WO2022150660A1 - Antigen-binding polypeptides specific for coronaviruses and uses thereof - Google Patents

Antigen-binding polypeptides specific for coronaviruses and uses thereof Download PDF

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Publication number
WO2022150660A1
WO2022150660A1 PCT/US2022/011721 US2022011721W WO2022150660A1 WO 2022150660 A1 WO2022150660 A1 WO 2022150660A1 US 2022011721 W US2022011721 W US 2022011721W WO 2022150660 A1 WO2022150660 A1 WO 2022150660A1
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Prior art keywords
antigen
antibody
txg
binding fragment
amino acid
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PCT/US2022/011721
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French (fr)
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WO2022150660A9 (en
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Wyatt James MCDONNELL
Bruce Alexander ADAMS
Michael John Terry STUBBINGTON
David Benjamin JAFFE
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10X Genomics, Inc.
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Publication of WO2022150660A1 publication Critical patent/WO2022150660A1/en
Publication of WO2022150660A9 publication Critical patent/WO2022150660A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 500 nM, less than 120 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 5 nM, less than 1 nM, or less than 1 pM. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM.
  • KD equilibrium dissociation constant
  • FIG. 8 illustrates another example of a barcode carrying bead.
  • compositions and methods of the diclosure can be useful for the identification and characterization of antibodies having specific binding affinity (e.g., ability to bind, with varying degrees of specificity) to different epitopes on a coronavirus target, e.g., a spike protein.
  • Antibodies having binding affinity for different epitopes on the target protein, e.g., a SARS-2 spike protein can advantageously be used in a therapeutic antibody cocktail or combination therapy regimen.
  • sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul etal, J Mol Biol (1990) 215:403).
  • sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.
  • the antibodies and antigen-binding fragments thereof of the disclosure include the light chain CDRs (LCDR1, LCDR2, and LCDR3) from the antibodies belonging to the same clonotype family, for example, from a clonotype family selected from the group consisting of clonotype families A, B, and C. Accordingly, in some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 11, 14, and 17, respectively.
  • the LCDR3 amino acid sequence is at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443- 522.
  • the LCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 16-18 and SEQ ID NOS: 443-522, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.
  • the LCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522.
  • Non-conservative substitutions when used in reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution ( e.g ., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain.
  • the antibody or antigen-binding fragment includes: a HCVR and a LCVR which respectively are 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to the HCVR and LCVR of an antibody of Table 1.
  • the antibody or antigen-binding fragment includes the HCVR and LCVR of an antibody of Table 1.
  • the antibody or antigen-binding fragment of the disclosure is selected from Table 1.
  • the L452 amino acid substitution is a non-conservative amino acid substitution.
  • the SARS-CoV-2 S protein includes an amino acid substitution at position E484.
  • the E484 amino acid substitution is a conservative amino acid substitution.
  • the E484 amino acid substitution is a non-conservative amino acid substitution.
  • the amino acid substitution at position E484 is E484K or E484Q.
  • the SARS-CoV-2 S protein includes an amino acid substitution at position N501.
  • the N501 amino acid substitution is a conservative amino acid substitution.
  • the N501 amino acid substitution is a non-conservative amino acid substitution.
  • binding ability e.g., binding affinity and/or specificity
  • ELISAs Western blots
  • RIAs e.g., RI-binding fragments of the present disclosure towards target antigens
  • flow cytometry analysis e.g., flow cytometry analysis.
  • the binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a BiacoreTM system, or KinExA.
  • SPR Surface Plasmon Resonance
  • an antibody or antigen-binding fragment that “specifically binds” a target antigen is an antigen-binding fragment that binds the target antigen but does not significantly bind other antigens.
  • the antibody or antigen-binding fragment “specifically binds” a target antigen if it does not significantly bind other antigens but binds the target antigen with high affinity, e.g., with an equilibrium dissociation constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less.
  • KD equilibrium dissociation constant
  • the antibodies or antigen binding fragments of the disclosure that specifically bind a target antigen such as a CoV-S protein (e.g., SARS-CoV-2 S protein)
  • a target antigen such as a CoV-S protein (e.g., SARS-CoV-2 S protein)
  • a binding affinity to the target antigen expressed as KD of at least about 1 (G 8 M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25° C. or 37°C, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORETM, or by solution-affinity ELISA.
  • the antibody or antigen-binding fragment includes a HCVR which includes an amino acid sequence having at least 100% sequence identity to the HCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG- 0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-
  • the antibody or antigen-binding fragment includes a LCVR which includes an amino acid sequence having at least 100% sequence identity to the LCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG- 0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-
  • the antibody or antigen-binding fragment has a sub nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof.
  • the antibody or antigen binding fragment has a binding affinity with a KD value of less than 500 pM, for example, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM.
  • the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S.
  • Exemplary antibodies having this binding affinity property include TXG-0115, TXG- 0140, TXG-0153, TXG-0154, TXG-0173, and TXG-0174.
  • the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the beta variant.
  • Exemplary antibodies having these binding affinity properties include TXG-0115, TXG-0153, TXG-0154, and TXG- 0174.
  • the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the gamma variant.
  • Exemplary antibodies having these binding affinity properties include TXG-0115, TXG-0153, TXG-0154, TXG-0173, and TXG- 0174.
  • the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S and beta, gamma, kappa variants.
  • Exemplary antibodies having these binding affinity properties include TXG-0115 and TXG-0154. In some embodiments, such antibodies also have binding affinity for the SARS-CoV-2 delta variant.
  • the binding affinity and/or binding specificity of an antigen binding molecule (e.g., antibody or antigen-binding fragment) to a target antigen can be determined based on the counts and/or amounts of target antigens and optionally non-target antigens that are associated with the antigen-binding molecule.
  • the counts and/or amounts of such antigens can be facilitated by the respective reporter oligonucleotides coupled to the antigens, wherein a reporter oligonucleotide coupled to an antigen comprises a reporter barcode sequence that identifies the antigen coupled thereto.
  • the antibodies and antigen-binding fragments of the disclosure have a neutralizing activity ⁇ e.g, antagonistic activity) against SARS-CoV-2, e.g, able to bind to and neutralize the activity of SARS-CoV-S, as determined by in vitro or in vivo assays.
  • a neutralizing activity ⁇ e.g, antagonistic activity
  • the ability of the antibodies of the disclosure to bind to, block and/or neutralize the activity of SARS-CoV-2 may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein.
  • binding affinity and dissociation constants of anti-SARS-CoV-2 antigen-binding polypeptides for SARS-CoV-2 can be determined by surface plasmon resonance (SPR) assay.
  • SPR surface plasmon resonance
  • neutralization assays were used to determine infectivity of SARS-CoV-2 S protein-containing virus-like particles.
  • the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 1 pg/mL or lower, 200 ng/mL or lower, or 40 ng/mL or lower. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 200 ng/mL to 1,000 ng/mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 40 ng/mL to 200 ng/mL.
  • the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 40 ng/mL to 200 ng/mL.
  • IC50 value 40 ng/mL to 200 ng/mL.
  • Exemplary antibodies having this neutralizing activity property include TXG-0115 and TXG-0140.
  • the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 40 ng/mL or lower.
  • exemplary antibodies having this neutralizing activity property include TXG-0154.
  • kits for producing an antibody or antigen-binding fragment thereof include rearing (i) a transgenic animal as disclosed herein, or culturing (ii) a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced.
  • the methods include rearing a transgenic animal as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced in the transgenic animal.
  • the methods include culturing a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced in the recombinant cell.
  • Non-limiting examples of IL-6 pathway target antibodies include Actemra (tocilizumab) (blocks IL-6 receptor) and Sirukumab (anti-IL-6 antibody candidate).
  • the antibody or antigen-binding fragment has a binding specificity to an epitope on a domain of the CoV-S protein.
  • the domain of the CoV-S protein is in the SI domain (i.e., subunit.
  • the domain of the CoV-S protein is the S2 domain (i.e., subunit).
  • the antibody or antigen binding fragment has a binding affinity to a RBD or a NTD of the S 1 subunit.
  • the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein.
  • the antibody or antigen-binding fragment has a sub nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof.
  • the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 500 pM, for example, less than 100 pM, less than 50 pM, less than 10 pM, less than 5 pM, less than 1 pM, less than 0.5 pM.
  • allocating individual particles (e.g., labelled engineered cells) to discrete partitions can, in one non-limiting example, be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non- aqueous fluid, such that droplets are generated at the junction of the two streams.
  • Fluid properties e.g., fluid flow rates, fluid viscosities, etc.
  • particle properties e.g, volume fraction, particle size, particle concentration, etc.
  • microfluidic architectures e.g, channel geometry, etc
  • other parameters can be adjusted to control the occupancy of the resulting partitions (e.g, number of biological particles per partition, number of beads per partition, etc.).
  • the various parameters can be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions.
  • the flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions.
  • the method further includes individually partitioning one or more single cells from a plurality of cells in a partition of a second plurality of partitions.
  • the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above.
  • the use of the systems and methods described herein creates resulting partitions that have multiple occupancy rates of less than 25%, less than 20%, less than 15%), less than 10%, and in some embodiments, less than 5%, while having unoccupied partitions of less than 50%), less than 40%, less than 30%, less than 20%, less than 10%, and in some embodiments, less than 5%.
  • a mixed, but known barcode sequences set can provide greater assurance of identification in the subsequent processing, e.g, by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.
  • a fluid flow unit can comprise compressors (e.g, providing positive pressure), pumps (e.g, providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.
  • the TEMED can diffuse from the second fluid 516 into the aqueous fluid 512 comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets 518, 520, resulting in the formation of gel (e.g ., hydrogel) microcapsules or cell beads, as solid or semi-solid beads or particles entraining the cells (e.g., labelled B cells or plasma cells) 514.
  • gel e.g ., hydrogel
  • cells e.g., labelled B cells or plasma cells
  • the biological particle can be encased inside of or comprise a gel or matrix (e.g., polymer matrix) to form a “cell bead.”
  • a cell bead can contain biological particles (e.g ., labelled B cell, memory B cell, or plasma cell) or macromolecular constituents (e.g., RNA, DNA, proteins, secreted antibodies or antigen-binding fragments thereof etc.) of biological particles.
  • a cell bead can include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example, after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads.
  • Systems and methods disclosed herein can be applicable to both (i) cell beads (and/or droplets or other partitions) containing biological particles and (ii) cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles.
  • the wells or microwells can assume an “open” configuration, in which the wells or microwells are exposed to the environment (e.g ., contain an open surface) and are accessible on one planar face of the substrate, or the wells or microwells can assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate.
  • the wells or microwells can be configured to toggle between “open” and “closed” configurations.
  • the well can be configured to hold a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 pL, at least 10 pL, at least 100 pL, at least 1000 pL, or more.
  • the well can be configured to hold a volume in a range of volumes listed herein, for example, from about 5 nL to about 20 nL, from about 1 nL to about 100 nL, from about 500 pL to about 100 pL, etc.
  • the well can be of a plurality of wells that have varying volumes and can be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.
  • the microwell array or plate includes different types of microwells that are located adjacent to one another within the array or plate. For example, a microwell with one set of dimensions can be located adjacent to and in contact with another microwell with a different set of dimensions. Similarly, microwells of different geometries can be placed adjacent to or in contact with one another.
  • the adjacent microwells can be configured to hold different articles; for example, one microwell can be used to contain a cell, cell bead, or other sample (e.g., cellular components, nucleic acid molecules, nucleic acid barcode molecules, etc.) while the adjacent microwell can be used to contain a microcapsule, droplet, bead, or other reagent.
  • the adjacent microwells can be configured to merge the contents held within, e.g., upon application of a stimulus, or spontaneously, upon contact of the articles in each microwell.
  • the reagents can also be loaded at operations interspersed with a reaction or operation step.
  • microcapsules, droplets, or beads including reagents for fragmenting polynucleotides (e.g, restriction enzymes) and/or other enzymes (e.g, transposases, ligases, polymerases, etc.) can be loaded into the well or plurality of wells, followed by loading of microcapsules, droplets, or beads including reagents for attaching nucleic acid barcode molecules to a sample nucleic acid molecule.
  • the bead includes nucleic acid barcode molecules that are releasably attached thereto, as described below.
  • the bead can degrade or otherwise release the nucleic acid barcode molecules into the well 1002; the nucleic acid barcode molecules can then be used to barcode nucleic acid molecules within the well 1002. Further processing can be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein.
  • a microcapsule, droplet, or bead can be partitioned into a well.
  • the droplets can be selected or subjected to pre-processing prior to loading into a well.
  • the droplets can include cells, and only certain droplets, such as those containing a single cell (or at least one cell), can be selected for use in loading of the wells.
  • Such a pre selection process can be useful in efficient loading of single cells, such as to obtain a non- Poissonian distribution, or to pre-filter cells for a selected characteristic prior to further partitioning in the wells.
  • the technique can be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.
  • Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g, change in pH or use of a reducing agent), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof.
  • a thermal stimulus e.g., a thermal stimulus, photo-stimulus, chemical stimulus (e.g, change in pH or use of a reducing agent), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof.
  • a bead cannot be degradable.
  • the bead can be a gel bead.
  • a gel bead can be a hydrogel bead.
  • a gel bead can be formed from molecular precursors, such as a polymeric or monomeric species.
  • a semi-solid bead can be a liposomal bead.
  • Solid beads can include metals including iron oxide, gold, and silver.
  • the bead can be a silica bead.
  • the bead can be rigid. In other cases, the bead can be flexible and/or compressible.
  • a bead can be of any suitable shape.
  • bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.
  • the unique molecular identifying sequence 716 can be a random sequence (e.g, such as a random N-mer sequence).
  • the UMI can provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA.
  • FIG. 7 shows three nucleic acid molecules 702, 718, 720 coupled to the surface of the bead 704, an individual bead can be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands, millions, or even billion of individual nucleic acid molecules.
  • Encapsulated species can be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the species from the bead.
  • species can be partitioned in a partition (e.g., droplet) during or subsequent to partition formation.
  • a partition e.g., droplet
  • Such species can include, without limitation, the abovementioned species that can also be encapsulated in a bead.
  • the partitions described herein can include small volumes, for example, less than about 10 microliters (pL), 5pL, lpL, 10 nanoliters (nL), 5 nL, 1 nL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.
  • partitioning species can generate a population or plurality of partitions.
  • any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions, or more partitions can be generated or otherwise provided.
  • the plurality of partitions can include both unoccupied partitions (e.g., empty partitions) and occupied partitions.
  • Biological particles e.g., cells, cell beads, cell nuclei, organelles, and the like
  • nucleic acid barcode molecules e.g., mRNA, cDNA, gDNA, etc.,
  • biological particles are co-partitioned with barcode carrying beads (e.g., gel beads) and the nucleic acid molecules of or derived from the biological particle are barcoded as described elsewhere herein.
  • reagents can also be co-partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids.
  • DNase and RNase inactivating agents or inhibitors such as proteinase K
  • chelating agents such as EDTA
  • the ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles.
  • Unique identifiers e.g., in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles.
  • the co-partitioned nucleic acid molecules can also include other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles (e.g labelled B cells, memory B cells, or plasma cells). These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences.
  • sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any
  • any reference to “reservoir,” as used herein, can also refer to a “chamber.”
  • an aqueous fluid 608 that includes suspended beads 612 can be transported along the channel segment 602 into the junction 606 to meet a second fluid 610 that is immiscible with the aqueous fluid 608 in the reservoir 604 to create droplets 616, 618 of the aqueous fluid 608 flowing into the reservoir 604.
  • droplets can form based on factors such as the hydrodynamic forces at the junction 606, flow rates of the two fluids 608, 610, fluid properties, and certain geometric parameters (e.g., w, ho, a, etc.) of the channel structure 600.
  • the first plurality of cells of the first sample can be provided a first plurality of fluorescent markers configured to label the first plurality of cells of the given type.
  • the second plurality of cells of the second sample can be provided a second plurality of fluorescent markers configured to label the second plurality of cells of the given type.
  • the first plurality of fluorescent markers can include a first identifying feature, such as a first barcode, while the second plurality of fluorescent markers can include a second identifying feature, such as a second barcode, that is different than the first identifying feature.
