WO2005007671A2 - Compositions et methodes de traitement du sras - Google Patents

Compositions et methodes de traitement du sras Download PDF

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WO2005007671A2
WO2005007671A2 PCT/US2004/013187 US2004013187W WO2005007671A2 WO 2005007671 A2 WO2005007671 A2 WO 2005007671A2 US 2004013187 W US2004013187 W US 2004013187W WO 2005007671 A2 WO2005007671 A2 WO 2005007671A2
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sars
methods
agent
antibody
seq
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PCT/US2004/013187
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WO2005007671A3 (fr
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Guoliang Yu
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Epitomics, Inc.
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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
    • C07K16/1002—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae

Definitions

  • the present invention is in the field of severe acute respiratory syndrome (SARS).
  • SARS severe acute respiratory syndrome
  • SARS severe acute respiratory syndrome
  • the illness generally begins with a fever greater than 38°C, which is sometimes accompanied by chills or other symptoms, including headache, malaise, or body aches.
  • the infected individual may develop a dry, nonproductive cough, and the infected person may experience difficulty breathing. In 10- 20%) of cases thus far, patients will require mechanical ventilation. Approximately 3-5%) of the individuals that contract this disease die. There is currently no treatment for this disease.
  • the SARS virus is a member of the coronavirus family of viruses.
  • Coronaviruses are characterized by: 1) irregularly shaped particles 60-220 nm in diameter, with an outer envelope bearing distinctive, club-shaped peplomers, the outer envelope giving the virus a crown-like appearance from which the family name is derived.
  • Coronaviridae includes the genuses Coronavirus and Toro virus.
  • the genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, murine hepatitis virus, rat coronavirus, porcine hemagglutinating encephalomyelitis virus, etc.; while the genus Torovirus includes Berne virus and Breda virus.
  • the present invention provides compositions and methods of treating severe acute respiratory syndrome (SARS), and methods of reducing SARS viral load, reducing the time to SARS viral clearance, and reducing morbidity or mortality in the clinical outcomes, in patients suffering from a SARS.
  • the present invention further provides methods of reducing the risk that an individual will develop SARS.
  • the methods generally involve administering a therapeutically effective amount of an agent, e.g. a peptide or a monoclonal antibody, that interacts with the E2 core structure and prevents viral entry into a susceptible cell.
  • the cell may be in vitro or in vivo.
  • Fig. 1 shows the sequence of an exemplary SARS E2 protein (SEQ ID NO:l).
  • Fig. 2 shows a prediction of the secondary structure of an exemplary SARS E2 protein (SEQ ID NO:l), showing alpha-helical structure at various positions in the protein.
  • Fig. 3 is a schematic showing an arrangement of E2 alpha-helices in an E2 complex that forms during viral entry.
  • Fig. 4 shows the sequence of two alpha-helices useful in the subject methods.
  • SEQ ID NO: 2 is a N-terminal-alpha helix and SEQ ID NO: 3 is a C-terminal-alpha helix.
  • Fig. 5 shows exemplary amino acid sequences that therapeutic peptides may comprise.
  • Fig. 6 shows exemplary multimer structures that may be produced recombinantly to make antigen suitable for use in the subject methods.
  • SARS virus includes any member of the family Coronaviridae, that is a causative agent of SARS.
  • SARS virus further includes naturally-occurring (e.g., wild-type) SARS virus; naturally-occurring SARS virus variants; and SARS virus variants generated in the laboratory, including variants generated by selection, variants generated by chemical modification, and genetically modified variants (e.g., coronavirus modified in a laboratory by recombinant DNA methods).
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • “treating" SARS refers to reduction of a symptom of SARS.
  • SARS symptoms include: a fever of greater than 38°C, headache, chills, body aches, a dry cough, shortness of breath, difficulty breathing, hypoxia, or radiographic or other findings of either pneumonia or acute respiratory distress syndrome. Death is also a symptom of SARS.
