WO2021236937A2 - Dosages pour détecter des composés qui se lient à la protéine de spicule du sras-cov-2 - Google Patents

Dosages pour détecter des composés qui se lient à la protéine de spicule du sras-cov-2 Download PDF

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WO2021236937A2
WO2021236937A2 PCT/US2021/033420 US2021033420W WO2021236937A2 WO 2021236937 A2 WO2021236937 A2 WO 2021236937A2 US 2021033420 W US2021033420 W US 2021033420W WO 2021236937 A2 WO2021236937 A2 WO 2021236937A2
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assay
cov
sars
fragment
spike protein
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WO2021236937A3 (fr
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Stephen Edward Paucha SMITH
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Seattle Children's Hospital D/B/A Seattle Children's Research Institute
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • C07K2317/70—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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number

Definitions

  • the assay is compatible with high-throughput screening systems.
  • SARS-CoV-2 spike glycoprotein is its viral surface protein that mediates host (e.g., human) cell entry. This spike protein has a trimeric structure with either none or one of three receptor binding domains (RBDs) in the “up” state, capable of binding to its target.
  • RBDs receptor binding domains
  • the SARS-CoV-2 spike glycoprotein’s target protein on host cells is angiotensin converting enzyme 2 (ACE2). Binding of the SARS-CoV-2 spike protein to ACE2 is necessary and sufficient for infection of the target cell. While knowledge regarding SARS-CoV-2 is expanding rapidly, a need to identify compounds that inhibit binding between the SARS-CoV-2 spike protein and ACE2 is needed. Such compounds could provide valuable prophylactic and/or therapeutic treatments against COVID19. SUMMARY OF THE DISCLOSURE [0004] The current disclosure provides microparticle-based vitro assays that detect antibodies that bind the SARS-CoV-2 spike protein and that detect binding between the SARS-CoV-2 spike protein and ACE2.
  • the disclosed assays can detect compounds that interfere with SARS-CoV-2 spike protein and ACE2 binding and can quantify the degree of interference.
  • the assays can be used for many purposes including, for example, assessing functional immunity in recovered patients, screening plasma donations for potency in the context of convalescent plasma therapy, screening drug libraries for compounds that inhibit binding of SARS-CoV-2 to ACE2, and assessing mutant spike proteins to test existing immunity against potentially emerging viral strains.
  • the assay does not require biosafety containment (i.e., can be safely run at biosafety level 0) and can be practiced in a high throughput manner.
  • FIGs. 1A-1D Graphical depictions of particle-based assays.
  • (1A) Ig-detection based assay. 5 ⁇ m immunoprecipitation detected by flow cytometry (IP-FCM) beads are coated with COVID-spike protein (triangles). Beads are then incubated with human serum, and antibodies within the serum (IgG) bind the spike protein.
  • IP-FCM flow cytometry
  • Phycoerythrin (PE)-labeled anti-human-IgG or -IgM antigen binding fragment (FAB) fragments are used to detect human antibodies binding the spike protein.
  • PE Phycoerythrin
  • FAB antigen binding fragment
  • FIGs. 3A-3E Detection of anti-SARS-CoV-2 IgG in serum from recovered COVID19 patients.
  • FIGs. 4A-4E Development of an in vitro Trimer-Spike binding assay.
  • (4A) ACE2- conjugated CML beads were mixed with increasing dilutions of biotinylated Trimer (left) or RBD (right) and detected with streptavidin-PE ( histograms). Soluable, unlabeled ACE2 was added to inhibit binding to establish specificity ( histograms), which inhibited binding by >90%.
  • COVID+ plasma samples inhibit Ace2-Spike interaction.
  • 5A Compared to a no-serum control ( ), pre-COVID control serum significantly inhibited Trimer-ACE2 binding (p ⁇ 0.05), but COVID+ serum inhibited binding to a much greater degree (P ⁇ 0.001 vs control serum and no serum).
  • 5B Same data as in 5A expressed as % inhibition vs the no-serum control average. An arbitrary cut-off of 85% inhibition captures all IgG-positive COVID samples. P ⁇ 0.001.
  • 5C Compared to a no-serum control ( ), pre-COVID control serum slightly and non-significantly inhibited RBD-ACE2 binding.
  • COVID+ serum inhibited in some samples, such that the group mean was significantly different from both pre-COVID and no serum controls (P ⁇ 0.005 vs control serum and p ⁇ 0.01 vs no serum). However, the population appeared to bifurcate into a responder and non-responder population (dotted line at MFI 20,000).
  • 5D Same data as in 5C expressed as % inhibition vs the no-serum control average. P ⁇ 0.005.
