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WO2006028634A2 - Pegylation specifique de site d'anticorps largement neutralisant diriges contre le vhc et leur utilisation pour le traitement d'infections chroniques par vhc - Google Patents

Pegylation specifique de site d'anticorps largement neutralisant diriges contre le vhc et leur utilisation pour le traitement d'infections chroniques par vhc Download PDF

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WO2006028634A2
WO2006028634A2 PCT/US2005/028168 US2005028168W WO2006028634A2 WO 2006028634 A2 WO2006028634 A2 WO 2006028634A2 US 2005028168 W US2005028168 W US 2005028168W WO 2006028634 A2 WO2006028634 A2 WO 2006028634A2
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antibody
fragment
antibody fragment
hcv
isolated
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WO2006028634A3 (fr
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Vijay Ramakrishnan
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Vijay Ramakrishnan
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to site-directed pegylation of broadly-neutralizing MAb derivatives and their use as therapeutic drugs for the treatment of chronic hepatitis C viral (HCV) infections.
  • HCV chronic hepatitis C viral
  • HCV infections lead to liver cirrhosis, liver cancer, and B-cell related immunodeficiencies in humans (Lauer, G. M., and Walker, B. D., N. Engl. J. Med. 345:41 , 2001 ; Sung ef. a/., J. Virol. 77:2134, 2003).
  • the only effective therapy available to date is the combined administration of pegylated interferon and ribavirin. However, this therapy is effective only in ⁇ 34- 42% of the patients infected with HCV strains of genotype 1a and 1 b, which are the most prevalent HCV genotypes in developed countries.
  • MAbs monoclonal antibodies
  • MAbs have emerged as "magic bullets" to treat chronic conditions such as cancers and rheumatoid arthritis because MAbs are highly selective to the targets (Gura, T., Nature 417:584-586, 2002; Walsh, G., Nature Biotechnol. 21 :865-870, 2003). More significantly MAbs have a serum half-life of several days to few weeks. SynagisTM is the first market- validated MAb that inhibits viral entry, and is administered to treat pediatric patients infected with Respiratory Synctial Virus (RSV) infections. Synagis has a serum half-life of 20 days.
  • RSV Respiratory Synctial Virus
  • HuMAbs human MAbs
  • MAbs which are the most advanced generation of MAb drugs
  • the issue of immunogenicity or side effects due to repeated administrations of protein/MAb therapies is resolved.
  • Entry inhibition is the process of stopping and neutralizing the virus before it penetrates human cells.
  • the mechanism of HCV attachment and entry into human cells is fairly understood, and is similar to that of the mechanism used by HIV and RSV.
  • the envelope proteins E2 and E1 of HCV are analogous to gp120 and gp41 of HIV 1 respectively.
  • HCV E2 binds to human CD81 (Pileri ef. a/., Science 282: 938, 1998), a tetraspannin receptor expressed on various cell types including hepatocytes and B lymphocytes. It is presumed that subsequent conformational changes in E2, E1 , and CD81 lead to membrane penetration and delivery of HCV RNA.
  • Neutralizing Abs are believed to act by binding to the exposed envelope (Env) surface of E2/E1 and arrest the subsequent set of events in the entry process.
  • HCV isolates are highly divergent, classified into six genotypes and eleven subtypes (Simmonds ef. al., Hepatology 19:1321 , 1994).
  • Our understanding on the humoral immune response to HCV is rather limited because the virus can be studied only in humans and chimpanzees, and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralization antibodies to HCV might be important in preventing infections. Using a recently described neutralization assay (Bartosch et. al., J. Exp. Med.
  • MAb fragments are considered effective in neutralizing HCV, unfortunately, because of their smaller size they are expected to have shorter serum half-life (in the order of minutes), and are cleared rapidly through glomerular filtration by kidneys.
  • a novel way to address this problem is to pegylate MAb fragments such that the apparent molecular size of the pegylated MAb fragments is dramatically increased leading to extended serum half-life while the broadly- neutralizing property of MAb fragments is still retained.
  • Pegylation is the process of covalently linking polyethylene glycol (PEG) of approximately 5-60 kilodaltons to antibodies or proteins, which leads to dramatic improvement in pharmacokinetic and pharmacodynamic properties (Harris and Chess, Nature Rev. Drug Discovery 2:214-220, 2003).
  • the present invention provides a substantially homogenous preparation of pegylated MAb fragments of anti-E2 MAbs and its derivatives thereof, and related methods.
  • the method provides high yield of a pegylated MAb fragment that is modified exclusively at defined site(s), thereby providing several processing and therapeutic advantages as compared to other species involving random modification.
  • pegylated MAb fragments are equally broadly-neutralizing, and their properties such as pharmacokinetics, pharmacodynamics, bioactivity and biocompatibility are substantially improved.
  • Pegylated MAb fragments prepared from 5-100 kDa PEG polymers are highly efficacious.
  • the HCV viral load may decrease for over 3-21 days.
  • the present invention also relates to optimization of anti-E2 MAb fragment by specific antibody engineering procedures wherein the affinity and broadly-neutralizing properties are significantly improved.
  • the amino acid residues of the complementarity determining residue (CDR) sequences of light and heavy chains of MAb fragment are mutated, and that the affinity improvements are additive.
  • a free cysteine residue is optionally engineered in the anti-E2 MAb fragment amino acid sequence, preferably away from the antigen binding site.
  • the present invention relates to human anti-E2 MAb fragment having cysteine mutations engineered into positions away from the epitope binding sequences of anti-E2 MAb fragment.
  • the present invention also relates to pegylated anti-E2 MAb fragments wherein PEG is conjugated to the modified cysteine residue.
  • the PEG has a molecular weight ranging from at least about 5 kDa to not more than about 100 kDa.
  • a particularly preferred PEG is at least about 20 and not more than about 60 kDa.
  • the present invention further relates to any of the pegylated anti-E2 MAb derivatives as above, in a pharmaceutically acceptable carrier.
  • the present invention further relates to processes for preparing pegylated antibody derivatives as described above.
  • the principal embodiment of the method for making the substantially homogenous preparation of pegylated-MAb fragment comprises: (a) engineering a cysteine residue into a specific amino acid position within the amino acid sequence of anti-E2 MAb derivative; (b) conjugating a polyethylene glycol to said MAb derivative at said cysteine residue to provide a mono-pegylated antibody conjugate; and (c) isolating said pegylated MAb conjugate.
  • the present invention also relates to methods of treating individuals using pegylated anti-E2 MAb fragment conjugates as above.