  • the first plurality of fluorescent markers and the second plurality of fluorescent markers can fluoresce at the same intensities and over the same range of wavelengths upon excitation with a same excitation source (e.g., light source, such as a laser).
  • Cell surface features can include, but are not limited to, a receptor, an antigen or antigen fragment (e.g, an antigen or antigen fragment that binds to an antigen-binding molecule located on a cell surface), a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen- presenting complex, a major histocompatibility complex, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof.
  • an antigen or antigen fragment e.g, an antigen or antigen fragment that binds to an antigen-binding molecule located on a cell surface
  • a surface protein e.g, an antigen or antigen fragment that binds to an antigen-binding molecule located on a cell surface
  • FIG. 11 describes exemplary labelling agents (1110, 1120, 1130) including reporter oligonucleotides (1140) attached thereto.
  • Labelling agent 1110 e.g., any of the labelling agents described herein
  • reporter oligonucleotide 1140 can include barcode sequence 1142 that identifies labelling agent 1110.
  • the preventive methods of the disclosure involve administering a composition comprising an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment of the present disclosure (e.g, of Table 1), to a subject to inhibit the manifestation of a disease or infection (e.g ., viral infection) in the body of a subject, for which the antigen-binding polypeptide is effective when administered to the subject at an effective or therapeutically effective amount or dose.
  • a composition comprising an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment of the present disclosure (e.g, of Table 1)
  • a disease or infection e.g ., viral infection
  • the kit can include a device (e.g ., an injection device or catheter) for performing such administration.
  • the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment thereof of the present disclosure (e.g., of Table 1).
  • This protein contains D614G mutation, which has become increasingly common in SARS-CoV-2 viruses from around the world.
  • This protein also carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using AvitagTM technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.
  • Solubilized antigens were each conjugated with, e.g., allowed to form a complex with (or bind to) one of the following Total SeqC reagents, supplied by BioLegend, which each contain a unique barcoded DNA oligonucleotide supplied by the vendor as follows:
  • Category 1 (e.g., Fluorophore-reactive): Antibodies are classified into this category if the mix of antigens includes target and non-target antigens linked to different fluorophores, and counts are detected for target and non-target antigen linked to fluorophore 1 but not fluorophore 2, which indicates that the antibodies bind to the fluorophore and not the target antigen. In this particular Example, antibodies were classified into this category if counts were detected for only one spike protein and the corresponding albumin labeled with the same fluorophore.
  • the antigens used in these experiments were purchased from ACROBiosystems (His-tagged wild-type SARS- CoV-2: Cat# SPN-C52H9; His-tagged SARS-CoV-2 gamma variant: Cat# SPN-C52Hg; His- tagged SARS-CoV-2 kappa variant: Cat# SPN-C52Hr;. His-tagged SARS-CoV-2 beta variant: Cat# SPN-C52Hk; His-tagged SARS-CoV-2 NTD: Cat# SPN-C52H6; and His-tagged HcoV- HKU1 (isolate N5) spike trimer: Cat# SPN-C52H5).
  • His-tagged human serum albumin (HSA) was also purchased from ACROBiosystems (Cat# HSA-H5220). Mutations identified in the beta, gamma, and kappa variants are as follows.
  • delta and kappa variants share two mutations E484Q and L452R. They were identified in India’s second COVID-19 wave, and have been reported to share significant similarity, presumably due to the fact that they are from the same lineage.
  • IC50 values varied from single digit ng/mL (9 ng/mL) to approximately 1.0 pg/mL. A precise IC50 value could not be generated for some of the Abs tested. Antibodies failing to neutralize the virus at any of the concentrations tested were given an IC50 value of greater than 1 pg/mL (> 1 pg/mL). A summary of the antibodies’ neutralization potency is also shown in FIG. 26. [0610] In these experiments, positive plasma controls (CS478 pi_vac_pfl, plasma of Pfizer vaccine) were run on every plate to determine the intra-plate and inter-day variation.
  • FIG. 25A is an Upset plot of the potently neutralizing antibodies selected from 239 antibodies identified in Example 6.
  • FIG. 25B is an Upset plot of the potently neutralizing antibodies selected from the antibodies of Table 3.
  • rows represent the binding of these neutralizing antibodies to pre-fusion spike trimers from major SARS-CoV-2 variants of concern, and the endemic HKU1 coronavirus spike protein as well as the SARS-CoV-2 N terminal domain.

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Abstract

The present disclosure relates generally to antigen-binding polypeptides, e.g., antibodies, and antigen-binding fragments thereof, that specifically bind to the spike (S) protein of a coronavirus, pharmaceutical compositions comprising the antibodies and methods of use. The antibodies of the disclosure are useful for inhibiting or neutralizing coronavirus activity, thus providing a means of treating or preventing coronavirus infection. Also provided are recombinant nucleic acids, recombinant cells, compositions and methods useful for identifying or producing such antibodies and antigen-binding fragments, as well as methods of using such antibodies and antigen-binding fragments for treating, preventing, or ameliorating health conditions associated with viral infections (e.g., coronavirus infections).

Description

ANTIGEN-BINDING POLYPEPTIDES SPECIFIC FOR CORONA VIRUSES AND
USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U. S. Provisional Patent Application Serial Nos. 63/135,504, filed on January 8, 2021; 63/170,353, filed on April 2, 2021; 63/235,651, filed on August 20, 2021; and 63/253,670, filed on October 8, 2021. The disclosures of the above-referenced applications are herein expressly incorporated by reference it their entireties, including any drawings
FIELD
[0002] The present disclosure relates to antibodies and antigen-binding fragments that bind specifically to coronavirus spike proteins, and therapeutic and diagnostic methods of using such antibodies and fragments.
INCORPORATION OF THE SEQUENCE LISTING
[0003] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing text file, named “Sequence Listing_057862-554001WO_ST25.txt,” was created on December 27, 2021 and is 1.37 MB.
BACKGROUND
[0004] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly identified emerging coronavirus causing an acute respiratory distress syndrome known as COVID-19 that is similar to severe acute respiratory syndrome (SARS) caused by the closely related SARS-CoV. To date, SARS-CoV-2 is continuing its spread across the world with more than 80 million confirmed cases in 190 countries and nearly two million deaths. In view of the continuing threat to human health, there is an urgent need for preventive and therapeutic antiviral therapies for SARS-CoV-2 control. Because this virus uses its spike glycoprotein for interaction with the cellular receptor ACE2 and the serine protease TMPRSS2 for entry into a target cell, this spike protein represents a target for antibody therapeutics. In particular, fully human antibodies that specifically bind to the SARS-CoV-2 spike protein (SARS-CoV-2-S) with high affinity and that inhibit virus infectivity could be important in the prevention and treatment of COVID-19.
[0005] There is a need for antibodies with specific binding affinity to the spike (S) protein of coronaviruses, including neutralizing therapeutic antibodies and their use for treating or preventing coronavirus infection. The present disclosure addresses this need, in part, by providing anti-SARS-CoV-2 S antibodies and antigen-binding fragments thereof, as well as therapeutic methods of using such antibodies and fragments for treating viral infections.
SUMMARY
[0006] The present disclosure relates generally to antigen-binding molecules, e.g.. antibodies and antigen-binding fragments that specifically bind to the spike (S) protein of a coronavirus. Also provided are nucleic acids encoding at least one antigen-binding molecule as disclosed herein, recombinant cells including at least one antigen-binding molecule or nucleic acid as disclosed herein, and methods for identifying such antigen-binding molecules (e.g., antibodies and fragments), as well as therapeutic and diagnostic methods of using such antigen binding molecules (e.g, antibodies and fragments).
[0007] In one aspect of the disclosure, provided herein are isolated antibodies or antigen binding fragments thereof, that bind specifically to a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the antibody is selected from the group consisting of TXG-0153, TXG-0115, TXG-140, and TXG-154, wherein: (a) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0153 have the amino acid sequences as set forth in SEQ ID NOS: 88, 168, 248, 328, 408, and 488, respectively; (b) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG- 0115 have the amino acid sequences as set forth in SEQ ID NOS: 72, 152, 232, 312, 392, and 472, respectively; (c) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0140 have the amino acid sequences as set forth in SEQ ID NOS: 83, 163, 243, 323, 403, and 483, respectively; and (d) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0154 have the amino acid sequences as set forth in SEQ ID NOS: 89, 169, 249, 329, 409, and 489, respectively.
[0008] In another aspect, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, and wherein the antibody or fragment thereof shares an epitope bin with an antibody or antigen-binding fragment thereof that binds to amino acids 332-337, 339-340, 343-346, 354, 356-361, 440-441, and 509 of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.
[0009] In another aspect of the disclosure, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibodies or antigen-binding fragments include: (a) a heavy chain complementary determining region 1 (HCDR1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3; (b) a HCDR2 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 4-6; and (c) a HCDR3 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 7-9.
[0010] Non-limiting exemplary embodiments of the antibodies and antigen-binding fragments thereof of the disclosure can include one or more of the following features. In some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure include the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 1, 4, and 7, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in in SEQ ID NOS: 2, 5, and 8, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 3, 6, and 9, respectively.
[0011] In one aspect, some embodiments of the disclosure provides isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibodies or antigen-binding fragments include: (a) a light chain complementary determining region 1 (LCDR1) including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 10-12; (b) a LCDR2 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 13-15; and (c) a LCDR3 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 16-18. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 11, 14, and 17, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 12, 15, and 18, respectively.
[0012] In another aspect, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibodies or antigen-binding fragments include: (a) a HCDR1 including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3; (b) a HCDR2 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 4-6; c) a HCDR3 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 7-9; (d) a LCDR1 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 10-12; e) a LCDR2 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 13-15; and (f) a LCDR3 including an amino acid sequence is selected from the group consisting of SEQ ID NOS: 16-18. In some embodiments, the antibody or antigen-binding fragment includes: (a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 1, 4, and 7, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively. In some embodiments, the antibody or antigen-binding fragment includes: (a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 2, 5, and 8, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 11, 14, and 17, respectively.
[0013] In some embodiments, the antibody or antigen-binding fragment includes: (a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 3, 6, and 9, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 12, 15, and 18, respectively.
[0014] In some embodiments, the HCDR1 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122. In some embodiments, the HCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3 and SEQ ID NOS: 43-122, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122. In some embodiments, the HCDR2 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-202. In some embodiments, the HCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 4-6 and SEQ ID NOS: 123-202, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-202. In some embodiments, the HCDR3 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282. In some embodiments, the HCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-9 and SEQ ID NOS: 203-282, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282.
[0015] In some embodiments, the LCDR1 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362. In some embodiments, the LCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 10-12 and SEQ ID NOS: 283-362, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362. In some embodiments, the LCDR2 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442. In some embodiments, the LCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-15 and SEQ ID NOS: 363-442, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442. In some embodiments, the LCDR3 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522. In some embodiments, the LCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 16-18 and SEQ ID NOS: 443-522, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522.
[0016] In some embodiments, an antibody or antigen-binding fragment of the disclosure includes: (a) a HCDR1 including an amino acid sequence having 100% sequence identity to SEQ ID NOS: 43-122; (b) a HCDR2 including an amino acid sequence having 100% sequence identity to SEQ ID NOS: 123-202; (c) a HCDR3 including an amino acid sequence having 100% sequence identity to SEQ ID NOS: 203-282; (d) a LCDR1 including an amino acid sequence having 100% sequence identity to SEQ ID NOS: 283-362; (e) a LCDR2 including an amino acid sequence having 100% sequence identity to SEQ ID NOS: 363-442; and (f) a LCDR3 including an amino acid sequence having 100% sequence identity to SEQ ID NOS: 443-522.
[0017] In some embodiments, the antibody or antigen-binding fragment includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-45; (b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-125; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-205; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-285; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-365; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-445.
[0018] In some embodiments, the antibody or antigen-binding fragment includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 46-47; (b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 126-127; (c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 206-207; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 286-287; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 366-367; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 446-447. In some embodiments, the antibody or antigen-binding fragment includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 117-120; (b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 197-200; (c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 277-280; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 357-360; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 437-440; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 517-520.
[0019] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes a framework region having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 523-1162. In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: (a) TXG-0021, TXG-0022, and TXG- 0023; (b) TXG-0027 and TXG-0028; or (c) TXG-0227, TXG-0228, TXG-0229, and TXG-0230.
[0020] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes one or more of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure includes all six HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes the heavy chain framework regions HFWR1, HFWR2, HFWR3, and HFWR4 of the same antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes the light chain framework regions LFWR1, LFWR2, LFWR3, and LFWR4 of the same antibody or anti gen -binding fragment. [0021] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region (HCVR) including an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163- 1322. In some embodiments, the HCVR includes an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163- 1322. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region (LCVR) including an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482. In some embodiments, the LCVR includes an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482.
[0022] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a HCVR including an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163-1322; and b) a LCVR including an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482. In some embodiments, the antibody or antigen-binding fragment includes: a HCVR and a LCVR which respectively are 90% identical to the HCVR and LCVR of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment includes the HCVR and LCVR of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure is selected from Table 1.
[0023] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a constant region. In some embodiments, the constant region is an IgA, IgD, IgE, IgG, or IgM heavy chain constant region. In some embodiments, wherein the constant region is a kappa type or lambda type light chain constant region.
[0024] In some embodiments, the antibody or antigen-binding fragment of the disclosure is a human antibody. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment (scFv), a F(ab), a F(ab'), a Fab'-SH, a F(ab')2, or a Fv fragment.
[0025] In some embodiments, the antibody or antigen-binding fragment has a binding affinity to an epitope in a domain of the SARS-CoV-2 S protein. In some embodiments, the domain of the SARS-CoV-2 S protein is in the SI subunit. In some embodiments, the antibody or antigen-binding fragment has a binding affinity to a receptor binding domain (RBD) or a N- terminal domain (NTD) of the SI subunit. In some embodiments, the SARS-CoV-2 S protein includes one or more amino acid substitutions. In some embodiments, the SARS-CoV-2 S protein includes one or more amino acid substitutions at a position selected from the group consisting of K417, L452, E484, N501, and D614. In some embodiments, the SARS-CoV-2 S protein includes one or more amino acid substitutions selected from the group consisting of K417T, K417N, L452R, E484K, E484Q, N501Y, and D614G. In some embodiments, the SARS- CoV-2 S protein includes the amino acid substitutions K417N, E484K, and N501Y. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein In some embodiments, the subunit of the S protein of SARS-CoV-2 is the S2 subunit. In some embodiments, the one or more amino acid substitutions includes D614G substitution.
[0026] In some embodiments, the antibody or antigen-binding fragment has binding affinity for a pre-fusion trimeric form of the CoV-S protein. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a stabilized prefusion spike protein (e.g, an S2P-stabilized pre-fusion spike protein) in monomeric or multimeric (e.g., trimeric) form. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a non prefusion spike protein in monomeric or multimeric (e.g, trimeric) form. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a non-S2P-stabilized pre-fusion spike protein in monomeric or multimeric (e.g., trimeric) form.
[0027] In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 500 nM, less than 120 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 5 nM, less than 1 nM, or less than 1 pM. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM.
[0028] In some embodiments, the antibody or antigen-binding fragment includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: TXG-0021, TXG- 0022, TXG-0023, TXG-0027, TXG-0028, TXG-0043, TXG-0049, TXG-0056, TXG-0061, TXG-0062, TXG-0068, TXG-0072, TXG-0083, TXG-0085, TXG-0089, TXG-0090, TXG-0098, TXG-0108, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0130, TXG-0134, TXG-0135, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0150, TXG-0152, TXG-0153, TXG-0154, TXG-0160, TXG-0165, TXG-0169, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0216, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, TXG-0229, and TXG-0230.
[0029] In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM and includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: TXG-0022, TXG- 0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG- 0230.
[0030] In some embodiments, the antibody or antigen-binding fragment includes a HCVR which includes an amino acid sequence having at least 90% sequence identity to the HCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG-0230. In some embodiments, the antibody or antigen-binding fragment includes a LCVR which includes an amino acid sequence having at least 90% sequence identity to the LCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG- 0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG-0230.
[0031] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM.
[0032] In some embodiments, the antibody or antigen-binding fragment with sub nanomolar binding affinity for a SARS-CoV-2 S protein is selected from the group consisting of TXG-0028, TXG-0049, TXG-0056, TXG-0072, TXG-0089, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0194, and TXG-0217.
