WO2021221668A1 - Compositions d'anticorps monoclonaux anti-sras-cov-2 - Google Patents

Compositions d'anticorps monoclonaux anti-sras-cov-2 Download PDF

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WO2021221668A1
WO2021221668A1 PCT/US2020/030770 US2020030770W WO2021221668A1 WO 2021221668 A1 WO2021221668 A1 WO 2021221668A1 US 2020030770 W US2020030770 W US 2020030770W WO 2021221668 A1 WO2021221668 A1 WO 2021221668A1
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seq
amino acid
acid sequence
variable region
chain variable
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PCT/US2020/030770
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English (en)
Inventor
Ling Chen
Jian Han
Xuefeng NIU
Song Li
Pingchao LI
Wenjing PAN
Qian Wang
Ying Feng
Xiaoneng MO
Qihong YAN
Xianmiao YE
Jia Luo
Linbing QU
Daniel Weber
Miranda L. BYRNE-STEELE
Zhe Wang
Fengjia YU
Fang Li
Nanshan Zhong
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iRepertoire, Inc.
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Priority to PCT/US2020/030770 priority Critical patent/WO2021221668A1/fr
Publication of WO2021221668A1 publication Critical patent/WO2021221668A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the virus mostly targets the lung, but it also affects multiple organs such as the kidney, liver, brain, gastrointestinal tract.
  • the virus spreads by respiratory droplets, urine, and feces.
  • Clinical symptoms of SARS-CoV-2 include fever, cough, shortness of breath, absence of other pathogens, and pneumonia characterized by bilateral ground-glass opacities on imaging of chest CT.
  • the majority of COVID-19 patients show mild or moderate syndrome and recovery after proper clinical care, while some COVID-19 patients rapidly developed severe pneumonia and multi-organ failure.
  • the present disclosure relates to isolated or non-naturally occurring monoclonal antibodies capable of binding to one or more SARS-CoV-2 epitopes.
  • the present disclosure relates to an isolated or non-naturally occurring monoclonal antibody comprising: a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-17; and a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-26.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-4; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-8; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 11-12; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising: a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 15-17; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26.
  • the present disclosure relates to an isolated or non- naturally occurring monoclonal antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-76.
  • the present disclosure is directed to a composition comprising any of the foregoing antibodies.
  • the present disclosure is directed to an immortalized B cell clone expressing any of the foregoing antibodies.
  • the present disclosure is directed to a pharmaceutical composition comprising any of the foregoing antibodies and a pharmaceutically acceptable carrier.
  • the present disclosure is directed to a method for detecting COVID- 19 in a biological specimen which comprises contacting the specimen with an antibody, wherein the antibody is a monoclonal antibody or antibody fragment disclosed herein, and determining the presence or absence of the SARS-CoV-2 virus.
  • FIG. 1 is a chart displaying a longitudinal analysis of TCR and BCR repertoires of
  • the 7 treemap plots each represent TCR-beta, -alpha, -delta, -gamma, and BCR-IgH, -IgK, -IgL.
  • a rectangle in a treemap plot represents a unique clonotype. The size of a rectangle denotes the relative frequency of an individual CDR3 sequence, and the varying square size reflects areas of clonal expansion within the immune repertoire sampled. From the left upper plot clockwise to the bottom plots: IgH, IgK, IgL, TRB, TRA, TRD, and TRG.
  • FIG. 2 displays treemap plots of TCR and BCR repertoires in PBMCs of ICU patients.
  • A Treemap plots containing 7 adaptive immune chains from patient 4 and patient
  • a rectangle in a treemap plot represents a unique clonotype. The size of a rectangle denotes the relative frequency of an individual CDR3 sequence.
  • FIG. 3 illustrates a graph of CDR3 node clusters in a control sample, wherein the node size corresponds with expression levels. Node sizes (i.e., expression level) is roughly uniform.