  • the invention is directed to a method of inhibiting HCV infections in humans, which method comprises administering to humans in need thereof a pegylated MAb fragment molecule comprising (a) a single chain variable fragment (scFv) or antibody fragment (FAb), collectively called as MAb fragment, and (b) a peg molecule, wherein the pegylated MAb fragment binds to an epitope of the HCV envelope protein that is inaccessible to whole immunoglobulin molecules due to molecular steric hindrance, whereupon the HCV infection is inhibited.
  • scFv single chain variable fragment
  • FAb antibody fragment
  • Figure 1 Amino acid sequence of anti-E2 heavy chains.
  • the anti-E2 heavy chain sequences are provided as SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:8.
  • the complementarity determining regions (CDRs 1-3) of the heavy chain are marked.
  • Figure 2 Linkers.
  • FIG 3 Engineering of anti-E2 MAb fragment by in vitro scanning saturation mutagenesis.
  • the 8 th and 6 th positions of CDR-2 and CDR-3, respectively, of the V H of HCV #4 are replaced with nineteen other amino acids (only four variants are shown here for the sake of clarity), and the affinity measurements for variants measured.
  • This illustration exemplifies that the affinity improvements are additive when the variants are combined (see Table-1). For example, approximately 500-fold affinity improvement can be obtained when the mutated sequences of variants 2-1 and 3-4 are combined.
  • Figure 4 Pegylation of anti-E2 MAb fragment. The linear sequence of the pegylated
  • MAb fragments can be constructed in different formats as shown in constructs A-E.
  • V L variable sequence of light chain
  • V H variable sequence of heavy chain
  • C L and C H i are the fragments of constant frames of light and heavy chains, respectively. Varying lengths of C L and C H i are added to optimize the binding of the pegylated MAb fragment to the CD4-inducible epitope of gp120.
  • Engineered "free" cysteine (Cys) residue is placed away from the antigen binding sites of V L and V H , which are connected by a (G 4 S) n linker. Addition of the flexible linker (F L ) relieves molecular steric hindrance due to pegylation, if any.
  • Construct E the peg molecule is directly linked to the terminal cysteine residue of the C m fragment through a disulfide bridge.
  • pegylation is meant the process by which polyethylene glycol (peg) chains are attached to therapeutic drugs such as proteins, peptides, antibodies, or antibody fragments.
  • peg polyethylene glycol
  • therapeutic drugs such as proteins, peptides, antibodies, or antibody fragments.
  • pegylation improves pharmacokinetics by increasing the molecular mass of proteins and shielding them form proteolytic enzymes.
  • these pegylated therapeutic drugs overcome such shortcomings as degradation to proteolytic enzymes, rapid clearance by the kidneys, and generation of neutralizing antibodies.
  • site specific pegylation is meant that the process by which a peg molecule is attached to a specific amino acid residue such as cysteine in an antibody or antibody fragment.
  • pharmacodynamics is meant that changes in measurable clinical parameters related to a drug, such as decrease in viral load.
  • half-life is meant that the amount of time it takes for one-half of the drug dose to be lost through biological processes.
  • shelf-life is meant that the amount of time a stored drug retains its activity.
  • an anti-HCV antibody of the invention physically associates with its target molecule (e.g. E2 of Env) to inhibit HCV entry into a cell and/or to inhibit or prevent HCV replication in humans.
  • the antibody does not substantially physically associate with other molecules.
  • a “broadly-neutralizing” antibody against HCV and similar terms is meant an antibody that can inhibit the activity (e.g., the ability to enter a target cell) of several HCV clinical (primary) isolates from more than one genotype. HCV isolates are classified under six genotypes and eleven subtypes.
  • hypervariable region-1 is meant that the region of HVR1 , which is located at the N terminus of the envelope glycoprotein 2 (E2) gene and consists of 34 amino acids spanning position 384-414; it is the most variable region of the HCV genome and contains linear epitopes that are recognized by patients' antibodies and mutates rapidly in vivo.
  • selected is meant that an antibody or antibody fragment of the invention is chosen or isolated from a group or library of candidate antibodies or antibody fragments using a screening assay for choosing or isolating antibodies with a desired characteristic (that is, the ability to bind a complex comprising HCV E2, E1 , CD81 receptor and preferably a co-receptor), as would be understood by a skilled practitioner in the art.
  • antibodies is used herein a broad sense includes monoclonal antibodies, fragments and multimers of immunoglobulin molecules (Hudson and Souriau, Nature Medicine 9:
  • single chain antibodies and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to broadly-neutralize HCV by binding a CD4-inducible HCV epitope that is enhanced by binding a HCV co-receptor, as described herein.
  • the antibodies are tested for their desired activity using their in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to established clinical testing methods.
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 (Burton et.al.,) and U.S. Patent No.
  • any "antibody or antibody fragment” of the invention can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acid residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bioavailability, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as binding to its epitope or antigen. Functional or active regions of the antibody or antibody fragment may be identified and/or improved by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • amino acid sequence variants of antibodies or antibody fragments can be generated and those that display equivalent or improved affinity for antigen can be identified using standard techniques and/or those described herein.
  • Methods for generating amino acid sequence variants are readily apparent to a skilled practitioner in the art and can include directed evolution technologies (US Patent No: 6,180,341) or random mutagenesis (e.g., by PCR) of the nucleic acid encoding the antibody or antibody fragment (Zoller, M.J. Curr. Opinion in Biotechnol. 3:348-354, 1992). Both naturally occurring and non-naturally occurring amino acids may be used to generate amino acid sequence variants of the antibodies and antibody fragments of the invention.
  • isolated polypeptide is meant a polypeptide (or a fragment thereof) that has been separated from the components that naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%, at least 70%, at least 80%, at least 90%, or more, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated.
  • a substantially pure polypeptide may be obtained, for example, by extraction from natural source by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by appropriate methods such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analyses.
  • a protein is substantially free of naturally associated components when it is separated from those contaminants, which accompany it in its natural state.
  • a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • substantially pure polypeptides not only include those derived from eukaryotic organisms but also those produced in E. coli or other prokaryotes.
  • the human antibodies of the invention can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole ef a/.,
  • Human antibodies of the invention can also be produced using phage display libraries (Hoogenboom ef a/., J. MoI. Biol., 227:381 , 1991 ; Marks ef a/., J. MoI. Biol., 222:581 , 1991 ; and C. F. Barbas, D. R. Burton, J. K. Scott, G. J. Silverman, Phage
  • Antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described elsewhere (Remington: The Science and Practice of Pharmacy [19th ed.] ed. A. R. Gennaro, Mack Publishing Company, Easton, PA 1995).