  • E2 is a peptide, sometimes named “spike glycoprotein” or "E2 glycoprotein” that is encoded by the SARS virus genome. E2 functions during viral entry into the cell. Exemplary E2 proteins are provided by the sequences of Genbank Accession number AAP13441, AAP13567, andNP_828851. Based on these sequences, one of skill in the art could recognize SARS virus E2 proteins as they become available.
  • antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • the recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgG ls IgG 2 , IgG 3 , IgG 4 ), delta, epsilon and mu heavy chains or equivalents in other species.
  • Full-length immunoglobulin "light chains” (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH 2 - terminus and a kappa or lambda constant region at the COOH-terminus.
  • Full-length immunoglobulin "heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein.
  • the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin- avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like.
  • Fab', Fv, F(ab') 2 and or other antibody fragments that retain specific binding to antigen.
  • Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab') 2 , as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol.
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called “complementarity determining regions" or CDRs.
  • the extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1983)).
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.
  • variable segments of the genes from a rabbit monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3.
  • a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a rabbit antibody and the constant or effector domain from a human antibody (e.g., the anti-Tac chimeric antibody made by the cells of A.T.C.C. deposit Accession No. CRL 9688), although other mammalian species may be used.
  • antibody domains, regions and fragments are accorded standard definitions as are well known in the art. See, e.g., Abbas, A.
  • humanized antibody or “humanized immunoglobulin” refers to an antibody comprising one or more CDRs from an animal antibody, the antibody having being modified in such a way so as to be less immunogenic in a human than the parental animal antibody.
  • An animal antibody can be humanized using a number of methodologies, including chimeric antibody production, CDR grafting (also called reshaping), and antibody resurfacing.
  • murinized antibody or “murinized immunoglobulin” refers to an antibody comprising one or more CDRs from an animal antibody, the antibody having being modified in such a way so as to be less immunogenic in a mouse than the parental animal antibody.
  • An animal antibody can be murinized using a number of methodologies, including chimeric antibody production, CDR grafting (also called reshaping), and antibody resurfacing.
  • polypeptide and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • fusion proteins including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, ⁇ -galactosidase, luciferase, etc.; and the like.
  • the term "isolated,” when used in the context of an isolated antibody, refers to an antibody of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95%) free, at least 98%) free, and even at least 99%) free from other components with which the antibody is associated with prior to purification.
  • Techniques for determining nucleic acid and amino acid "sequence identity" also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981).
  • This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Davhoff Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
  • An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, WI) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, WI).
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%>-85%>, preferably at least about 85%- 90%, more preferably at least about 90%-95%, and most preferably at least about 95%>-98%> sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra. Two nucleic acid fragments are considered to "selectively hybridize" as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule.
  • Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency.
  • the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30%) sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a hybrid molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70%> sequence identity with the sequence of the selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
  • Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B.D.
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
  • blocking agents in the hybridization solutions e.g., formamide, dextran sulfate, and polyethylene glycol
  • hybridization conditions The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • An example of stringent hybridization conditions is hybridization at 50°C or higher and OJXSSC (15 mM sodium chloride/1.5 mM sodium citrate).
  • Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90%) as stringent as the above specific stringent conditions.
  • a first polynucleotide is "derived from” a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above.
  • a first polypeptide is "derived from” a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.
  • the present invention provides compositions and methods of treating severe acute respiratory syndrome (SARS), and methods of reducing SARS viral load, reducing the time to SARS viral clearance, and reducing morbidity or mortality in the clinical outcomes, in patients suffering from a SARS.
  • the present invention further provides methods of reducing the risk that an individual will develop SARS.
  • the methods generally involve administering a therapeutically effective amount of an agent, e.g. a peptide or a monoclonal antibody, that interacts with the E2 core structure and prevents viral entry into a susceptible cell.
  • the agent binds to the SARS virus E2 protein to prevent the formation of a trimer of E2 proteins that is essential for viral entry to a cell.
  • compositions The SARS virus E2 protein is encoded by the SARS genome and is described in general by Genbank Accession numbers AY274119, AY278741, AY278554, NC_004718, AY278491.