  • 5E If the COVID+ population is split by the dashed lines in 5C and 5D, the non-responder population is not significantly different from pre-COVID or no serum controls, while the responder population is (P ⁇ 0.001). [0011] FIGs.6A, 6B.
  • trimer-conjugated beads prevents inhibition of Ace2 binding.
  • (6B) RBD-ACE2 inhibition from undepleted serum or serum depleted with BSA-coated beads is not significantly different.
  • FIGs.7A-7C Trimer IgG levels correlate with inhibition, while RBD IgG levels do not.
  • 7A Trimer MFI plotted against % inhibition on the Trimer-ACE2 inhibition assay for all COVID+ samples.
  • 7B RBD MFI plotted against % inhibition on the RBD-ACE2 inhibition assay for all COVID+ samples.
  • 7C % inhibition on the Trimer-ACE2 inhibition assay plotted against % inhibition on the RBD-ACE2 inhibition assay.
  • IP-FCM Immunoprecipitation detected by flow cytometry
  • ELISA sandwich enzyme-linked immunosorbent assays
  • microparticle-based assays disclosed herein include a plurality of microparticles coated with a capture protein, wherein the capture protein is a form of the SARS- CoV-2 spike protein or ACE2.
  • the protein of interest (capture protein or target molecule) an antibody that binds the SARS-CoV-2 spike protein or ACE2.
  • the capture protein is ACE2
  • the protein of interest is a form of the SARS-CoV-2 spike.
  • the capture protein-conjugated microparticles are incubated with a sample that can include the target molecule and a detection molecule that specifically binds to the target molecule.
  • the detection molecule is labeled with a detectable label.
  • microparticle-based assays include a plurality of microparticles coated with a capture protein that specifically binds to a target molecule.
  • the capture protein- conjugated microparticles are incubated with a labeled target molecule. Additional substances may be added to the reaction that may or may not inhibit the binding of the labeled target molecule to the capture protein-coated microparticle.
  • a sandwiched configuration is formed in which the capture protein-conjugated microparticle is bound to the labeled target molecule which is also bound by a detectable label. This configuration may or may not be prevented from forming by the additional substances added. For a depiction of this type of assay, see FIG. 1C.
  • a sample containing a potential binding inhibitor can be included in the mixture with the capture protein-conjugated microparticles, and the labeled target molecule.
  • a decrease in observed binding based on the presence of the sample containing the potential binding inhibitor indicates the presence of an actual binding inhibitor (e.g., effective antibody, protein, peptide, nucleic acid, or small molecule drug).
  • an actual binding inhibitor e.g., effective antibody, protein, peptide, nucleic acid, or small molecule drug.
  • Other protein, peptide, nucleic acid, and/or small molecule drug libraries can also be screened.
  • the capture protein-conjugated microparticles, samples, labeled target molecules, and/or detection molecules e.g., antibody, tag binder-detectable label conjugate (e.g., streptavidin-PE)
  • the capture protein-conjugated microparticles and the sample can be mixed and incubated first.
  • Microparticles refer to small discrete particles including microbeads, nanobeads, nanoshells, or nanodots. Microparticles can include, for example, latex beads, polystyrene beads, fluorescent beads, and/or colored beads, and can be made from organic matter and/or inorganic matter. They can be made of any suitable materials that allow for the conjugation of capture proteins to their surface. Examples of suitable materials include: ceramics, glass, polymers, and magnetic materials.
  • Suitable polymers include polystyrene, poly-(methyl methacrylate), poly- (lactic acid), (poly-(lactic-co -glycolic acid)), polyesters, polyethers, polyolef ⁇ ns, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon cross- linked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Additional examples include carbon graphited, titanium dioxide, and paramagnetic materials.
  • microparticles can be made of one or more materials.
  • microparticles are paramagnetic microparticles.
  • Particular embodiments utilize carboxy-modified polystyrene latex (CML) flow cytometry beads and/or magnetic MagPlex® (Luminex, Austin, TX) flow cytometry beads.
  • CML carboxy-modified polystyrene latex
  • MagPlex® Luminex, Austin, TX flow cytometry beads.
  • microparticles used within an assay are homogeneous in size and absorbing ability.
  • the microparticles are solid and insoluble in a sample of interest to facilitate separation from the sample.
  • the microparticle material is inert to components in the sample and the reagent.
  • the microparticle can range in size from having a radius or diameter of from, for example, 20 nm to 1000 ⁇ m, 200 nm to 200 ⁇ m, or 1.0 ⁇ m to 10 ⁇ m.
  • the capture protein is conjugated to the surface of the microparticle by any method known in the art.
  • the capture protein is conjugated to the surface of the microparticle according to the chemistry of the microparticle’s surface.
  • the capture protein can be conjugated to the surface of the microparticle using adsorption or covalent attachment.