  • an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7.0 to about 7.5. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered.
  • the antibodies can be administered to the subject or patient by injection (e.g., but not limited to, intravenous, intradermal, subcutaneous, intramuscular), or by other methods such as infusion that ensures its delivery to the bloodstream in an effective form. Local or intravenous injection is preferred.
  • Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, particular type of antibody used and other drugs being administered.
  • an antibody of the invention is efficacious in treating or inhibiting HCV infections in a subject by observing that the antibody reduces viral load or delays or prevents a further increase in viral load.
  • Viral loads can be measured by methods that are known in the art, for example, using PCR assays to detect the presence of HCV nucleic acid or antibody assays to detect the presence of HCV protein in a sample (e.g., but not limited to, blood or another body fluid) from a subject or patient, or by measuring the level of circulating anti-HCV antibodies in the patient.
  • An antibody treatment that decreases HCV viral load in an HCV-positive patient is considered an efficacious antibody treatment.
  • the broadly-neutralizing antibodies of the invention can also be administered prophylactically to patients or subjects who are at risk for being exposed to HCV or who have been newly exposed to HCV.
  • patients include, but are not limited to, healthcare workers, fetuses, neonates, or infants (or nursing infants) whose mothers are infected or at risk for being infected, intravenous drug users, recipients of blood transfusions, blood products, or transplantation tissue, and other individuals who have been exposed to a body fluid that contains or may contain HCV.
  • efficacious treatment with an antibody of the invention partially or completely inhibits or delays the appearance of the virus or minimizes the level of the virus in the blood or other body fluid(s) of the exposed individual.
  • an effective amount is meant the amount of an anti-HCV antibody of the invention that is useful for treating, partially or completely inhibiting, or preventing an HCV infection in a patient or subject as described herein.
  • Effective dosages and schedules for administering the antibodies of the invention may be determined empirically, and making such determinations is routine to one of ordinary skill in the art.
  • An effective dose of HCV-antibody of the invention generally will range between about 1 ⁇ g/kg of body weight and 25 mg/kg of body weight.
  • the invention is directed to a method of inhibiting HCV infection in humans, which method comprises administering to a human in need thereof a pegylated MAb fragment that comprises (a) single chain variable fragment (scFv) or antibody fragment (FAb) comprising about 200-400 amino acids and including the epitope binding site, and (b) a polyethylene glycol molecule of size ranging from at least about 5 to not more than about 60 kDa.
  • the pegylated MAb fragment binds to an epitope of the HCV envelope protein that may not be readily accessible to whole immunoglobulin molecule due to molecular steric hindrance.
  • the peptide or polypeptide is a pegylated MAb fragment.
  • the invention is directed to a method of inhibiting HCV infection in humans, which method comprises administering to humans in need thereof a pegylated MAb fragment molecule comprising a) a MAb fragment and b) a peg molecule, wherein the pegylated MAb fragment molecule binds to an epitope of the HCV envelope protein E2 that may not be readily accessible to whole immunoglobulin molecules due to steric hindrance, whereupon the HCV infection is inhibited.
  • Inhibiting HCV infection refers to the inhibition in the onset of HCV infection, the inhibition of an increase in an existing viral infection, or a reduction in the severity of the infection.
  • Inhibition of HCV infection can be assayed by methods that are known in the art, such as by the assessment of viral load.
  • HCV loads can be measured by methods that are known in the art, for example, using polymerase chain reaction assays to detect the presence of HCV RNA, or antibody assays to detect the presence of HCV protein in a sample (e.g. blood) from HCV-infected humans.
  • HCV is an enveloped RNA virus that often establishes chronic infection leading to liver cirrhosis and hepatocellular carcinoma (Lauer, G. M., and Walker, B. D. N. Engl. J. Med. 345:41 , 2001).
  • HCV infection leads to loss of liver function, and, without a liver transplantation, ultimately to death.
  • Post-transplant patients are still at risk from re-infection of the new liver from circulating HCV.
  • the HCV genome encodes a single polyprotein that is processed by viral and cellular proteases into structural and non-structural proteins.
  • the structural proteins include the envelope glycoproteins E1 and E2, which mediate viral binding and entry into host cells.
  • E1 and E2 form heterodimers and undergo extensive post translational modifications by N-linked glycosylation (Lauer and Walker, supra). Both E1 and E2 associate to form heterodimers, which accumulate in the endoplasmic reticulum, the proposed site for HCV assembly and budding (Op De Beeck ef. al., J. Gen. Virol. 82:2589, 2001). HCV purified from plasma has been reported to exist in association with plasma lipoproteins, suggesting that the virus may use the low-density-lipoprotein receptor (LDLr) to gain entry into cells (Aguello et. al., Proc. Natl. Acad. Sci. USA 96:12766, 1999; Wunschmann et. al., J. Virol. 74:10055, 2000; Bartosch et. al., J. Biol. Chem. 278:41624, 2003).
  • LDLr low-density-lipoprotein receptor
  • HCV is primarily a hepatotropic virus
  • HCV infection is frequently associated with mixed cryoglobulinemia, non-Hodgkin's B-cell lymphoma, and Sjogren's syndrome, all of which involve B-cell proliferation (Ferri ef. al., Br. J. Haemotol. 88:392, 1994; Selva-O'Callaghan ef. al., Arthritis Rheum. 42:2489, 1999; Zuckerman ef. al., Ann. Internal Med. 127:423, 1997), suggesting that HCV may infect B cells or affect B-cell functions in natural infection.
  • Minus-strand HCV RNA has been detected by reverse transcriptase PCR in peripheral lymphocytes, bone marrow, lymph nodes, and central nervous system of some HCV patients (Radkowski ef. al., J. Virol. 76: 600, 2002). Analysis of plus-strand HCV RNA sequences and quasispecies patterns suggested that HCV RNAs in these cells are different from those in the serum (Laskus ef. al., J. Virol. 74:1014, 2000). Several investigators have shown that HCV can infect B-cells (Morsica ef. al., Blood 94:1138, 1999), and T- cells (Shimizu ef. al., Proc. Natl. Acad. Sci.
  • HCV shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% similarity to one another (Ramadori, G., and Meier, V., Eur. J. Gastro. and Hepatol. 13:465, 2001 ).
  • Phylogenetic analyses of nucleotide sequences derived from part of the gene encoding a nonstructural protein (NS5) has provided six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients infected with chronic hepatitis. Many of these HCV types comprise a number of more closely related subtypes, leading to a current total of eleven genetically distinct viral populations (Simmonds ef.