  • SARS E2 proteins may be identified by sequence comparison, e.g., by using the BLAST algorithm to the E2 proteins identified in these Genbank accessions.
  • the sequence of an exemplary SARS E2 protein, SEQ ID NO:l is provided in Fig. 1.
  • the SARS E2 protein complex mediates endocytosis and membrane fusion during SARS viral entry to a susceptible cell.
  • each E2 protein has two alpha helices that participate in E2 complex formation: a N-terminal alpha helix and a C-terminal alpha helix that are predicted (see Fig. 2 for the prediction for SEQ ID NO:l).
  • the alpha helices of each polypeptide interact with each other to form an anti-parallel structure, and the anti-parallel structures of three E2 proteins interact to form an E2 complex that is a cylinder (schematically shown in Fig. 3).
  • the sequence of the predicted N-terminal alpha helix and C-terminal alpha helix, SEQ ID NOS: 2 and 3 are described in Fig. 4.
  • the two alpha-helices that participate in E2 complex formation are known collectively as "E2 -multimerization regions”.
  • Therapeutic agents The invention provides agents for treating SARS.
  • the compositions are either derived from an E2-multimerization region or are compositions that specifically bind to an E2-multimerization region and/or E2 core structure. In many embodiments, the agents bind to E2 and prevent E2 multimerization.
  • Peptides Subject therapeutic peptides are based on the sequences of the E2-multimerization regions described above.
  • the peptides usually contain at least 10 contiguous amino acids (e.g. about 11, about 15, about 20, about 25, about 30, or the entire length) of an E2- multimerization region, and, in certain embodiments, are at least 15 (e.g.
  • the subject peptides may contain naturally occurring or non-naturally occurring amino acids, and the peptide may be modified, e.g., PEGylated, fused to other moieties, or oligomerized (e.g., is a tetramer or dimer).
  • Subject therapeutic peptides may also be based on variants of the E2-multimerization regions described, which variants include mutants, fragments, and fusions of a E2- multimerization region. Mutants can include amino acid substitutions, additions or deletions.
  • amino acid substitutions can be conservative amino acid substitutions (gly/ala; val/ile/leu; asp/glu; asn/gln; ser/thr; lys/arg; and phe/tyr) or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function.
  • Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/ hydrophilicity, and/or steric bulk of the amino acid substituted.
  • Subject therapeutic peptides may comprise any of the amino acid sequences shown in Fig. 5 (SEQ ID NOS:4-70).
  • Antibodies Subject therapeutic antibodies usually specifically bind to an E2-multimerization region and/or to the core structure of an E2 complex. The subject therapeutic antibodies may bind to a particular structure that is formed by non-contiguous amino acids.
  • the subject therapeutic antibodies may not specifically bind to a particular sequence of contiguous amino acids.
  • the subject antibodies may bind one of the peptides set forth in Fig. 5, for example.
  • the antibodies may bind to a structure formed by a non-contiguous sequence of amino acids (e.g., a structure that only occurs when a subject polypeptide is folded or when two amino acid sequences interact), and, accordingly, may not significantly bind to a peptide (e.g., any of the peptides listed in the sequence listing) while it is in denatured form.
  • Suitable antigens for antibody production may be made using a number of methods.
  • a polypeptide is produced recombinantly in a host cell, and purified.
  • Methods for producing recombinant proteins are well known in the art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995 and Sambrook, et al, Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.). and, as such, are not described in any detail herein.
  • a suitable antigen is a peptide containing at least 10 contiguous amino acids (e.g. about 11, about 15, about 20, about 25, about 30, or the entire length) of an E2-multimerization region.
  • an antigen contains a mixture of peptides containing of at least 10 contiguous amino acids of each of the C- andN- E2-multimerization regions (e.g. SEQ ID NOS:2 and 3).
  • the peptides are produced using methods that allow the peptides to form secondary structure, e.g. alpha-helices that interact to form a three dimensional structure similar to the E2 core structure shown in Fig. 3.