[0033] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for HCOV and/or for a SARS-CoV-2 S variant selected from the group consisting of beta, gamma, delta, and kappa.
[0034] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S and beta, gamma, kappa variants. Exemplary antibodies having these binding affinity properties include TXG-0115 and TXG-0154.
[0035] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S. Exemplary antibodies having these binding affinity properties include TXG-0140.
[0036] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S, as well as the trimeric forms of the beta and gamma variants. Exemplary antibodies having these binding affinity properties include TXG-0153.
[0037] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2, and has binding affinity to the N-terminal domain (NTD) of the SI subunit and to the trimeric forms of wild-type SARS-CoV-2 S, as well as to trimeric forms of the gamma and kappa variants. Exemplary antibodies having these binding affinity properties include TXG-0173. [0038] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2, and has binding affinity to the N-terminal domain (NTD) of the SI subunit and to the trimeric forms of wild-type SARS-CoV-2 S, as well as to trimeric forms of the beta, gamma, and kappa variants. Exemplary antibodies having these binding affinity properties include TXG-0174.
[0039] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment neutralizes at least 50% of 200 times the tissue culture infectious dose (200xTCID50) of the coronavirus.
[0040] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 1 pg/mL or lower, 200 ng/mL or lower, or 40 ng/mL or lower. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 200 ng/mL to 1,000 ng mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 40 ng/mL to 200 ng/mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 8 ng/mL to 40 ng/mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 1 pg/mL or lower. Exemplary antibodies or antigen-binding fragment thereof having this neutralizing activity property include TXG-0153, TXG-0173, TXG-0115, TXG- 0140, TXG-0154, and TXG-0174.
[0041] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 200 ng/mL or lower. Exemplary antibodies having this neutralizing activity property include TXG-0115, TXG-0140, and TXG- 0154.
[0042] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 200 ng/mL to 1000 ng/mL. Exemplary antibodies having this neutralizing activity property include TXG-0153, TXG-0173, and TXG- 0174. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value of 100 ng/mL or lower. Exemplary antibodies having this neutralizing activity property include TXG-0140 and TXG-0154. [0043] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 40 ng/mL or lower. Exemplary antibodies having this neutralizing activity property include TXG-0154.
[0044] In some embodiments, the isolated antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.
[0045] In some embodiments, the antibody or antigen binding-fragment has a binding affinity to the N-terminal domain of (NTD) of a SARS-CoV-2 S protein and potently neutralizes live SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0072, TXG-0136, TXG-0137, TXG-0173, and TXG-0174. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0173, and TXG-0174. In some embodiments, the antibody or antigen-binding fragment has a binding affinity to the N-terminal domain of (NTD) of an S protein from a SARS- CoV-2 delta variant. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0072, TXG-0137, and TXG-0174. In some embodiments, the antibody or antigen-binding fragment is TXG-0174.
[0046] In some embodiments, the antibody or antigen-binding fragment has a binding affinity primarily to the RBD of a SARS-CoV-2 S protein and potently neutralizes live SARS- CoV-2. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0115, TXG-0140, TXG-0153, and TXG-0154. In some embodiments, the antibody or antigen-binding fragment is TXG-0153. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0115, TXG-0140, and TXG-0154.
[0047] In some embodiments, the antibody or antigen-binding has a binding affinity primarily to the RBD of an S protein from a SARS-CoV-2 delta variant. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0140 and TXG-0154. In some embodiments, the antibody or antigen-binding fragment has a binding affinity for a SARS-CoV-2 S protein and is selected from the group consisting of TXG-0085, TXG-0112, TXG-0192, TXG-0227, TXG-0228, TXG-0229, and TXG-0230.
[0048] In some embodiments, the antibody or antigen-binding fragment has a binding affinity for an S protein of a SARS-CoV-2 delta variant and is selected from the group consisting of TXG-0115, TXG-0136, TXG-0192, and TXG-0230. In some embodiments, such antibodies and antigen-binding fragments have a neutralizing activity against live SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment is TXG-0115.
[0049] In some embodiments, the antibody or antigen-binding fragment has a binding affinity for an S protein from a SARS-CoV-2 delta variant and is selected from the group consisting of TXG-0085, TXG-0112, TXG-0173, TXG-0227, TXG-0228, and TXG-0229. In some embodiments, such antibody or antigen-binding fragment potently neutralizes live SARS- CoV-2. In some embodiments, the antibody or antigen-binding fragment is TXG-0173. In one aspect, provided herein are recombinant nucleic acids encoding an antibody of the disclosure or an antigen-binding fragment thereof. In some embodiments, the nucleic acids include a nucleic acid sequence encoding a HCVR comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163- 1322. In some embodiments, the nucleic acids include a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1487-1886. In some embodiments, the nucleic acids include a nucleic acid sequence encoding a LCVR comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482. In some embodiments, the nucleic acids include a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1887- 2286. In some embodiments, the nucleic acids include a first nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1487-1886; and a second nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1887- 2286.
[0050] In another aspect, provided herein are vectors including a recombinant nucleic acid of the disclosure. Non-limiting exemplary embodiments of the vectors as described herein can include one or more of the following features. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a plasmid vector or a viral vector.
[0051] In yet another aspect, provided herein are recombinant cells including a) an antibody or antigen-binding fragment as disclosed herein; or b) a recombinant nucleic acid as disclosed herein; or c) a vector as disclosed herein. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. Also provided, in another aspect, are animals including a vector as disclosed herein. In some embodiments, the disclosure provides a transgenic animal that is a non-human animal. In some embodiments, the transgenic animal produces an antibody or antigen-binding fragment as disclosed herein.
[0052] In another aspect, provided herein are methods for producing an antibody or antigen-binding fragment thereof, including: growing (i) a transgenic animal as disclosed herein, or (ii) a recombinant cell as disclosed herein under conditions such that the antibody or antigen binding fragment is produced. In some embodiments, the methods for producing an antibody or antigen-binding fragment thereof as described herein further include isolating the produced antibody or antigen-binding fragment from (i) the transgenic animal or (ii) recombinant cell and/or the medium in which the recombinant cell is cultured.
[0053] In another aspect, provided herein are compositions, for example, pharmaceutical compositions including one or more antibodies or antigen-binding fragment as disclosed herein, and a pharmaceutically acceptable carrier or diluent. Non-limiting exemplary embodiments of the pharmaceutical compositions as described herein can include one or more of the following features.
[0054] In some embodiments, the composition includes: (a) a first antibody or antigen binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein); (b) a first antibody or antigen-binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to a NTD of a SARS-CoV-2 S protein; or (c) a first antibody or antigen-binding fragment having a binding affinity to a NTD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein). In some embodiments, the composition is a sterile composition. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further includes an adjuvant. In some embodiments, the pharmaceutical composition of the disclosure further includes a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of: (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. [0055] In another aspect, provided herein are methods for treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the methods including administering to the subject a therapeutically effective amount ( e.g ., a composition comprising a therapeutically effective amount) of an antibody or antigen-binding fragment as disclosed herein. In a related aspect, provided herein are methods for reducing binding of SARS- Co-2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject, the method including administering to the subject a therapeutically effective amount (e.g., a composition comprising a therapeutically effective amount) of an antibody or antigen-binding fragment as disclosed herein. In some embodiments, the method includes administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment as disclosed herein. In some embodiments, the composition includes: (a) a first antibody or antigen-binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein); (b) a first antibody or antigen binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to aNTD of a SARS-CoV-2 S protein; or (c) a first antibody or antigen-binding fragment having a binding affinity to a NTD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein).
[0056] Non-limiting exemplary embodiments of the methods as described herein can include one or more of the following features. In some embodiments, the antibody or antigen binding fragment is administered in combination with a SARS-Co-2V S protein conjugated to a therapeutic agent. In some embodiments, the subject is administered one or more further therapeutic agents. In some embodiments, the one or more further therapeutic agents includes an antiviral drug or a vaccine. In some embodiments, the one or more further therapeutic agents is selected from the group consisting of: (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds TMPRSS2, and (iv) an antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. In some embodiments, the antibody or antigen-binding fragment is administered to the subject subcutaneously, intravenously, and/or intramuscularly.
[0057] In another aspect, provided herein are methods for detecting the presence of SARS- CoV-2 S protein and/or SARS-CoV-2 in a biological sample, the methods including contacting an antibody or antigen-binding fragment as disclosed herein with a biological sample from an individual infected with or suspected of being infected with SARS-CoV-2.
[0058] In yet another aspect, provided herein are methods for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the methods including: (a) contacting a plurality of B cells obtained from a subject who has been exposed to a coronavirus with a plurality of antigens, wherein the plurality of antigens includes a CoV-S antigen and a non-CoV-S antigen, and wherein each of the antigens include a reporter oligonucleotide, wherein the contacting provides a B cell bound to a CoV-S antigen; (b) partitioning the B cell bound to the CoV-S antigen in a partition of a plurality of partitions, wherein the partitioning provides a partition including (i) the B cell bound to the CoV-S antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the CoV-S antigen; and d) assessing the binding affinity of the barcoded antibody or antigen-binding fragment to a CoV-S protein; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the CoV-S protein if the barcoded antibody specifically binds to the CoV-S protein.
[0059] Non-limiting exemplary embodiments of the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein can include one or more of the following features. In some embodiments, the reporter oligonucleotide may include (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence. In some embodiments, such reporter sequence may be useful to identify the target antigen or fragment to which the reporter oligonucleotide is coupled/bound. In some embodiments, the methods of the disclosure further include coupling a barcode moiety to the antibody or antigen-binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment. In some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the reporter oligonucleotide and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide is complementary to the capture handle sequence of the reporter oligonucleotide. In instances wherein the capture sequence configured to couple to an mRNA analyte, it may include a polyT sequence. In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI).
[0060] In some embodiments, the antibody or antigen-binding fragment has a binding specificity to an epitope on a domain of the CoV-S protein. In some embodiments, the domain of the CoV-S protein is in the SI domain (i.e., subunit). In some embodiments, the antibody or antigen-binding fragment has a binding affinity to a RBD or a NTD of the SI domain (i.e, subunit). In some embodiments, the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a pre-fusion trimeric form of the CoV-S protein. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a stabilized prefusion spike protein ( e.g ., an S2P-stabilized pre-fusion spike protein) in monomeric or multimeric (e.g., trimeric) form. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a non-prefusion spike protein in monomeric or multimeric (e.g., trimeric) form. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a non-S2P-stabilized pre-fusion spike protein in monomeric or multimeric (e.g., trimeric) form.
[0061] In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 500 nM, less than 120 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 5 nM, less than 1 nM, or less than 1 pM. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM. In some embodiments, the antibody or antigen-binding fragment has a sub-nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM. In some embodiments, the antibody or antigen-binding fragment as a neutralizing activity against a CoV-S protein. In some embodiments, the subunit of the CoV-S protein is the S2 subunit.
[0062] In some embodiments, the CoV-S protein is a spike protein of SARS-CoV-1, SARS-CoV-2, or MERS-CoV. In some embodiments, the subject is suspected of being infected with a coronavirus, has been infected with a coronavims, has been vaccinated, or has been recovered from a coronavirus infection. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human.
[0063] In some embodiments, the antigens are each coupled to a fluorescent label identifying the antigens. In some embodiments, the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein further include isolating and/or enriching the plurality of B cells prior to (b). In some embodiments, the enriching further including sorting of the B cells bound to the CoV-S antigen and/or non-CoV-S antigen based on detection of one or more of the fluorescent labels coupled to the antigens. In some embodiments, the CoV-S protein is coupled to a barcode moiety. In some embodiments, the methods further include purifying/isolating the antibody or antigen-binding fragment that has been identified as having a binding specificity for the CoV-S protein.
[0064] In one aspect, provided herein are antibodies identified by a method disclosed herein.
[0065] In another aspect, provided herein are kits identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the kits including: (a) a plurality of CoY-S antigens and non-CoV-S antigens, and wherein each of the antigens include a reporter oligonucleotide including (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and (b) instructions for performing a method of identifying an antibody having binding affinity for a CoV-S protein as described herein.
[0066] Also provided are kits for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the kits including instructions for use thereof and one or more of the following: (a) an antibody or antigen-binding fragment as disclosed herein; (b) a recombinant nucleic acid or a vector as disclosed herein; and (c) a recombinant cell as disclosed herein
[0067] In some embodiments, provided are kits for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the kits including instructions for use thereof and a pharmaceutical composition as disclosed herein.
[0068] In some embodiments, provided are kits for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the kits including instructions for use thereof and one or more of the following: (a) an antibody or antigen-binding fragment as disclosed herein; (b) a pharmaceutical composition as disclosed herein; (c) a recombinant nucleic acid or a vector as disclosed herein; and (d) a recombinant cell as disclosed herein.
[0069] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1 shows an exemplary scheme for antigen-specific enrichment of B cells by using fluorescence-activated cell sorting (FACS) technique. In these experiments, cells were initially gated on being single, live (7AADnegatlve) and PE-Cy7-CD19+ and then sorted on their PE and/or APC status directly into master mix and water. In this figure, Y axis represents PE signal (trimerized SARS-2 glycoprotein S antigen+ and/or HSA+ control antigen-binding cells). X axis represents APC signal (pre-fusion trimerized SARS-2 S glycoprotein D614G antigen+ and/or HSA+ control antigen-binding cells). Numbers adjacent to each gate name represent the fraction of events relative to the parent population (single, live, CD 19+ cells) for that gate.
[0071] FIG. 2 schematically illustrates that the new scoring system described herein allowed for determining relative KD values which in turn facilitate identification of binding antibodies with good dynamic range.
[0072] FIGS. 3A-3B schematically depict the results of representative analysis performed to illustrate that the new scoring system described herein allow for selection of high-affinity antibodies with a data set. In this analysis, BEAM scores are approximately normally distributed, increase exponentially as target antigen-binding relative to expressed antibody and control antigen increases, are correlated with generation probability of the HCDR3 junction, e.g., following the known general relationship of somatic hypermutation (SHM) and increasing affinity, and also reveal that class switching increases predicted relative affinity in concordance with the literature.
[0073] FIG. 4 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on relative KD. BEAM scores were found to be also generally higher within sublineages that contain more daughter antibodies than narrow sublineages.
[0074] FIG. 5 shows an exemplary microfluidic channel structure for partitioning individual biological particles in accordance with some embodiments of the disclosure.
[0075] FIG. 6 shows an exemplary microfluidic channel structure for the controlled partitioning of beads into discrete droplets.
[0076] FIG. 7 shows an exemplary barcode carrying bead.
[0077] FIG. 8 illustrates another example of a barcode carrying bead.
[0078] FIG. 9 schematically illustrates an example microwell array.
[0079] FIG. 10 schematically illustrates an example workflow for processing nucleic acid molecules.
[0080] FIG. 11 schematically illustrates examples of labelling agents.
[0081] FIG. 12 depicts an example of a barcode carrying bead.
[0082] FIG. 13A, 13B, and 13C schematically depict an example workflow for processing nucleic acid molecules.
[0083] FIG. 14 shows an exemplary microfluidic channel structure for delivering barcode carrying beads to droplets.
[0084] FIGS. 15A and 15B depict the amino acid sequences of a wild-type SARS-CoV spike protein (FIG. 15A) and a variant SARS-CoV spike protein (FIG. 15B). Various mutations have been introduced and indicated by the original amino acids above the mutated amino acids. These mutations fall in 3 classes: 1) proline stabilization/S2P mutations (F817P, A892P, A899P, A942P, K986P, V987P), 2) alanine stabilization mutations (R683A, R685A), and 3) viral variant mutations (D614G). The asterisks in the sequences indicate the start and end of the sequences used to produce the antigens used in the experiments described in the Examples below. The C- terminal end of the antigens (ending at the 2nd asterisk) is fused to the T4 trimerization domain and the His tag.
[0085] FIGS. 16A-16B schematically summarize the results of representative SPR analyses performed to evaluate binding affinity of exemplary antibodies of the disclosure to the following antigens: (1) a trimerized wild-type SARS-CoV-2 S protein (FIG. 16A), (2) a SARS- CoV-2 S protein variant with D614G substitution (FIG. 16B).
