WO2002059340A1 - Immunopolypeptides to hepatitis c virus - Google Patents

Abstract

Infection with hepatitis C virus (HCV) causes chronic infection and lead to liver cirrhosis and hepatocellular carcinoma. E2 glycoprotein and NS3 protein are thought to be responsible for the binding of HCV to target cells and contains neutralizing epitopes. E2 also may be involved in the pathogenesis of sialadenitis among subset of patients with chronic HCV infection who develop Sjögren's syndrome (SS). To study the antibody reactivity to E2 glycoprotein and NS3 protein in these patients, a phage display library was constructed from the bone marrow of a patient with HCV and SS. The resulting antibody derived sequences were isolated and sequenced to provide information about the CDR and framework regions. The invention provides immunopolypeptides incorporating the discovered CDR's and in preferred embodiments provides immunopolypeptides incorporating triplets of CDR's with framework regions. Further preferred embodiments include antibody fragments, single chain fragments, and complete antibodies especially having sequences of human origin. The phage library, corresponding nucleotide sequences, transfected host cells and the like are also provided. Methods for diagnosis, treatment and processing are also provided.

Description

IMMUNOPOLYPEPTIDES TO HEPATITIS C VIRUS

Cross Reference to Related Applications

This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application 60/264,451, filed January 26, 2001, the disclosure of which is herein incorporated by reference.

Field of the Invention

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.

Background of the Invention

Descriptions of hepatitis diseases causing jaundice and icterus have been known to man since antiquity. 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). It can be distinguished from other forms of viral-associated liver diseases, including that caused by known hepatitis viruses, i.e., hepatitis A virus (HAV), hepatitis B virus (HBV), and delta hepatitis virus (HDV), as well as the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Hepatitis C virus (HCV) was first identified in transfused individuals. Transmission from man to chimpanzee and serial passage in chimpanzees provided evidence that this form of hepatitis is due to a transmissible infectious agent, namely a virus.

The prevalence of HCV in the United States is 1.8% (estimated 3.9 million) and estimated 2.7 million are chronically infected (74%): 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). Among the methods used to detect hepatitis C antigens and antibodies are agar-gel diffusion, counter- immunoelectrophoresis, immuno-fluorescence microscopy, immune electron microscopy, radioimmunoassay, and enzyme- linked immunosorbent assay. Recently 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). The prolonged interval between exposure to HCV and antibody detection, and the lack of adequate information regarding the profile of immune response to various structural and non- structural proteins of hepatitis C virus creates problems regarding the infectious state ofthe patient in the latent and antibody negative phase and the ability to treat such a patient. 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. Recently, CD81 has been found to be the receptor for E2 (Science 1998, 282:939-941). A neutralization assay can be performed by NOB (neutralization of binding) protocol. When a high titer of serum Abs shows a neutralization by this protocol, that serum also correlates with natural resolution of chronic hepatitis C (Hepatol 1998, 28:1117-1120).

E2 glycoprotein also may be involved in the pathogenesis of sialadenitis among subset of patients with chronic HCV infection who develop Sjδgren'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).

It is self-evident that an early diagnosis and treatment of a patient infected with HCV is of prime importance. Therefore, there is a need to develop methods for detection and treatment of chronic and acute hepatitis C. There is a further need to develop specific binding proteins, such as antibodies, to certain envelop and nonstructural proteins of hepatitis C, such as E2 and NS3. There is also a need to develop fully human antibodies to hepatitis C. Further needs include development of an antibody library, development of a screening method for hepatitis C, and development of recombinant processes for production of antibodies to hepatitis C.

Summary of the Invention These and other needs are achieved by present invention, which is directed to an immunopolypeptide, which binds to hepatitis C. 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. In particular, 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. Preferably, the CDR's for the immunopolypeptide are selected as triplets so that the immunopolypeptide will contain at least three CDR sequences. Preferably, the immunopolypeptide amino acid sequence also includes any ofthe framework regions having the amino acid sequences given in Fig's 12 and 13 . Preferably, 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. Preferably, the phage library incorporates nucleotide sequences encoding the individual variable region sequences V_H and V_L ofthe immunopolypeptide ofthe invention. These 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. In particular, 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.

Brief Description of Drawings 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.