  • FIG. 4 illustrates a graph of CDR3 node clusters in a subject diagnosed with
  • FIG. 5 displays (A) an exemplary ELISA plate loading layout for testing various combinations of heavy and light chains against (B) Spike protein and (C) the RBD domain of the Spike protein.
  • the present disclosure relates to isolated or non-naturally occurring anti-SARS-
  • CoV-2 monoclonal antibodies relate to an isolated or non-naturally occurring monoclonal antibody comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1- 17 (see Table 1) and a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18-26 (see Table 2).
  • the present disclosure also relates to an isolated or on-naturally occurring monoclonal antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18-76 (see Table 2).
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies such that the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or by using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries.
  • the antibodies disclosed herein are produced recombinantly, using vectors and methods available in the art, as described further below.
  • the antibodies disclosed herein may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. No. 5,567,610).
  • the antibodies disclosed herein may also be produced in transgenic animals, such as mice, that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • JH antibody heavy-chain joining region
  • Amino acid substitutions may be made to the antibodies disclosed herein on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five or fewer amino acids.
  • variant polypeptides may also or alternatively be modified by deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • Immortalized B cell clones can be generated using the alternative EBV immortalization method described in W02004/076677. With this method, B cells producing a presently disclosed antibody can be transformed with EBV in the presence of a polyclonal B cell activator. Transformation with EBV can also be adapted to include polyclonal B cell activators. Additional stimulants of cell growth and differentiation, such as IL-2 and IL-15, may be added during the transformation step to enhance efficiency.
  • the present disclosure further provides antibody fragments comprising a polypeptide of the present disclosure.
  • antibody fragments comprising a polypeptide of the present disclosure.
  • the smaller size of the fragments allows for rapid clearance, and may lead to improved access to certain tissues.
  • the antibody fragments are screened to ensure that antigen binding has not been disrupted. This may be accomplished by any of a variety of means known in the art, but one method involves the use of a phage display library.
  • an “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody and that retains an acceptable percentage of binding activity to the target antigen. As will be understood by one of skill in the art, what constitutes an acceptable percentage depends on the particular intended use. Examples of antibody fragments include: Fab, Fab’, F(ab’)2, and Fv fragments; single-chain antibody molecules; multispecific antibodies formed from antibody fragments; and other fragments known to those of skill in the art.
  • antibody fragments were traditionally derived via proteolytic digestion of intact antibodies, antibody fragments can now be produced directly by recombinant host cells.
  • Fab, Fv and ScFv antibody fragments for example, can all be expressed in and secreted from E. coli , thus allowing the facile production of large amounts of these fragments.
  • Other techniques for the production of antibody fragments are known to those of skill in the art.
  • any of the above-described antibodies or antibody fragments thereof may be formulated into a pharmaceutical treatment for providing passive immunity for individuals suspected of, or at risk of developing, COVID-19 comprising a therapeutically effective amount of said antibody.
  • the pharmaceutical preparation may include a suitable excipient or carrier.
  • the total dosage will vary according to the weight, health and circumstances of the individual and the efficacy of the antibody.
  • Formulations to be used for in vivo administration are preferably sterile, which can be accomplished by filtration of the formulation through sterile filtration membranes.
  • carriers include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin, ge
  • Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases. Therefore, passive immunization using the presently disclosed antibodies and antibody fragments thereof disclosed herein could provide a treatment strategy for COVID-19.
  • antibodies disclosed herein are intrinsically therapeutically active.
  • antibodies disclosed herein can be conjugated to a cytotoxic agent or growth inhibitory agent, such as a radioisotope or toxin, that is used in treating infected cells bound or contacted by the antibody.
  • Subjects at risk for COVTD-19 include patients who have come into contact with an infected person, particularly those with an underlying health condition, such as diabetes, heart disease, or a respiratory condition such as asthma or COPD. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of COVID-19, such that COVID-19 is prevented or, alternatively, delayed in its progression.