[0086] FIG. 17A depicts binding kinetics of hypothetical antibodies having the same KD value 10 nM, with varying kon and koff rates. Dashed and solid curves depict optimal binding kinetics of antibodies having high therapeutic potential due to binding stability, while dash-dot and heavy solid curves depict less optimal binding kinetics.
[0087] FIG. 17B depicts binding kinetics of exemplary TXG antibodies and FDA- approved or late clinical development stage spike antibodies (data from each antibody shown in triplicate). Antibodies having optimal binding kinetics are depicted in FIG. 17B as boxes with asterisk symbols (*). Antibodies having less optimal binding kinetics are depicted in FIG. 17B as boxes with solid circle (·). FDA-approved or late clinical development stage spike antibodies used as positive controls are depicted as boxes with solid triangle (A).
[0088] FIG. 17C depicts the relationship between Koff of a given TXG antibody to the pre-fusion trimeric spike and its binding kinetics. Koff is shown here for the purpose of brevity as half-life and mean-life kinetics of a receptor-ligand pair are determined by the Koff of the interaction and not the Kon or the ratio of Koff to Kon (KD). Box plots are shown for each kinetic profile described above and shown in FIG. 17C. Twenty-seven (27) antibodies are shown as having a Koff rate of le-05, indicating they have surpassed the lower limit of detection from the SPR data and therefore have lower estimated KD values than those reported in the provided data.
[0089] FIG. 17D depicts the relationship between Koff and Kon of given TBS-antibodies to the pre-fusion trimeric spike, color coded by kinetic profile.
[0090] FIG. 18 schematically depicts the general procedure of live virus neutralization assay employed to determine the anti-SARS-CoV-2 activity of various antibodies described herein.
[0091] FIG. 19 depicts representative raw data from neutralization assay described in FIG. 18. CTRL-0004: Casirivimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL- 0008: Sotrovimab; CS478 pi_vac_pfl, positive plasma control of Pfizer vaccine.
[0092] FIG. 20 depicts representative neutralization percentage from neutralization assay described in FIG. 18. CTRL-0004: Casirivimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; CS478 pi vac pfl , positive plasma control of Pfizer vaccine.
[0093] FIG. 21 schematically depicts representative neutralization curves (ID50) of four FDA-approved antibodies or antibodies in late clinical development (controls). CTRL-0004: Casirivimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; CS478 pi vac pf l , positive plasma control of Pfizer vaccine.
[0094] FIG. 22 schematically depicts representative neutralization curves (ID50) of six exemplary antibodies in accordance with some embodiments of the disclosure.
[0095] FIG. 23 schematically depicts an UpSet plot wherein antibodies are binned into antigen bins based on two rounds of SPR binding affinity data. For an antibody to be placed into a bin a detectable kinetic fit at all concentrations of antigen was required from at least one of the SPR experiments described in Examples 9 and 12, or orthogonal neutralization data.
[0096] FIG. 24 schematically depicts an CfpSet plot of antibodies identified as having neutralization activity against live SARS-COV-2, wherein the antibodies are binned into antigen bins as described in FIG. 23.
[0097] FIGS. 25A and 25B depict EipSet plots of the potently (IC50 <= 1000 ng / ml) neutralizing antibodies retrieved in this BEAM-seq workflow. FIG. 25A is an Upset plot of the potently neutralizing antibodies selected from 239 antibodies identified in Example 6. FIG. 25B is an Upset plot of the potently neutralizing antibodies selected from the antibodies of Table 3. In these figures, rows represent the binding of these neutralizing antibodies to pre-fusion spike trimers from major SARS-CoV-2 variants of concern, and the endemic HKU1 coronavirus spike protein as well as the SARS-CoV-2 N terminal domain.
[0098] FIG. 26 schematically summarizes of the neutralization potency of the antibodies as determined in testing against SARS-CoV-2 (see, also Table 8).
[0099] FIG. 27 is a heat map summarizing results of the epitope binning assays described in Example 14, wherein antibodies were tested against one another in a pairwise and combinatorial fashion for binding to a specific target antigen, i.e., pre-fusion trimerized spike protein from SARS-CoV-2 USA-WA1/2020 isolate.
[0100] FIG. 28 is a heat map summarizing results of the epitope binning assays described in Example 14, wherein antibodies were tested against one another in a pairwise and combinatorial fashion for binding to spike protein from SARS-CoV-2 delta variant. DETAILED DESCRIPTION OF THE DISCLOSURE
[0101] The present disclosure generally relates to, inter alia , compositions, methods, kits, and systems for the diagnosis and/or treatment of various health conditions associated with viral infection, e.g ., coronavirus infection. In particular, some embodiments of the disclosure relate to isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some embodiments of the disclosure relate to compositions and methods useful for producing such antibodies and antigen-binding fragments, including recombinant nucleic acid molecules, recombinant cells, and transgenic animals that have been engineered to produce an antibody as disclosed herein or an antigen-binding fragment thereof. Some embodiments of the disclosure relate to compositions and methods for reducing binding of SARS-Co-2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject. Some embodiments of the disclosure relate to compositions and methods useful for treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject. Additional embodiments of the disclosure relate to methods, kits, and systems for the prevention, amelioration, and/or treatment of a health condition in individuals in need thereof. Also provided are compositions and methods for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S).
[0102] As described in greater detail below, antibodies binding the S glycoprotein, including neutralizing antibodies, are one of the central determinants of effective immunity against human-infecting coronaviruses such as SARS-1, SARS-2, MERS, and endemic coronaviruses 229E, NL63, OC43, and HKU1. Antibodies isolated from human survivors of SARS-2 and other coronavirus infection are ideal therapeutics as the natural selective pressure of somatic hypermutation drives the production of strongly neutralizing antibodies which are inherently low in immunogenicity. Successful isolation of such antibodies can be achieved using other methods including phage display, immunization of humanized mice or other mammals, structural homology search, and more. However, the actual process of selecting antibodies from such approaches can be daunting, time consuming, and often excludes biologically relevant information. As illustrated in greater detail below, the approaches described in the present disclosure allow for the identification and isolation of potent neutralizing antibodies from a naturally infected human survivor of SARS-2 within a short time period, e.g., within one week, using “barcode-enabled antigen mapping by sequencing” (BEAM-seq). In particular, by staining survivor peripheral blood mononuclear cells (PBMCs) with antigens labeled with 1) Feature Barcode Technology-compatible DNA oligonucleotides, 2) the PE or APC fluorophores, and 3) biotin and tetramerizing these antigens, it was demonstrated herein the ability to successfully identify antibodies that specifically bound the S glycoprotein of SARS-2, while excluding antibodies that bound biotin, the PE or APC fluorophores, or an irrelevant control antigen (human serum albumin). As discussed in greater detail in the experiments described in the Examples, the final antigens tested were tetramers of trimers ( e.g ., four trimers coupled on each streptavidin). Additionally, it was demonstrated herein the ability to account for biological covariates of antigen binding that are missed in other experimental approaches, including the expression of the antibody / B cell receptor itself. Furthermore, the results of epitope binning assays described herein (e.g., Example 14) demonstrated that the compositions and methods of the diclosure can be useful for the identification and characterization of antibodies having specific binding affinity (e.g., ability to bind, with varying degrees of specificity) to different epitopes on a coronavirus target, e.g., a spike protein. Antibodies having binding affinity for different epitopes on the target protein, e.g., a SARS-2 spike protein, can advantageously be used in a therapeutic antibody cocktail or combination therapy regimen. For example, a neutralizing antibody with binding affinity for an N-terminal domain (NTD) of a SARS-2 spike protein can be effectively combined with a neutralizing antibody with binding affinity for a receptor binding domain (RTD) of the same target as the two antibodies do not bind in the same epitope.
[0103] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.
[0104] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.
[0105] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. etal. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. etal. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. etal. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY : Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.
DEFINITIONS
[0106] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.
[0107] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”
[0108] The term “analyte carrier,” as used herein, generally refers to a discrete biological system derived from a biological sample. The analyte carrier may be or comprise a biological particle. The analyte carrier, e.g., biological particle, may be a macromolecule. The analyte carrier, e.g ., biological particle, may be a small molecule. The analyte carrier, e.g ., biological particle, may be a virus, e.g., a phage. The analyte carrier, e.g., biological particle, may be a cell or derivative of a cell. The analyte carrier, e.g., biological particle, may be an organelle. The analyte carrier, e.g., biological particle, may be a rare cell from a population of cells. The analyte carrier, e.g., biological particle, may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The analyte carrier, e.g., biological particle, may be a constituent of a cell. The analyte carrier, e.g., biological particle, may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The analyte carrier, e.g., biological particle, may be or may include a matrix (e.g., a gel or polymer matrix) including a cell or one or more constituents from a cell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The analyte carrier, e.g., biological particle, may be obtained from a tissue of a subject. The analyte carrier, e.g., biological particle, may be a hardened cell. Such hardened cell may or may not include a cell wall or cell membrane. The analyte carrier, e.g., biological particle, may include one or more constituents of a cell, but may not include other constituents of the cell. An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix, or cultured when including a gel or polymer matrix.
[0109] The terms “cell,” “cell culture,” “cell line,” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers, or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation ( e.g ., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell or cell line.
[0110] An "equivalent amino acid residue" refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side-chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, “equivalent amino acid residues” can be regarded as “conservative amino acid substitutions” to each other.
[0111] Within the meaning of the term “equivalent amino acid substitution” as applied herein, one amino acid may be substituted for another without substantially altering the structure and/or functionality of the polypeptide. Exemplary equivalent or conserved amino acid substitutions are within the groups of amino acids indicated herein below:
[0112] i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys);
[0113] ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, He, Phe, Trp, Pro, and Met);
[0114] iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu, lie);
[0115] iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro);
[0116] v) Amino acids having aromatic side chains (Phe, Tyr, Trp);
[0117] vi) Amino acids having acidic side chains (Asp, Glu);
[0118] vii) Amino acids having basic side chains (Lys, Arg, His);
[0119] viii) Amino acids having amide side chains (Asn, Gin);
[0120] ix) Amino acids having hydroxy side chains (Ser, Thr);
[0121] x) Amino acids having sulphur-containing side chains (Cys, Met); [0122] xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr);
[0123] xii) Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp); and.
[0124] xiii) Hydrophobic amino acids (Leu, lie, Val).
[0125] In some embodiments, a Point Accepted Mutation (PAM) matrix is used to determine equivalent amino acid substitutions. In some embodiments, a BLOck Substitution Matrix (BLOSUM) is used to determine equivalent amino acid substitutions.
[0126] As used herein, “isolated” antigen-binding polypeptides, antibodies or antigen binding fragments thereof, polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium.
An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or antigen-binding fragments.
[0127] The term “recombinant” when used with reference to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been altered or produced through human intervention such as, for example, has been modified by or is the result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of the protein or can be include amino acid residues that have been modified, e.g., labeled. The term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and/or substitution of one or more of amino acids in the peptide or protein; creation of a fusion protein, e.g., a fusion protein comprising an antibody fragment; and addition, deletion, and/or substitution of one or more of nucleic acids in the nucleic acid sequence. The term ’’recombinant” when used in reference to a cell is not intended to include naturally-occurring cells but encompass cells that have been engineered/modified to include or express a polypeptide or nucleic acid that would not be present in the cell if it was not engineered/modified.
[0128] As used herein, a “subject” or an “individual” includes animals, such as human ( e.g ., human individuals) and non-human animals. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit; and non-mammals, such as amphibians, reptiles, etc. A subject can be a mammal, preferably a human or humanized animal, e.g., an animal with humanized VDJC loci. The subject may be non-human animals with humanized VDJC loci and knockouts of a target of interest. The subject may be in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer. The subject may have a viral infection, e.g., a coronavirus infection, or be predisposed to developing an infection. Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection (e.g., of coronavirus), include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant), subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g., 5 years of age or younger) or old age (e.g, 65 years of age or older) are at increased risk.
Moreover, a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g, subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.
[0129] A “variant” of a polypeptide, such as an immunoglobulin chain (e.g, VH, VL, HC, or LC), refers to a polypeptide comprising an amino acid sequence that has at least about 70- 99.9% (e.g, 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity or similarity to a referenced amino acid sequence that is set forth herein. In some embodiments, the term “percent identity,” as used herein in the context of two or more proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acids that are the same, e.g., about 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9%9, 99.5%, 99.9%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST. Similarly, a “variant” of a nucleic acid molecule refers to a nucleic acid molecule comprising a nucleic acid sequence that has at least about 70-99.9% (e.g., 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9%9, 99.5%, 99.9%) sequence identity or similarity to a referenced nucleic acid sequence that is set forth herein. In some embodiments, this definition also refers to, or may be applied to, the complement of a query sequence. This definition includes sequence comparison performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. In some embodiments, this definition also includes sequences that have modifications such as deletions and/or additions (e.g., insertions), as well as those that have substitutions. Such modifications can occur naturally or synthetically. Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. In some embodiments, sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul etal, J Mol Biol (1990) 215:403). In some embodiments, the sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine structural similarity or identity amino acid sequences include those relying on position-specific structure scoring matrix (P3SM) that incorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g., Setcliff et al, Cell Host & Microbe 23(6), May 2018.
[0130] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
[0131] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.
[0132] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
CORONA VIRUSES
[0133] Coronaviruses are a family of large, enveloped, positive-sense single- stranded RNA viruses. They infect humans, other mammals and avian species, including livestock and companion animals (such as dogs, cats, chicken, cattle, pigs, and birds), and are therefore not only a challenge for public health but also a veterinary and economic concern. Coronaviruses include the genera of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. Whereas alphacoronaviruses and betacoronaviruses exclusively infect mammalian species, gammacoronaviruses and deltacoronaviruses have a wider host range that includes avian species. Coronaviruses cause respiratory, gastrointestinal, and neurological disease. The most common coronaviruses in clinical practice are 229E, OC43, NL63, and HKU1, which typically cause common cold symptoms in immunocompetent individuals. Human and animal coronavirus infections mainly result in respiratory and enteric diseases.
[0134] Human coronaviruses, such as HCoV-229E and HCoV-OC43, have long been known to circulate in the population and they, together with the more recently identified HCoV- NL63 and HCoV-HKUl, cause seasonal and usually mild respiratory tract infections associated with symptoms of the “common cold.” In strong contrast, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoY) and SARS-CoV-2, which have emerged in the human population over the past 20 years, are highly pathogenic. By infecting bronchial epithelial cells, pneumocytes and upper respiratory tract cells in humans, SARS-CoV, MERS-CoV and SARS-CoV-2 infections can develop into severe, life- threatening respiratory pathologies and lung injuries.
[0135] The initial steps of coronavirus infection involve the specific binding of the coronavirus spike (S) protein to the cellular entry receptors, which have been identified for several coronaviruses and include human aminopeptidase N (APN; HCoV-229E), angiotensin converting enzyme 2 (ACE2; HCoV-NL63, SARS-CoV and SARS-CoV-2) and dipeptidyl peptidase 4 (DPP4; MERS-CoV). The expression and tissue distribution of entry receptors consequently influence viral tropism and pathogenicity. During the intracellular life cycle, coronaviruses express and replicate their genomic RNA to produce full-length copies that are incorporated into newly produced viral particles. Coronaviruses possess large RNA genomes flanked by 5' and 3' untranslated regions that contain cis-acting secondary RNA structures essential for RNA synthesis. At the 5' end, the genomic RNA features two large open reading frames (ORFs; ORFla and ORFlb) that occupy two-thirds of the capped and polyadenylated genome. ORFla and ORFlb encode 15-16 non-structural proteins (nsp), of which 15 compose the viral replication and transcription complex (RTC) that includes, amongst others, RNA- processing and RNA-modifying enzymes and an RNA proofreading function necessary for maintaining the integrity of the >30 kb coronavirus genome. ORFs that encode structural proteins and interspersed ORFs that encode accessory proteins are transcribed from the 3' one- third of the genome to form a nested set of subgenomic mRNAs (sg mRNAs). Coronavirus accessory proteins are highly variable sets of virus-specific proteins that display limited conservation even within individual species but they are principally thought to contribute to modulating host responses to infection and are determinants of viral pathogenicity. Nevertheless, the molecular functions of many accessory proteins remain largely unknown owing to the lack of homologies to accessory proteins of other coronaviruses or to other known proteins.