  • genotypes are independent predictors of the antiviral response to peginterferon-ribavirin therapy. While genotype 1 (a and b) exhibits a poor response to this treatment with a response rate of 35-42%, genotypes 2 and 3 respond much better with a response rate of 80% (Manns ef. al., Lancet 358:958, 2001).
  • HVR1 hypervariable region-1
  • Therapeutic MAbs are beginning to find routine applications in therapies for human diseases, as demonstrated by the large number of MAbs approved for sale or in final stages of clinical trials (Walsh, G., Nature Biotechnol. 21 :865-870, 2003).
  • therapeutic applications of these drugs are largely limited to acute indications such as rheumatoid arthritis and cancers because of several shortcomings, which include their susceptibility to destruction by proteolytic enzymes, short circulating half-life, short shelf-life, low solubility, rapid kidney clearance and their propensity to generate neutralizing antibodies.
  • most MAbs must be delivered by intravenous injections, which is an expensive procedure involving frequent hospital visits. Chronic therapy usually requires large doses (3-5 mg/kg) of antibody repeatedly administered over months to years.
  • antibody fragments such as FAb or MAb fragment that can be readily expressed in microbial systems with the potential for large-scale and cost-efficient production.
  • antibody fragments tend to have short in vivo half-life, making them unsuitable for clinical therapies that require antibody circulation over extended periods of time.
  • E. coli is currently the host of choice for production of FAb and MAb fragments (Better ef. al., Science 240:1041-1043, 1988).
  • human immunoglobulins have long circulating half-lives, with f 1/2 ⁇ (distribution-phase half-life) values of 18 ⁇ 22 h and t V2 ⁇ (terminal elimination-phase half-life) values of 21 ⁇ 23 days for human IgGI 1 2 or 4 (Mariani, G. and Strober, W., in Fc Receptors and the Action of Antibodies (Metzger, H., ed.), pp.94-177, American Society for Microbiology, Washington DC).
  • f 1/2 ⁇ distributed-phase half-life
  • t V2 ⁇ terminal elimination-phase half-life
  • the cellular receptor responsible for maintaining half-life has been identified as the neonatal Fc receptor, FcRn (King, D.J., in Applications and Engineering of Monoclonal Antibodies, pp, 67-75, Taylor & Francis, London). Mice deficient in this receptor have reduced plasma IgG levels and clear administered IgG or Fc with an abnormally short half-life (Ghetie ef. al., Eur. J. Immunol. 26:690-696, 1996). Once internalized into cells, IgG is salvaged from the endosome during acidification through binding to FcRn, protecting the antibody from degradation. The IgG is then recycled to the cell surface where the higher pH leads to dissociation and return of the IgG to the circulation.
  • FcRn neonatal Fc receptor
  • Pegylation is an alternative method that overcomes these deficiencies by attaching polyethylene glycol (PEG) chains to MAb fragments and FAbs (Harris and Chess, supra).
  • FDA has approved PEG for use as a vehicle or base in foods, cosmetics and pharmaceuticals, including injectable, topical, rectal and nasal formulations.
  • PEG shows little toxicity, and is eliminated from the body intact by either kidneys if the PEGs are less than 30 kDa, or in the faeces if the PEGs are larger than 20 kDa (Yamaoka ef. al., J. Pharm. ScL 83:601-606, 1994).
  • PEG lacks immunogenicity (Working ef. al., ACS Symposium Series 680:45-57, 1997). Generation of antibodies to PEG under routine clinical administration of pegylated proteins is not known.
  • Pegylation is a well established and validated approach for the modification of a range of MAbs, proteins and peptides (Chapman, A., Adv. Drug Deliv. Rev. 54:531-545, 2002; and Harris and Chess, supra).
  • the benefits include: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics and decreased viral load; (d) improved solubility-PEG has been found to be soluble in many different solvents, ranging from water to many organic solvents such as toluene, methylene chloride, ethanol and acetone (Harris and Chess, supra); (e) pegylated antibody fragments can be concentrated to 200 mg/ml, and the ability to do so opens up formulation and dosing options such as subcutaneous administration of a high protein dose; this is in contrast to many other therapeutic antibodies which are typically administered intravenously; (f) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles e
  • first-generation pegylation methods were fraught with difficulties.
  • first generation pegylation the PEG polymer was generally attached to the epsilon ( ⁇ ) amino groups of lysine amino acid residues. This resulted in the modification of multiple lysines, and gave mixtures of PEG isomers with different molecular masses (Zaplinsky, S. Adv. Drug Delivery Rev. 16:157-182, 1995). The existence of these isomers makes it difficult to reproduce drug batches, and can contribute to the antigenicity of the drug and poor clinical outcomes.
  • first generation methods mainly used linear PEG polymers with molecular masses of 12 kDa or less.
  • Second-generation pegylation chemistry strives to avoid the pitfalls associated with mixtures of isomers, diol contamination, unstable bonds and low molecular mass PEG.
  • An overall goal of second-generation pegylation methods is to create larger PEG polymers to improve the pharmacokinetic and pharmacodynamic effects. In some cases the changes are dramatic, such as the pegylation of interleukin-6 (IL-6), which increases the half-life of IL-6 100-fold, which in turn results in 500-fold rise in its thrombopoietic potency (see Harris and Chess, supra).
  • Site-specific pegylation can minimize the loss of biological activity and reduce immunogenicity.
  • cysteines can be optionally added to polypeptides precisely where they are required by site directed mutagenesis. Although many proteins might not benefit from site-specific pegylation, in others, such as MAb fragments and FAbs, it is crucial that the PEG is attached at a site distant from the antigen binding site (Chapman et. al., Nature Biotechnol. 17:780-783, 1999).
  • Branched PEGs of greatly increased molecular masses — up to 100 kDa or more, compared with the 12 kDa or less found in first-generation PEGs — have been prepared.
  • a branched PEG acts as if it were much larger than a corresponding linear PEG of the same molecular mass.
  • Branched PEGs are also better at cloaking the attached polypeptide from the immune system and proteolytic enzymes, thereby reducing immunogenicity and likelihood of destruction.
  • the present invention relates to substantially homogenous preparations of chemically modified proteins, and methods thereof.
  • substantially homogenous as used herein means that the only chemically modified proteins observed are those having one "modifier” (e.g., PEG) moiety.
  • the preparation may contain unreacted (i.e., lacking modifier moiety) protein as ascertained by peptide mapping and N-terminal sequencing.