  • the capture protein is covalently attached to the surface of the microparticle using amine-reactive coupling, sulfhydryl-reactive coupling, carbonyl-reactive coupling, active hydrogen immobilization, and/or the Mannich reaction.
  • Amine-reactive coupling includes NHS-ester reactive groups formed by EDC, reductive amination (aldehyde coupling), azlactone ring reactivity, and carbonyl diimidazole (CDI) to activate hydroxyls.
  • Sulfhydryl-reactive coupling includes maleimide-activated supports, iodoacetyl-activated supports, and pyridyl disulfide supports.
  • Carbonyl-reactive coupling includes hydrazide-activated supports, carboxyl-reactive supports, and carbodiimide (EDC)-mediated crosslinking.
  • beads e.g., 50 ⁇ L of CML beads (Invitrogen #C37255, USA) or 250ul of MagPlex Microspheres (Luminex #MC100XX-01, USA)
  • EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl; Pierce, USA
  • the beads can then be washed with PBS.
  • the activated bead pellet can be resuspended in a solution containing a capture protein (e.g., 25 ⁇ g of the receptor binding domain (RBD) fragment, Trimer or ACE2 protein in PBS) and gently mixed. Coupled beads can then be washed (e.g., in PBS) and stored for later use.
  • a capture protein e.g., 25 ⁇ g of the receptor binding domain (RBD) fragment, Trimer or ACE2 protein in PBS
  • Coupled beads can then be washed (e.g., in PBS) and stored for later use.
  • capture proteins within assays disclosed herein can include ACE2 and/or a SARS-CoV-2 spike protein. Particular embodiments utilize a full-length trimeric form of the spike protein.
  • the reference sequence can be the sequence derived from the first virus isolate, Wuhan- Hu-1, released on January 10, 2020 (Wu et al., A new coronavirus associated with human respiratory disease in China. Nature (2020). See also GenBank: MN908947.3. Additional embodiments can utilize a modified and/or stabilized version of the full-length spike protein. Stabilizing mutations can include K986P and V987P according to wild type numbering. In certain examples, at amino acid P1213, the sequence can be fused to a thrombin cleavage site, a T4 foldon sequence for proper trimerization can be utilized, and a C-terminal histidine tag can be used for purification.
  • DMS variants include a complete set of possible protein variants, with 19 possible amino acid substitutions at each amino acid position.
  • DMS variants include all possible amino acids at less than all positions of a protein, for example at 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of positions.
  • DMS libraries can be synthetically constructed by and/or obtained from a synthetic DNA company such as Twist Bioscience (San Francisco, CA).
  • methods to generate a codon-DMS library include: polymerase chain reaction (PCR) mutagenesis (Dingens et al. Cell Host and Microbe. 2017;21(6):777-787; 1968ns et al. Immunity. 2019 Jan 29); nicking mutagenesis as described in Wrenbeck et al. (Nature Methods 13: 928-930, 2016) and Wrenbeck et al.
  • PCR polymerase chain reaction
  • test samples can be used to test for compounds that inhibit binding between the SARS-CoV-2 spike protein and hACE2.
  • the test samples include human serum or plasma. Human serum or plasma samples can be heat-inactivated (e.g., for 30 min.
  • conjugate binding interactions are used for detection of binding.
  • Conjugate binding interactions include at least two elements with high affinity and fidelity molecular recognition to each other such as in protein/ligand, antigen/antibody, sugar/lectin, and RNA/ribosome interactions.
  • a particularly useful example of such an interaction pair is the biotin- avidin (or streptavidin) system.
  • conjugate binding pairs include a tag and a tag binder, such as biotin and streptavidin, biotin and avidin, glutathione and glutathione S- transferase, maltose and maltose-binding protein, chitin and chitin-binding protein, fluorescein isothiocyanate (FITC) and anti-FITC, or a protein and its antibody.
  • the tag binder is linked to a detectable label.
  • Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • detectable labels include fluorochrome, fluorescent protein, a chromophore, an enzyme, a linker molecule, a biotin molecule, an electron donor, an electron acceptor, a dye, a metal, or a radionuclide.
  • the tag includes biotin.
  • the tag binder includes streptavidin.
  • the streptavidin is linked to phycoerythrin (PE) to form the tag binder-detectable label conjugate, streptavidin-PE.
  • tags include His tag, Flag tags, Xpress tag, Avi tag, Calmodulin binding peptide (CBP) tag, Polyglutamate tag, HA tags, Myc tag, Strep tag (which refers to the original STREP ® tag, STREP ® tag II (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1, Softag 3, and V5.
  • Associated tag binders are commercially available.
  • His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript.
  • Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich.
  • Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies, and GenScript.
  • Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia.
  • Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abcam, and Pierce Antibodies.
  • HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal, and Abcam.
  • Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abcam, and Cell Signal.