  • an underlying health condition such as diabetes, heart disease, or a respiratory condition such as asthma or COPD.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of COVID-19, such that COVID-19 is prevented or, alternatively, delayed in its progression.
  • the subject is usually administered or provided a pharmaceutical formulation comprising at least one antibody disclosed herein.
  • the disclosed antibodies are administered to the subject in therapeutically effective amounts.
  • therapeutically effective amounts means amounts that eliminate or reduce the subject’s viral burden.
  • the antibodies can be administered to a human subject, in accord with known methods, such as intravenous administration (as a bolus or by continuous infusion) or by intra-muscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the antibodies may be administered parenterally, when possible, at the target cell site, or intravenously. Intravenous or subcutaneous administration of the antibody is preferred in certain embodiments.
  • Therapeutic compositions of the invention are administered to a subject systemically, parenterally, or locally.
  • the antibodies disclosed herein are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle.
  • a pharmaceutically acceptable, parenteral vehicle examples include water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used.
  • Liposomes can be used as carriers.
  • the vehicle can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives.
  • the antibodies can be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml, although other concentrations are also available.
  • the dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection and the characteristics of the particular cytotoxic agent or growth inhibitory agent conjugated to the antibody (if used), such as the therapeutic index and the subject’s medical history.
  • a therapeutically effective amount of an antibody is administered to a subject.
  • the amount of antibody administered is in the range of about 0.1 mg/kg to about 50 mg/kg of subject body weight.
  • about 0.1 mg/kg to about 50 mg/kg body weight (approximately 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion.
  • an immunoconjugate comprising an antibody disclosed herein conjugated with a cytotoxic agent is administered to a subject.
  • the immunoconjugate is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cell to which it binds.
  • the cytotoxic agent targets or interferes with the nucleic acid in the infected cell. Examples of such cytotoxic agents are described above and include, but are not limited to, maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
  • Other therapeutic regimens can be combined with the administration of one or more of the anti-SARS-CoV-2 antibodies of the present disclosure.
  • the combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both or all active agents simultaneously exert their biological activities.
  • Preferably such combined therapy results in a synergistic therapeutic effect.
  • the present disclosure provides methods of administration of the antibody by gene therapy.
  • Such administration of nucleic acid encoding the antibody is encompassed by the expression “administering a therapeutically effective amount of an antibody”. See, for example, PCT Patent Application Publication WO96/07321 concerning the use of gene therapy to generate intracellular antibodies.
  • the anti-SARS-CoV-2 antibodies disclosed herein are used to determine the structure of bound antigen, such as conformational epitopes, the structure of which is then used to develop a vaccine having or mimicking this structure, such as through chemical modeling and SAR methods. Such a vaccine could then be used to prevent COVID-19.
  • the anti-SARS-CoV-2 antibodies disclosed herein are capable of differentiating between subjects with and subjects without COVID-19, and determining whether or not a patient has an infection.
  • a biological sample is obtained from a subject suspected of being infected or known to be infected with SARS-CoV-2.
  • the biological sample includes cells from the subject.
  • the sample is contacted with an anti-SARS-CoV-2 antibody disclosed herein for a time and under conditions sufficient to allow the anti-SARS-CoV-2 antibody to bind to infected cells present in the sample.
  • the sample can be contacted with an anti-SARS-CoV-2 antibody for 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 3 days or any point in between.
  • the amount of bound anti-SARS-CoV-2 antibody is determined and compared to a control value, which may be a pre-determined value or a value determined from a normal tissue sample.
  • a control value which may be a pre-determined value or a value determined from a normal tissue sample.
  • An increased amount of antibody bound to the subject’s sample as compared to the control sample is indicative of the presence of infected cells in the subject’s sample. Bound antibody is detected using procedures described herein and known in the art.
  • diagnostic methods of the present disclosure are practiced using anti-SARS-CoV-2 antibodies disclosed herein that are conjugated to a detectable label, such as a fluorophore, to facilitate detection of bound antibody.
