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WO2005027847A2 - Anticorps destines a lutter contre le sras - Google Patents

Anticorps destines a lutter contre le sras Download PDF

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Publication number
WO2005027847A2
WO2005027847A2 PCT/US2004/030557 US2004030557W WO2005027847A2 WO 2005027847 A2 WO2005027847 A2 WO 2005027847A2 US 2004030557 W US2004030557 W US 2004030557W WO 2005027847 A2 WO2005027847 A2 WO 2005027847A2
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WIPO (PCT)
Prior art keywords
sars
cov
antibodies
phage
library
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Application number
PCT/US2004/030557
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English (en)
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WO2005027847A3 (fr
Inventor
Katherine S. Bowdish
Toshiaki Maruyama
Martha Wild
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Alexion Pharmaceuticals, Inc.
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Application filed by Alexion Pharmaceuticals, Inc. filed Critical Alexion Pharmaceuticals, Inc.
Publication of WO2005027847A2 publication Critical patent/WO2005027847A2/fr
Publication of WO2005027847A3 publication Critical patent/WO2005027847A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C12N2770/20051—Methods of production or purification of viral material
    • 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
    • C12N2770/20061—Methods of inactivation or attenuation
    • C12N2770/20063—Methods of inactivation or attenuation by chemical treatment

Definitions

  • SARS-CoV SARS-associated coronavirus
  • WHO World Health Organization
  • the illness spread to more than two dozen countries in North America, South America, Europe, and Asia.
  • the illness usually begins with a high fever (measured temperature greater than 100.4°F).
  • the fever is sometimes associated with chills or other symptoms, including headache, general feeling of discomfort and body aches.
  • Some people also experience mild respiratory symptoms at the outset. Diarrhea is seen in approximately 10 percent to 20 percent of patients.
  • SARS patients may develop a dry, nonproductive cough that might be accompanied by or progress to a condition (hypoxia) in which insufficient oxygen is getting to the blood.
  • a condition hyperoxia
  • Most patients develop pneumonia.
  • SARS is caused by a previously unrecognized coronavirus, called SARS-associated coronavirus (SARS-CoV).
  • SARS-CoV SARS-associated coronavirus
  • Patients with SARS currently receive the same treatment that would be used for any patient with serious community- acquired atypical pneumonia. To date, no effective treatment for SARS-CoV infection has been found. It would be advantageous to identify and produce fully human neutralizing immunoglobulin (Ig) against SARS-CoV.
  • the antibodies are fully human antibodies derived, for example, from convalescent human donors or vaccinated individuals.
  • the antibodies are derived from non-human primates that have been exposed to the SARS-CoV.
  • the antibodies described herein are useful in both therapeutic applications
  • the antibodies are used to identify epitopes on the SARS-CoV to develop vaccines against the SARS-CoV.
  • DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Fully human antibodies that bind to and/or neutralize SARS-associated coronavirus (SARS-CoV) are described herein.
  • the antibodies and functional fragments of antibodies can be used to prevent and/or neutralize viral infection.
  • Antibodies that can be identified in accordance with the methods set forth herein include monoclonal Abs and antibody fragments such as Fab, Fab 1 , F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains and/or antibody fragments derived from phage or phagemid display technologies.
  • Functional antibody fragments are those fragments of antibodies which are capable of binding to an antigen notwithstanding the absence of regions normally found in whole antibodies.
  • Single chain antibodies scFv are included in functional antibody fragments.
  • Ig is intended to include immunoglobulins of all types, including IgG, IgA, etc.
  • the strategy for identifying, isolating, purifying and testing antibodies against the SARS-CoV includes the following steps: 1. Identify convalescent donors with neutralizing activity to SARS-CoV. In this step sera from convalescent donors can be tested in a plaque reduction neutralization test (PRNT).
  • PRNT plaque reduction neutralization test
  • libraries are constructed from the RNA derived from the blood and/or bone marrow of positive donors.
  • the libraries are panned on inactivated virus preparation (e.g., UV irradiated whole virion) and/or in vitro expressed SARS- CoV antigens.
  • inactivated virus preparation e.g., UV irradiated whole virion
  • in vitro expressed SARS- CoV antigens e.g., SARS- CoV antigens.
  • human Fabs are produced by fermentation and purified by column chromatography, following removal of the coding sequence for any fusion protein used in phage display. 5. Characterize purified Fabs. In this step Fabs are titrated on the antigen and sub-grouped by epitope specificity as determined by competitive ELISA. Candidate Fabs of varying epitope specificities can be selected for use in characterization experiments. 6. Identify a panel of human Fabs that neutralize SARS virus. In this step, in vitro PRNT can be used for identification of the relevant Fabs. 7. Produce and purify Ig from Fabs identified as neutralizing. In this step full-length human Igs are generated from selected Fabs.
  • in vitro PRNT can be used to confirm neutralization by the full length human Igs.