[0136] Coronavirus virions are spherical with diameters of approximately 125 nm. The most prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family. The initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor. The sites of RBD within the SI region (often referred to as SI subunit) of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-terminus of S 1. The S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptor. Following receptor binding, the virus must next gain access to the host cell cytosol. This is generally accomplished by acid-dependent proteolytic cleavage of S protein by a cathepsin, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes. Additional information regarding coronavirus biology, pathophysiology, diagnosis, and treatment can be found in recent reviews by V’kovski P. el al. (Nature Rev. Microbiol. Oct. 28, 2020) and Wiersinga WJ etal. (JAMA. 2020;324(8):782-793).
[0137] It has been noted that SARS-CoV and SARS-CoV-2 have similar structural proteins including the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. In SARS- CoV, the S protein is the most immunogenic and contains a RBD that is a target for neutralizing antibodies. The RBD binds to the angiotensin-converting enzyme 2 (ACE2), which serves as a functional receptor to mediate cell entry of the virus. The RBD-ACE2 interaction is of high affinity and is highly specific. Although the SARS-CoV-2 and SARS-CoV proteins share a high degree of homology, the RBD has a relatively low degree of homology. Thus, many neutralizing antibodies developed against SARS-CoV that target the RBD cannot neutralize SARS-CoV-2. [0138] Coronavirus S proteins are class I fusion glycoproteins which assemble into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle. The proteins are divided into two parts (region or subunit) with distinct functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (SI) and C-terminal (S2) halves of the S proteins. The surface-exposed SI includes the NTD and RBD that specifically engages the host cell receptor, thereby determining virus cell tropism and pathogenicity. The transmembrane S2 domain contains heptad repeat regions, e.g., heptad repeat 1 (HR1) and heptad repeat 2 (HR2), central helix (CH), connector domain (CD), transmembrane domain (TM), and cytoplasmic tail (CT), the fusion peptide (FP), which mediate the fusion of viral and cellular membranes upon extensive conformational rearrangements. The function of S2 subunit is to fuse the membranes of viruses and host cells. The cleavage site at the border between the SI and S2 subunits is called S1/S2 protease cleavage site. For all the coronaviruses, host proteases cleave the spike glycoprotein at the S2’ cleavage site to activate the proteins which allows subsequent fusion of the membranes of viruses and host cells through irreversible conformational changes. CoV-S binds to its cognate receptor, angiotensin-converting enzyme 2 (ACE2), via a receptor binding domain (RBD) present in the SI subunit. The amino acid sequence of full- length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in SEQ ID NO: 1483 and FIG. 15A. The term “CoV-S” as used herein includes protein variants of CoV spike protein isolated from different CoV isolates as well as recombinant CoV spike protein or a fragment thereof. CoV spike protein variants include CoV spike fusion proteins and CoV spike proteins with one or more substitutions, as exemplified by the amino acid sequence provided in SEQ ID NO: 1484 and FIG. 15B. In some embodiments, the CoV spike protein is a synthetic CoV spike protein.
COMPOSITIONS OF THE DISCLOSURE
[0139] As described in greater detail below, one aspect of the present disclosure relates to anti-CoV-S antigen-binding polypeptides, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or an antigenic fragment thereof. Also provided, in other related aspects of the disclosure, are nucleic acids encoding the antibodies and antigen-binding fragments as disclosed herein, recombinant cells and transgenic animals engineered to produce the antibodies and antigen-binding fragments as disclosed herein, pharmaceutical compositions containing one or more of the nucleic acids, recombinant cells, and antibodies and antigen-binding fragments as disclosed herein.
Antigen-binding proteins
[0140] One aspect of the present disclosure relates to antigen-binding polypeptides, such as antibodies and antigen-binding fragments thereof identified by a method disclosed herein, for example, e.g., antibodies and antigen-binding fragments thereof that specifically bind to CoV spike protein or an antigenic fragment thereof.
[0141] An antibody is generally understood by the skilled artisan in the art to refer to immunoglobulin molecules including four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g., IgM). Exemplary antibodies include, for example, those listed in Table 1. Each heavy chain includes a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (which is comprised of domains CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FWR). Each VH and VL includes three CDRs and four FWRs, arranged from amino-terminus to carboxy-terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. Heavy chain CDRs can also be referred to as HCDRs, and numbered as described above (e.g., HCDR1, HCDR2, and HCDR3). Likewise, light chain CDRs can be referred to as LCDRs, and numbered LCDR1, LCDR2, and LCDR3. In some embodiments of the disclosure, the FWRs of the antibodies or antigen-binding fragments thereof are identical to the human germline sequences, or are naturally or artificially modified. Thus, the present disclosure provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g, anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) including HCDR and LCDR sequences of Table 1 within a variable heavy chain or light chain region of human germline immunoglobulin sequences.
[0142] In some embodiments, the assignment of amino acids to each domain is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al. National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991). The amino acid sequence boundaries of an antibody CDR can also be determined by one of skill in the art using any of a number of numbering schemes, including those described by Kabat (1978) Adv. Prof Chem. 32:1-75; Kabat, etal. , (1977) J. Biol. Chem. 252:6609-6616 (“Kabat” numbering scheme, which derives CDR definitions and a residue numbering scheme based purely on antibody sequence information); Chothia, etal. , (1987) J Mol. Biol. 196:901-917, Chothia, etal., (1989) Nature 342:878-883, or Al-Lazikani etal, 1997, J. Mol. Biol., 273:927- 948 (“Chothia” numbering scheme, which defines defined CDRs from the earliest structures of antibodies); MacCallum etal., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc etal, Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pldckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme). In addition or alternatively, the amino acid sequence boundaries of an antibody CDR can also be determined by one of skill in the art using an “enhanced Chothia” scheme as described previously in, e.g., Abhinandan KR and Martin AC, Mol Immunol 2008 Aug;45(14):3832-9; or using a more recent methodology of distance-function clustering of antibody CDR loop conformations based on directional statistics and clustering algorithm using affinity propagation (see, e.g., North B. etal. J Mol Biol. 2011 Feb 18; 406(2): 228-256.
[0143] In some embodiments, CDR and FWR regions are determined using evolutionarily conserved motifs. See, e.g., U.S. Application Ser. No. 63/163,292, which is hereby incorporated by reference in its entirety.
Table 1: Exemplary antigen-binding polypeptides, e.g., antibodies, of the disclosure. HCVR(l) and LCVR(l) correspond to the sequences of heavy chain variable region and light chain variable regions, respectively, without a leader peptide sequence. HCVR(2) and LCVR(2) correspond to the sequences of heavy chain variable region and light chain variable regions, respectively, including a leader peptide sequence. Exemplary nucleic acid sequences encoding the HCVRs and LCVRs are listed in SEQ ID NOS: 1487-1886 and 1887-2286, respectively.
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
[0144] The term “antigen -binding fragment” of an antibody or antigen-binding polypeptide, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody ( e.g ., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FWR3-CDR3-FWR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMFPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein. In some embodiments of the disclosure, the antigen binding fragment includes three or more CDRs of an antibody of Table 1 (e.g., HCDR1, FICDR2 and HCDR3; or LCDR1, CDR2 and LCDR3).
[0145] An antigen-binding fragment of an antibody, in some embodiment of the disclosure, include at least one variable domain. The variable domain can be of any size or amino acid composition and will generally include at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH- VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.
[0146] In some embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that can be found within an antigen binding fragment of an antibody of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may include a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). Antigen-binding proteins ( e.g ., antibodies and antigen -binding fragments) can be mono-specific or multi-specific (e.g., bi- specific).
[0147] In one aspect of the disclosure, provided herein are isolated isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the antibody is selected from the group consisting of TXG-0153, TXG-0115, TXG-140, and TXG-154, wherein: (a) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0153 have the amino acid sequences as set forth in SEQ ID NOS: 88, 168, 248, 328, 408, and 488, respectively; (b) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0115 have the amino acid sequences as set forth in SEQ ID NOS: 72, 152, 232, 312, 392, and 472, respectively; (c) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0140 have the amino acid sequences as set forth in SEQ ID NOS: 83, 163, 243, 323, 403, and 483, respectively; and (d) the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0154 have the amino acid sequences as set forth in SEQ ID NOS: 89, 169, 249, 329, 409, and 489, respectively.
[0148] In some embodiments, the antibody or antigen-binding fragment thereof all six CDRs from TXG-0153. In some embodiments, the antibody or antigen-binding fragment thereof all six CDRs from TXG-0115. In some embodiments, the antibody or antigen-binding fragment thereof all six CDRs from TXG-0140. In some embodiments, the antibody or antigen-binding fragment thereof all six CDRs from TXG-0154.
[0149] In another aspect, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibody or fragment thereof as disclosed herein competes with a reference antibody for binding to the same epitope on the SARS-CoV-2 spike protein. In some embodiments, the reference antibody is an FDA approved antibody selected from the group consisting of Casirivimab, Imdevimab, Bamlanivimab, Etesevimab, Sotrovimab, and Tixagevimab. In some embodiments, the antibody or fragment thereof as disclosed herein competes with Sotrovimab for binding to the same epitope in the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In some embodiments, the Sotrovimab epitope in RBD of the SARS-CoV-2 spike protein includes amino acid residues 332-337, 339-340, 343-346, 354, 356-361, 440-441, and/or 509 of the RBD of the SARS-CoV-2 spike protein. See, e.g., Pinto D. etal. Nature , Volume 583, pages 290-295 (2020). In some embodiments, the antibody or fragment thereof as disclosed herein shares an epitope bin with Sotrovimab. In some embodiments, the antibody or fragment thereof as disclosed herein competes with Sotrovimab for binding to amino acid residues 332-337, 339- 340, 343-346, 354, 356-361, 440-441, and/or 509 of the RBD of the SARS-CoV-2 spike protein.
[0150] In one aspect of the disclosure, provided herein are isolated antibodies or antigen binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibodies or antigen-binding fragments include: (a) a heavy chain complementary determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3; (b) a HCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 4-6 and c) a HCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 7-9.
[0151] Non-limiting exemplary embodiments of the antibodies and antigen-binding fragments thereof of the disclosure can include one or more of the following features. In some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure include the heavy chain CDRs (HCDR1, HCDR2, and HCDR3) from the antibodies belonging to the same clonotype family, for example, from a clonotype family selected from the group consisting of clonotype families A, B, and C. Accordingly, in some embodiments, the antibodies and antigen binding fragments thereof of the disclosure include the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 1, 4, and 7, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in in SEQ ID NOS: 2, 5, and 8, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 3, 6, and 9, respectively
[0152] In one aspect, some embodiments of the disclosure provides isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibodies or antigen-binding fragments include: (a) a light chain complementary determining region 1 (LCDR1) comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 10-12; (b) a LCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 13-15; and (c) a LCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 16-18.
[0153] In some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure include the light chain CDRs (LCDR1, LCDR2, and LCDR3) from the antibodies belonging to the same clonotype family, for example, from a clonotype family selected from the group consisting of clonotype families A, B, and C. Accordingly, in some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 11, 14, and 17, respectively. In some embodiments, the antibodies and antigen-binding fragments include the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 12, 15, and 18, respectively. In another aspect, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of SARS-CoV-2, wherein the antibodies or antigen-binding fragments include: (a) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3; (b) a HCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 4-6; c) a HCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 7-9; (d) a LCDR1 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 10-12; (e) a LCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 13- 15; and (f) a LCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 16-18.
[0154] In some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure include (a) the heavy chain CDRs (HCDR1, HCDR2, and HCDR3), and (b) the light chain CDRs (LCDR1, LCDR2, and LCDR3) from the antibodies belonging to the same clonotype family, for example, a clonotype family selected from the group consisting of clonotype families A, B, and C. Accordingly, in some embodiments, the antibody or antigen binding fragment comprises: (a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 1, 4, and 7, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively. In some embodiments, the antibody or antigen-binding fragment comprises: (a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 2, 5, and 8, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 11, 14, and 17, respectively. In some embodiments, the antibody or antigen-binding fragment comprises: (a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 3,
6, and 9, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 12, 15, and 18, respectively.
[0155] Non-limiting exemplary embodiments of the antibodies and antigen-binding fragments thereof of the disclosure can include one or more of the following features. In some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure can include a polypeptide including an amino acid sequence that is set forth herein except for one or more ( e.g ., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations ( e.g ., conservative substitutions), non-sense mutations, deletions, or insertions. For example, the present disclosure includes antigen-binding polypeptides which include an immunoglobulin light chain variant comprising an LCVR amino acid sequence set forth in Table 1 and Sequence Listing but having one or more of such mutations and/or an immunoglobulin heavy chain variant comprising an HCVR amino acid sequence set forth in Table 1 and Sequence Listing but having one or more of such mutations. As described in greater detail below, in some embodiments, an anti-CoV-S antibody or antigen-binding fragment of the disclosure can include an immunoglobulin light chain variant comprising LCDR1, LCDR2 and LCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) and/or an immunoglobulin heavy chain variant comprising HCDR1, HCDR2 and HCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g, conservative substitutions). Such substitutions can be in a CDR, framework, and/or constant region of an antibody or antigen-binding fragment.
[0156] Accordingly, in some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure can include one or more variant CDRs (e.g, any one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) that are set forth herein with at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to, e.g., the heavy chain and light chain CDRs of Table 1 and Sequence Listing.
[0157] In some embodiments, the HCDR1 amino acid sequence is at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122. In some embodiments, the HCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3 and SEQ ID NOS: 43-122, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122. In some embodiments, the HCDR2 amino acid sequence is at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123- 202. In some embodiments, the HCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 4-6 and SEQ ID NOS: 123-202, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-202. In some embodiments, the HCDR3 amino acid sequence is at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282. In some embodiments, the HCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-9 and SEQ ID NOS: 203-282, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282.
[0158] In some embodiments, the LCDR1 amino acid sequence is at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362. In some embodiments, the LCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 10-12 and SEQ ID NOS: 283-362, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362. In some embodiments, the LCDR2 amino acid sequence is at least 90%, e.g ., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442. In some embodiments, the LCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-15 and SEQ ID NOS: 363-442, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442. In some embodiments, the LCDR3 amino acid sequence is at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443- 522. In some embodiments, the LCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 16-18 and SEQ ID NOS: 443-522, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522.
[0159] In some embodiments, an antibody or antigen-binding fragment of the disclosure includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 43-122; (b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 123-202; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 203-282; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 283-362; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 363-442; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 443-522.
[0160] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies belonging to a clonotype family. One skilled in the art will appreciate that when an antibody is said to include a plurality of HCDRs and/or LCDRs that “are independently selected from a group of antibodies,” this can mean that each of the HCDRs and/or LCDRs of the antibody or antigen-binding fragment can be independently selected from the HCDRs and LCDRs of the antibodies belonging to said group of antibodies. As such, in some embodiments, an antibody or antigen-binding fragment as disclosed herein can include three HCDRs and three LCDRs each of which can be independently selected from the HCDRs and LCDRs of different antibodies belonging to the same group of antibodies. As such, in some embodiments, an antibody or antigen-binding fragment as disclosed herein can include the three HCDRs and three LCDRs of any one of the antibodies of the group.
[0161] Some embodiments of the disclosure provide an antibody or antigen-binding fragment including three HCDRs and three LCDRs each of which are independently selected from the HCDRs and LCDRs of the antibodies belonging to a clonotype family. In some embodiments, an antibody or antigen-binding fragment as disclosed herein can include three HCDRs and three LCDRs each of which can be independently selected from the HCDRs and LCDRs of different antibodies belonging to the same clonotype family. In some embodiments, an antibody or antigen-binding fragment as disclosed herein can include the three HCDRs and the three LCDRs of a selected antibody of a clonotype family. As described in greater detail in Examples 6-7 below, antibodies and antigen-binding fragments exhibiting a binding affinity to a target antigen above a cut-off threshold were selected for further analysis using lOx Genomics “Enclone” (available at https://bit.ly/enclone), which is a computational tool developed for clonal grouping to study the adaptive immune system. In this computational tool, the lOx Genomics Chromium Single Cell V(D)J data containing B cell receptor (BCR) and T cell receptor (TCR) RNA sequences are entered as input data to Enclone. Based on the input, Enclone finds and organizes cells arising from the same progenitors into groups ( e.g ., clonotype families) and compactly displays each clonotype along with its salient features, including mutated amino acids. In some embodiments, the antibodies belong to clonotype family A, B, and C. In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following clonotype family of antibodies: (a) clonotype family A: TXG-0021, TXG-0022, and TXG-0023; (b) clonotype family B: TXG-0027 and TXG-0028; or (c) clonotype family C: TXG-0227, TXG-0228, TXG-0229, and TXG-0230..