  • Strep tag antibodies are commercially available from suppliers including Abcam, Iba, and Qiagen.
  • a detection antibody or other detection molecule ACE2, or a SARS-CoV-2 spike protein (RBD or Trimer) is conjugated to a detectable label.
  • the detectable label includes a fluorochrome. Any art- recognized fluorochrome can be used.
  • the detectable label includes a fluorescent protein.
  • Exemplary fluorescent proteins include blue fluorescent proteins (e.g.
  • eBFP eBFP2, eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire
  • cyan fluorescent proteins e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise
  • green fluorescent proteins e.g.
  • the detectable label includes an enzyme.
  • exemplary enzyme labels include horseradish peroxidase, hydrolases, and alkaline phosphatase.
  • Exemplary fluorescence labels include rhodamine, phycoerythrin, and fluorescein.
  • diluted supernatant plasma can be added into wells of a plate with RBD- or Trimer-conjugated beads. The wells can be capped and the plate can be left to rotate at 4°C, for example overnight. The next day, the plate can be spun to pellet the beads and the supernatant can be discarded.
  • the pelleted beads can be washed and incubated with a test sample that may include antibodies that bind the SARS-CoV-2 spike protein and a detection molecule (e.g., anti-Human IgG-PE (Jackson ImmunoResearch, #709-116-149, lot 145536, USA) or streptavidin-PE (Biolegend, # 405203)) for 30 minutes at room temperature.
  • a detection molecule e.g., anti-Human IgG-PE (Jackson ImmunoResearch, #709-116-149, lot 145536, USA) or streptavidin-PE (Biolegend, # 405203)
  • the sandwiched target molecule within the capture protein- conjugated microparticle and detection label can be detected by any method known in the art.
  • the detection method includes a flow cytometer, microplate reader, Luminex reader, BeadXpress® System (Illumina, Inc., San Diego, CA) or other detection instrument.
  • the detection method includes flow cytometry.
  • Flow cytometry instrumentation and techniques are well-known in the art. In general, flow cytometry relies on the passage of a stream of microparticle suspension through a light beam and electro-optical sensors in such a manner that only one microparticle at a time passes through the beam-sensor region. As each microparticle passes this region, the light beam is perturbed by the microparticle, and the resulting scattered and fluorescent light are detected.
  • the detected optical signals are used by the instrumentation to identify the subgroup to which each microparticle belongs, along with the presence and amount of label, so that individual microparticle results are achieved.
  • the intensity of the detectable label emitted from the detectable label on the microparticles upon irradiation of the fluorochrome with an excitation light is determined. If the intensity of the florescent emitted from the fluorochrome on one of the microparticles is above a predetermined value (i.e., a threshold or cutoff value), the test sample is determined to contain the target molecule.
  • a predetermined or threshold value can be obtained by various suitable methods.
  • RBD- Trimer or BSA-coupled beads can be read on an Acea Novocyte flow cytometer with the following gating strategy: gate beads using FSC-H vs SSC-H, eliminate doublets using FSC-H vs FSC-A, and detect PE fluorescence using FL2 (488nm excitation, 572/28nm detection).
  • Background-subtracted MFI can be calculated by subtracting the BSA-coupled bead MFI from the RBD- or trimer-coupled bead MFI.
  • ACE2, RBD or trimer protein can be tagged.
  • the tag is biotin.
  • biotinylated RBD or Trimer protein can be added to ACE2-coupled beads in a microwell plate.
  • soluble unlabeled ACE2 or diluted plasma samples or other potential inhibitors of binding can be included in the incubation.
  • Each well of the plate can be capped and mixed end- over-end at 4 °C overnight.
  • Antibodies or binding fragments thereof can be used within embodiments of the disclosure.
  • Antibody binding fragments refer to at least one portion of an antibody, that retains the ability to specifically bind an antigen.
  • antibody fragments include Fab, Fab′, F(ab′) 2 , Fv fragments, single chain variable (scFv) fragments, disulfide-linked Fvs (sdFv), a Fd fragment including VH and constant CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid variable heavy only (VHH) domains, and an isolated CDR or other epitope binding fragments of an antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc.
  • the microparticle-based assay is a singleplex assay.
  • the microparticle-based assay is a multiplex assay.
  • the microparticle-based assay is high-throughput.
  • high-throughput refers to the ability to rapidly process multiple test samples, for example, arrays or microarrays, in an automated and/or massively parallel manner.
  • multiplex refers to the concurrent performance of multiple experiments on a single device or in a single assay.
  • a multiplex assay using an array allows the simultaneous detection and/or measurement of a plurality of different potential spike protein variants and/or potential binding inhibitors.