  • protein production in a cell e.g., a bacterial, yeast or mammalian cell, is sufficient to provide a protein with sufficient structure for use in these methods.
  • Such peptides may be produced separately and then mixed, denatured, and renatured, or, alternatively, may be produced in a suitable cell, allowing cellular machinery to correctly fold the polypeptides into a suitable E2 core protein structure.
  • Strategies for expressing two or more polypeptides in a single cell are known in the art.
  • these, peptides are produced as multimers fused to a flexible "linker" peptide that permits the peptides to form a "hairpin" structure. Suitable exemplary arrangements of multimers are shown in Fig. 6.
  • trimers of "C-C-C" and "N-N-N" may be mixed, e.g., equimolarly, to produce an E2 core structure.
  • C-N fusion may be mixed, e.g., equimolarly, to produce an E2 core structure.
  • C and N in the previous embodiments refer to peptides having at least 10 contiguous amino acids of the C and N E2-multimerization regions, and the arrows represent the polarity, e.g., C to N, or N to C of the polypeptide.
  • the subject polypeptides typically include at least one sequence that is flexible, allowing an amino acid chain to bend or rotate freely.
  • Suitable linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n and (GGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art. Glycine and glycine-serine polymers are preferred since both of these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
  • Glycine polymers are the most preferred as glycine accesses significantly more phi-psi space than even alanine, and is much less restricted tan residues with longer side chains (see Scheraga, Rev. Computational Chem. III73-142 (1992)).
  • serine is hydrophilic and therefore able to solubilize what could be a globular glycine chain. Similar chains have been shown to be effective in joining subunits of recombinant proteins such as single chain antibodies.
  • a flexible linker comprises about 6, about 8, about 10, about 12, about 15, about 20, about 25 or about 30 amino acids or more. Suitable antibodies are obtained by immunizing a host animal with these compositions.
  • Suitable host animals include mouse, rat, sheep, goat, hamster, rabbit, etc.
  • the compositions may be used as a vaccine, and, as such, may be used to immunize a human directly .
  • the first step is immunization of the host animal with an antigen, where the antigen will preferably be in substantially pure form, comprising less than about 1% contaminant.
  • the antigen may comprise complete E2 multimerization region or E2 core structure, fragments or derivatives thereof.
  • the antigen may be combined with an adjuvant, where suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like.
  • suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like.
  • the antigen may also be conjugated to synthetic carrier proteins or synthetic antigens.
  • a variety of hosts may be immunized to produce the polyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats, sheep, goats, and the like.
  • the antigen is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages.
  • Monoclonal antibodies are produced by conventional techniques. Generally, the spleen and/or lymph nodes of an immunized host animal provide a source of plasma cells. The plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells. Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desired specificity. Suitable animals for production of monoclonal antibodies include mouse, rat, hamster, etc.
  • the antibody may be purified from the hybridoma cell supernatants or ascites fluid by conventional techniques, e.g. affinity chromatography using the antigen bound to an insoluble support, protein A sepharose, etc.
  • the antibody may be produced as a single chain, instead of the normal multimeric structure. Single chain antibodies are described in Jost et al. (1994) J.B.C. 269:26267-73, and others.
  • DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine.
  • the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.
  • An immune response of a recipient against the blocking agent will potentially decrease the period of time that the therapy is effective.
  • Methods of humanizing antibodies are known in the art.
  • the humanized antibody may be the product of an animal having transgenic human immunoglobulin constant region genes (see for example International Patent Applications WO 90/10077 and WO 90/04036).
  • the antibody of interest may be engineered by recombinant DNA techniques to substitute the CHI, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92/02190).
  • Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521).
  • mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
  • the cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Patent nos. 4,683,195 and 4,683,202).
  • a library is made and screened to isolate the sequence of interest.
  • the DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences.
  • the sequences of human constant regions genes may be found in Kabat et al.
  • a truncated gene is designed.
  • a chimeric gene encoding a portion of the F(ab')2 fragment would include DNA sequences encoding the CHI domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.