Figure 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 μg/ml) and ovalbumin (4 μg/ml) determined by ELISA. B IF is a flag- tagged Bl. Ovalbumin (4 μg/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 μg/ml) was captured by goat anti-GST Ab (10 μg/ml) to microtiter wells. Ovalbumin (4 μg/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 μg/ml, respectively. Ovalbumin (4 μg/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 μg/ml and 2 μg/ml, respectively).

Figure 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 μg/ml) to microtiter wells and preincubated with H53 (0.032, 016, 0.8, 4, 20 and 100 μg/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.

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 μg/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 μg/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.

Definitions

Certain terms used to describe the present invention are defined in the section ofthe Detailed Description immediately preceding the Examples. Undefined terms have the ordinary, typical definitions provided in the art.

Detailed Description of the 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. Although 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

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. These amino acid sequences are grouped into light chain CDR's having SEQ ID NO's 171-275 and 291-308 and the heavy chain CDR's having SEQ ID NO's 78-170 and 276-290. Preferably, 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. Preferably, 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.

Preferably, 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. Preferably the triplet of CDR's is appropriately spaced so as to provide a trifunctional binding site. Preferably, the spaced triplet binds to the E2 glycoprotein or the NS3 protein. Especially preferably, 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. Although any spacer peptide sequence may be used, such as a short chain sequence of amino acid units having little or no ionic or lipophilic side chain properties, 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. When the CDR triplets are combined with such a framework sequence, the immunopolypeptide mimics the variable region of a single chain of an antibody. Preferably, the selected CDR's ofthe immunopolypeptide are spaced with a human framework. Preferably, the human framework is a consensus human framework of a human immunoglobulin, especially an IgG. More preferably, 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. Most preferably, 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. In addition, 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. Preferably, 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. The preferred version of this most preferable combination is the 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.

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. With the addition ofthe appropriate constant region sequences from an Fab, an Fab' and or an Fc fragment to this single light or heavy variable region chain, 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. When the last combination includes the variable region and the option constant region, it has a construction like that of an Fab or Fab' fragment. When this combination provides an Fab' fragment and two of such fragments are combined, the resulting immunopolypeptide is an F(ab')₂ monoclonal antibody fragment. When 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 μg/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 Encoding the Immunopolypeptide

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

Fab sequences of Figures 12 and 13. These nucleotide sequences are given in

Figures 14 and 15.

Using known techniques such as solid phase synthesis of oligonucleotides or endonuclease digestion and recombinant DNA technology, the 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.

Additionally, 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

Spring Harbor Laboratory Press, Cold Spring Harbor, NN. 1989, the disclosures of which is incoφorated herein by reference. 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. In particular, for the CDR's ofthe invention, mononuclear cells from a patient infected with hepatitis C are collected. Preferably, 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. Acad. Sci., USA 88, 7978-7982 (1991); and in U.S. Patent No's 5,580,717; 5,972,656; 6,113,898; and 6,140,470; the disclosures of which are incorporated herein by reference. Briefly, either total RNA or RNA that has been processed to obtain the poly A- RNA is obtained from cells. After hybridization of an oligo-d(T) primer, the RNA (mRNA) is reverse transcribed to yield the corresponding cDNA. This cDNA provides the stock DNA coding material leading to development ofthe immunopolypeptide sequences ofthe invention. 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. For efficient recombination, panning and bacterial transfection, 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.

Following selection of bacterial cultures exhibiting expression of polypeptide having specific immunoreactivity, the 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. In particular, the 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. Alternatively, 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. Similarly, 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.

In addition to use of bacterial host cells for expression ofthe immunopolypeptide 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. Mixing the nucleotide sequences for the selected immunospecific light and heavy chains and insertion into a phage followed by bacterial transfection will provide both chains. 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 incoφorate 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 incoφorated into these primers to allow for the cloning ofthe amplified fragment into a suitable vector (i.e. a phagemid or a λ phage) in a predetermined reading frame for expression. Phage assembly proceeds via an extrusion-like process through the bacterial membrane. For example 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. The two functional properties of cpIII, infectivity and noπnal (nonpolyphage) moφhogenesis 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 moφhogenic 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. USA, 88, 7978 (1991)). 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.