  • human donor fully human antibodies are obtained by the methods described herein. The donor is selected from patients that have been exposed to SARS-CoV and have recovered from SARS. Such individuals are most likely to have developed antibodies within his/her immunological repertoire that, alone or in combination, effectively neutralize SARS-CoV. Any known technique can be used to test the blood of potential donors to determine if the individual possesses antibodies having the desired neutralizing properties with respect to SARS-CoV.
  • PRNT is one of many such tests suitable for determining if a candidate's blood will render SARS-CoV non-infectious. It is also contemplated that antibodies can be derived from non-human primates that have been exposed to the SARS-CoV. Spleen and bone marrow can be harvested from non-human primates that have been virally infected and have had an opportunity to develop an immune response. Non-human primates that have recovered from SARS are particularly useful sources for antibodies, since such subjects are most likely to have developed an immunological repertoire of antibodies that effectively neutralize SARS-CoV. Once one or more suitable donors are selected, samples of their cells
  • RNA is isolated from the cells using techniques known to those skilled in the art and a combinatorial antibody library is prepared.
  • techniques for preparing a combinatorial antibody library involve amplifying target sequences encoding antibodies or portions thereof, such as, for example the light and/or heavy chains using the isolated RNA of an antibody.
  • target sequences encoding antibodies or portions thereof, such as, for example the light and/or heavy chains using the isolated RNA of an antibody.
  • first strand cDNA can be produced to provide a template.
  • Conventional PCR or other amplification techniques can then be employed to generate the library.
  • phage libraries expressing antibody Fab fragments can be constructed in plasmid vectors using the methods described in U.S. Application No. 10/251 ,085 (the disclosure of which is incorporated herein in its entirety by this reference).
  • one or more specific human antibodies are chosen based on a number of criteria including one or more of: high expression and high affinity, specificity and/or activity for the SARS-CoV.
  • the target ligand can be immobilized, e.g., on plates, beads, such as magnetic beads, sepharose, etc., beads used in columns.
  • the target ligand can be "tagged", e.g., using tags such as biotin, 2-fluorochrome, for detection of bound materials using, e.g., FACS sorting.
  • Screening a library of phage or phagemid expressing antibodies utilizes phage and phagemid vectors where antibodies are fused to a gene encoding a phage coat protein.
  • target ligands can be conjugated to magnetic beads according to manufacturers' instructions. To block non-specific binding to the beads and any unreacted groups, the beads may be incubated with excess bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the beads are then washed with numerous cycles of suspension in phosphate buffered saline (PBS)-0.05% Tween 20 and recovered with a strong magnet along the sides of a plastic tube.
  • PBS phosphate buffered saline
  • the beads are then stored with refrigeration until needed.
  • an aliquot of the library may be mixed with a sample of resuspended beads.
  • the tube contents are tumbled at cold temperatures (e.g., 4-5°C) for a sufficient period of time (e.g., 1-2 hours).
  • the magnetic beads are then recovered with a strong magnet and the liquid is removed by aspiration.
  • the beads are then washed by adding PBS-0.05% Tween 20, inverting the tube several times to resuspend the beads, and then drawing the beads to the tube wall with the magnet. The contents are then removed and washing is repeated 5- 10 additional times. 50 mM glycine-HCI (pH 2.2), 100 ⁇ g/ml BSA solution are added to the washed beads to denature proteins and release bound phage. After a short incubation time, the beads are pulled to the side of the tubes with a strong magnet and the liquid contents are then transferred to clean tubes. An alkali buffer, such as, for example 2M Tris-base (pH9), is added to the tubes to neutralize the pH of the phage sample. The phage are then diluted, e.g., 10 "3 to 10 "6 , and aliquots plated with E. coli cells to determine the number of plaque forming units of the sample. In certain cases, the platings are done in the
  • D-thiogalactopyranoside for color discrimination of plaques (i.e., lacZ+ plaques are blue, lacZ-plaques are white).
  • the titer of the input samples is also determined for comparison (dilutions are generally 10 "6 to 10 "9 ).
  • screening a library of phage expressing antibodies can be achieved, e.g., as follows using microtiter plates.
  • Target ligand is diluted and a small aliquot of ligand solution is adsorbed onto wells of microtiter plates (e.g. by incubation overnight at 4°C). An aliquot of BSA solution is added and the plate incubated at room temperature for 1 hr.
  • the contents of the microtiter plate are removed and the wells washed carefully with PBS-0.05% Tween 20.
  • the plates are washed free of unbound targets repeatedly.
  • a small aliquot of phage solution is introduced into each well and the wells are incubated at room temperature for 1-2 hrs.
  • the contents of microtiter plates are removed and washed repeatedly.
  • the plates are incubated with wash solution in each well for 5 minutes at room temperature to allow bound phage with rapid dissociation constants to be released.
  • the wells are then washed multiple, e.g., 5, times to remove all unbound phage.