  • C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.
  • Monoclonal antibodies may be humanized, if desired.
  • Suitable antibodies may be screened for using a variety of assays, including binding assays and in vitro assays for SARS.
  • Antibodies that have an activity to reduce levels of the SARS virus in in vitro cultured cells infected with the SARS virus, or an in vitro SARS virus protective activity have desirable properties.
  • antibodies are tested for their ability to bind specifically to a substrate.
  • the term "specifically" in the context of antibody binding refers to high avidity and/or high affinity binding of an antibody to a specific antigen i.e., a polypeptide, or epitope.
  • the specific antigen is an antigen (or a fragment or subtraction of an antigen) used to immunize the animal host from which the antibody-producing cells were isolated.
  • Antibody specifically binding an antigen or fragment thereof is stronger than binding of the same antibody to other antigens.
  • Antibodies which bind specifically to a polypeptide may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10%> or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to a subject polypeptide, e.g. by use of appropriate controls.
  • specific antibodies bind to an antigen with a binding affinity of 10 "7 M or more, e.g., 10 "8 M or more (e.g., 10 "9 M, 10 "10 , 10 "11 , etc.).
  • an antibody with a binding affinity of 10 "6 M or less is not useful in that it will not bind an antigen at a detectable level using conventional methodology currently used.
  • the therapeutic agents described above find use in a variety of methods, represented protocols of which are described below. Methods The therapeutic agents described above find use in various in vitro and in vivo methods.
  • the methods involve contacting cells that are infected with SARS virus or cells that are to be infected with SARS virus, and determining a SARS virus related phenotype, e.g., virus titer, number of cells infected, protection against SARS virus, etc., of the cells.
  • a SARS virus related phenotype e.g., virus titer, number of cells infected, protection against SARS virus, etc.
  • the therapeutic compositions described above also find use as a treatment for SARS.
  • the subject treatment is administered to an individual prophylactically, e.g., is initiated before the appearance of symptoms.
  • Such prophylactic treatment is administered in the case of individuals who are asymptomatic and who may or may not yet be infected, but who have come into close contact with an individual who has been diagnosed with SARS; individuals who are asymptomatic and who are not yet be infected, but who expect to come into contact with an individual who has been diagnosed with SARS (e.g., health care workers working in a facility in which individuals who have been diagnosed with SARS are being cared for); individuals who are asymptomatic and who are not yet be infected, and who are traveling to a location known to have a relatively high incidence of SARS cases; and the like.
  • the subject treatment is initiated after the appearance of clinical signs of SARS, e.g., the appearance of a fever often exceeding 38°C.
  • An advantage of the subject methods is that the severity of SARS symptoms is reduced, e.g., the viral load is reduced, and/or the time to viral clearance is reduced, and/or the morbidity or mortality is reduced.
  • a subject treatment method is prophylactic, the methods reduce the risk that an individual will develop pathological infection with a SARS virus.
  • Effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce the risk or reducing the probability that an individual will develop a pathological infection with a SARS virus.
  • an effective amount reduces the risk that an individual will develop a pathological infection by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%>, at least about 90%, or more, compared to the risk of developing a pathological infection with the virus in the absence of the treatment.
  • effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce SARS viral load by at least about 10%, at least about 20%>, at least about 25%), at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the viral load in the absence of treatment.
  • effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce the time to viral clearance, by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the time to viral clearance in the absence of treatment.
  • effective amounts of a subject agent are amounts that, alone or in combination therapy, reduce morbidity or mortality due to a SARS infection by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%>, at least about 60%, at least about 70%, at least about 80%, at least about 90%», or more, compared to the morbidity or mortality in the absence of treatment.
  • a subject treatment method is effective in reducing the risk of a pathological coronavirus infection, reducing viral load, reducing time to viral clearance, or reducing morbidity or mortality due to a coronavirus infection is readily determined by those skilled in the art.
  • Niral load is readily measured by measuring the titer or level of virus in serum.