[0162] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies belonging to a clonotype family A. Accordingly, in some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-45;
(b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-125; (c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-205; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-285; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-365; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-445.
[0163] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies belonging to a clonotype family B. Accordingly, in some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 46-47;
(b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 126-127; (c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 206-207; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 286-287; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 366-367; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 446-447.
[0164] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies belonging to clonotype family C. Accordingly, in some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 117-120; (b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 197-200; (c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 277-280; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 357-360; (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 437-440; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 517-520.
[0165] Variations in amino acid sequences of the CoV-S antibodies and antigen-binding fragments described herein may be naturally occurring, such as splicing variants or allelic variants. In addition or alternatively, variations in amino acid sequences of the CoV-S antibodies and antigen-binding fragments may be introduced by substitution, deletion or insertion of one or more codons into the nucleic acid sequences encoding the antibodies that results in a change in the amino acid sequences of the antibodies. Optionally, the variation may be resulted from substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids with any other amino acid in the antibodies. Amino acid substitutions in variants of CoV-S antibodies and antigen-binding fragments may be conservative or non-conservative. Those of skill in the art will understand that a “non-conservative substitution,” when used in reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution ( e.g ., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. A non-limiting exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
[0166] Conservatively modified variant anti-CoV-S antibodies and antigen-binding fragments thereof are also contemplated as part of the present disclosure. A “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc. ). Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment. 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. In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.
[0167] Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Exemplary conservative amino acids substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 1443 45.
[0168] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes a framework region having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 523- 1162. [0169] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a heavy chain framework region 1 (HFWR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 523-602; (b) a heavy chain framework region 2 (HFWR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 603-682; (c) a heavy chain framework region 3 (HFWR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 683-762; and (d) a heavy chain framework region 4 (HFWR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 763-842.
[0170] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a light chain framework region 1 (LFWR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 843-922; (b) a light chain framework region 2 (LFWR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 923-1002; (c) a light chain framework region 3 (LFWR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1003-1082; and (d) a light chain framework region 4 (LFWR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1083-1162.
[0171] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes the heavy chain framework regions HFWR1, HFWR2, HFWR3, and HFWR4 of the same antibody or antigen-binding fragment as shown in Table 2. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes the light chain framework regions LFWR1, LFWR2, LFWR3, and LFWR4 of the same antibody or antigen binding fragment as shown in Table 2.
Table 2: Exemplary antibodies of the disclosure and corresponding framework regions.
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
[0172] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163-1322. In some embodiments, the HCVR comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163-1322. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region (LCVR) comprising an amino acid sequence having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482. In some embodiments, the LCVR comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482.
[0173] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a HCVR comprising an amino acid sequence having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163-1322; and b) a LCVR comprising an amino acid sequence having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482. In some embodiments, the antibody or antigen-binding fragment includes: a HCVR and a LCVR which respectively are 90%, e.g., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to the HCVR and LCVR of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment includes the HCVR and LCVR of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure is selected from Table 1.
[0174] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a constant region (e.g., an Fc region). In some embodiments, the constant region is an IgA, IgD, IgE, IgG, or IgM heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment of the disclosure includes a constant region of the type IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3 and IgG4) or IgM. In some embodiments, the constant region is an IgG constant region.
[0175] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a kappa type light chain constant region. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a lambda type light chain constant region.
[0176] In some embodiments, the antibody or or antigen-binding fragment of the disclosure comprises a hinge domain. In some embodiments, the hinge domain comprises a stabilizing mutation.
[0177] In some embodiments, the antibody or or antigen-binding fragment of the disclosure comprises a variant Fc region. The variant Fc region may comprise one or more amino acid modifications that reduce the affinity of the variant Fc Region for an FcyR receptor. Exemplary amino acid modifications that reduce the affinity of the variant Fc Region for an FcyR receptor include L234A; L235A; or L234A and L235A, according to Kabat (EU index) numbering system. The variant Fc region may comprise one or more amino acid modifications that enhance the serum half-life of the variant Fc region. Exemplary amino acid modifications that enhance the serum half-life of the variant Fc region include M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, according to Kabat (EU index) numbering system.
[0178] In some embodiments, the antibody or antigen-binding fragment of the disclosure is a human antibody or antigen-binding fragment. One of ordinary skill in the art will understand that the term “human” antibody includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non human cell, e.g., a mouse cell. The human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences ( e.g ., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, such as CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FWR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal.
[0179] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody, a chimeric antibody, or a hybrid antibody, or an antigen-binding fragment of any thereof. The term “humanized antibody” as used herein encompasses antibodies comprising heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human like,” i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Another type of humanized antibody is a FWR-grafted antibody in which human FWR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human FWR sequences. In some embodiments, the antibodies or antigen-binding fragments of the disclosure include a murine antibody, phage display antibody, or nanobody / VHH containing the frameworks and/or CDRs described in this disclosure ( e.g ., Table 1 and Sequence Listing). As used herein, the term “chimeric antibody” encompasses antibodies having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different species. As used herein, the term “hybrid antibody” encompasses antibodies having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different animals, or wherein the variable domain, but not the constant region, is from a first animal. For example, a variable domain can be taken from an antibody isolated from a human and expressed with a fixed constant region not isolated from that antibody. Hybrid antibodies are synthetic and non- naturally occurring because the variable and constant regions they contain are not isolated from a single natural source. In some embodiments, the hybrid antibodies of the disclosure includes a light chain from a first antibody and a heavy chain from a second antibody, wherein the first and second antibodies are from different species. In some embodiments, the chimeric antibodies of the disclosure includes a non-human light chain which is combined with a heavy chain or set of heavy chain CDRs disclosed in this application.
[0180] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment is an engineered antibody or engineered antibody fragment. Non-limiting examples of engineered antibody fragment include a single chain variable fragment (scFv), a nanobody, a diabody, a triabody, a minibody, an F(ab’)2 fragment, an F(ab) fragment, a VH domain, a VL domain, a single chain variable fragment (scFv), a single domain antibody (sdAb), a VNAR domain, and a VHH domain. In some embodiments, the antibody or antigen-binding fragment of the disclosure is a single-chain antibody fragment (scFv), a F(ab) fragment, a F(ab') fragment, a Fab'-SH, a F(ab')2 fragment, or a Fv fragment.
[0181] In some embodiments, provided herein are recombinant immune receptors comprising one or more antigen-binding fragments of the disclosure. Such recombinant immune receptors can immunologically recognize and/or specifically bind to a CoV-S protein, or an epitope thereof. In some embodiments, the CoV-S-specific receptor is a chimeric antigen receptor (CAR). Generally, a CAR includes an antigen-binding domain as disclosed herein, e.g ., a single-chain variable fragment (scFv) of an antibody having a binding affinity to an antigen (e.g., a CoV-S protein) or an epitope thereof, fused to a transmembrane domain and an intracellular domain of a T cell receptor (TCR). In this case, the antigenic specificity of a CAR can be encoded by a scFv which specifically binds to the antigen, or an epitope thereof. CARs, and methods of making them, are known in the art.
[0182] In some embodiments, the antibody or antigen-binding fragment has a binding affinity (e.g., ability to bind, with varying degrees of specificity) to an epitope in a subunit of the SARS-CoV-2 S protein. One skilled in the art will understand that the term “epitope” refers to an antigenic determinant (e.g, a CoV-S polypeptide) that interacts with a specific antigen-binding site of an antigen-binding polypeptide, e.g., available region of an antibody molecule, known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can be linear or conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural characteristics, and/or specific charge characteristics.
[0183] Methods for determining the epitope of an antigen-binding polypeptide, e.g., antibody or antigen-binding fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis, peptide cleavage analysis, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed. Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding polypeptide ( e.g ., antibody or fragment or polypeptide) interacts is hydrogen/deuterium exchange detected by mass spectrometry.
[0184] In some embodiments, the antibody or antigen-binding fragment has a binding affinity to an epitope the SI subunit of the SARS-CoV-2 S protein. In some embodiments, the antibody or antigen-binding fragment has a binding affinity to a RBD of the SI subunit. In some embodiments, the subunit of the S protein of SARS-CoV-2 is the S2 subunit. In some embodiments, the antibody or antigen-binding fragment has a binding affinity to a NTD of the SI subunit.
[0185] In some embodiments, the SARS-CoV-2 S protein may include one or more amino acid substitutions. In some embodiments, the SARS-CoV-2 S protein includes one or more of the following Proline substitutions: F817P, A892P, A899P, A942P, K986P, and V987P. In some embodiments, the SARS-CoV-2 S protein includes one or more of the following Alanine substitutions: R683A and R685A. In some embodiments, the one or more amino acid substitutions includes D614G substitution. In some embodiments, the SARS-CoV-2 S protein has the amino acid sequence provided in SEQ ID NO: 1484. In some embodiments, the S protein of SARS-CoV-2 has the amino acid sequence provided in SEQ ID NO: 1485.
[0186] In some embodiments, the SARS-CoV-2 S protein includes one or more amino acid substitutions at a position selected from the group consisting of K417, L452, E484, N501, and D614. In some embodiments, the SARS-CoV-2 S protein includes an amino acid substitution at position K417. In some embodiments, the K417 amino acid substitution is a conservative amino acid substitution. In some embodiments, the amino acid substitution at position K417 is K417T or K417N. In some embodiments, the K417 amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the L452 amino acid substitution is a conservative amino acid substitution. In some embodiments, the amino acid substitution at position L452 is L452R. In some embodiments, the L452 amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the SARS-CoV-2 S protein includes an amino acid substitution at position E484. In some embodiments, the E484 amino acid substitution is a conservative amino acid substitution. In some embodiments, the E484 amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the amino acid substitution at position E484 is E484K or E484Q. In some embodiments, the SARS-CoV-2 S protein includes an amino acid substitution at position N501. In some embodiments, the N501 amino acid substitution is a conservative amino acid substitution. In some embodiments, the N501 amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the amino acid substitution at position N501 is N501Y. In some embodiments, the SARS-CoV-2 S protein includes an amino acid substitution at position D614. In some embodiments, the D614 amino acid substitution is a conservative amino acid substitution. In some embodiments, the D614 amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the amino acid substitution at position D614 is D614G.
[0187] In some embodiments, the SARS-CoV-2 S protein includes one or more amino acid substitutions selected from the group consisting of K417T, K417N, L452R, E484K, E484Q, N501Y, and D614G. In some embodiments, the SARS-CoV-2 S protein includes a combination of the following amino acid substitutions: K417N, E484K, and N501Y. In some embodiments, the SARS-CoV-2 S protein includes a combination of the following amino acid substitutions: K417T, E484K, andN501Y. In some embodiments, the SARS-CoV-2 S protein includes a combination of the following amino acid substitutions: L452R and E484Q. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a pre-fusion trimeric form of the CoV-S protein. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a stabilized prefusion spike protein ( e.g ., an S2P-stabilized pre-fusion spike protein) in monomeric or multimeric (e.g., trimeric) form. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a non-prefusion spike protein in monomeric or multimeric (e.g., trimeric) form. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a non-S2P- stabilized pre-fusion spike protein in monomeric or multimeric (e.g., trimeric) form. Pre-fusion and non-prefusion conformations of the spike protein are described in, e.g., Cai Y. et al. Science Vol. 369, Issue 6511, pp. 1586-1592, 2020; Xu C. etal. Science Advances, Vol. 7, no. 1, Jan 2021; Wrobel AG etal. Nat Comms 11, 5337, 2020; and Zhang J. etal. Science March 16, 2021; all of which are hereby incorporated by reference in their entirety.
[0188] In some embodiments, the methods disclosed herein include assessing the specific binding affinity (e.g., ability to bind, with varying degrees of specificity) of the antibody or antigen-binding fragment to the target antigen. In some embodiments, the methods disclosed herein optionally include identifying the antibody or antigen-binding fragment as having a binding specificity for a CoV-S protein if the antibody or antigen-binding fragment specifically binds to the CoV-S protein. Generally, binding affinity can be used as a measure of the strength of a non-covalent interaction between two molecules, e.g. , an antibody or antigen-binding fragment thereof and an antigen (e.g., coronavirus S protein antigen). In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules can be quantified by determination of the equilibrium dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasm on resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or k0n) and dissociation rate constant kd (or koff), respectively. KD is related to ka and kd through the equation KD = kd / ka. The value of the dissociation constant can be determined directly by various methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340- 362). For example, the KD can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). As shown in Examples 9 and 12 below, binding affinity of the antibodies and fragments described herein can also be assayed using a Carterra LSA SPR biosensor equipped with a HC30M chip.
[0189] Other assays to evaluate the binding ability (e.g., binding affinity and/or specificity) of the antibodies and antigen-binding fragments of the present disclosure towards target antigens include, for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of an antibody or an antigen-binding fragment for a target antigen (e.g., coronavirus S protein antigen) can be calculated by the Scatchard method described by Frankel etal.,Mol. Immunol, 16: 101-106, 1979. It will be understood that the binding affinity of an antibody or antigen-binding fragment for a target antigen is the strength of interaction between the antibody or antigen-binding fragment with the target antigen, whereas the binding specificity of an antibody or antigen-binding fragment for a target antigen relates to the affinity to the target antigen relative to other antigens. It will also be understood that an antibody or antigen-binding fragment that “specifically binds” a target antigen (such as S protein) is an antigen-binding fragment that binds the target antigen but does not significantly bind other antigens. In some embodiments, the antibody or antigen-binding fragment “specifically binds” a target antigen if it does not significantly bind other antigens but binds the target antigen with high affinity, e.g., with an equilibrium dissociation constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less. In some embodiments, the antibodies or antigen binding fragments of the disclosure that specifically bind a target antigen, such as a CoV-S protein (e.g., SARS-CoV-2 S protein), have a binding affinity to the target antigen expressed as KD, of at least about 1 (G8 M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25° C. or 37°C, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA. In some embodiments, the antibody or antigen-binding fragment has a binding affinity and/or binding specificity with an equilibrium dissociation constant (KD) value of less than 500 nM, for example, less than 400 nM, less than 300 nM, less than 200 nM, less than 150 nM, less than 120 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 5 nM, or less than 1 nM.
[0190] In some embodiments, the binding affinity and/or binding specificity of an antigen binding molecule (e.g., antibody or antigen-binding fragment) to a CoV-S target antigen is determined based on a quantity/number of target and optionally non-target antigen sequence reads and/or unique molecular identifiers (UMIs) associated with the antigen binding molecule via a process termed “barcode-enabled antigen mapping by sequencing” (BEAM-seq) (see, e.g., Examples 6 and 7 and FIGS. 2, 3, and 4 below). Antigen sequence reads and/or UMIs can be associated bioinformatically with antigen binding molecule sequences via shared partition barcode sequences. For example, binding affinity and/or binding specificity of an antigen binding molecule to the CoV-S antigen can be determined based on independent observations of quantity/number of UMIs associated with the CoV-S antigen and optionally non-target antigen from one or more partitions, wherein each of the one or more partitions comprise a cell expressing the same antigen-binding molecule. For other example, binding affinity and/or binding specificity of an antigen binding molecule to the CoV-S antigen can be determined based on independent observations of quantity/number of UMIs associated with the antigen from one or more partitions, wherein each of the one or more partitions comprise a cell expressing an antigen-binding molecule belonging to the same clonotype group. For example, high ( e.g ., over 40) target antigen UMI counts can be used to predict high binding affinity. As demonstrated in the Examples below, an antibody can be predicted to have specific binding affinity for the target CoV-S antigen if it is associated with high target antigen counts and low non-target antigen counts.
[0191] In some embodiments, the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies of following group of antibodies: TXG-0021, TXG-0022, TXG-0023, TXG-0027, TXG-0028, TXG-0043, TXG-0049, TXG-0056, TXG-0061 , TXG-0062, TXG-0068, TXG-0072, TXG-0083, TXG-0085, TXG-0089, TXG-0090, TXG-0098, TXG-0108, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0130, TXG-0134, TXG-0135, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0150, TXG-0152, TXG-0153, TXG-0154, TXG-0160, TXG-0165, TXG-0169, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0216, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, TXG-0229, and TXG-0230.