  • a multiplex assay includes microparticles having distinguishing features to identify the class of microparticle so that a mixture of multiple sets of distinguishable microparticles is included in the assay.
  • the microparticles are distinguishable according to their fluorescent emission spectrum, size, bar code, or other reporting entities.
  • the microparticles are distinguishable according to their size. Microparticles of different sizes generate different forward light scatters (FSC), which can be picked up and recorded by flow cytometry.
  • FSC forward light scatters
  • the multiplex assay includes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more classes of microparticle.
  • the multiplex assay includes 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more classes of microparticle.
  • High throughput multiplexed bead-based assays are advantageous because they can be run in a short time period (e.g., less than 3 hours) and require little test antigen. Further, multiple test compounds can be assessed simultaneously. Additionally, pre-coated beads can be formed and stored for later use, increasing the efficiency of testing procedures. For example, particular embodiments include coupling beads to COV-2 RBD and/or COV-2 trimer and storing them for later use at 4oC.
  • coupled beads can be washed and added to wells including a tag binder- detectable label conjugate, tagged hACE2, and optionally, one or more test compounds. Wells can incubate for 1 hr followed by detection of binding based on the detectable label using flow cytometry.
  • coupled beads can be washed and added to wells including a test sample that may include an antibody that binds the COV-2 RBD and/or COV-2 trimer. Wells can incubate for 1 hr followed by addition of a detectable label conjugated to anti-IG antibody.
  • the microparticles are distinguishable according to their fluorescent emission spectrum.
  • the microparticles of each class have a fluorescent emission spectrum that is different from the emission spectra of the other microparticle classes.
  • each different class of microparticle is pre- coated with a reagent that is specific for a different capture protein of interest, and thus different target proteins are captured from the sample to different classes of microparticles.
  • microparticles can be coupled to the trimeric spike protein, variants of the spike protein (e.g., deep mutational scanning variants), and/or truncated RBD segments of the spike protein.
  • Different classes of microparticles could also be coupled different ACE2 variants.
  • the target proteins are then labeled with a detection antibody with a detectable label as described previously.
  • the detectable label includes a fluorochrome which has an emission different from any fluorescent emissions by the microparticles.
  • the assay is read in a flow cytometer or similar instrument: each microparticle is identified as a member of a particular class, e.g., on the basis of its fluorescent emission spectrum, and whether that microparticle has the target protein captured on it is determined, e.g., by detecting the presence or absence of detectable label on the microparticle. Since the relationship between a particular microparticle class and a particular target protein is predetermined by the choice of capture protein used to coat those particles, the presence of detectable label on a given particle is indicative of the presence of a given target protein in the original sample even when a single detectable label is used to label all the analytes. See, e.g., U.S. Pat.
  • the multiplex Luminex assay format differs from conventional enzyme-linked immunosorbent assay (ELISA) in that the multiplex capture antibody is attached to a polystyrene bead whereas the ELISA capture antibody is attached to the microplate well.
  • the use of the suspension bead-based technology enables the multiplexing capabilities of the Luminex assays.
  • the xMAP® technology uses 5.6 micron polystyrene microbeads, which are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number, or bead region, allowing differentiation of one bead from another.
  • Beads covalently bound to different specific antibodies can be mixed in the same assay, utilizing a 96- well microplate format.
  • beads can be read, using the Luminex 100TM or Luminex 200 detection system, in single-file by dual lasers for classification and quantification of each analyte.
  • Particular embodiments include use of a microbead-based assay wherein fluorochrome- labeled beads coupled to the COV-2 RBD and/or COV-2 trimer are incubated with tagged hACE2, a tag binder-detectable label conjugate, and test compounds.
  • Readouts can be obtained using an analyzer, such as a laser-based instrument (e.g., a Luminex® analyzer).
  • Ace2 is directly conjugated to microparticles using a covalent coupling reaction.
  • a tagged SARS-CoV-2 spike “probe” is added to the Ace2-bead/analyte mixture.
  • a tag binder-detectable label conjugate is added, which binds to the tag on the spike and produces fluorescence if binding between Ace2 and the SARS-CoV-2 spike probe is present.
  • one embodiment of the current disclosure provides coupling human ACE2 to microparticles (e.g., microbeads) and tagging the COV-2 receptor binding domain (RBD) and/or the COV-2 trimer.
  • microparticles e.g., microbeads
  • RBD COV-2 receptor binding domain
  • COV-2 trimer tagging the COV-2 receptor binding domain
  • binding between the ACE2-coupled microparticles and the COV-2 RBD and/or COV-2 trimer can be quantified using flow cytometry to set a baseline.
  • Experimental conditions can further include a test compound to determine if the test compound inhibits binding between ACE2-coupled microparticles and the tagged COV-2 RBD and/or COV-2 trimer as evidenced by a decreased flow cytometry signal from baseline.