  • the number of virus e.g., the number of viral particles or the number of viral genomes
  • the number of virus in the serum can be determined using any known assay, including, e.g., a quantitative polymerase chain reaction assay using oligonucleotide primers specific for the SARS virus being assayed. Whether morbidity is reduced can be determined by measuring any symptom associated with a SARS infection, including, e.g., fever, respiratory symptoms (e.g., cough, ease or difficulty of breathing, and the like).
  • the invention provides formulations, including pharmaceutical formulations, that include an agent which inhibits multimerization of the SARS virus E2 protein.
  • a formulation comprises an effective amount of an agent that inhibits SARS virus E2 multimerization in a host.
  • An "effective amount” refers to an amount that is sufficient to produce a desired result, e.g., reduction SARS virus E2 multimerization, reduction of a SARS symptom, reduction of viral load, reduced time for viral clearance, etc.
  • the desired result is at least a reduction or increase in a phenotype as compared to a control such that the phenotype is more similar to normal.
  • the active agent(s) may be administered to the host using any convenient means capable of resulting in the desired reduction in of a SARS phenotype.
  • the agent can be incorporated into a variety of formulations for therapeutic administration.
  • the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • the agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such' as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the agents can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. Other modes of administration will also find use with the subject invention.
  • an agent of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions.
  • the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.
  • suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • An agent of the invention can be administered as injectables.
  • injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985; Remington: The Science and Practice of Pharmacy, A.R. Gennaro, (2000) Lippincott, Williams & Wilkins.
  • the composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. Dosages Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1 ⁇ g to about 1,000 ⁇ g or about 10,000 ⁇ g of an agent that reduces a symptom of SARS a subject animal. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • routes of administration include intranasal, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect.
  • the composition can be administered in a single dose or in multiple doses.
  • the agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes.
  • routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.
  • Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal.
  • Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
  • the agent can also be delivered to the subject by enteral administration.
  • Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.
  • Methods of administration of the agent through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
  • a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
  • Iontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
  • treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as an sebaceous gland disorder and psychological trauma associated therewith.
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.
  • a subject polynucleotide can be delivered as a naked polynucleotide, or associated with (complexed with) a delivery vehicle. "Associated with”, or “complexed with”, encompasses both covalent and non-covalent interaction of a polynucleotide with a given delivery vehicle.
  • Additional therapeutic agents can be used in conjunction with administration of an additional antiviral agents, e.g., a specific antiviral agent that is effective in treating a pathological SARS infection.
  • Additional antiviral agents that are suitable for use in combination therapy include, but are not limited to, nucleotide and nucleoside analogs. Non-limiting examples include AZT (zidovudine), DDI (didanosine), DDC (dideoxycytidine), D4T (stavudine), combivir, abacavir, adefovir dipoxil, cidofovir, ribavirin, ribavirin analogs, and the like.
  • the method further includes administration of ribavirin.
  • Ribavirin l- ⁇ -D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif, is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U.S. Pat. No. 4,211,771.
  • the invention also contemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No. 6,277,830).
  • the ribavirin may be administered orally in capsule or tablet form, or in the same or different administration form and in the same or different route as the therapeutic compositions described above.
  • Ribavirin is generally administered in an amount ranging from about 30 mg to about 60 mg, from about 60 mg to about 125 mg, from about 125 mg to about 200 mg, from about 200 mg to about 300 gm, from about 300 mg to about 400 mg, from about 400 mg to about 1200 mg, from about 600 mg to about 1000 mg, or from about 700 to about 900 mg per day, or about 10 mg/kg body weight per day.
  • an additional antiviral agent is administered during the entire course of treatment.
  • an additional antiviral agent is administered for a period of time that is overlapping with that of the subject treatment, e.g., the additional antiviral agent treatment can begin before the subject treatment begins and end before the subject treatment ends; the additional antiviral agent treatment can begin after the treatment begins and end after the subject treatment ends; the additional antiviral agent treatment can begin after the treatment begins and end before the subject treatment ends; or the additional antiviral agent treatment can begin before the subject treatment begins and end after the subject treatment ends.