Inclusion ofthe phage FI intergenic region in the vector allows for packaging of single-stranded phagemid with the aid of helper phage. The use of helper phage superinfection may result in expression of two forms of cpIII. Consequently, normal phage moφhogenesis may be perturbed by competition between the cpIII/Fd fragment fusion protein and the native cpIII ofthe helper phage for incoφoration 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. These cloning sites are compatible with known mouse and human PCR primers (see, e.g., Huse et al., Science, 246, 1275-1281 (1989)). The nucleotide sequences ofthe pelB leader sequences are recruited from the λ HC2 and λ 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 10¹¹ cfu/ml. By using phagemids encoding different antibiotic resistances, ratios of clonally distinct phage may easily be determined by titering on selective plates. In single- pass enrichment experiments, 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.

Diagnosis and Treatment With The Immunopolypeptides

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. Preferably, from about 100 to about 100,000 units of antibody per day can be administered. Alternatively, 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.

Pharmaceutical formulations 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.

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. In particular, radioimmunoassay (RIA), radioallergosorbent test (RAST), radioimmunosorbent test (RIST), immunradiometric assay (IRMA) Fair assay, fluorescence immunoassay (FIA), sandwich assay, enzyme linked immunosorbent assay (ELISA) assay, northern or southern blot analysis, and 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 incoφorated herein by reference. Definitions

Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. The following terms are to be given the particular definitions given below. The term " 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.

The phrase "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. For immunopolypeptides, the length ofthe comparison sequences will generally be at least 25 amino acids. For nucleic acids, the length will generally be at least 75 nucleotides.

The term "identity" or "homology" 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). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. The term "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. 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); Νovotny and Haber, Proc. Νatl. Acad. Sci. USA 82 4592- 4596 (1985). The term "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. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al. (1989), Nature 342: 877).

The more highly conserved portions of variable regions are called the framework (FR). The variable domains of native heavy and light chains each comprise three FR regions, largely adopting a β-Sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-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. Normally, 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. The term "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%>.

The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of 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. As used herein, "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. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. 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 (λ), based on the amino sequences of their constant domain.

Depending on the amino acid sequences ofthe constant domain of their heavy chains, "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 prefeπed 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. In additional to their specificity, 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. For example, 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 coπesponding 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 In some instances, Fv framework residues ofthe human immunoglobulin are replaced by coπesponding non-human residues. Furthermore, 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. Generally, 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. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994).

The term "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). By using a linker that is too short to allow pairing between the two domains on the same chain, 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).

The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, 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.

The term "amino acid" and "amino acids" refer to all naturally occurring L-α-amino acids.

The term "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.

The terms "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.

The terms "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.

The terms "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.

The term "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. Such 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. In the present application, "phage" and "vector" are sometimes used interchangeably, as the phage is the form of vector used in the present invention. However, the term 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, for example, are based on pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen).

The expression "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, for example, 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. However, 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). For example, 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. Generally, "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 puφoses 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.

The word "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.

As used herein, "solid phase" means a non-aqueous matrix to which the antibody ofthe present invention can adhere. Example of 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. In certain embodiments, depending on the context, 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. As used herein, "affinity maturation using phage display" (AMPD) 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). As used herein, the term "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.

As used herein, "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.

The following examples further illustrated the invention. They are not meant to be general limitations ofthe invention, however, as the invention is fully set forth in the foregoing description.

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.

Library construction. Approximately 2 ml bone marrow was obtained from a thirty five year old female patient with chronic hepatitis C virus (HCV) infection and Sjδgren's syndrome (See FIG 1 for the reactivity of these cells against E2). Bone marrow aspiration was performed for the evaluation of neutropenia in this patient. Mononuclear cells (7xl0⁷ cells) were separated by Ficoll (histopaque) (SIGMA) gradient and homogenized in 10 ml TRI reagent (Molecular Research Center, Cincinnati, OH). RNA was extracted and first strand cDNA was synthesized using Superscript cDNA amplification kit (GIBCO BRL). 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 1.3xl0⁷ for IgGl, kappa library and 2.1xl0⁶ for IgGl, lambda library.

Library panning on HCV E2 glycoprotein. The following antigens were used for panning: (a) recombinant E2 glycoprotein (genotype la, amino acids 388-644) (JMed Virol 1995, 45:415-422) (b) recombinant E1/E2 complex fused to glutathione S transferase (GST-E1/E2) (See FIG 2). Fragments encoding HCV envelope protein El and E2 were generated by PCR from a full- length cDNA clone of hepatitis C virus type la. Recombinant E2 was directly coated overnight at 4°C to a microtiter plate (Costar, Cambridge, MA) and GST- E1/E2 complex was captured with goat anti-GST Ab. After the antigens were coated 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.