  • detectable label such as a fluorophore
  • diagnostic methods of the present disclosure are practiced using anti-SARS-CoV-2 antibodies disclosed herein that are conjugated to a detectable label, such as a fluorophore, to facilitate detection of bound antibody.
  • methods of secondary detection of the anti-SARS-CoV-2 antibody include, for example, RIA, ELISA, precipitation, agglutination, complement fixation and immuno-fluorescence.
  • kits suitable for performing diagnostic and prognostic assays using the antibodies disclosed herein include a suitable container comprising an anti-SARS-CoV-2 antibody of the present disclosure in either labeled or unlabeled form.
  • the kit further includes reagents for performing the appropriate indirect assay.
  • the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions are also included.
  • a longitudinal assessment of CDR3 expression in a cohort of ten COVID-19 patients was performed.
  • the cohort of patients included 4 males and 6 females, with a median age of 57 years old (ranging from 33-81 years).
  • Peripheral blood samples from 15 Asian healthy donors were collected as normal controls, which included 9 males and 6 females, with ages ranging from 22-67 years (Table 51).
  • the inclusion criteria for healthy donors are: 1) without any past diagnosis of chronic diseases; and 2) without any diagnosis of acute diseases in the past three months.
  • Peripheral blood mononuclear cells (PBMCs) samples from a 57-year-old healthy seasonal influenza vaccinee were also used as a longitudinal healthy control.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs were collected from each patient and the CDR3 repertoire was amplified using Arm-PCR with unique molecular identifiers for noise cleanup, as disclosed in US20150132754 (Wang, et al.) and US20120171725 (Han), which are incorporated herein by reference, and were then sequenced.
  • the five most influential (by eigenvector centrality) CDR3 clusters i.e., CDR3 sequences varying by one amino acid from each other sequence in the same cluster) were analyzed at each of the PBMC collection time points.
  • TCR- beta was extremely low in the early disease stage. TCR-beta gradually increased with the improvement throughout the disease, especially at the convalescent stage (the fourth time point). The percentage of TCR-beta increased to the same level as health control (FIG. 1). The applicants also discovered that the IgH in the repertoires gradually reduced as the disease regressed. It was the highest on the day 4-7 after symptom onset and returned to the level of about 20% on the 19th or 20th day (FIG. 1).
  • the proportion of IgL was at the highest on day 4-7 after symptom onset, and there was a downward trend along the course of the disease (see FIG. 1, patients 2 and 3).
  • the proportions of TCR and BCR chains were relatively stable before vaccination (day 0) and after vaccination over 28 days (FIG. 1).
  • FIG. 2 treemap plots containing total TCR-beta unique CDR3 clonotypes are shown for patient 4, patient 5, and a healthy control. Each rectangle in a treemap plot represents a unique clonotype. The size of a rectangle denotes the relative frequency of an individual CDR3 sequence.
  • PBMCs were isolated by density gradient separation on a Ficoll-Hypaque gradient as previously described by Niu et al., Emerg Microbes Infect 9, 111-123 (2020). (GE Healthcare, Chicago, IL, USA). Total RNA was extracted using TRIzolTM LS reagent according to the manufacturer’s protocol Invitrogen.
  • iR-RepSeq-plus 7-Chain Cassette (iRepertoire) was used to generate NGS libraries covering all TCR and BCR chains including TCR-beta, -alpha, -delta, -gamma, and BCR- IgH, -IgK, - IgL. All 7 chains were amplified in a single assay using a strategy which allows the incorporation of unique molecular identifiers during the reverse transcription (RT) step.
  • RT reverse transcription
  • One disposable cassette was used for one sample’s library preparation; all necessary reagents for amplification and purification were preloaded into the cassette.
  • RNA 1000 ng extracted RNA with an appropriate volume of RT mix and nuclease-free water were added into the cassette, which was processed by the iR-Processor.