[0192] In some embodiments, the antibody or antigen-binding fragment has a binding affinity and/or binding specificity with an equilibrium dissociation constant (KD) value of less than 500 nM, for example, less than 400 nM, less than 300 nM, less than 200 nM, less than 150 nM, less than 120 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 5 nM, or less than 1 nM.
[0193] In some embodiments, the antibody or antigen-binding fragment has a binding affinity with a KD value lower than the binding affinity between a SARS-CoV-2 S protein and its receptor ACE2, which has been previously estimated to have a KD value of about 120 nM. Accordingly, in some embodiment of the disclosure, the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 120 nM. In some embodiments, the antibody or antigen-binding fragment has a KD value of less than 120 nM and includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies of Table 5.
[0194] In some embodiments, the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM and includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: TXG-0022, TXG- 0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG- 0230.
[0195] In some embodiments, the antibody or antigen-binding fragment includes a HCVR which includes an amino acid sequence having at least 90% sequence identity to the HCVR of an antibody of Table 5. In some embodiments, the antibody or antigen-binding fragment includes a HCVR which includes an amino acid sequence having at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the HCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG- 0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG-0230. In some embodiments, the antibody or antigen-binding fragment includes a HCVR which includes an amino acid sequence having at least 100% sequence identity to the HCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG- 0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG-0230.
[0196] In some embodiments, the antibody or antigen-binding fragment includes a LCVR which includes an amino acid sequence having at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the LCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG-0230. In some embodiments, the antibody or antigen-binding fragment includes a LCVR which includes an amino acid sequence having at least 100% sequence identity to the LCVR of an antibody selected from the group consisting of TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG- 0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, and TXG-0230.
[0197] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof. For example, in some embodiments of the disclosure, the antibody or antigen binding fragment has a binding affinity with a KD value of less than 500 pM, for example, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM. In some embodiments, the antibody or antigen-binding fragment with sub-nanomolar binding affinity for a SARS-CoV-2 S protein is selected from the group consisting of TXG-0028, TXG-0049, TXG-0056, TXG-0072, TXG-0089, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0194, and TXG-0217. One skilled in the art will appreciate that when an antibody is said to be “selected from a group,” this can mean that the antibody comprises all six CDRs from any one of the antibodies selected from the group. In some embodiments, the antibody can comprise the HCVR and LCVR from any one of the antibodies selected from the group. In some embodiments, the antibody can be any one of the antibodies in the group.
[0198] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for HCOV and/or for a SARS-CoV-2 S variant selected from the group consisting of beta, gamma, delta, and kappa.
[0199] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for HCOV. Exemplary antibodies having this binding affinity property include TXG-0136.
[0200] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for the SARS-CoV-2 S beta variant. Exemplary antibodies having this binding affinity property include TXG-0136, TXG-0145, TXG-0153, TXG-0154, TXG- 0174, TXG-0182, TXG-0194, and TXG-0217.
[0201] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for the SARS-CoV-2 S gamma variant. Exemplary antibodies having this binding affinity property include TXG-0136, TXG-0137, TXG-0145, TXG-0147, TXG- 0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0194, and TXG-0217.
[0202] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for the SARS-CoV-2 S kappa variant. Exemplary antibodies having this binding affinity property include TXG-0136, TXG-0137, TXG-0145, TXG-0154, TXG- 0165, TXG-0173, TXG-0174, TXG-0182, TXG-0194, and TXG-0217. In some embodiments, such antibodies also have binding affinity for the SARS-CoV-2 delta variant.
[0203] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S. Exemplary antibodies having this binding affinity property include TXG-0115, TXG- 0140, TXG-0153, TXG-0154, TXG-0173, and TXG-0174.
[0204] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the beta variant. Exemplary antibodies having these binding affinity properties include TXG-0115, TXG-0153, TXG-0154, and TXG- 0174. In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the gamma variant. Exemplary antibodies having these binding affinity properties include TXG-0115, TXG-0153, TXG-0154, TXG-0173, and TXG- 0174. [0205] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the kappa variant. Exemplary antibodies having these binding affinity properties include TXG-0115, TXG-0154, TXG-0173, and TXG- 0174. In some embodiments, such antibodies potently neutralize live SARS-CoV 2 and have binding affinity to the delta variant.
[0206] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the N-terminal domain of the SI subunit. Exemplary antibodies having these binding affinity properties include TXG-0173, and TXG-0174.
[0207] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S and beta, gamma, kappa variants. Exemplary antibodies having these binding affinity properties include TXG-0115 and TXG-0154. In some embodiments, such antibodies also have binding affinity for the SARS-CoV-2 delta variant.
[0208] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S. Exemplary antibodies having these binding affinity properties include TXG-0140.
[0209] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2 and has binding affinity to the trimeric forms of wild-type SARS- CoV-2 S, as well as the trimeric forms of the beta and gamma variants. Exemplary antibodies having these binding affinity properties include TXG-0153.
[0210] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2, and has binding affinity to the N-terminal domain (NTD) of the SI subunit and to the trimeric forms of wild-type SARS-CoV-2 S, as well as to trimeric forms of the gamma and kappa variants. Exemplary antibodies having these binding affinity properties include TXG-0173. In some embodiments, such antibodies also have binding affinity for the SARS-CoV-2 delta variant.
[0211] In some embodiments, the antibody or antigen-binding fragment potently neutralizes live SARS-CoV 2, and has binding affinity to the N-terminal domain (NTD) of the SI subunit and to the trimeric forms of wild-type SARS-CoV-2 S, as well as to trimeric forms of the beta, gamma, and kappa variants. Exemplary antibodies having these binding affinity properties include TXG-0174. In some embodiments, such antibodies also have binding affinity for the SARS-CoV-2 delta variant.
[0212] In some embodiments, the binding affinity and/or binding specificity of an antigen binding molecule ( e.g ., antibody or antigen-binding fragment) to a target antigen (such as S protein) can be determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen-binding molecule bound to the antigen (see, e.g., Example 6 and Table 3 below).
[0213] In some embodiments, the binding affinity and/or binding specificity of an antigen binding molecule (e.g., antibody or antigen-binding fragment) to a target antigen (such as S protein) can be determined based on the counts and/or amounts of target antigens and optionally non-target antigens that are associated with the antigen-binding molecule. The counts and/or amounts of such antigens can be facilitated by the respective reporter oligonucleotides coupled to the antigens, wherein a reporter oligonucleotide coupled to an antigen comprises a reporter barcode sequence that identifies the antigen coupled thereto.
[0214] In some embodiments, the binding affinity and/or binding specificity of an antigen binding molecule (e.g., antibody or antigen-binding fragment) to a target antigen (such as S protein) can be determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen bound to one or more cells expressing the antigen-binding molecule via a process termed “barcode-enabled antigen mapping by sequencing” (BEAM-seq) (see, e.g., Example 7 and FIGS. 2, 3, and 4 below). For example, binding affinity and/or binding specificity of an antigen binding molecule to a target antigen can be determined based on independent observations of quantity/number of UMIs associated with the antigen in two or more partitions, wherein each of the two or more partitions comprise a cell expressing the same antigen-binding molecule.
[0215] In some embodiments, the antibodies and antigen-binding fragments of the disclosure bind to a target antigen, such as a CoV-S protein (e.g., SARS-CoV-2 S protein), and compete for binding with another antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof) to the target antigen. Accordingly, also provided herein are antibodies or antigen-binding fragments thereof that compete for binding with an antibody disclosed herein, e.g, in Table 1.
[0216] The term “competes” as used herein, refers to an antibody or antigen-binding fragment that binds to a target antigen, and inhibits or blocks the binding of another antigen binding polypeptide ( e.g ., antibody or antigen-binding fragment thereof) to the target antigen.
The term also includes competition between two antigen-binding polypeptides e.g., antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice versa. In some embodiments, the first antigen-binding polypeptide {e.g., antibody or antigen binding fragment) and second antigen-binding polypeptide {e.g., antibody or antigen-binding fragment thereof) may bind to the same epitope. Alternatively, the first and second antigen binding polypeptides {e.g., antibodies or antigen-binding fragments) may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Competition between antigen-binding polypeptides {e.g., antibodies or antigen-binding fragments) may be measured by methods known in the art, for example, by a real-time, label -free bio-layer interferometry assay. Epitope mapping {e.g., via alanine scanning or hydrogen-deuterium exchange (HDX)) can be used to determine whether two or more antibodies are non-competing {e.g., on a spike protein RBD monomer), competing for the same epitope, or competing but with diverse micro-epitopes {e.g., identified through HDX). In some embodiments, competition between a first and second anti-CoV-S antigen binding polypeptide {e.g, antibody or antigen-binding fragment thereof) is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding polypeptide {e.g, antibody) (not initially complexed with CoV-S protein) to bind to soluble CoV-S protein complexed with a second anti-CoV-S antigen-binding polypeptide {e.g, antibody or antigen binding fragment thereof). A reduction in the ability of the first anti-CoV-S antigen-binding polypeptide {e.g, antibody or antigen-binding fragment thereof) to bind to the complexed CoV-S protein, relative to uncomplexed CoV-S protein, indicates that the first and second anti-CoV-S antigen-binding polypeptides {e.g., antibodies or antigen-binding fragments thereof) compete.
The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label -free bio-layer interferometry assay, e.g, on an Octet RED384 biosensor (Pall ForteBio Corp ), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).
[0217] In some embodiments, the antibodies and antigen-binding fragments of the disclosure have a neutralizing activity {e.g, antagonistic activity) against SARS-CoV-2, e.g, able to bind to and neutralize the activity of SARS-CoV-S, as determined by in vitro or in vivo assays. The ability of the antibodies of the disclosure to bind to, block and/or neutralize the activity of SARS-CoV-2 may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein. For example, the binding affinity and dissociation constants of anti-SARS-CoV-2 antigen-binding polypeptides for SARS-CoV-2 can be determined by surface plasmon resonance (SPR) assay. Alternatively, neutralization assays were used to determine infectivity of SARS-CoV-2 S protein-containing virus-like particles. One of ordinary skill in the art will understand that a neutralizing or antagonistic CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment, generally refers to a molecule that inhibits an activity of CoV-S to any detectable degree, e.g., inhibits or reduces the ability of CoV-S to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or mediate viral reproduction in a host cell. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have a neutralization activity IC50 value of less than 150 ng/ml for viral neutralization, as determined by a quantitative focus reduction neutralization test (FRNT) described previously by Zost etal. (Nature, 584:443-449, 2020). In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 150 ng/ml for blocking ACE2. In some embodiments, the antibodies and antigen binding fragments of the disclosure have blocking activity IC50 value of less than 10 ng ml for S2P ectodomain binding. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 10 ng/ml for RBD ectodomain binding. In some embodiments, the antibody or antigen-binding fragment neutralizes at least 50% of 200 times the tissue culture infectious dose (200><TCID50) of the coronavirus at an antibody concentration of 12.5 pg/ml or less. Here, TCID50 represents the viral load at which 50% of cells are infected when a solution containing the virus is added to cell culture. In some embodiments, neutralizing antibodies are effective at antibody concentrations of <3.125 pg/ml, <.8 pg/ml, <.2 pg/ml, or <.l pg/ml.
[0218] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 1 pg/mL or lower, 200 ng/mL or lower, or 40 ng/mL or lower. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 200 ng/mL to 1,000 ng/mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 40 ng/mL to 200 ng/mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 8 ng/mL to 40 ng mL. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 1 pg/mL or lower. Exemplary antibodies or antigen-binding fragment thereof having this neutralizing activity property include TXG-0153, TXG-0173, TXG-0115, TXG- 0140, TXG-0154, and TXG-0174.
[0219] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 200 ng/mL or lower. Exemplary antibodies having this neutralizing activity property include TXG-0115, TXG-0140, and TXG- 0154.
[0220] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 200 ng/mL to 1000 ng/mL. Exemplary antibodies having this neutralizing activity property include TXG-0153, TXG-0173, and TXG- 0174. In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value of 100 ng/mL or lower. Exemplary antibodies having this neutralizing activity property include TXG-0140 and TXG-0154.
[0221] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 40 ng/mL to 200 ng/mL. Exemplary antibodies having this neutralizing activity property include TXG-0115 and TXG-0140.
[0222] In some embodiments, the antibody or antigen-binding fragment has a neutralizing activity against SARS-CoV-2 with an IC50 value that is 40 ng/mL or lower. Exemplary antibodies having this neutralizing activity property include TXG-0154.
[0223] In some embodiments, the antibody or antigen binding-fragment has a binding affinity to the N-terminal domain of (NTD) of a SARS-CoV-2 S protein and potently neutralizes live SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0072, TXG-0136, TXG-0137, TXG-0173, and TXG-0174. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0173, and TXG-0174. In some embodiments, the antibody or antigen-binding fragment has a binding affinity to the N-terminal domain of (NTD) of an S protein from a SARS- CoV-2 delta variant. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0072, TXG-0137, and TXG-0174. In some embodiments, the antibody or antigen-binding fragment is TXG-0174.
[0224] In some embodiments, the antibody or antigen-binding fragment has a binding affinity primarily to the RBD of a SARS-CoV-2 S protein and potently neutralizes live SARS- CoV-2. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0115, TXG-0140, TXG-0153, and TXG-0154. In some embodiments, the antibody or antigen-binding fragment is TXG-0153. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0115, TXG-0140, and TXG-0154.
[0225] In some embodiments, the antibody or antigen-binding has a binding affinity primarily to the RBD of an S protein from a SARS-CoV-2 delta variant. In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of TXG-0140 and TXG-0154. In some embodiments, the antibody or antigen-binding fragment has a binding affinity for a SARS-CoV-2 S protein and is selected from the group consisting of TXG-0085, TXG-0112, TXG-0192, TXG-0227, TXG-0228, TXG-0229, and TXG-0230.
[0226] In some embodiments, the antibody or antigen-binding fragment has a binding affinity for an S protein of a SARS-CoV-2 delta variant and is selected from the group consisting of TXG-0115, TXG-0136, TXG-0192, and TXG-0230. In some embodiments, such antibodies and antigen-binding fragments have a neutralizing activity against live SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment is TXG-0115.
[0227] In some embodiments, the antibody or antigen-binding fragment has a binding affinity for an S protein from a SARS-CoV-2 delta variant and is selected from the group consisting of TXG-0085, TXG-0112, TXG-0173, TXG-0227, TXG-0228, and TXG-0229. In some embodiments, such antibody or antigen-binding fragment potently neutralizes live SARS- CoV-2. In some embodiments, the antibody or antigen-binding fragment is TXG-0173.
[0228] In some embodiments, the isolated antibodies or antigen-binding fragments as described herein are recombinant antibodies and antigen-binding fragments. In some embodiments, the antibodies or antigen-binding fragments as described herein are isolated ( e.g ., purified) antibodies and antigen-binding fragments. As described above, when referring to polypeptides, e.g., antigen-binding polypeptides, antibodies, and antigen-binding fragments, one skilled in the art will understand that the term “isolated protein”, “isolated polypeptide” or “isolated antibody” is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is subtantially free of other proteins from the same species, (3) is expressed by a recombinant cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or biosynthesized in a recombinant cellular system different from the cell from which it naturally originates can be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally-associated components by isolation or purification, using one or more protein purification techniques.
[0229] Examples of isolated antibodies include anti- SARS-CoV S protein antibodies that have been purified using SARS-CoV S protein or a portion thereof, anti- SARS-CoV S protein antibodies that have been synthesized by a hybridoma or other recombinant cell line in vitro, and a human anti-SARS-CoV S protein antibody derived from a transgenic mouse. Examples of purification techniques suitable for the purification of the antibodies and antigen-binding fragments disclosed herein include affinity chromatography, anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydroxyapatite chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), metal affinity chromatography, mixed mode chromatography (MMC), centrifugation, diafiltration, and ultrafiltration.