Screening of Fab/phage. Single individual clones were isolated from titration plates after the 2^nd, the 3^rd, and the 4^th round and grown in Super Broth (SB) medium with carbenicillin and tetracycline. Then, Fab/phage production was induced with the addition of helper phage overnight at 30°C. Recombinant E2 or anti-GST Ab and a control antigen, ovalbumin (4 μg/ml) (Pierce, Rockford, IL) were coated overnight at 4°C to microtiter wells. GST-E1/E2 was captured with anti-GST Ab coated plates and Fab/phage was tested by enzyme linked immunosorbent assay (ELISA). FIG 1. Reactivity of Patient Serum to E2 Glycoprotein (genotype la, aa 388-644) (JMed 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. El was cloned into the baculovirus transfer vector pAcsG2T 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 #: m62321) represented in the final transfer vectors were; nucleotides (nt) 904-1421 (for El), (nt) 1471-2754 (for E2) as shown in Figure 2.

DNA sequencing. 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 μg/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 μg/ml) and ovalbumin (4 μg/ml) determined by ELISA. B IF is a flag-tagged Bl. Ovalbumin (4 μg/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 μg/ml) was captured by goat anti-GST Ab (10 μg/ml) to microtiter wells. Ovalbumin (4 μg/ml) was used as a control antigen.

TABLE 1. NOB (Neutralization of binding) Assay.

Fab 50%) neutralization at"

A 0.5 μg/ml

Bl 0.6 μg/ml

B3 1.0 μg/ml

Cl 0.8 μg/ml

KZ52 (control Ab) (no inhibition) ^inhibition of binding of E2 to CD81 (Abrignani's group, Chiron, Italy).

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 μg ml, respectively. Ovalbumin (4 vg/ml) was used as a control antigen.

TABLE 2 Summary of Epitope Masking Panning against GST-E1/E2 Complex

Fabs used for Positive Positive Positive epitope clones/total clones clones/total clones clones/total clones masking after 2^nd round after 3^rd round after 4th round

(10 μg/ml) (titer) (titer) (titer)

Bl 1/10 (2.3x101 19/20 (5xl0³) 20/20 (1.4x10°)

C 1/10 (4.7x10⁴) 15/20 (5xl0⁵) 20/20 (2.6xl0⁵) l & C 1/20 (2.9xl0⁴) 15/20 (7.5xl0⁴) 18/20 (1.4x10^s)

TABLE 3 Fabs that were Isolated after Each Round

Preparation of Cl IgG. To convert Fab Cl to a whole IgG molecule, heavy chain and the light chain gene of Cl were inserted into a PDR1 vector, which has the leader sequences and constant region of human IgG gene. First, the heavy chain and the light chain gene of Cl were cut out by restriction enzyme digestion. They were sequentially put in to the vector and digested with Sal I. Chinese hamster ovarian (CHO) cells were transformed with this DNA and after selection with L-methionine sulfoxide (MSX) and serial dilution, the proper clone which secretes Cl IgG was chosen and propagated for production of large amount of Cl IgG. Cl IgG was purified through a protein A column (Pharmacia). Cl IgG showed good neutralizing ability by NOB assay (FIG 6). 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-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 μg/ml and 2 μg/ml, respectively). 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 μg/ml and 2 μg/ml, respectively).

Competition ELISA. To determine whether the isolated antibodies recognize different epitopes on E2 or not, competition ELISA between mouse monoclonal Ab, H53 (gift from Dr. Jean Dubuisson, Journal of Virology, 72:2183-2191, 1998). GST-E1/E2 was captured with goat anti-GST Ab (10 μg/ml) to microtiter wells and preincubated with H53 (0.032, 0.16, 0.8, 4, 20, 100 μg/ml). Human Fabs were added and the detection of human Fabs were performed with alkaline-phosphatase (AP) conjugated goat anti-human IgG F(ab')₂ Ab (1 :500) in 1% BSA/PBS. Fabs, Bl, B2, B3, Dl, D2, D3, D4, and E shared epitope with H53. Another competition ELISA was performed between Cl IgG and Fabs which did not share epitope with H53. In this assay, captured GST-E1/E2 was preincubated with human Fabs and Cl IgG (0.16, 0.8, 4, and 20 μg/ml) was added later. The detection of Cl IgG was done with AP conjugated anti-human IgG Fc (1:1,000). Binding of Cl IgG was completely blocked by Fabs A, Cl, H2, 1, J3, L4 and M.