  • the test compound can include serum or plasma from subjects, including human subjects before infection with COVID-19, during active infection with COVID-19, and following recovery from infection with COVID-19.
  • the assay may detect antibodies for potential therapeutic use when the test compound is from subjects following recovery from infection with COVID-19.
  • the test compound can also include proteins, nucleotides, and/or or small molecules selected for screening as potential therapeutic or prophylactic compounds. [0048] Exemplary Embodiments. 1.
  • a microparticle-based assay to: identify compounds that inhibit binding between human angiotensin-converting enzyme 2 (hACE2) and severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) spike protein or a variant or fragment thereof, the microparticle-based assay including microparticles coupled to hACE2 or microparticles coupled to the SARS-CoV-2 spike protein or a variant or fragment thereof. 2.
  • hACE2 human angiotensin-converting enzyme 2
  • SARS-CoV-2 severe acute respiratory syndrome corona virus 2
  • the SARS-CoV-2 spike protein or variant or fragment thereof is tagged with biotin, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin binding peptide (CBP) tag, Polyglutamate tag, HA tag, Myc tag, or Strep tag. 4.
  • tag binder-detectable label conjugate includes streptavidin-PE or a binding molecule that binds His tag, Flag tag, Xpress tag, Avi tag, Calmodulin binding peptide (CBP) tag, Polyglutamate tag, HA tag, Myc tag, or Strep tag bound to a detectable label. 5.
  • the hACE2 is tagged with biotin, His tag, Flag tag, Xpress tag, Avi tag, Calmodulin binding peptide (CBP) tag, Polyglutamate tag, HA tag, Myc tag, or Strep tag. 7.
  • tag binder-detectable label conjugate includes streptavidin-PE or a binding molecule that binds His tag, Flag tag, Xpress tag, Avi tag, Calmodulin binding peptide (CBP) tag, Polyglutamate tag, HA tag, Myc tag, or Strep tag bound to a detectable label.
  • microparticles within the assay are coupled to the SARS-CoV-2 spike protein or a variant or fragment thereof and the assay identifies antibodies that bind the SARS-CoV-2 spike protein or a variant or fragment thereof and wherein the assay further includes an anti-immunoglobulin (Ig) antibody or binding fragment thereof linked to a detectable label.
  • Ig anti-immunoglobulin
  • the anti-Ig antibody or binding fragment thereof is an anti-IgG or anti-IgM antibody or binding fragment thereof.
  • the fragment of the SARS-CoV-2 spike protein is the receptor binding domain (RBD) fragment.
  • the stabilized variant includes K986P and V987P mutations according to wild type numbering.
  • the microparticles include beads. 16.
  • embodiment 21 wherein the high throughput manner simultaneously tests at least 50, at least 100, at least 500, or at least 1000 samples for: the presence of compounds that bind the SARS-CoV-2 spike protein or a variant or fragment thereof and/or that inhibit binding between the SARS-CoV-2 spike protein or a variant or fragment thereof and hACE2.
  • the samples include human serum or plasma samples or candidate prophylactic and/or therapeutic drug compounds.
  • the human serum or plasma samples are heat- inactivated.
  • the candidate prophylactic and/or therapeutic drug compounds include proteins, peptides, nucleic acids, and/or small molecules.
  • embodiment 25 wherein the proteins include antibodies or binding fragments thereof.
  • embodiment 26 wherein the antibodies or binding fragments thereof are recombinantly produced and/or engineered.
  • 28 The use of embodiment 27, wherein the engineered antibody includes multiple binding domains.
  • 29 Use of the assays of any of embodiments 2-19, including co-incubating the microparticles, the tagged SARS-CoV-2 spike protein or variant or fragment thereof, and a test sample potentially including a compound that inhibits binding between the hACE2 coupled to the microparticles and the tagged SARS-CoV-2 spike protein or variant or fragment thereof. 30.
  • embodiment 29 further including co-incubating tag binder-detectable label conjugate and detecting bound tagged SARS-CoV-2 spike protein or variant or fragment thereof.
  • 31 The use of embodiment 30, wherein the tagged SARS-CoV-2 spike protein or variant or fragment thereof is a biotinylated SARS-CoV-2 spike protein or variant or fragment thereof.
  • 32. The use of embodiment 31, wherein the tag binder-detectable label conjugate includes streptavidin-PE. 33.
  • any of embodiments 5-19 including co-incubating the microparticles, the tagged hACE2, and a test sample potentially including a compound that inhibits binding between the SARS-CoV-2 spike protein or variant or fragment thereof coupled to the microparticles and the tagged hACE2.
  • a test sample potentially including a compound that inhibits binding between the SARS-CoV-2 spike protein or variant or fragment thereof coupled to the microparticles and the tagged hACE2.