  • a pH change may be used.
  • the platings are done in the presence of XGal and IPTG for color discrimination of plaques (i.e., lacZ+ plaques are blue, lacZ- plaques are white).
  • the titer of the input samples is also determined for comparison (dilutions are generally 10 "6 to 10- 9 ).
  • screening a library of antibodies can be achieved using a method comprising a first "enrichment” step and a second filter lift step as follows.
  • Antibodies from an expressed combinatorial library e.g., in phage
  • capable of binding to a given ligand (“positives") are initially enriched by one or two cycles of affinity chromatography.
  • a microtiter well is passively coated with the ligand of choice (e.g., about 10 ⁇ g in 100 ⁇ l).
  • the well is then blocked with a solution of BSA to prevent non-specific adherence of antibodies to the plastic surface.
  • About 10 11 particles expressing antibodies are then added to the well and incubated for several hours. Unbound antibodies are removed by repeated washing of the plate, and specifically bound antibodies are eluted using an acidic glycine-HCI solution or other elution buffer.
  • the eluted antibody phage solution is neutralized with alkali, and amplified, e.g., by infection of £. coli and plating on large petri dishes containing broth in agar.
  • the ligand can be covalently coupled to agarose or acrylamide beads using commercially available activated bead reagents.
  • the antibody solution is then simply passed over a small column containing the coupled bead matrix which is then washed extensively and eluted with acid or other eluant.
  • the goal is to enrich the positives to a frequency of about >1/10 5 .
  • a filter lift assay is conducted. For example, when antibodies are expressed in phage, approximately 1-2 x 10 5 phage are added to 500 ⁇ l of log phase £.
  • coli and plated on a large LB-agarose plate with 0.7% agarose in broth.
  • the agarose is allowed to solidify, and a nitrocellulose filter (e.g., 0.45 ⁇ ) is placed on the agarose surface.
  • a series of registration marks is made with a sterile needle to allow re-alignment of the filter and plate following development as described below.
  • Phage plaques are allowed to develop by overnight incubation at 37°C. (the presence of the filter does not inhibit this process).
  • the filter is then removed from the plate with phage from each individual plaque adhered in situ.
  • the filter is then exposed to a solution of BSA or other blocking agent for 1-2 hours to prevent non-specific binding of the ligand (or "probe").
  • the probe itself is labeled, for example, either by biotinylation (using commercial NHS-biotin) or direct enzyme labeling, e.g., with horse radish peroxidase or alkaline phosphatase. Probes labeled in this manner are stable indefinitely and can be re-used several times.
  • the blocked filter is exposed to a solution of probe for several hours to allow the probe to bind in situ to any phage on the filter displaying a peptide with significant affinity to the probe.
  • the filter is then washed to remove unbound probe, and then developed by exposure to enzyme substrate solution (in the case of directly labeled probe) or further exposed to a solution of enzyme-labeled avidin (in the case of biotinylated probe).
  • Positive phage plaques are identified by localized deposition of colored enzymatic cleavage product on the filter which corresponds to plaques on the original plate.
  • the developed filter is simply realigned with the plate using the registration marks, and the "positive" plaques are cored from the agarose to recover the phage. Because of the high density of plaques on the original plate, it is usually impossible to isolate a single plaque from the plate on the first pass. Accordingly, phage recovered from the initial core are re-plated at low density and the process is repeated to allow isolation of individual plaques and hence single clones of phage.
  • screening a library of plasmid vectors expressing antibodies on the outer surface of bacterial cells can be achieved using magnetic beads as follows.
  • Target ligands are conjugated to magnetic beads essentially as described above for screening phage vectors.
  • a sample of bacterial cells containing recombinant plasmid vectors expressing a plurality of antibodies expressed on the surface of the bacterial cells is mixed with a small aliquot of resuspended beads.
  • the tube contents are tumbled at 4°C for 1-2 hrs.
  • the magnetic beads are then recovered with a strong magnet and the liquid is removed by aspiration.
  • the beads are then washed, e.g., by adding 1 ml of PBS- 0.05% Tween 20, inverting the tube several times to resuspend the beads, and drawing the beads to the tube wall with the magnet and removing the liquid contents.
  • the beads are washed repeatedly 5-10 additional times.
  • the beads are then transferred to a culture flask that contains a sample of culture medium, e.g., LB containing ampicillin.
  • a sample of culture medium e.g., LB containing ampicillin.
  • the bound cells undergo cell division in the rich culture medium and the daughter cells will detach from the immobilized targets.
  • inducer is added again to the culture to generate more antibodies.
  • These cells are then harvested by centrifugation and rescreened.
  • Successful screening experiments are optimally conducted using multiple rounds of serial screening.
  • the recovered cells are then plated at a low density to yield isolated colonies for individual analysis. The individual colonies are selected and used to inoculate LB culture medium containing ampicillin. After overnight culture at 37°C, the cultures are then spun down by centrifugation.
