Human Fabs to E2 Glycoprotein. Reactivity of anti-E2 Fabs with recombinant E2 (4 ⁇ g/ml) and ovalbumin (4 ⁇ g/ml) determined by ELISA.
  • B IF is a flag-tagged Bl.
  • Ovalbumin (4 ⁇ g/ml) is included as a control antigen. OD405, optical density at 405 nm.
  • FIG 4 shows the test results for reactivity of human Fabs to GST-El /E2.
  • GST-El /E2 (8 ⁇ g/ml) was captured by goat anti-GST Ab (10 ⁇ g/ml) to microtiter wells.
  • Ovalbumin (4 ⁇ g/ml) was used as a control antigen.
  • Binding profile of human Fabs Panning on GST-E1/E2 complex yielded 36 distinct Fabs to E2 glycoprotein, including the ones isolated in the panning on E2, belonging to 13 groups according to their heavy chain sequences (See TABLE 2). Binding pattern of 31 distinct Fabs were analyzed. The Fabs were tested on GST-E1/E2 captured with anti-GST-Ab, E2 alone, ovalbumin with or without anti-GST Ab (as control antigens). Table 3 shows the immunoreactive Fabs that were isolated after each round. FIG 5 shows the test results of reactivity of Fabs to GST-E1/E2 complex and E2 alone. GST-E1/E2 and E2 alone were coated at 8 and 4 ⁇ g ml, respectively. Ovalbumin (4 vg/ml) was used as a control antigen.
  • FIG 6 shows the results ofthe inhibition of binding of GST-El /E2 and CD81 by the so constructed whole human anti-E2 antibodies.
  • CD81 was coated at 4°C overnight and blocked with 4% nonfat dry milk PBS.
  • GST-E1/E2 was preincubated with human anti-E2 Abs and KZ52 IgG (a negative control Ab) for 1 hour at room temperature and added to the wells. Detection of bound GST-El /E2 was performed with rat anti-E2 antibody and AP conjugated anti-rat IgG (H+L) Ab (1 :500).
  • Percentage was calculated by 100-(OD405 (human anti-E2 Abs at each concentration)/OD405 (KZ52 IgG at each concentration)). Cl IgG and Bl Fab effectively blocked the binding of GST-E1/E2 and CD81 (50% at 0.1 ⁇ g/ml and 2 ⁇ g/ml, respectively).
  • FIG. 7 shows the results ofthe competition assay performed to see the inhibition of bindings of human monoclonal Fabs to E2 by mouse monoclonal conformational Ab, H53 (gift from Dr.
  • GST-E1/E2 was captured with goat anti-GST Ab (10 ⁇ g/ml) to microtiter wells and preincubated with H53 (0.032, 016. 0.8, 4, 20, and 100 ⁇ g/ml).
  • H53 0.032, 016. 0.8, 4, 20, and 100 ⁇ g/ml.
  • Detection of human Fabs was performed with alkaline-phosphatase conjugated goat anti-human IgG F(ab') 2 Ab (1:500) in 1% BSA/PBS.
  • FIG 8 shows the results ofthe inhibition of binding of Cl IgG to GST- E1/E2 by Fabs. Detection of Cl IgG was done with AP conjugated anti-human IgG Fc (1 :1000). Binding of Cl IgG was completely blocked by Fabs A, Cl, H2, I, J3, L4, and M. Fab Bl and Cl was used as a negative and a positive control, respectively.
  • Figure 13 presents the single chain amino acid sequences ofthe Fab fragments identified by this procedure to have El binding.
  • the chains include the CDR sequences and the framework sequence ofthe variable light and heavy chain regions and truncated Fab constant regions.
  • NS-3 nonstructural protein 3
  • HCV hepatitis C virus
  • the libraries were panned on recombinant NS-3 for four rounds (See FIG's 9 and 10 for the results).
  • the antigen was coated on microtiter wells at 4°C overnight and the subsequent panning was done as described above for panning on E2 glycoprotein.
  • FIG 9 shows the results of titration of patient sera on NS-3 antigen.
  • Library patient serum (Y) (genotype la) and a Japanese patient serum (J) (genotype lb) with chronic HCV infection were titrated on recombinant NS-3.
  • Normal human serum (NHS) was included as a negative control.
  • Ovalbumin (4 ⁇ g/ml) was included as a control antigen.
  • FIG 10 shows the results of isotyping of patient serum IgG reactivity to
  • NS-3 Detection of human IgGl, 2, 3, and 4 was performed with mouse IgG anti-human IgG isotype specific antibodies (PharMingen). Reactivity of sera of both library patient (Y) and another patient (J) sera was restricted to IgGl .
  • FIG 11 shows the results of a reactivity test of Fab/phage clones isolated after 4 rounds of panning on NS-3. They showed specific reactivity to NS-3.