  • the instrument was automatically set up to carry out all amplification and purification. Briefly, RT was performed using Qiagen OneStep RT-PCR mix (Qiagen). First-strand cDNA was selected, and unused primer was removed by SPRIselect bead selection (Beckman Coulter) followed by the second round of binding and extension with the V-gene primer mix. After binding and extension, SPRIselect beads were used to purify the first and second strand synthesis products.
  • the final constructed library included Illumina dual index sequencing adapters, a 10-nucleotide unique molecular identifier region, and an 8- nucleotide internal barcode associated with the C-gene primer.
  • Amplified libraries were multiplexed and pooled for sequencing on the Illumina NovaSeq platform with a 500-cycle kit (250 paired-end reads) through a commercial sequencing service lab (Personal Biotechnology Co., Ltd, Shanghai, China).
  • the output of the immune receptor sequence covered within the first framework region through the beginning of the constant region including CDR1, CDR2, and CDR3.
  • sequence reads were de-multiplexed according to barcode sequences at the 5’ end of reads from the constant region. Reads were then trimmed according to their base qualities with a 2-base sliding window. If either quality value in this window was lower than 20, the sequence stretching from the window to the 3’ end was trimmed out from the original read. Trimmed pair-end reads were joined together through overlapping alignment with a modified Needleman-Wunsch algorithm. If paired forward and reverse reads in the overlapping region were not perfectly matched, both forward and reverse reads were thrown out without further consideration.
  • the merged reads were mapped using a Smith- Waterman algorithm to germline V, D, J and C reference sequences downloaded from the IMGT web site (Lefranc, 2003).
  • To define the CDR3 region the position of CDR3 boundaries of reference sequences from the IMGT database was migrated onto reads through mapping results, and the resulting CDR3 regions were extracted and translated into amino acids.
  • the dataset was condensed by the combination of unique molecular identifiers (UMIs) and CDR3 regions to remove incorrect CDR3s introduced by sequencing and amplification. Reads with the same combination of CDR3 and UMI were condensed into one UMI count.
  • UMIs unique molecular identifiers
  • FIG. 3 illustrates a graph of CDR3 node clusters in a control sample, wherein the node size corresponds with expression levels. Node sizes (i.e., expression level) are roughly uniform.
  • FIG. 4 illustrates a graph of CDR3 node clusters in one patient from the cohort, five days after onset of symptoms, again wherein the node size corresponds with expression levels. Compared with the control in FIG. 2, clusters of elevated expression levels are apparent. The most influential CDR3 sequences from the largest clusters were obtained from the cohort of COVID-19 patients and are listed in Tables 1 and 2.

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Abstract

La présente invention concerne des anticorps monoclonaux anti-SARS-CoV-2 isolés ou non naturels. En particulier, la présente invention concerne un anticorps monoclonal isolé ou non naturel comprenant une région variable de chaîne légère comprenant une séquence d'acides aminés choisie dans le groupe constitué par les SEQ ID NOS : 1-17 et une région variable de chaîne lourde comprenant une séquence d'acides aminés choisie dans le groupe constitué par les SEQ ID NOS : 18-26. La présente invention concerne également un anticorps monoclonal isolé ou non naturel comprenant une région variable de chaîne lourde comprenant une séquence d'acides aminés choisie dans le groupe constitué par les SEQ ID NOS : 18-76. Les anticorps de l'invention peuvent être utilisés comme agent thérapeutique pour traiter la COVID-19 ou comme outil de diagnostic pour évaluer une infection par la COVID-19 chez un sujet.
PCT/US2020/030770 2020-04-30 2020-04-30 Compositions d'anticorps monoclonaux anti-sras-cov-2 WO2021221668A1 (fr)

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WO2023138658A1 (fr) * 2022-01-24 2023-07-27 卡瑞济(北京)生命科技有限公司 Récepteur de lymphocytes t spécifique de 2019-ncov et son utilisation
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CN115975019B (zh) * 2022-09-30 2024-02-02 中国医学科学院病原生物学研究所 一种抗新型冠状病毒的抗体及其用途

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