  • screening a library of plasmid vectors expressing antibodies on the surface of bacterial cells can be achieved as follows.
  • Target ligand is adsorbed to microtiter plates as described above for screening phage vectors. After the wells are washed free of unbound target ligand, a sample of bacterial cells is added to a small volume of culture medium and placed in the microtiter wells. After sufficient incubation, the plates are washed repeatedly free of unbound bacteria.
  • a large volume, approximately 100 ⁇ I of LB containing ampicillin is added to each well and the plate is incubated at 37°C for 2 hrs.
  • the bound cells undergo cell division in the rich culture medium and the daughter cells detach from the immobilized targets.
  • the contents of the wells are then transferred to a culture flask that contains about 10ml of LB containing ampicillin.
  • inducer is added again to the culture to generate more antibodies.
  • These cells are then harvested by centrifugation and rescreened. Screening can be conducted using rounds of serial screening as described above, with respect to screening using magnetic beads.
  • the libraries expressing antibodies as a surface protein of either a vector or a host cell can be screened by passing a solution of the library over a column of a ligand immobilized to a solid matrix, such as sepharose, silica, etc., and recovering those phage that bind to the column after extensive washing and elution.
  • a solid matrix such as sepharose, silica, etc.
  • One important aspect of screening the libraries is that of elution. For clarity of explanation, the following is discussed in terms of antibody expression by phage. It is readily understood, however, that such discussion is applicable to any system where the antibodies are expressed on a surface fusion molecule.
  • the conditions that disrupt the peptide-target interactions during recovery of the phage are specific for every given peptide sequence from a plurality of proteins expressed on phage. For example, certain interactions may be disrupted by acid pHs but not by basic pHs, and vice versa.
  • a variety of elution conditions should be tested (including but not limited to pH 2-3, pH 12-13, excess target in competition, detergents, mild protein denaturants, urea, varying temperature, light, presence or absence of metal ions, chelators, etc.) to compare the primary structures of the antibodies expressed on the phage recovered for each set of conditions to determine the appropriate elution conditions for each ligand/antibody combination.
  • Some of these elution conditions may be incompatible with phage infection because they are bactericidal and will need to be removed by dialysis.
  • the ability of different expressed proteins to be eluted under different conditions may not only be due to the denaturation of the specific peptide region involved in binding to the target but also may be due to conformational changes in the flanking regions.
  • flanking sequences may also be denatured in combination with the actual binding sequence; these flanking regions may also change their secondary or tertiary structure in response to exposure to the elution conditions (i.e., pH 2-3, pH 12- 13, excess target in competition, detergents, mild protein denaturants, urea, heat, cold, light, metal ions, chelators, etc.) which in turn leads to the conformational deformation of the peptide responsible for binding to the target.
  • elution conditions i.e., pH 2-3, pH 12- 13, excess target in competition, detergents, mild protein denaturants, urea, heat, cold, light, metal ions, chelators, etc.
  • Any panning method suitable for recovery of antibodies demonstrating desired characteristics e.g. good expression and desired effect on SARS-CoV infection
  • Any selection display system may be used in conjunction with a library according to the present disclosure.
  • Selection protocols for isolating desired members from large libraries are known in the art, as typified by phage display techniques.
  • Such systems in which diverse polypeptide sequences are displayed on the surface of filamentous bacteriophage (Scott and Smith (1990) Science, 249: 386), have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encode them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen.
  • the nucleotide sequences encoding the VH and VL regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E.
  • phage- or phagemid-based display systems are that, because they are biological systems where the antibody protein is linked to its encoding gene, the selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • scFv libraries displayed on bacteriophage coat proteins have been described.
  • phage display approaches are also known, for example as described in WO96/06213 and WO92/01047 and WO97/08320, which are incorporated herein by reference.
  • the display of Fab libraries is also known, for instance as described in WO92/01047 and WO91/17271.
  • Other systems for generating libraries of antibodies or polynucleotides involve the use of cell-free enzymatic machinery for the in vitro synthesis of the library members.
  • RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818).
  • a similar technique may be used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92/05258 and WO92/14843).
  • in vitro translation can be used to synthesize antibody molecules as a method for generating large libraries.
  • the light chain and heavy chain Fd products are under the control of a lac promoter, and each chain has a leader signal fused to it in order to be directed to the periplasmic space of the bacterial host. It is in this space that the antibody fragments will be able to properly assemble.
  • the heavy chain fragments are expressed as a fusion with a phage coat protein domain which allows the assembled antibody fragment to be incorporated into the coat of a newly made phage or phagemid particle.
  • Generation of new phagemid particles requires the addition of helper phage which contain all the necessary phage genes.
