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Title:  Monoclonal antibodies specific for the E2 glycoprotein of hepatitic C virus and their use in the diagnosis, treatment, and prevention of hepatitis C

United States Patent:  6,924,362

Issued:  August 2, 2005

Inventors:  Schofield; Darren (Royston, GB); Emerson; Suzanne U. (Kensington, MD); Purcell; Robert H. (Boyds, MD); Alter; Harvey J. (Chevy Chase, MD)

Assignee:  The United States of America as represented by the Department of Health & Human Services (Washington, DC)

Appl. No.:  432006

Filed:  November 30, 2001

PCT Filed:  November 30, 2001

PCT NO:  PCT/US01/45221

371 Date:  December 15, 2003

102(e) Date:  December 15, 2003

PCT PUB.NO.:  WO02/05556

PCT PUB. Date:  July 18, 2002

The present invention describes the identification and characterization of five human HC E1-specific monoclonal antibodies isolated from a phage display library and their use in the diagnosis, treatment, and prevention of HCV in mammals, preferably humans.

SUMMARY OF THE INVENTION

The present invention relates to human monoclonal antibodies which exhibit immunological binding affinity for hepatitis C virus (HCV) E2 polypeptide antigen, and are cross-reactive against different HCV strains.

The present invention also relates to the amino acid sequences of the FRs and CDRs of the γ1 chains of these antibodies and to nucleic acid molecules that encode these amino acid sequences.

The present invention further relates to the combinatorial library, from which the specific antibodies described herein are obtained, since the library may provide a repository of additional monoclonal antibodies against HCV proteins.

The invention therefore also relates to monoclonal antibodies, which may be isolated from this library, wherein such antibodies may include antibodies to HCV E2 polypeptide as well as antibodies to other HCV polypeptides.

Also, the present invention relates to the use of the monoclonal antibodies of the invention in the development of prophylactic, therapeutic, and diagnostic agents for the prevention, treatment, and detection of hepatitis in mammals, preferably humans.

Further, the present invention relates to a kit comprising the antibodies of the present invention for use as a therapeutic, prophylactic, or diagnostic agent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to five human monoclonal antibodies which exhibit immunological binding affinity for HCV E2, wherein the antibodies are isolated as Fab fragments from a phage display library prepared from bone marrow lymphocyte and plasma cell RNAs isolated from an asymptomatic chronically HCV infected patient (patient H). In particular, the antibodies of the instant invention were selected using a panning procedure that utilized a soluble form of the HCV E2 glycoprotein to select for Fab molecules specific for the HCV E2 antigen.

Methods for producing an intact immunoglobulin from isolated Fab fragments by combining Fab fragments with an Fc domain are known to those skilled in the art. The term "antibody" is used herein to refer to intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and portions of an immunoglobulin molecule, including antigen binding fragments i.e. Fab, Fab′, F(ab′)₂, Fd, F(v), and sFv, as well as chimeric antibody molecules thereof, which exhibit immunological binding properties of the parent antibody molecule.

The terms "immunological binding affinity" and "immunoreactive" as used interchangeably herein, refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The affinity of immunological binding interactions may be expressed in terms of the dissociation constant (K_d) of the interaction, wherein a smaller K_d represents a greater affinity. Table 2 shows that of the five monoclonal antibodies directed against HCV-E2, two have particularly high affinity (HCV#4 and HCV#7). Immunological binding properties of selected antibodies may be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex association and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. (See, Davies et al. (1990) Ann. Rev. Biochem., 59:439-73).

The amino acid sequences of the variable regions of the γ1 chain of the five clones of the present invention (HCV#1, HCV#4, HCV#7, HCV#12, and HCV#13) are shown in FIG. 1 as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. Each of SEQ ID NOs:1-5 consists of a continuous amino acid sequence comprising three consecutive CDRs flanked by four FRs, wherein the amino acid sequence begins at the N-terminal with FR1 and continues through CDR1, FR2, CDR2, FR3, CDR3, ending at the C-terminal with FR4.