  • biologically active agents refers to recombinant or naturally occurring proteins, useful for prophylactic and therapeutic applications. One skilled in the art will readily be able to adapt a desired biologically active agent to the compositions of present invention.
  • the epitope of the HCV envelope protein to which the pegylated MAb fragment molecule binds can be any epitope that is inaccessible to larger molecules, such as whole IgGs, due to molecular steric hindrance.
  • the viral epitope is preferably a conserved HCV epitope (i.e., the amino acid sequence of the epitope is shared by one or more strains and clades of HCV).
  • the epitope is an epitope of HCV, such as an HCV envelope glycoprotein.
  • the pegylated MAb fragment molecule can be any pegylated MAb fragment molecule that can bind to an epitope of HCV envelope protein.
  • the pegylated MAb fragment molecule can comprise any suitable MAb fragment, any suitable peg molecule, any suitable constant fragments of light and heavy chains, any suitable Fc region, and, optionally, any suitable long flexible linker.
  • the pegylated MAb fragment molecule comprises a flexible linker ( Figure 2), desirably and preferably the linker is positioned between the MAb fragment and peg ( Figure 4).
  • the pegylated MAb fragment can be any suitable pegylated MAb fragment that binds to an epitope of HCV envelope protein that may not be readily accessible to whole IgG molecules due to molecular steric hindrance.
  • the pegylated MAb fragment molecule is sufficiently flexible so as to avoid any steric, size, or orientation effects that prevent larger molecules (e.g., whole IgG molecules) from accessing the epitope of the HCV envelope protein.
  • the MAb fragment comprises about 200 to about 400 amino acids.
  • the MAb fragment molecule of the pegylated MAb fragment can be any suitable MAb fragment.
  • a MAb fragment comprises a heavy chain variable domain (V H ) joined via a short linker peptide to a light chain variable domain (V L ), and is responsible for antigen binding.
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the MAb fragment to be used in the method of the invention is preferably broadly cross-reactive (e.g., can bind to a broad range of viral isolates from different genotypes and subtypes) with a high neutralization activity (e.g., typically with an IC 50 of less than 100 ⁇ g/ml).
  • the MAb fragment is a MAb fragment of anti-E2 FAbs (including any one of SEQ ID NO:1 , SEQ ID NO:7-12; for example see International Patent Application WO 02/055560).
  • a variant of an aforementioned MAb fragment can be used.
  • the variant of the MAb fragment retains the ability to bind to the same epitope of the HCV envelope protein.
  • a variant of an MAb fragment can be obtained by any suitable method, including random and site-directed mutagenesis of the nucleic acid encoding the MAb fragment and sequential antigen panning (see, e.g., PCT/US03/14292) and/or in vitro scanning saturation mutagenesis (US Patent No: 6,180,341). While a variant of the nucleic acid can be generated in vivo or in vitro and then isolated and purified, alternatively, a variant of the nucleic acid can be synthesized.
  • any antibody or antibody fragment may be used according to the present invention.
  • the preferred construct comprises a single chain antibody (scFv) format or FAb fragment, since no chain association event must take place following the translation. This facilitates the in vitro system for transcription/translation.
  • Antibody or “antibody fragment” refers to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE or any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as FAb, single domain antibodies (DAbs), Fv, MAb fragment (single chain Fv) and the like.
  • DAbs single domain antibodies
  • Fv single domain antibodies
  • MAb fragment single chain Fv
  • the specificity of an antibody is determined by the complementarity determining regions (CDRs) within the light chain variable regions (V L ) and the heavy chain variable regions (V H ).
  • CDRs complementarity determining regions
  • the FAb fragment of an antibody which is about one-third the size of a complete antibody contains the heavy and light chain variable regions, the complete light chain constant region and a portion of the heavy chain constant region.
  • FAb molecules are stable and associate well due to the contribution of the constant region sequences.
  • the yield of functional FAb expressed in bacterial systems is lower than that of the smaller Fv fragment which contains only the variable regions of the heavy and light chains.
  • the Fv fragment is the smallest portion of an antibody that still retains a functional antigen binding site.
  • the Fv fragment has the same binding properties as the FAb, however without the stability conferred by the constant regions, the two chains of the Fv can dissociate relatively easily in dilute conditions.
  • V H and V L regions may be fused via a polypeptide linker (Huston et. al., Meth. in Enzymol. pp. 46-99, 1991) to stabilize the antigen binding site.
  • This single polypeptide Fv fragment is known as a single chain antibody (MAb fragment).
  • the VH and V L can be arranged with either domain first.
  • the (G 4 S) n linker joins the carboxy terminus of the first chain to the amino terminus of the second chain ( Figure 4).
  • SSM In vitro scanning saturation mutagenesis
  • SSM represents as a systematic new tool for exploring in vitro antibody affinity evolution, analogous to somatic hypermutation in vivo.
  • interesting single mutants can be used as a starting point for subsequent rounds of SSM at other sites, so that multiple mutations with synergistic effects on binding may be identified.
  • This same sequential mutation approach should be useful to optimize properties such as affinity, potency, efficacy, altered specificity, reduced immunogenicity, and removal of proteolytic cleavage sites (US Patent No: 6,180,341).
  • the ability of the variants of the above-described MAb fragments to bind to the same epitope of the HCV envelope protein can be assessed by any suitable manner known in the art, such as by the enzyme-linked immunosorbent assay (ELISA).
  • the variant of the above-described MAb fragment includes molecules that have about 50% or more identity to the above-described MAb fragments. More preferably, the variant includes molecules that have 85% to about 99% identity with the above-described MAb fragments.
  • the variant of the MAb fragment contains from 1 to about 40 amino acid substitutions, deletions, inversions, and/or insertions thereof. More preferably, the variant of the above-described MAb fragments contains from 1 to about 10 amino acid substitutions, deletions, inversions, and/or insertions thereof.
  • substitutions, deletions, inversions, and/or insertions of the MAb fragment preferably occur in non-essential regions.
  • the identification of essential and non-essential amino acids in the MAb fragment can be achieved by methods known in the art, such as by site-directed mutagenesis (for example, SSM) and AlaScan analysis (Moffison et. a/., Chem. Biol. 5:302-307, 2001).
  • Essential amino acids have to be maintained or replaced by conservative substitutions in the variants of the MAb fragment fragments, such that the pegylated MAb fragment maintains the ability to bind to an epitope of HCV envelope protein.
  • Non-essential amino acids can be deleted, or replaced by a spacer or by conservative or non-conservative substitutions.