  • the antibodies displayed on the surface of phage or phagemid particles are bound to the desired antigen, ii) non-binders are washed away, iii) bound particles are eluted from the antigen, and iv) eluted particles are exposed to fresh bacterial hosts in order to amplify the enriched pool for an additional round of selection. Typically three or four rounds of panning are performed prior to screening antibody clones for specific binding. In this way phage/phagemid particles allow the linkage of binding phenotype (antibody) with the genotype (DNA) making the use of antibody display technology very successful.
  • vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and/or screening.
  • lytic phage vector modified T7 or Lambda Zap systems
  • selection of desired human antibodies and/or functional antibody fragments it is contemplated that they can be produced in large volume by any technique known to those skilled in the art, e.g., in vitro synthesis, recombinant DNA production and the like.
  • antibodies and/or functional antibody fragments may be produced by using conventional techniques to construct an expression vector encoding the antibody.
  • suitable control sequences appropriate for expression of the antibody in a desired host will readily envision suitable control sequences appropriate for expression of the antibody in a desired host.
  • the expression vectors may then be transferred to a suitable host cell by conventional techniques to produce a transfected host cell for expression of antibodies and/or functional antibody fragments.
  • the transfected host cell is then cultured using any suitable technique known to those skilled in the art to produce antibodies and/or functional antibody fragments.
  • host cells may be co-transfected with two expression vectors, the first vector containing an operon encoding a heavy chain derived polypeptide and the second containing an operon encoding a light chain derived polypeptide.
  • the two vectors may contain different selectable markers but, with the exception of the heavy and light chain coding sequences, are preferably identical. This procedure provides for equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA or both.
  • the host cell used to express antibodies and/or functional antibody fragments may be either a bacterial cell such as Escherichia coli, or preferably a eukaryotic cell including, but not limited to Chinese hamster ovary, NSO or 293EBNA cells.
  • the choice of expression vector is dependent upon the choice of host cell, and may be selected so as to have the desired expression and regulatory characteristics in the selected host cell.
  • RNA may be obtained from blood or bone marrow cells of convalescent human donors that have been exposed to the SARS-CoV and have recovered from SARS, or from vaccinated donors, or from the spleen and bone marrow of virally infected non-human primates, for example the use of Tri reagent (Molecular research center, Cincinnati, Ohio, USA).
  • single-stranded DNA complementary to the RNA can be synthesized by using the RNA as a template and treating with reverse transcriptase using random hexamers or oligo(dT) complementary to the polyA chain on the 3' terminus as primer (Larrik, J. W. et al., Bio/Technology, 7, 934-938, 1989).
  • gene specific primers can be used with oligonucleotides that bind to sequences specific to one or more antibodies (e.g., lgG1 constant region). Kits for cDNA synthesis are widely available in the art.
  • PCR polymerase chain reaction
  • Primers such as those described in Barbas III, et al. (2001) Phage Display, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, or Jones, S. T. et al., Bio/Technology, 9, 88-89, 1991 , may be used for amplification of the originating species antibody variable region genes.
  • PCR may also be performed with gene-specific primers.
  • Single primer amplification can also be employed, such as, for example, the processes described in U.S. Application Nos.
  • Variable region genes may be cloned into phage or phagemids, or another suitable selection system, and presented as a library for selection against a target. Library construction and panning techniques are well known in the art.
  • the antibodies and/or functional antibody fragments may be used in conjunction with, or attached to other antibodies (or parts thereof) such as other human or humanized monoclonal antibodies.
  • These other antibodies may be catalytic antibodies and/or reactive with other markers (epitopes) characteristic for a disease against which the antibodies are directed or may have different specificities chosen, for example, to recruit molecules or cells of the target species, e.g., receptors, target proteins, diseased cells, etc.
  • the antibodies (or parts thereof) may be administered with such antibodies (or parts thereof) as separately administered compositions or as a single composition with the two agents linked by conventional chemical or by molecular biological methods.
  • the diagnostic and therapeutic value of the present antibodies and/or functional antibody fragments may be augmented by labeling the antibodies with labels that produce a detectable signal (either in vitro or in vivo) or with a label having a therapeutic property.
  • radionuclides may produce a detectable signal and have a therapeutic property.
  • radionuclide labels include 125 l, 131 l, 14 C.
  • detectable labels include a fluorescent chromophore such as green fluorescent protein, fluorescein, phycobiliprotein or tetraethyl rhodamine for fluorescence microscopy, or an enzyme which produces a fluorescent or colored product for detection by fluorescence, absorbance, visible color or agglutination, or an enzyme which produces an electron dense product for demonstration by electron microscopy; or an electron dense molecule such as ferritin, peroxidase or gold beads for direct , or indirect electron microscopic visualization.
  • the antibodies and/or functional antibody fragments herein may typically be administered to a patient in a composition comprising a pharmaceutical carrier.
  • a pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivery of the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be included in the carrier. Pharmaceutically accepted adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. It should be understood that compositions can contain both entire antibodies and/or functional antibody fragments. It should be understood that combinations of two or more antibodies that bind the SARS-CoV can be administered in combination to combat and/or prevent SARS-CoV infection.