  • embodiment 43 further including co-incubating tag binder-detectable label conjugate and detecting bound tagged hACE2.
  • 35 wherein the tagged hACE2 is biotinylated hACE2.
  • the tag binder-detectable label conjugate includes streptavidin-PE. 37.
  • any of embodiments 30-36 wherein the detecting includes detecting a median fluorescent intensity (MFI) signal indicative of the amount of binding between hACE2 and the SARS-CoV-2 spike protein or variant or fragment thereof following the incubating.
  • MFI median fluorescent intensity
  • embodiment 37 further including detecting the anti-immunoglobulin (Ig) antibody or binding fragment thereof bound to the microparticles through the SARS-CoV-2 spike protein or variant or fragment thereof.
  • the detecting includes detecting a median fluorescent intensity (MFI) signal indicative of the amount of binding between the anti-immunoglobulin (Ig) antibody or binding fragment thereof and an antibody bound to the microparticle through the SARS-CoV-2 spike protein or variant or fragment thereof.
  • MFI median fluorescent intensity
  • SARS-CoV-2 severe acute respiratory syndrome corona virus 2
  • hACE2 human angiotensin- converting enzyme 2
  • RBD receptor binding domain
  • biotinylated variant of the SARS-CoV-2 spike protein is a stabilized biotinylated variant.
  • biotinylated variant of the SARS-CoV-2 spike protein is a stabilized biotinylated variant.
  • the stabilized biotinylated variant includes K986P and V987P mutations according to wild type numbering.
  • biotinylated variant of the SARS-CoV-2 spike protein is an alanine scanning biotinylated variant or a deep mutational scanning biotinylated variant.
  • the bead-based assay of embodiment 68 where different classes of beads within the multiplex assay are coupled to the full-length SARS-CoV-2 spike protein versus a fragment of the SARS-CoV-2 spike protein.
  • 70. Use of a bead-based assay of any of embodiments 42-69 to assess functional immunity in recovered COVID19 patients, screen plasma donations for potency in the context of convalescent plasma therapy, screen drug libraries for compounds that inhibit binding of SARS-CoV-2 to hACE2, or assess mutant spike proteins to test existing immunity against potentially emerging viral strains.
  • 71 The use of embodiment 70, practiced in a high throughput manner. 72.
  • embodiment 71 wherein the high throughput manner simultaneously tests at least 50, at least 100, at least 500, or at least 1000 samples for the presence of compounds that inhibit binding between SARS-CoV-2 to hACE2.
  • the samples include human serum or plasma samples or candidate prophylactic and/or therapeutic drug compounds.
  • the human serum or plasma samples are heat- inactivated.
  • the candidate prophylactic and/or therapeutic drug compounds include proteins, peptides, nucleic acids, and/or small molecules.
  • embodiment 75 wherein the proteins include antibodies or binding fragments thereof. 77.
  • IP-FCM assays were designed to detect the presence of anti- COVID antibodies in human serological samples by covalently coupling either the recombinant RBD fragment or a spike trimer construct to carboxy-modified polystyrene latex microbeads (CML).
  • FIG.4A shows strong binding of both RBD and Trimer to ACE2 beads, which is inhibited by unlabeled ACE2.
  • the serum of one precovid control and three COVID+ samples (2 RBD-“responders”, one RBD “non-responder”) were depleted by incubating them with large numbers of Trimer-conjugated beads for three consecutive overnight incubations until the level of IgG detected on the beads plateaued at a background level, or with bovine-serum-albumin (BSA)-coated beads as a negative control.
  • BSA bovine-serum-albumin
  • CML beads Invitrogen #C37255, USA
  • MagPlex Microspheres Luminex #MC100XX-01, USA
  • 50 ⁇ L of CML beads Invitrogen #C37255, USA
  • 250ul of MagPlex Microspheres Luminex #MC100XX-01, USA
  • 500 uL of room temperature MES 50 mM MES (2-(N- morpholino)ethanesulfonic acid), pH 6.0, 1 mM EDTA, in ddH20, stored at 4 ⁇ C, used at RT.
  • Beads were spun down for 1 minute at 13,000 x g at 4 °C and supernatant discarded after each wash.
  • the CML beads were then resuspended in 50uL of MES and activated with 20 ⁇ L of 50mg/mL EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl; Pierce, USA), freshly dissolved in MES from powder stored at -20 ⁇ C.
  • EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl
  • Pierce, USA freshly dissolved in MES from powder stored at -20 ⁇ C.
  • This 70 ⁇ L of CML bead, MES, and EDAC was mixed for 15 minutes by continuously pipetting gently up and down at room temperature.
  • the beads were washed three times with 0.5 mL of PBS.