The term "FR" refers to "framework regions," which are conserved sequences flanking the three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions". In an antibody molecule, the N-terminal antigen-binding surface is complementary to the three-dimensional surface of a bound antigen and comprises the three hypervariable regions of the heavy chain; the three hypervariable regions of the light chain form relative to each other in three-dimensional space. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs".

As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR (framework region) residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs.

The term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these complementarity-determining regions are denoted as "CDR1", "CDR2," and "CDR3," respectively. CDRs are involved in antigen-antibody binding, and the CDR3 comprises a particularly unique region specific for antigen-antibody binding. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

Currently, there are six known distinct genotypes of HCV with multiple distinct subtypes which have been identified based on phylogenetic analyses (Houghton, M. (1996) Fields Virology, 3^rd Edition, Fields et al., eds., Lippencott-Raven Publishers, Philadelphia, Pa.; Simmonds et al., J. Gen. Virol., (1993) 74:2391-99). Of the five antibody molecules where γ1 chain amino acid sequences are shown in FIG. 1, four novel human monoclonal antibody molecules specific to the HCV E2 envelope glycoprotein, are "cross-genotype reactive", i.e. the antibody molecule which specifically binds to an E2 antigenic determinant of HCV isolates from at least two genotypes.

The instant invention therefore also relates to nucleic acid molecules encoding the CDR and FR sets of the γ1-chain amino acid sequences shown in SEQ ID NO:1 through SED ID NO:5. The DNA constructs encoding five monoclonal antibodies, wherein each plasmid contains the γ1 heavy chain (Fd portion) and κ light chain genes in pComb3H vector, have been deposited with the American Type Culture Collection (ATCC) on Nov. 30, 2000 and have ATCC Patent Deposit Designation numbers: PTA-2747; PTA-2748; PTA-2749; PTA-2745; PTA-2746.

The following biological material has been deposited in accordance with the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA, on the date indicated:

The present invention also relates to variations of these nucleic acid sequences due to degeneracy of the genetic code. The coding sequences for the heavy and light chain portions of the Fab molecules may be isolated or synthesized and may be cloned into any suitable vector or replicon for expression. Examples of suitable vectors include bacterial, mammalian, yeast, and viral expression systems.

The Fab molecules of the present invention may also be produced using conventional methods of protein synthesis, based on the ascertained amino acid sequences.

The invention further relates to the phage display library described herein from which these human monoclonal antibodies are obtained. This library has been deposited with the American Type Culture Collection (ATCC) on Nov. 30, 2000 and has ATCC Patent Deposit Designation number: PTA-2750. The invention further relates to methods of making human monoclonal antibodies from the deposited phage display library. One skilled in the art has knowledge of the method for isolating a monoclonal antibody from the phage display library. In a preferred embodiment, the method involves (1) using immunoaffinity techniques such as panning to select phage particles that immunoreact with a pre-selected antigen; (2) infecting bacteria with the selected phage particles; (3) preparing and analyzing the phagemid DNA from the colonies recovered; and (4) expressing and purifying soluble Fab fragments from clones of interest for further analysis. Of course, the skilled artisan would readily understand that the pre-selected antigen used in screening could be an HCV E2 protein or peptide fragment, or any other protein of HCV (or peptide fragment thereof) other than E2 such as the E1 and core structural proteins or the non-structural proteins. By using the method disclosed above, additional human monoclonal antibodies to HCV, or to any other pathogens that may exist in the individual from whose RNA the library was prepared, may be isolated from the library of the instant invention.

The invention also relates to the use of the five monoclonal antibodies, whose γ1 chain sequences are shown in SEQ ID NOs:1-5, or those antibodies directed against HCV obtained from the deposited phage display library, as diagnostic agents. The antibodies may be used as an in vitro diagnostic agent to test for the presence of HCV in biological samples. In particular, the novel specific binding molecules of the present invention may be used in highly sensitive methods for screening and identifying individuals carrying and/or infected with HCV, as well as for screening for HCV-contaminated blood or blood products. The present binding molecules may also be used in assays for monitoring the progress of anti-HCV therapies in treated individuals, and for monitoring the growth rate of HCV cultures used in research and investigation of the HCV agent.