According to the data by competition ELISA and binding profile on E2 and GST-E1/E2, these Fabs were grouped into 3 types as shown on TABLE 4. Type I does not share epitope with H53 and binds stronger to GST-El/e2 complex. Type II shares epitope with H53 and binds stronger to E2 alone. Type III does not share epitope with H53 and binds stronger to E2 alone. Many of Fabs that belong to type I shared common amino acids in their heavy chain CDR3 as shown on TABLE 5. 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. Jean Dubuisson, Journal of Virology, 72:2183-2191, 1998). GST-E1/E2 was captured with goat anti-GST Ab (10 μg/ml) to microtiter wells and preincubated with H53 (0.032, 016. 0.8, 4, 20, and 100 μg/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.

TABLE 4 Epitope Mapping of Anti-HCV E2 Fabs Determined by Competition Assay and Bindings to GST-E1/E2 and E2 Alone.

Type Fab share epitope bindings to with

H53^a GST-E1/E2 >E2 alone

A^b, Cl, C2, C3, C4, C5, C6, Hl,

H2, H3, I, J1, J2, J3, J4, L1, L2, L3, L4, M No yes

II B1, B2, B3, D1, D2, D3, D4, E yes No

III F, G No

No etermined by competition assay with H53. ^bFabs that showed neutralizing ability by NOB (50% at 0.5-1.0 μg/ml) are shown in bold case. TABLE 5

Comparison of Heavy Chain CDR3

A ENKFRYCRGGSCYSGAFDM

C1,C1*,C4,C5,C6 PETP- • -S- -F- •

^•E- -N

C2,C2* PETP F- •

•E- -N C3 PETP- • -S- -V- •

^•E- -N

H1,H1*,H1**,H2 SVTP-H-G- -F- •

^•E- -Y

H3 SVTP ^• • ^• G ^• • F ^{• ■} -E- -Y

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.

Example 2 Monoclonal Antibody Fragments To NS-3

It has been discovered that nonstructural protein 3 (NS-3) is involved in the replication of hepatitis C virus (HCV). IgGl antibodies against this protein are found to be present in high titer in a library patient serum (patient Y, chronic HCV infection/SS) as shown in FIGs 9 and 10). Libraries of IgGl were panned on recombinant NS-3 for four rounds.

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. After screening by ELISA and DNA sequencing, as described above for E2 glycoprotein, five distinct IgGl K antibodies were isolated (See FIG.13). These Fabs shared common amino acids in their heavy chain CDR3:

(SEQ ID NO: 689) (TABLE 5).

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 μg/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.

Ovalbumin (4 μg/ml) was included as a control antigen. All publications, patents and patent applications are incoφorated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and may details have been set forth for the puφosed of illustration, it will be apparent to those skilled in the art that the invention includes additional embodiments and that certain of the details described therein may be varied considerable without departing from the basic principles ofthe invention.

Claims

What is claimed is:

1. An immunopolypeptide comprising at least one CDR sequence selected from the group consisting of SEQ ID NO's 78-308.

2. An immunopolypeptide according to claim 1 having a triplet of CDR sequences.

3. An immunopolypeptide according to claim 2 wherein each CDR ofthe triplet is separated from other CDR's by a spacer amino acid sequence.

4. An immunopolypeptide according to claim 3 wherein the spacer amino acid sequence is a framework region sequence having an amino acid sequence selected from the group consisting of SEQ ID NO's 309-401, 433-537, 573-587 and 593-605.

5. An immunopolypeptide according to claim 3 wherein the spacer amino acid sequence is a constant region sequence having an amino acid sequence selected from the group consisting of SEQ ID NO's 402-432, 538-572, 588-592 and 606.

6. An immunopolypeptide according to claim 4 wherein the CDR's ofthe triplet are selected from either a light chain group or a heavy chain group.

7. An immunopolypeptide according to claim 6 wherein the CDR's are matched according to their Fab source.

8. An immunopolypeptide according to claim 7 wherein the framework region sequence is matched to the Fab source ofthe CDR triplet.