  • the antibodies and/or functional antibody fragment compositions may be administered to a patient in a variety of ways.
  • compositions for parenteral administration may include a solution of the antibodies and/or functional antibody fragments, or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • concentration of antibodies and/or functional antibody fragments in these formulations can vary widely, e.g., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • the antibodies described herein which are found to neutralize the SARS-CoV can be used to identify epitopes on the SARS-CoV for use in developing vaccines. Any suitable technique can be used to identify epitopes on the SARS-CoV with which the antibody interacts. For example, epitope mapping (i.e.
  • determination of epitopes on the surface of a macromolecule can be achieved by means of monoclonal antibodies and blotting technique (as described by Van Leuven et al. in J. Immunol. 90 (1986) 125-130) or by means of monoclonal antibodies and solid-phase RIA technique (as described by Mazza M. M and Retegui L. A. in Molec. Immun. 26 (1989) 231- 240).
  • the antibodies (or scFvs thereof) are used to immunoprecipitate the epitopes from lysates prepared from the microsomal fraction of the SARS-CoV.
  • the immunoprecipitated epitopes are purified (for example by SDS-PAGE) and identified using known techniques (including, for example, matrix assisted laser desorption ionization mass spectrometry (MALD1-MS) or microcapillary reverse-phase HPLC nano- electrospray tandem mass spectrometry ( ⁇ LC/MS/MS)). Parameters for performing these various techniques are within the purview of one skilled in the art. Specifically, in one such method to identify the antigens for these antibodies, scFvs are used to immunoprecipitate the antigens from lysates prepared from the microsomal fraction of surface-biotinylated SARS-CoV.
  • MALD1-MS matrix assisted laser desorption ionization mass spectrometry
  • ⁇ LC/MS/MS microcapillary reverse-phase HPLC nano- electrospray tandem mass spectrometry
  • SARS-CoV is labeled with a solution of 0.5mg/ml sulfo-NHS-LC- biotin in PBS, pH ⁇ .O for 30 seconds. After washing with PBS to remove unreacted biotin, the SARS-CoV is disrupted by nitrogen cavitation and the microsomal fraction is isolated by differential centrifugation. The microsomal fraction is resuspended in NP40 Lysis Buffer and extensively precleared with normal mouse serum and protein A sepharose. Antigens are immunoprecipitated with HA-tagged scFv antibodies coupled to Rat Anti-HA agarose beads.
  • antigens are separated by SDS- PAGE and detected by Western blot using streptavidin-alkaline phosphatase(AP) or by Coomassie G-250 staining. An antibody which does not bind to the SARS- CoV is used as a negative control. Antigen bands are excised from the Coomassie-stained gel and identified by mass spectrometry (MS). The immunoprecipitated antigens can also be identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) or microcapillary reverse- phase HPLC nano-electrospray tandem mass spectrometry ( ⁇ LC/MS/MS).
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometry
  • ⁇ LC/MS/MS microcapillary reverse- phase HPLC nano-electrospray tandem mass spectrometry
  • the antigens identified can then be used as an immunogen to elicit additional antibodies thereto using techniques within the purview of those skilled in the art.
  • the epitopes can be formulated into suitable compositions and administered as a vaccine prophylactically to subjects at risk of exposure to SARS.
  • Vero E6 cells (ATCC CRL 1586) are inoculated with isolates of SARS- CoV on Dulbecco's Modified Eagle's Medium supplemented with penicillin/streptomycin, glutamine and 2% fetal calf serum. The culture is incubated at 37°C. The virus is passaged into newly seeded Vero E6 cells. A virus stock is prepared from passage 2 of these cells and preserved at -70°C. The titer of the virus stock is determined by plaque assay on Vero E6 cells.
  • Vero E6 cells For virus propagation, 10 flasks of Vero E6 cells are infected with a multiplicity of infection of 10 "2 .
  • the cultures When infected cells show a cytopathic effect of 4+ at approximately 48hr, the cultures are frozen and thawed to lyse the cells, and the supernatants are clarified from cell debris by centrifugation at 10,000 rpm in a Beckman high-speed centrifuge.
  • the supernatants are ultra-centrifuged through a 5%/40% glycerol step gradient at 151 ,000 x g for 1 hr at 4°C.
  • the virus pellet is resuspended in phosphatase-buffered saline (PBS).