  • the methods described above find use in treating an individual in need thereof.
  • Individuals who are to be treated according to the methods of the invention include individuals who have been clinically diagnosed with SARS, as well as individuals who exhibit one or more of the signs and the symptoms of SARS but have not yet been diagnosed with SARS.
  • Individuals who are to be treated according to the methods of the invention also include individuals with anticipated exposure to individuals diagnosed with SARS (e.g., health care professionals; individuals traveling to areas with a relatively high incidence of SARS; and the like); individuals with suspected exposure to an individual diagnosed with SARS; and individuals with known exposure to an individual with SARS.
  • "Exposure” includes contact that is sufficiently close as to allow the etiologic agent to be transmitted from an infected individual to the exposed individual. Animal models for SARS, e.g.
  • kits Also provided by the subject invention are kits containing therapeutic compositions for practicing the subject methods, as described above.
  • the subject kits at least include one or more of: a monoclonal antibody that specifically bind to an E2-multimerization region and/or E2 core structure or a peptide derived from an E2-multimerization region.
  • the various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.
  • kits with unit doses of the active agent e.g. in oral or injectable doses, are provided.
  • the subject kits typically further include instructions for using the components of the kit to practice the subject methods.
  • the instructions for practicing the subject methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

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Abstract

L'invention concerne des compositions et des méthodes permettant de traiter le syndrome respiratoire aigu sévère (SRAS), et des méthodes permettant de réduire la charge virale du SRAS, de réduire le temps de clairance virale, et de réduire la morbidité ou la mortalité dans les issues cliniques, chez les patients souffrant du SRAS. L'invention concerne en outre des méthodes permettant de réduire le risque d'apparition d'un SRAS chez un individu. Ces méthodes consistent à administrer une dose thérapeutiquement efficace d'un agent, p. ex. un peptide ou un anticorps monoclonal, qui interagit avec la structure centrale de la protéine E2 et empêche la pénétration du virus dans une cellule sensible.
PCT/US2004/013187 2003-04-29 2004-04-28 Compositions et methodes de traitement du sras WO2005007671A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009053512A1 (fr) * 2007-10-23 2009-04-30 Consejo Superior De Investigaciones Científicas Protéines n, m, he du torovirus porcin, procédé d'obtention et leurs utilisations dans le diagnostic et le traitement de torovirus porcin
US8986692B2 (en) 2009-08-28 2015-03-24 Jiangsu Simcere Pharmaceutical R & D Co., Ltd. Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody
EP4101864A4 (fr) * 2020-02-05 2023-09-20 Shanxi Jinbo Bio-Pharmaceutical Co., Ltd. Polypeptide, son procédé de préparation et son utilisation

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US20050186575A1 (en) * 2001-05-17 2005-08-25 Rottier Petrus J.M. Corona-virus-like particles comprising functionally deleted genomes

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US20050186575A1 (en) * 2001-05-17 2005-08-25 Rottier Petrus J.M. Corona-virus-like particles comprising functionally deleted genomes

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009053512A1 (fr) * 2007-10-23 2009-04-30 Consejo Superior De Investigaciones Científicas Protéines n, m, he du torovirus porcin, procédé d'obtention et leurs utilisations dans le diagnostic et le traitement de torovirus porcin
ES2339728A1 (es) * 2007-10-23 2010-05-24 Consejo Superior De Investigaciones Cientificas Proteinas n, m y he de torovirus porcino, procedimiento de obtencion y sus aplicaciones en diagnostico y tratamiento de torovirus porcino.
US8986692B2 (en) 2009-08-28 2015-03-24 Jiangsu Simcere Pharmaceutical R & D Co., Ltd. Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody
EP4101864A4 (fr) * 2020-02-05 2023-09-20 Shanxi Jinbo Bio-Pharmaceutical Co., Ltd. Polypeptide, son procédé de préparation et son utilisation

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