An immunopolypeptide according to claim 8 wherein the amino acid sequence is a V OΓ V_H fragment ofthe Fab source ofthe matched CDR triplet and framework regions.

10. An immunopolypeptide according to claim 9 having an amino acid sequence selected from the group consisting of SEQ ID NO's 1-77.

11. An immunopolypeptide according to claim 9 which is a combination of VLand V_H.

12. An immunopolypeptide according to claim 9 which further includes at least one constant consensus region.

13. An immunopolypeptide according to claim 12 which is a combination of a light and heavy chain fragment.

14. An immunopolypeptide according to claim 13 which is an Fab, Fab', F(ab')₂, Fd, or Fv fragment.

15. An immunopolypeptide according to claim 13 which is a complete monoclonal antibody.

16. An immunopolypeptide as in any of claims 1-15 which is derived from a human.

17. A immunopolypeptide expressed by a phage listed in Figure 12 or 13.

18. A human immunopolypeptide that specifically binds to an E2 glycoprotein of human hepatitis C.

19. An immunopolypeptide according to claim 1 that specifically binds to an epitope ofthe E2 glycoprotein.

20. An immunopolypeptide according to claim 1 that specifically binds to an NS3 protein of hepatitis C.

21. An immunopolypeptide Fab fragment having a light variable chain amino acid sequence selected from the group consisting of SEQ ID NO's 32-66 and 72-77 and a heavy variable amino acid sequence selected from the group consisting of SEQ ID NO's 1-31 and 67-71.

22. An immunopolypeptide Fab fragment having its CDR amino acid sequences of its light chain selected from the group consisting of SEQ ED NO's 171-275 and 291-308 and its CDR amino acid sequence of its heavy chain sequence selected from the group consisting of SEQ ID No's 78-170 and 276-290.

23. An immunopolypeptide Fab fragment expressed by a bacterial phage listed in Figure 12 or 13.

24. A human immunopolypeptide Fab fragment that specifically binds to an E2 glycoprotein of human hepatitis C.

25. A human immunopolypeptide Fab fragment of claim 24 that specifically binds to an epitope ofthe E2 glycoprotein.

26. A human immunopolypeptide Fab fragment that specifically binds to an NS3 protein of hepatitis C .

27. An immunopolypeptide single chain fragment expressed by a phage listed in Figure 12 or 13.

28. An immunopolypeptide single chain fragment that specifically binds to an E2 glycoprotein of human hepatitis C.

29. An immunopolypeptide single chain fragment that specifically binds to an NS3 protein of hepatitis C.

30. A method of forming a human immunopolypeptide of claim 1 comprising a) obtaining a group of bacteria infected with a recombinant phage containing cDNA transcribed from immune cell RNA from a human or non-human primate infected with hepatitis C; b) selecting from the group, a bacterium producing a protein that binds to an E2 glycoprotein of human hepatitis C; c) producing the human immunopolypeptide from the selected bacterium.

31. The method of claim 30 wherein the immune cell is a bone marrow cell .

32. A phage library containing phage encoding at least a CDR sequence of an immunopolypeptide of claim 1.

33. A phage library obtained by inserting into a bacterial phage host, cDNA encoding an anay of antibody amino acid sequences obtained from the immune cells of a patient carrying hepatitis C.

34. The phage library of claim 32 wherein the cDNA encodes a CDR that is specific for an epitope on an NS3 or an E2 protein of a hepatitis C virus.

35. A pharmaceutical composition comprising an immunopolypeptide of claim 1 in combination with a pharmaceutically acceptable carrier.

36. A method of treating a patient infected with hepatitis C comprising administering to the patient an effective amount of an immunopolypeptide of claim 1.

37. The method according to claim 33 wherein the immunopolypeptide is combined with a pharmaceutically acceptable carrier.

38. A method of diagnosing a patient to determine whether the patient has hepatitis C virus comprising treating blood ofthe patient with an effective amount of an immunopolypeptide of claim 1 to form an immunoreactive mixture and combining the immunoreactive mixture with a labeled non-human antibody for an immunocomplex ofthe immunopolypeptide and glycoprotein E2 or non structural protein 3 of human hepatitis virus C.

39. A phage as listed in Figure 12 or 13.