In one embodiment, a sample such as biological fluid or tissue obtained from an individual is contacted with a diagnostically effective amount of one or more of the human monoclonal antibodies of this invention under conditions which will allow the formation of an immunological complex between the antibody and the HCV antigen that may be present in the sample. The formation of an immunological complex, which indicates the presence of HCV in the sample, is then detected by immunoassays. Such assays include, but are not limited to, radioimmunoassays, Western blot assays, immunofluorescence assays, enzyme immunoassays, chemiluminescence assays, immunohistochemical assays, and the like, wherein a "label", as used herein referring to a detectable compound or composition which is conjugated directly or indirectly to the antibody, may be used for diagnostic purposes. 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 immunotherapies and immunoprophylactics are based on virus-neutralizing antibodies, the antibodies of the instant invention are preferably neutralizing antibodies and more preferably, neutralizing antibodies which are immunoreactive with E2 antigens from HCV isolates belonging to different genotypes. There are several in vitro assays known in the art which may be used to assess the capacity of a given antibody to have neutralizing activity. For example, the inhibition of binding of E2 to CD81 by the monoclonal antibodies of the present invention (See Table 3), is an indicator of potential neutralizing activity in vitro. In addition, a neutralization of binding (NOB) assay such as that described in Example 6 may be used to estimate HCV neutralizing antibodies, and to evaluate inhibition of the binding of HCV to human cells. FIG. 2 shows the results of a NOB assay in which the HCV#4 monoclonal antibody and experimental NOB positive controls all inhibit binding of HCV to cells.

The invention therefore also relates to the use of the antibodies of the present invention as pharmaceutical compositions for either prophylactic or therapeutic purposes. Such compositions are thus used as immunoprophylactic or immunotherapeutic agents.

When supplied prophylactically, a pharmaceutical composition(s) of the invention is provided in advance of any exposure to any one or more of the HCV strains or in advance of any symptoms due to infection of the viruses. The prophylactic administration of a pharmaceutical composition(s) of the invention serves to prevent or attenuate any subsequent infection of these viruses in a mammal. For therapeutic use, a pharmaceutical composition(s) of the invention is provided at (or shortly after) the onset of infection or at the onset of any symptom of infection or any disease or deleterious effects caused by these viruses. The therapeutic administration of the pharmaceutical composition(s) serves to attenuate the infection or disease. The pharmaceutical composition(s) of the present invention may, thus, be provided either prior to the anticipated exposure to the hepatitis C virus or after the initiation of infection.

Alternatively, genes encoding the recombinant antibodies may be introduced into a suitable mammalian host cell for expression or co-expression using a number of virus-based systems which have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient means for gene delivery systems. A selected nucleotide sequence encoding a single chain V_H or V_L domain polypeptide may be inserted into a vector and packaged in retroviral particles using techniques known in the art (Marasco et al. (1999) J. Immunol. Meth. 231:223-238). The recombinant virus may then be isolated and delivered to a subject. A number of suitable retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller (1990) Hum. Gene Therapy, 1:5-14; Scarpa et al (1991) Virology. 180:849-52; Burns et al. (1993) Proc. Natl. Acad. Sci. USA, 90:8033-7; and Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop., 3:102-9. Additionally, expression vectors derived from adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids can be used for the delivery of nucleotide sequences to a target organ, tissue, or cell population. Methods, which are well known to those skilled in the art, may be used to construct expression vectors containing sequences encoding one or more recombinant antibodies along with appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

The preparation of pharmaceutical compositions containing one or more antibodies, antibody fragments, sFv molecules, or combinations thereof, as the active ingredient is generally known to one skilled in the art. Typically, such compositions are prepared as injectables (e.g. either as liquid solutions or suspensions, or as solid forms, suitable for solution or suspension in liquids prior to injection). The compositions will also generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