  • PBS phosphatase-buffered saline
  • Plaque reduction neutralizing test All dilutions are made in Eagle's minimal essential medium supplemented with 5% heat-inactivated fetal bovine serum. The challenge virus is diluted to contain 100 PFU per 0.1 ml. The sera of convalescent patients and control sera from healthy volunteers are serially diluted (twofold) at 0.5 ml/tube. Virus and serum are incubated at 37°C for 1 hr. Following the incubation, they are placed on ice. Infectious virus remaining in the virus-serum mixture is quantitated by counting PFU on Vero E6 cell monolayers. A 0.2 ml volume of each mixture is adsorbed to cells grown in 10 cm 2 wells of plastic plates (37°C for 1 hr). Each mixture is assayed in two wells. Following adsorption, the cells are overlaid with
  • Fab libraries expressing antibody Fab fragments (kappa and lambda light chains complexed to IgG heavy chain Fd) are constructed in plasmid pAX243hG vectors (see International application PCT/US02/21680 filed July 9, 2002) by the methods described in WO03/025202A2, the disclosures of which is incorporated herein by reference. Two Fab libraries are generated for
  • each donor lgG ⁇ and IgG ⁇ .
  • Panning The library phage are then panned (selection procedure) on recombinant antigens, S (spike glycoprotein), M glycoprotein and UV irradiated virus preparations (Lane et al., J Immunol 160(2), 970-8, 1998) where surface glycoproteins are effectively coated to microtiter wells via wheat-germ agglutinin.
  • the panning is repeated four times for enrichment against selected viral antigens.
  • the wells are coated with 100 ⁇ l NeutrAvidin (Pierce) (10 ⁇ g/ml) in PBS at 4°C overnight.
  • the wells are washed with PBS and blocked with 4% non-fat dry milk (BioRad)/PBS for 30 min-1 hr at 37°C.
  • the wells are incubated with 100 ⁇ l Biotinylated Wheat germ agglutinin (Sigma) (50 ⁇ g/ml) in PBS at 37°C 1 hr.
  • the wells are washed with PBS and incubated with 100 ⁇ l of an inactivated virus preparation (1 :100) in PBS at 37°C for 1 hr.
  • the wells are washed with PBS and blocked again with 4% non-fat dry milk/PBS for ⁇ 30 min.
  • blocking solution is removed and the wells are incubated with 100 ⁇ l of library phage for 1-2 hrs at 37°C.
  • the wells are washed with PBS to remove non-specific Fab-bearing phage, increasing stringency at each round of panning by increasing the number of washes (2x, 5x, 10x and 10x).
  • the remaining phage are eluted with 0.1 M glycine-HCI buffer, pH 2.2, 1 mg/ml bovine serum albumin (BSA). After neutralization with 2M Tris base, eluted phage are propagated in E. coli strain ER2738 cells overnight with helper phage
  • Library Screening Screening is performed in high-throughput by picking 1150 colonies using a Q-pix instrument, and performing ELISAs using a Tecan robot. Programs for performing these activities are readily available. Individual colonies on titration plates from each panning round are grown overnight in deep-well microtiter culture dishes in a Hi-Gro high-speed incubator shaker. After centrifugation, supernatants containing Fab attached to phage by coat protein III (Fab-phage) are incubated with specific antigens or a control antigen such as ovalbumin coated on microtiter wells (Costar).
  • Fab-phage coat protein III
  • Fab-phage supernatant containing phage expressing Fabs from specific clones are used to infect E. coli strain TOP10F' cells for preparation of phagemid DNA.
  • Phagemid DNA of each clone is prepared by QIAprep 96 Turbo Miniprep Kit (Qiagen) according to the manufacturer's protocol.
  • Purified DNA is sequenced by automated dye terminator sequencing (Retrogen, San Diego). DNA sequences are analyzed using DNAstar software to divide Fabs into groups of related clones generally based on the homology of heavy chain complementarity determining region (CDR) 3 and to identify candidates for soluble Fab production and purification.
  • CDR heavy chain complementarity determining region
  • Protein A (Pharmacia) by fast performance liquid chromatography (FPLC).
  • Fabs are titered against antigen in ELISA to compare the antigen- binding characteristics of Fabs within related groups established by DNA sequence analysis. Determination of epitope specificity by competitive ELISA. Epitope specificity is determined by competitive ELISA. Competition between Fab-phage and purified Fab is assessed by anti-M13 phage antibody in this assay. Briefly, 50 ⁇ l of antigen is coated overnight at 4°C on microtiter wells and blocked with 4% non-fat dry milk/PBS. Then blocker is replaced with dilutions of purified Fab and allowed to bind to its epitope at 37°C for 1 hr. To this, 50 ⁇ l of Fab-phage is added and incubation proceeds for another hour at
  • Bound Fab-phage are detected with horse radish peroxidase-conjugated anti-M13 antibody (Pharmacia) via coat protein III. Decrease of Fab-phage binding by increasing concentration of purified Fab indicates identical or overlapping epitopes. If binding of Fab-phage remains the same at any concentration of purified Fab, the epitopes are well separated on the antigen.