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Abstract

L'infection par le virus de l'hépatite C (VHC) provoque une infection chronique et entraîne une cirrhose du foie et un carcinome hépatocellulaire. La glycoprotéine E2 et la protéine NS3 sont identifiées comme étant responsables de la liaison du VHC à des cellules cibles et elles contiennent des épitopes neutralisant. La glycoprotéine E2 peut également être impliquée dans la pathogène de la sialadénite parmi un sous-ensemble de patients atteints d'une infection au VHC chronique développant le syndrome Sjögren (SS). Afin d'étudier la réactivité des anticorps à la glycoprotéine E2 et à la protéine NS3 chez ces patients, une bibliothèque d'expressions des phages a été élaborée à partir de la moelle osseuse d'un patient atteint du VHC et du SS. Les séquences provenant des anticorps ainsi obtenues ont été isolées et séquencées de manière à obtenir des informations concernant les régions déterminantes de la complémentarité (CDR) et les régions d'infrastructure. La présente invention concerne des immuopolypeptides contenant lesdites régions CDR. Des modes de réalisation privilégiés de l'invention concernent des immunopolypeptides contenant des triplets de CDR accompagnés de régions d'infrastructure. D'autres modes de réalisation décrits dans cette invention comprennent des fragments d'anticorps, des fragments monocaténaires, et des anticorps complets, plus spécialement ceux qui présentent des séquences d'origine humaine. Cette invention concerne également la bibliothèque de phages, des séquences nucléotidiques correspondantes, des cellules hôtes transfectées et autres éléments analogues. En outre, cette invention concerne des méthodes permettant le diagnostic, le traitement et la maturation moléculaire.
PCT/US2002/002303 2001-01-26 2002-01-25 Immunopolypeptides diriges contre le virus de l'hepatite c WO2002059340A1 (fr)

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EP2333067A1 (fr) * 2005-03-25 2011-06-15 National Research Council of Canada Procédé d'isolation de polypeptides solubles
US9718883B2 (en) 2003-09-10 2017-08-01 Amgen Fremont Inc. Antibodies to M-CSF
CN109100507A (zh) * 2017-06-20 2018-12-28 江苏先思达生物科技有限公司 慢性肝炎肝损伤的血清糖蛋白n-糖组图谱模型的建立方法

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9718883B2 (en) 2003-09-10 2017-08-01 Amgen Fremont Inc. Antibodies to M-CSF
US10280219B2 (en) 2003-09-10 2019-05-07 Amgen Fremont Inc. Antibodies to M-CSF
EP1621546A1 (fr) * 2004-07-30 2006-02-01 Tecnogen S.C.P.A. Ligands peptidiques avec affinité spécifique pour les immunoglobulines
EP2333067A1 (fr) * 2005-03-25 2011-06-15 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
CN109100507A (zh) * 2017-06-20 2018-12-28 江苏先思达生物科技有限公司 慢性肝炎肝损伤的血清糖蛋白n-糖组图谱模型的建立方法

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