A therapeutically effective amount of a monoclonal antibody for individual patients may be determined by titrating the amount of antibody given to the individual to arrive at the therapeutic or prophylactic effect while minimizing side effects. The amount of composition to be delivered depends on the subject being treated, the capacity of the subject's immune system to mount its own immune responses, and the degree of protection desired. The exact amount necessary will vary depending on the age and general condition of the individual to be treated, the severity of the condition being treated and the particular anti-HCV agent selected and its mode of administration, among other factors. One skilled in the art may readily determine an appropriate effective amount. Therefore, a "therapeutically effective amount" of the composition will be sufficient to bring about treatment or prevention of HCV disease symptoms. The effective amount may be determined by measuring the amount of HCV following administration of the composition. Levels of HCV may be measured by in vitro assays known in the art such as RT-PCR. The plasma concentration of antibodies for individuals receiving the treatment is typically between 0.1 μg/ml to 100 μg/ml. In general, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 5 mg/kg to about 20 mg/kg body weight of the mammal, although a lower or higher dose may be administered. Additionally, inocula typically contain peptide concentrations of about 1 microgram to about 50 milligrams per inoculation (dose), preferably about 10 micrograms to about 10 milligrams per dose, most preferably about 100 micrograms to about 5 milligrams per dose.

The monoclonal antibodies of this invention may be administered via one of several routes including, but not limited to, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, and the like. In addition, the pharmaceutical compositions may be administered as an immunoprophylactic in a single or multiple dose schedule or as an immunotherapy in a multiple dose or continuous dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, preferably 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgement of the administering practitioner.

The invention therefore also relates to the use of neutralizing monoclonal antibodies of the invention in passive immunotherapy of HCV infection. In addition to antibodies comprising intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of immunoglobulin molecules, and chimeric antibody molecules thereof, there are several therapeutically useful molecules known in the art which comprise antigen-binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. One such molecule is a Fab molecule which comprises a heterodimer that includes an intact antigen-binding site. The enzyme pepsin may cleave IgG into several fragments including, a "F(ab′)₂" fragment which comprises two antigen-binding sites. An "Fv" fragment may be produced by preferential proteolytic cleavage of an IgM immunoglobulin molecule, and sometimes of IgG or IgA immunoglobulin molecules. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V_H::V_L heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. (See Inbar et al. (1972) Proc. Natl. Acad. Sci. USA, 69:2659-62; Hochman et al. (1976) Biochem., 15:2706-10; and Ehrlich et al. (1980) Biochem., 19:4091-6). When used in passive immunotherapy, the patient is administered a therapeutically effective amount of one or more neutralizing human monoclonal antibodies. The passive immunotherapy of this invention may be practiced on individuals infected with HCV, or individuals at risk of HCV infection.

The present invention therefore relates to pharmaceutical compositions comprising at least one antibody of the group comprising the antibodies of the invention and additional antibodies derived from the phage display library, and a pharmaceutically acceptable carrier where such carriers may include physiologically acceptable buffers, for example, saline or phosphate buffered saline.

The present invention further relates to anti-idiotypic antibodies to the monoclonal antibodies of this invention. In one embodiment, an anti-idiotypic antibody may be prepared by immunizing a host animal with a monoclonal antibody of this invention by methods known to those of skill in the art. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal may be used or the Fc region of the administered antibodies may be removed. The anti-idiotypic antibodies produced may be used to prepare pharmaceutical compositions rather than using the monoclonal antibodies of this invention.

The antibodies of the invention and/or those obtained from the phage display library may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition.

The practice of the present invention employs, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, (2^nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis, (N. Gait, ed., 1984); Nucleic Acid Hybridization, (B. Hames & S. Higgins, eds., 1985); Transcription and Translation, (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture, (R. Freshney, ed. 1986); Perbal, A Practical Guide to Molecular Cloning, (1984); Fundamental Virology, 2^nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.).
 

Claim 1 of 16 Claims

1. A human monoclonal antibody that exhibits immunological binding affinity for a hepatitis C virus (HCV) E2 antigen, where said monoclonal antibody has a γ chain CDR3 region amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:1 through SEQ ID NO:5.

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