  • PRNT Plague reduction neutralizing test
  • Fab anti-hepatitis B virus surface antigen specific antibody
  • Virus and Fab are incubated at 37°C for 1 hr and then placed on ice. Infectious virus remaining in the virus-Fab mixture is quantitated by determining PFU on Vero E6 cell monolayers. A 0.2 ml volume of each mixture is adsorbed to cells grown in 10 cm 2 wells of tissue culture plates (37°C for 1 hr). Each mixture is assayed in two wells. Following adsorption, the cells are overlaid with 2 ml of Eagle's minimal essential medium containing 5% fetal bovine serum, 25 mM HEPES buffer, 50 ⁇ g of gentamycin per ml, and 1% agarose.
  • the cells are incubated at 37°C in a humidified CO 2 incubator until plaques are visible under an inverted phase microscope. After incubation, 2 ml of neutral red (1 :6,000 final concentration) is added to each well, and the plaques are counted after an additional 24 hr incubation.
  • Vectors have been developed that create a full-length lgG1 (e.g., the pEE vectors available from Lonza Biologies) or lgG2/G4 heavy chain. See, provisional U.S. application 60/475,202 filed May 30, 2003 the disclosure of which is incorporated herein in its by this reference. These vectors utilize a glutamine synthetase gene as a selectable marker, permitting growth of transfected cells in glutamine-free medium (Bebbington et al., Biotechnology (N Y) 10(2), 169- 75,1992).
  • amino acids encoded by artificial restriction sites (Xba I and Xho I) at the start of both the light chain and heavy chain framework region 1 of Fab candidates are eliminated by an overlap PCR that connects the eukaryotic leader sequences with the framework 1 region of each light chain and heavy chain fragment.
  • the amplified fragments are subcloned into the vector using restriction sites (Hind III and Asc I) in the vector sequence and the Not I site immediately after the end of light chain constant
  • Vectors are transfected by electroporation using standard methods into the NSO mouse myeloma cell line. Stable cell lines are selected in glutamine-free medium and are isolated by limiting dilution. Transient transfections can also be performed with this vector in CHO-K1 cells in order to examine smaller quantities of Ig prior to selecting a stable cell line. For purification of Ig, transiently infected cells or stable cell lines expressing Ig candidates are grown in flasks or miniPerm bioreactors

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Abstract

La présente invention concerne des anticorps et certains de leurs fragments fonctionnels, et notamment des anticorps complètement humains dérivés de donneurs humains convalescents ou d'individus vaccinés. Se liant au coronavirus associé au SRAS, ils peuvent s'utiliser pour prévenir et/ou neutraliser l'infection virale, pour identifier des épitopes sur le coronavirus associé au SRAS, et pour mettre au point des vaccins contre le coronavirus associé au SRAS.
PCT/US2004/030557 2003-09-18 2004-09-18 Anticorps destines a lutter contre le sras WO2005027847A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054469A1 (fr) * 2003-12-05 2005-06-16 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Anticorps monoclonaux anti-sars
WO2021195308A1 (fr) * 2020-03-27 2021-09-30 Back of the Yards Algae Sciences LLC Utilisation de la phycobiliprotéine (pbp) pour inhiber ou traiter une infection par sras-cov-2
WO2022101877A1 (fr) * 2020-11-13 2022-05-19 Targetalent, Lda Composition de cryoconservation d'échantillons biologiques pour pcr
WO2023019019A3 (fr) * 2021-08-13 2023-06-15 Abwiz Bio, Inc. Humanisation, maturation d'affinité et procédés d'optimisation pour des protéines et des anticorps

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAVANAGH D.: 'Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus' AVIAN PATHOLOGY vol. 32, no. 6, December 2003, pages 567 - 582, XP008062275 *
'News Feature. SARS, What have we learned?' NATURE vol. 424, 10 July 2003, pages 121 - 126, XP002996045 *
PEIRIS J.S.M. ET AL: 'Coronavirus as a possible cause of severe acute respiratory syndrome' THE LANCET vol. 361, 19 April 2003, pages 1319 - 1325, XP002295043 *
VIRET J.-F. ET AL: 'Development of a SARS vaccine: an industrial perspective on the global race angainst a global disease' EXPERT REV. VACCINES vol. 2, no. 4, August 2003, pages 465 - 467, XP008062274 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054469A1 (fr) * 2003-12-05 2005-06-16 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Anticorps monoclonaux anti-sars
US7622112B2 (en) 2003-12-05 2009-11-24 Jody Berry Anti-SARS monoclonal antibodies
WO2021195308A1 (fr) * 2020-03-27 2021-09-30 Back of the Yards Algae Sciences LLC Utilisation de la phycobiliprotéine (pbp) pour inhiber ou traiter une infection par sras-cov-2
WO2022101877A1 (fr) * 2020-11-13 2022-05-19 Targetalent, Lda Composition de cryoconservation d'échantillons biologiques pour pcr
WO2023019019A3 (fr) * 2021-08-13 2023-06-15 Abwiz Bio, Inc. Humanisation, maturation d'affinité et procédés d'optimisation pour des protéines et des anticorps

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