  • the variants can be obtained by substitution of any of the amino acids present in the MAb fragment. As can be appreciated, there are positions in the sequence that are more tolerant to substitutions than others, and some substitutions can improve the binding activity of the native MAb fragment.
  • the amino acids that are essential should either be identical to the amino acids present in the MAb fragment, or substituted by conservative substitutions.
  • the amino acids that are non ⁇ essential can be identical to those in the MAb fragment, can be substituted by conservative or non- conservative substitutions, and/or can be deleted.
  • Conservative substitution refers to the replacement of an amino acid in the MAb fragment with a naturally or non-naturally occurring amino acid having similar steric properties. Where the side-chain of the amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally or non-naturally occurring amino acid that is also polar or hydrophobic.
  • a non-conservative substitution is a substitution in which the substituting amino acid (naturally or non-naturally occurring) has significantly different size, configuration and/or electronic properties compared to the amino acid being substituted.
  • the side chain of the substituting amino acid can be significantly lower (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • the MAb fragment or MAb fragment and peg are joined together by a long flexible linker
  • the linker can be any suitable long flexible linker, such that the MAb fragment of the pegylated MAb fragment can bind to the epitope of the HCV envelope protein (i.e., the molecule is not excluded from binding by molecular steric hindrance).
  • the linker can be any suitable length, but is preferably at least about 15 to about 50 amino acids in length.
  • the long flexible linker is an amino acid sequence that is naturally present in IgG molecules of the host, such that the presence of the linker would not result in an immune response against the linker sequence by the mammal.
  • the linker is the long flexible linker of SEQ ID NO: 6 or SEQ ID NO: 7.
  • the pegylated MAb fragment molecule encompassed by the invention comprise an epitope binding MAb fragment, a peg molecule, and, optionally, a flexible linker.
  • the pegylated MAb fragment molecules of the invention comprise any one of the sequences set forth in Figure 1 , or variants thereof, which retain the ability to bind to the same HCV epitope with high affinity.
  • Variants of the pegylated MAb fragment molecules can be obtained by any suitable method, including those methods discussed here.
  • the ability of a variant to bind to the same epitope of the HCV envelope protein can be assessed by any suitable manner known in the art, such as by ELISA.
  • the variants of the above-described pegylated MAb fragment molecules include molecules that have about 90% or about 99% identity with the above-described pegylated MAb fragment molecules.
  • the variants of the pegylated MAb fragment molecules contain from 1 to about 30 amino acid substitutions, deletions, inversions, and/or insertions thereof.
  • the variants of the pegylated MAb fragment molecules contain molecules of peg whose size range from at least about 5 kilodalton (kDa) to not more than about 100 kDa.
  • the peg molecule is either linear or branched and whose size range from at least about 5 kilodalton (kDa) to not more than about 100 kDa.
  • the pegylated MAb fragment molecule preferably recognizes one or more strains of HCV.
  • the molecule is broadly cross-reactive and can bind to a wide range of isolates of HCV from different genotypes with high affinity.
  • the invention encompasses any pegylated MAb fragment molecule that binds to an epitope of HCV envelope protein
  • the molecule is preferably an antibody to HCV E2 envelope glycoprotein.
  • the binding of the pegylated MAb fragment molecule preferably is enhanced by the presence of CD81 receptor (and preferably a co-receptor).
  • the enhancement is exemplified by at least a two-fold increase in the binding affinity, such as a two-fold decrease in EC 50 as measured by ELISA.
  • the anti-E2 MAb fragment is expected to be capable of pharmaceutical use in humans.
  • cysteine residue already present in the native sequence as the site for pegylation.
  • a select cysteine mutation can be optionally introduced by site-directed mutagenesis into the protein sequence ( Figure 4).
  • the purpose of the cysteine point mutation is to allow a pegylation conjugation site.
  • cysteine protein analogs can be easily prepared using conventional methods well known to one of ordinary skill in the art. Subject to considerations for optimization as discussed below, the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is at least about 5 kDa and not more than about 100 kDa, usually at least about 10 kDA and not more than about 60 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight).
  • Various sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • the present invention includes pharmaceutical compositions comprising effective amounts of the modified antibodies and fragments described herein, together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers needed for administration.
  • pharmaceutically acceptable diluents preservatives, solubilizers, emulsifiers, adjuvants and/or carriers needed for administration.
  • the optimal pharmaceutical formulation for a desired biologically active agent will be determined by one skilled in the art depending upon the route of administration and desired dosage. Exemplary pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton, PA., pgs.1435-1712 (1990)).
  • One skilled in the art will be able to ascertain effective dosages by administration and observing the desired therapeutic effect.
  • the formulation of the conjugate will be such that at least about 0.01 ⁇ g pegylated anti-E2 MAb fragment/kg body weight/day and not more than about 10 mg pegylated anti-E2 MAb fragment/kg body weight/day will yield the desired therapeutic effect.
  • the effective dosages may be determined using diagnostic tools over time. The therapeutic dosages are determined depending on the severity of the infection, viral load (sustained virological response) over the course of therapy. The dosages may therefore vary over the course of therapy, with, for example, a relatively high dosage being used initially, until therapeutic benefit is seen, and lower dosages used to maintain the therapeutic benefits.
  • PEG-MAL purchased from Nektar Therapeutics, San Carlos, CA; formerly Shearwater Corp.
  • Maleimide undergoes alkylation reactions with sulfhydryl groups to form stable thioether bonds.
  • the availability of synthetic PEG polymers having one maleimide group at a single terminus allows thiol- directed pegylations.
  • rapid and specific reaction with cysteine and not with lysine or histidine at pH 6.0 can be achieved (Yang et. a/., supra).
  • the free cysteine residue at the C-terminus or linker of the MAb fragment proteins is reduced prior to reaction with MAL-PEG.
  • the reduction solution contained 3 mg/ml MAb fragment, 2 mM DTT, 2 mM EDTA, 100 mM sodium phosphate, pH 7.8. The reduction is performed at 37 0 C for 2 h. Free DTT is removed on a HiPrep or PD-10 desalting column. The column is equilibrated with 100 mM sodium phosphate, pH 6.0, 2 mM EDTA. Near quantitative reduction of one thiol per MAb fragment molecule is achieved as measured by DTNB assays (Creighton, 1989).
  • the typical pegylation reaction buffer contains 1 mg/ml reduced MAb fragment protein, 100 mM sodium phosphate pH 6.0, 2 mM EDTA and PEG-maleimide compound at a reaction molar ratio of 10:1 (PEG:MAb fragment).