  • the activated bead pellet was resuspended in a 100 ⁇ L solution containing 25 ⁇ g of either RBD, Trimer or ACE2 protein in PBS and gently mixed for 3 hours at room temperature at 1400 rpm on a pulsing vortexer.
  • the coupled beads were then washed 3 times in 0.5 mL of PBS and stored in 100 uL of Blocking Storage Solution (1% BSA in PBS, 0.01% Sodium Azide) at 4 ⁇ C until use.
  • Successful coupling was confirmed by staining 0.5 uL coupled beads with PE-conjugated anti-HIS tag antibodies (Santa Cruz) and compared to positive and negative controls by flow cytometry.
  • IP-FCM was performed as described in Smith et al., PLoS One 2012;7(9):e45722 (doi: 10.1371/journal.pone.0045722).
  • Patient plasma from both SARS- CoV-2 positive and negative patients was heated to 56°C for 1 hour, then spun at 13,000 x g for 10 minutes at 4°C.
  • the supernatant plasma was then diluted (typically at 1:1000) in cold Fly-P Buffer (50 mM Tris pH 7.4, 100 mM NaCl, 1% bovine serum albumin, and 0.01% sodium azide) and distributed into wells of a 96-well plate at a volume of 50 ⁇ L per well, in duplicate.
  • the plate was washed 2 more times with 125 ⁇ L of ice-cold Fly-P Buffer and the samples were resuspended in 50 ⁇ L of cold Fly-P Buffer. All plasma samples were also run in parallel using BSA-coupled CML beads to determine the baseline nonspecific signal generated from each sample, which varied greatly between individuals from 10 3 -10 5 MFI units, but was consistent for each individual. RBD- Trimer or BSA-coupled beads were then read on an Acea Novocyte flow cytometer with the following gating strategy: gate beads using FSC-H vs SSC-H, eliminate doublets using FSC-H vs FSC-A, and detect PE fluorescence using FL2 (488nm excitation, 572/28nm detection).
  • ACE2-Spike binding assay RBD or trimer protein was biotinylated by adding 1ul of freshly- dissolved sulfo-NHS-Biotin (ThermoScientific, #21217, USA) for 30 minutes on ice. The reaction was quenched with TRIS-HCL and excess biotin removed by three PBS washes in a 10K MWCO Amicon spin filter (Millipore).
  • Biotinylated RBD or Trimer protein was added to 5 x 10 4 ACE2- coupled CML beads in a total volume of 50 ⁇ L, in duplicate.
  • soluble unlabeled ACE2 or diluted plasma samples prepared as above were included in the incubation, maintaining a final reaction volume of 50uL.
  • Plasma was diluted 1:50 in FlyP buffer unless otherwise indicated.
  • Each well of the plate was capped mixed end-over-end at 4 °C overnight. The next day the plate was washed twice with 125 ⁇ L of cold Fly-P Buffer and incubated with 50 ⁇ L of 1:200 Streptavidin conjugated-PE (BioLegend, #405204, USA) in Fly-P Buffer protected from light for 30 minutes at room temperature.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).
  • Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser,
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.
  • amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
  • Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
  • “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity” also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences.
  • GCG Genetics Computer Group
  • BLASTP BLASTN
  • BLASTX Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin)
  • FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y..
  • variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence.
  • Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M -1 , while not significantly associating with any other molecules or components in a relevant environment sample.
  • Ka i.e., an equilibrium association constant of a particular binding interaction with units of 1/M
  • Binding domains may be classified as “high affinity” or “low affinity”.
  • “high affinity” binding domains refer to those binding domains with a Ka of at least 10 7 M-1, at least 10 8 M-1, at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 .
  • "low affinity" binding domains refer to those binding domains with a Ka of up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 .
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 -5 M to 10 -13 M).
  • Kd equilibrium dissociation constant
  • a binding domain may have "enhanced affinity,” which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain.
  • Ka Equilibrium association constant
  • Kd dissociation constant
  • Koff off- rate
  • the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of” excludes any element, step, ingredient or component not specified.
  • the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.
  • each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e.

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Abstract

L'invention décrit des dosages in vitro à base de microparticules qui détectent des anticorps qui se lient à la protéine de spicule du SARS-CoV-2 et qui détectent la liaison entre la protéine de spicule du SARS-CoV-2 et l'enzyme de conversion de l'angiotensine humaine 2 (hACE2). Les dosages peuvent quantifier l'inhibition d'une telle liaison par l'un ou l'autre du sérum/plasma humain ou de candidats médicaments. Le dosage est compatible avec des systèmes de criblage à haut débit.
PCT/US2021/033420 2020-05-20 2021-05-20 Dosages pour détecter des composés qui se lient à la protéine de spicule du sras-cov-2 WO2021236937A2 (fr)

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