  • the reaction is conducted at 25 0 C under nitrogen atmosphere for 2 h.
  • the typical conjugation yield is about 80% as analyzed by SDS-PAGE.
  • Unreacted MAb fragment protein could be successfully reprocessed in a second reduction and conjugation reaction with comparable yields.
  • Pegylated MAb fragment is then purified from native MAb fragment, high molecular weight impurities, and unreacted free PEG by HS chromatography (see e.g., Yang et. al, supra).
  • the column equilibration buffer contained 10 mM sodium phosphate, pH 5.0, and the gradient elution buffer is 10 mM sodium phosphate, pH 5.0, 1 M NaCI.
  • Ion exchange chromatographic procedures are used to further purify the pegylated MAb fragment.
  • Analytical characterization of pegylated MAb fragment and MAb fragment Protein concentrations, Western blot analysis, SDS-PAGE analysis and staining procedures are according to established procedures (Yang et. al., supra).
  • Mass values of pegylated MAb fragment and MAb fragment are determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker Daltronics OmniFlex NT) using an internal standard with a similar molecular weight. Apparent molecular weights (Stokes radius) of the MAb fragment proteins are estimated using Superdex 200 HR 10/30 gel filtration column chromatography (Amersham Biosciences) in 50 mM sodium phosphate buffer, pH 6.5, 150 mM NaCI.
  • MALDI-TOF-MS matrix-assisted laser desorption ionization/time-of-flight mass spectrometry
  • HCVpp in vitro pseudoparticle
  • 293T cells are transferred with expression vectors encoding the viral components (HCV E1/E2, retroviral core/packaging component, and GFP/integration signal).
  • HCV E1/E2 retroviral core/packaging component
  • GFP/integration signal GFP/integration signal.
  • Expression plasmids encoding E1 E2 glycoproteins of HCV strain H77 (genotype 1a) (Op De Beeck ef. al., J. Biol. Chem. 275:31428, 2000) or strain J (genotype 1 b) (Thomson et. al., Gastroenterology 121 :1226, 2001) are used.
  • an expression vector encoding the E1 E2 glycoproteins of strains H77 from which the hypervariable region (HVR1) of E2 had been deleted is used (Bartosch ef. al., J. Biol. Chem. 278:41624, 2003).
  • the medium is replaced 16 h after transfection.
  • Supematants containing the HCVpp are harvested 24 h later, filtered through 0.45 ⁇ filters, and used to infect Huh-7 cells, which had been seeded the day before at a density of 8 x 10 4 cells per well in 12-well plates. Dilutions of sera to be tested for neutralization are mixed with dilutions of viral supematants, preincubated in regular medium for 96 h at 37 0 C.
  • HCVpp transduced ⁇ 2-4% of cells.
  • the infectivity of HCVpp in the presence of sera from healthy seronegative human donors is standardized to 100%. All dilutions of test sera are compared with the same dilutions of these negative control sera.
  • the positive control sample is from a patient who is chronically infected with HCV.
  • Control neutralization experiments are performed by using HCVpp bearing glycoproteins derived from the feline endogenous retrovirus RD114 as described (Bartosch ef. al., J. Exp. Med.197:633, 2003).
  • EXAMPLE III PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES IN SCID MOUSE MODEL
  • PCR for up to 35 weeks with titers ranging from 3 x10 4 to 3 x 10 6 copies/ml. These viral titers are similar to those found in infected humans. Moreover, an approximately 3-log rise in viral titers after inoculation, detection of viral minus-strand RNA in the liver, and the ability to serially passage the virus through several generations of animals provide convincing evidence for active replication and production of infectious viral progeny in this system.
  • Alb-uPA SCID chimeric mouse model offers a number of important advantages. HCV infection and replication occur in human hepatocytes, as opposed to animal models such as the chimpanzees, the tamarin, and possibly tupaias. Thus, HCV replication takes place in its authentic environment. Moreover, successful infection could be achieved with viral isolates from different HCV-positive human donors. Therefore, the susceptibility of the system to wild-type virus could be shown. Most importantly, this system allows studying the natural infection process with an easily measurable read out and, therefore, may serve as a robust "in vivo neutralization assay". Once-a- week dosing regimens are compared for pegylated versus non-pegylated MAb fragment. Alb-uPA SCID mice are administered subcutaneously with 2.5-50 mg/kg of the MAb fragments on days 0, 7, 14, and 21. HCV viral load is monitored relative to a buffer control over 1-3 weeks.
  • the entry mechanism and its sequence of events leading to receptor binding and cell entry is well understood in HIV.
  • the binding of the HIV-1 envelope glycoproteins (gp120 and gp41) to CD4 and a co-receptor initiates a series of conformational changes that are at the heart of the fusion machinery (Doms, R.W., Virology 276:229, 2000; Dimitrov D.S., Cell 101 :697, 2000).
  • Interaction of receptor and co-receptor(s) with gp120 lead to intermediate Env conformations that may include structures conserved among various HIV isolates spanning several clades that could be used as potent entry inhibitors.
  • a broadly-neutralizing FAb (X5) has been isolated that targets a well- conserved inducible epitope in gp120 (Moulard ef. a/., Proc. Natl. Acad. ScL USA 99:6913, 2002; International Patent Application WO 03/033666).
  • X5 FAb bound with high affinity (nM) to a variety of Envs, including approximately fifty primary clinical isolates representing various clades.
  • HCV-E2 analogous to receptor-mediated fusion and entry pathway in HIV, there is an "inducible epitope" in HCV-E2 and that it is highly conserved among primary clinical isolates of HCV across genotypes 1-6.
  • This epitope is induced or "opens up” upon binding CD81 , and preferably the co-receptor SR-B1 binds to a distinct epitope in E2 that is not overlapping with this induced epitope or the CD81 binding epitope.
  • a human FAb (or scFv) that binds to this "induced" epitope will be isolated from a phage display library constructed from a HCV seropositive individual (e.g., PTA-2750 as described in International Patent Application WO 02/055560).
  • Purified E1 ⁇ 2-CD81 complex will be purified and used according to the established procedures (Moulard ef. al., supra). Two separate panels (A and B) of E1 :E2-CD81 will be constructed.
  • Panel A consists of E1 :E2 isolated from approximately fifty primary isolates representing genotypes 1-6
  • Panel B consists of E1 :E2 isolated from approximately fifty primary isolates representing genotypes 1a and 2a, which are the two most genetically divergent genotypes of HCV (see Yu ef. al., Proc. Natl. Acad. Sci. USA 101 :7705, 2004).
  • Example V IMPROVING THE BREADTH AND POTENCY OF HCV NEUTRALIZING ANTIBODIES BY SCANNING
  • HCV #4 A human FAb (HCV #4) has been isolated from a phage display library constructed from a seropositive individual. HCV #4 binds representative isolates of all genotypes of HCV (International Patent Application WO 02/055560).
  • An in vitro assay for neutralizing antibodies has been developed and retrospectively validated for its biological significance (Bartosch et. al., Proc. Natl. Acad. Sci. USA 100:14199, 2003; Yu ef. al., Proc. Natl. Acad. Sci. USA 101 :7705, 2004). Taken these results together, this in vitro assay should be of tremendous significance to isolate neutralizing antibodies that target broadly-conserved neutralization epitopes in HCV E2.
  • a human FAb (or scFv) will be selected that is broadly cross-reactive against several HCV clinical isolates. Any attempt to increase the affinity of MAbs will compromise its breadth of neutralization. Therefore, it is necessary to simultaneously improve the potency and breadth of neutralization of the drug candidate.
  • a recently developed procedure called sequential antigen panning (SAP; Zhang et. al., J. MoI. Biol. 335:209, 2004) will be used in combination with in vitro scanning saturation mutagenesis (SSM) to develop such a MAb therapeutic candidate.
  • any antibody or antibody fragment of the invention may be varied in order to generate variant antibodies with equivalent or improved affinity for E2-receptor complexes (Figure 3).
  • variant antibodies can be created and tested for their relative affinity using well-known methods (Burks ef. al., Proc. Natl. Acad. Sci. USA 94:412-417, 1997; Daugherty ef. al., Proc. Natl. Acad. ScL USA 97:2029-34, 2000; Zhang ef. al., supra).
  • in vitro scanning saturation mutagenesis is carried out (US Patent No: 6,180,341). Briefly, at each site, twenty-one genes encoding all possible amino acid substitutions as well as a double stop codon (control) are constructed by overlap extension PCR. The final products of the overlap extension PCR reaction contain a T7 promoter and ribosome binding site in front of the MAb fragment gene. An HSV sequence is also present at the C-terminal end of the MAb fragment gene, so that the MAb fragment protein can be detected by ELISA using an anti-HSV monoclonal antibody. The PCR overlap extension products are used as templates for coupled in vitro transcription-translation reactions to produce functional MAb fragment proteins. An E. coli S30 ribosomal extract, as opposed to mammalian or plant cell extracts, is used for in vitro translation.
  • SSM in vitro scanning saturation mutagenesis
  • the protein products from the coupled in vitro transcription-translation step are analyzed by ELISA.
  • ELISA 96-well microtiter plates are coated with the BSA conjugate of E2/receptor complexes. The plates were then incubated with equal amounts from each of the in vitro synthesis reactions.
  • the construct prepared with the wild-type sequence was used on each ELISA plate.
  • the wild-type construct was produced by the overlapping PCR method alongside the mutants, thereby providing an accurate calibration for all stages of the procedure.
  • the ELISA results for the different mutants are recorded.
  • the affinity improvements can be additive such that the improvement in affinity could be up to 500 fold.
  • EXAMPLE Vl PASSIVE IMMUNOPROPHYLAXIS AGAINST HCV IN A CHIMPANZEE
  • a na ⁇ ve chimpanzee is treated with one or more pegylated neutralizing HCV MAb fragments.
  • the chimpanzee is challenged subcutaneously with 64 CID 50 of HCV.
  • the chimpanzee is followed weekly for six months for biochemical evidence of hepatitis (e.g. ALT, ICD, GGTP), virologic evidence of infection (RT-PCR of viremia), and serologic evidence of antibody to HCV. If the chimpanzee remains free of infection, passive immunoprophylaxis is considered successful.
  • a chronically infected chimpanzee is treated with one or more pegylated neutralizing HCV MAbs.
  • the course of the HCV infection in the chimpanzee is monitored weekly for biochemical evidence of hepatitis (e.g. ALT, ICD, GGTP), virologic evidence of infection (RT-PCR of viremia), and serologic evidence of antibody to HCV. If there is no change in the level of replication, the dose of infused antibody is increased. If a clinical response (a decrease in the titer of viral genome in the blood, as measured by PCR) is detected, the dose of pegylated MAb fragments is adjusted to determine its minimum effective titer.

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Abstract

La présente invention concerne la pégylation d'anticorps thérapeutiques destinés au traitement d'infections par le Virus de l'Hépatite C (VHC). L'invention concerne plus particulièrement la pégylation spécifique de site de fragments d'anticorps E2 sur des restes d'acides aminés spécifiquement définis qui ont éventuellement été obtenus par génie génétique à distance des sites de liaison des antigènes. Les dérivés de MAb pégylé présentent des propriétés pharmacocinétiques et pharmacodynamiques sensiblement accrues, et ils font preuve d'une action largement neutralisante contre plusieurs isolats cliniques du VHC grâce à une liaison sélective avec les épitopes de neutralisation largement conservés de la glycoprotéine E2 du VHC.
PCT/US2005/028168 2004-09-01 2005-08-09 Pegylation specifique de site d'anticorps largement neutralisant diriges contre le vhc et leur utilisation pour le traitement d'infections chroniques par vhc WO2006028634A2 (fr)

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CN110357966A (zh) * 2018-04-09 2019-10-22 非同（成都）生物科技有限公司 具有延长的半衰期和增强的抗肿瘤效果的双特异性抗体

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CA2429946A1 (fr) * 2000-12-01 2002-07-18 The Government Of The United States Of America, As Represented By The Se Cretary, Department Of Health And Human Services Anticorps monoclonaux specifiques a la glycoproteine e2 du virus de l'hepatite c et leurs utilisations dans le diagnostic, le traitement et la prevention de l'hepatite c
TW200408407A (en) * 2001-11-30 2004-06-01 Dana Farber Cancer Inst Inc Methods and compositions for modulating the immune system and uses thereof

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CN106749645A (zh) * 2016-11-14 2017-05-31 广州泰诺迪生物科技有限公司 一种全人源抗丙型肝炎病毒的中和抗体
CN106749645B (zh) * 2016-11-14 2019-12-03 广州泰诺迪生物科技有限公司 一种全人源抗丙型肝炎病毒的中和抗体
CN110357966A (zh) * 2018-04-09 2019-10-22 非同（成都）生物科技有限公司 具有延长的半衰期和增强的抗肿瘤效果的双特异性抗体

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