[0230] Generally, a polypeptide (e.g., antibody or antigen-binding fragment) is “substantially pure,” “substantially homogeneous,” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide may be monomeric or multimeric. A substantially pure polypeptide generally includes about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, 96%, 97%, 98%, or in some embodiments, over 99% pure. Protein purity or homogeneity may be indicated by a number of means available in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a suitable stain available in the art. For certain purposes, higher resolution may be provided by using HPLC or other means available in the art for purification. Accordingly, in some embodiments, the isolated antibodies and antigen-binding fragments of the disclosure have a purity of greater than 80% such as, for example, a purity of greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
[0231] In some embodiments, an anti-SARS-CoV-S antigen-binding polypeptide (e.g., antibody or antigen-binding fragment) described herein is not an antibody or antigen-binding fragment described in the following patent publications CN111620946A, CN111690059A, US10787501, and WO2015179535. In some embodiments, the an anti-SARS-CoV-S antigen binding polypeptide ( e.g ., antibody or antigen-binding fragment) described herein is not an antibody or antigen-binding fragment described in the following documents Jakob Kreye et al., 2020; Seth Zost et al., (Nature Medicine, July 10, 2020); Xiaojian Han et al., (BioRxiv, Aug 21, 2020); Tal Noy-Porat et al., (Nature Comm., Aug. 27, 2020); Edurne Rujas et al. (BioRxiv, Oct 16, 2020), 2020; Renhong Yan et al., (BioRxiv, 2020); Christoph Kreer et al., (Cell, Aug 20, 2020; Vol. 182, Issue 4, pp. 843-854); Yunlong Cao et al., (Cell, July 9, Vol. 182, Issue 1, 2020); and Thomas Rogers et al., (Science Aug 21, 2020: Vol. 369, Issue 6506, pp. 956-963) .
Nucleic acids
[0232] In discussed above, an antibody described herein and antigen-binding fragments thereof can be obtained by expression of a nucleic acid molecule. Accordingly, one aspect of the disclosure relates to recombinant nucleic acids encoding an antibody described herein or an antigen-binding fragment thereof. Such nucleic acids can encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In some embodiments, the recombinant nucleic acids of the disclosure include a nucleic acid sequence that encodes an antibody of the disclosure or an antigen-binding fragment thereof. In some embodiments, the recombinant nucleic acids of the disclosure can be configured as expression cassettes or vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which allow in vivo expression of the receptor in a host cell.
[0233] Nucleic acid molecules of the present disclosure can be of any length, including for example, between about 1 Kb and about 50 Kb, e.g., between about 1.2 Kb and about 10 Kb, between about 2 Kb and about 15 Kb, between about 5 Kb and about 20 Kb, between about 10 Kb and about 20 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.
[0234] Accordingly, in some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding an antibody of the disclosure or an antigen-binding fragment thereof.
[0235] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. It will be understood by the skilled artisan that an expression cassette generally includes a construct of genetic material that contains coding sequences of the antibody or antigen-binding fragment thereof and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and/or into an individual. As such, in some embodiments, an expression cassette of the disclosure include a coding sequence for an antibody of the disclosure or an antigen-binding fragment thereof, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.
[0236] An expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, as a linear or circular, single-stranded or double- stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g., operably linked.
[0237] In some embodiments, the nucleic acid molecule of the disclosure is incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that can be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.
[0238] In some embodiments, the expression vector can be a viral vector. As will be appreciated by one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector can refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.
[0239] The nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the antibodies and antigen-binding fragment disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell. In some embodiments, the nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in human cells. Non-limiting examples of nucleic acid sequences optimized for expression in human cells are listed in SEQ ID NOS: 1647-1723 and 2047-2126. In some embodiments, the nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in hamster cells. Non-limiting examples of nucleic acid sequences optimized for expression in hamster cells are listed in SEQ ID NOS: 1727-1806 and 2127-2206. In some embodiments, the nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in prokaryotic cells. In some embodiments, the nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in E. coli cells. Non-limiting examples of nucleic acid sequences optimized for expression in E. coli cells are listed in SEQ ID NOS: 1807- 1886 and 2207-2286.
[0240] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any antibody or an antigen-binding fragment thereof as disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed/transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, T, & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, L, & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. etal. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. etal. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction . Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. etal. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).
[0241] DNA vectors can be introduced into cells, e.g ., eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.
[0242] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed ), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
[0243] For example, an antibody or an antigen-binding fragment thereof as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells ( e.g ., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans can consult P. Jones, “Vectors: Cloning Applications,” John Wiley and Sons, New York, N.Y., 2009).
[0244] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g., antibody. For example, just as polypeptide variants can be described in terms of their identity with a referenced amino acid sequence, the nucleic acid molecules encoding the polypeptide variants can have a certain identity with those that encode the referenced amino acid sequence. For example, nucleic acid molecule variants encoding an antibody of the disclosure, a variant thereof, or an antigen-fragment thereof can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, and most preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) identical to the nucleic acid encoding a referenced antibody or an antigen-fragment thereof. Thus, in some embodiments, the nucleic acid molecule encoding an antibody of the disclosure, a variant thereof, or an antigen-fragment thereof can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, and most preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) identical to the nucleic acid encoding an antibody or antigen-binding fragment thereof having an amino acid sequence set forth in the Sequence Listing.
[0245] In some embodiments, the nucleic acid molecule includes a nucleotide sequence encoding an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, and most preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to a HCVR of an antibody of the disclosure or an antigen-binding fragment thereof. Non-limiting examples of such nucleic acid sequences are listed in SEQ ID NOS: 1487-1886 of the Sequence Listing. In some embodiments, the nucleic acid molecule includes a nucleotide sequence encoding an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, and most preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to an LCVR of an antibody of the disclosure or an antigen-binding fragment thereof. Non-limiting examples of such nucleic acid sequences are listed in SEQ ID NOS: 1887-2286 of the Sequence Listing. In some embodiments, the nucleic acids include a first nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1487-1886; and a second nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1887-2286.
[0246] These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g, either a sense or an antisense strand).
[0247] The nucleic acid molecules are not limited to sequences that encode polypeptides (e.g, antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g, the coding sequence of an antibody) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.
[0248] The nucleic acid molecules of the dislosure, including variants and naturally- occuring nucleic acid sequences, can be produced using a number of methods including, for example, those described in Sambrook 2012, supra. For example, the sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules, and combinations thereof.
Recombinant cell and cell cultures
[0249] The nucleic acid of the present disclosure can be introduced into a host cell, such as, for example, a Chinese hamster ovary (CHO) cell, to produce a recombinant cell containing the nucleic acid molecule. Introduction of the nucleic acid molecules ( e.g ., DNA or RNA, including mRNA) or vectors of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery. For example, methods for introduction of heterologous nucleic acid molecules into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the nucleic acid molecule(s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. As discussed in greater detail below, in some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form (e.g, DNA or RNA, including mRNA), such that the subject's own cells produce the antibody. The present disclosure further provides modifications to nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibodies for the CoV spike protein.
[0250] Accordingly, in some embodiments, the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (. Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini-circle expression vector for transient expression.
[0251] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.
[0252] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene- delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.
[0253] In some embodiments, host cells can be genetically engineered ( e.g ., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
[0254] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. Suitable host cells for cloning or expression of antibodies and antigen-binding fragments include prokaryotic or eukaryotic cells described herein. In some embodiments, the recombinant cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is an E. coli cell. For example, antibodies and antigen-binding fragments may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
[0255] In some embodiments, the recombinant cell is a eukaryotic cell. Suitable host cells for the expression of glycosylated antibody can be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. Examples of such plant culture techniques include PLANTIBODIES™ technology for producing antibodies in transgenic plants.
[0256] Vertebrate cells can also be used as hosts. In this regards, mammalian cell lines that are adapted to grow in suspension can be useful. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non human animal cell. In some embodiments, the cell is a non-human primate cell.
[0257] Addtional examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line ( e.g. , 293 or 293 cells), baby hamster kidney cells (BHK), mouse sertoli cells (e.g., TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells, and myeloma cell lines such as Y0, NS0 and Sp2/0. In some embodiments, the recombinant cell is selected from the group consisting of a baby hamster kidney (BHK) cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, a SO murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HEK- 293 cell, a human HeLa cell, a human HepG2 cell, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a mouse 3T3 cell, a mouse connective tissue cell, a mouse muscle cell, and a rabbit kidney cell. In some embodiments, the recombinant cell is a Pichia pastoris cell or a Saccharomyces cerevisiae cell, both of which are also suitable for production of scFv, scFvFc, Fab, and F(ab’)2.
[0258] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.
[0259] The recombinant cell as disclosed herein, when cultured under appropriate conditions, expresses an antibody or binding fragment which can subsequently be collected from the culture medium (if the host cell secretes it into the medium ) or directly from the host cell producing it (if it is not secreted).
[0260] Antibodies can be recovered from the culture medium using standard protein purification methods. Examples of purification techniques suitable for the purification of the antibodies and antigen-binding fragments disclosed herein include affinity chromatography, anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydroxyapatite chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), metal affinity chromatography, mixed mode chromatography (MMC), centrifugation, diafiltration, and ultrafiltration.
[0261] Further, expression of antibodies of the invention from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the present disclosure, regardless of the glycosylation of the antibodies.
[0262] Also provided, in another aspect, are animals including a recombinant nucleic acid or a vector as disclosed herein. In some embodiments, the disclosure provides a transgenic animal that is a non-human animal. In some embodiments, the transgenic animal produces an antibody or antigen-binding fragment as disclosed herein.
[0263] The transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In some embodiments, the non-human animals of the disclosure are mice. Other animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to rats, rabbits, chickens, goats, pigs, sheep and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm ( e.g ., in pig), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.
[0264] In some embodiments, the animal is a vertebrate animal or an invertebrate animal.
In some embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the transgenic animals of the disclosure can be made using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). IN some embodiments, the transgenic animals of the disclosure can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination.
[0265] In another aspect, provided herein are methods for producing an antibody or antigen-binding fragment thereof, wherein the methods include rearing (i) a transgenic animal as disclosed herein, or culturing (ii) a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced. In some embodiments, the methods include rearing a transgenic animal as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced in the transgenic animal. In some embodiments, the methods include culturing a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced in the recombinant cell.
[0266] In some embodiments, the methods for producing an antibody or antigen-binding fragment thereof as described herein further include isolating ( e.g ., purifying) the produced antibody or antigen-binding fragment from (i) the transgenic animal or (ii) recombinant cell and/or the medium in which the recombinant cell is cultured. In some embodiments, the mammalian animal is a non-human primate. Accordingly, the antibodies or antigen-binding fragments produced by the methods disclosed herein are also within the scope of the disclosure.
[0267] Post-translational modifications of therapeutic antibodies are important product quality attributes that can potentially impact drug safety, efficacy, and safety. In some embodiments, antibodies and antigen-binding fragments of the present disclosure include immunoglobulin chains having the amino acid sequences set forth herein as well as cellular modifications and in vitro post-translational modifications to the antibody and antigen-binding fragment. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to CoV-S comprising heavy and/or light chain amino acid sequences set forth herein (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g., Met, Trp and/or His) is oxidized, the N-terminal Gin is pyroglutamate (pyroE) and/or the C-terminal Lysine is missing. Pharmaceutical compositions
[0268] The antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures of the disclosure can be incorporated into compositions, including pharmaceutical compositions. In some embodiments, such compositions include one or more of the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures as disclosed herein in an amount, a combination, or in a form that is not found in nature. For example, in some embodiments, the compositions of the disclosure include one or more of the antibodies, antigen-binding fragments, nucleic acids, recombinant cells as disclosed herein that have been isolated (e.g., purified) to an extent that they no longer in a form in which they would be found be nature. In another example, in some embodiments, the compositions of the disclosure include the antibodies, antigen-binding fragments, nucleic acids, recombinant cells of the disclosure in amounts that do not occur in nature. In yet another example, in some embodiments, the antibodies, antigen-binding fragments, nucleic acids, recombinant cells of the disclosure are combined in formulations and/or combinations that do not occur in nature. In some embodiments, such compositions include a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof in admixture with a suitable excipient, e.g., a pharmaceutically acceptable carrier. In some embodiments, an effective amount of a composition sufficient for achieving a therapeutic or prophylactic effect, ranges from about 0.000001 mg per kilogram body weight per administration to about 10,000 mg per kilogram body weight per administration. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per administration to about 100 mg per kilogram body weight per administration.
[0269] Generally, pharmaceutically acceptable carriers suitable for use in the compostions of the disclosure include, but are not limited to, means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some embodiments, the compositions of the disclosure include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. Additional pharmaceutically acceptable carriers suitable for use in the compostions of the disclosure include, but are not limited to, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
[0270] In one aspect, the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures of the disclosure can be incorporated into compositions suitable for various downstream applications, for example, pharmaceutical compositions. Exemplary compositions of the disclosure include pharmaceutical compositions which generally include one or more of the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures as described herein and a pharmaceutically acceptable excipient, e.g., carrier or diluent. In some embodiments, the composition is a sterile composition. In some embodiments, the composition is a lyophilized, desiccated, or freeze-dried composition. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further includes an adjuvant.
[0271] In some embodiments, the composition includes at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven antibodies or antigen-binding fragments as disclosed herein.
[0272] As discussed above, the compositions and methods of the di closure can be useful for the identification and characterization of antibodies having specific binding affinity (e.g., ability to bind, with varying degrees of specificity) to different epitopes on a coronavirus target, e.g., a spike protein. Antibodies having binding affinity for different epitopes on the target protein, e.g., a SARS-2 spike protein, can advantageously be used in a therapeutic antibody cocktail or combination therapy regimen. For example, a neutralizing antibody with binding affinity for an N-terminal domain (NTD) of a SAR.S-2 spike protein can be effectively combined with a neutralizing antibody with binding affinity for a receptor binding domain (RTD) of the same target as the two antibodies do not bind in the same epitope.
[0273] In some embodiments, the composition includes (a) a first antibody or antigen binding fragment having a binding affinity to a RBD and (b) a second antibody or antigen binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein. In some embodiments, the composition includes (a) a first antibody or antigen-binding fragment having a binding affinity to a RBD and (b) a second antibody or antigen-binding fragment having a binding affinity to a NTD of a SARS-CoV-2 S protein. In some embodiments, the composition includes (a) a first antibody or antigen-binding fragment having a binding affinity to a NTD and (b) a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein.
[0274] In some embodiments, an antibody or antigen-binding fragment as disclosed herein competes with another antibody or antigen-binding fragment for binding to the same epitope on the SARS-CoV-2 spike protein in a dose-dependent manner. As described in greater detail below, e.g., Example 14 and Table 9, the antibodies and antigen-binding fragments of the disclosure can be categorized in multiple epitope bins, wherein antibodies that share an epitope bin are antibodies which compete for binding to the same epitopes in a dose-dependent manner.
[0275] In some embodiments, the composition of the disclosure includes (a) a first antibody or antigen-binding fragment belonging to bin 1 or bin 2 described in Example 14, both of which represent NTD-binding antibodies and (b) a second antibody or antigen-binding fragment belonging to bin 3, bin 4, or bin 3/4 described in Example 14, which represent antibodies targeting primarily RBD. For example, in some embodiments, the composition of the disclosure includes (a) a first antibody or antigen-binding fragment belonging to bin 1 and (b) a second antibody or antigen-binding fragment belonging to bin 3, bin 4, or bin 3/4. In some embodiments, the composition of the disclosure includes (a) a first antibody or antigen-binding fragment belonging to bin 2 and (b) a second antibody or antigen-binding fragment belonging to bin 3, bin 4, or bin 3/4.
[0276] In some embodiments, the composition of the disclosure includes a first RBD- binding antibody or antigen-binding fragment thereof and a second RBD-binding antibody or antigen-binding fragment thereof, that have binding affinity for distinct epitopes on the spike protein. For example, in some embodiments, the composition of the disclosure includes (a) a first RBD-binding antibody or antigen-binding fragment belonging to bin 3 (sotrovimab-like antibodies) and (b) a second RBD-binding antibody or antigen-binding fragment belonging to bin 4. For example, in some embodiments, the composition of the disclosure includes (a) a first RBD-binding antibody or antigen-binding fragment belonging to bin 3, and (b) a second RBD- binding antibody or antigen-binding fragment belonging to bin 3/4. For example, in some embodiments, the composition of the disclosure includes (a) a first RBD-binding antibody or antigen-binding fragment belonging to bin 4, and (b) a second RBD-binding antibody or antigen binding fragment belonging to bin 3/4.
[0277] In some embodiments, the composition of the disclosure includes a first RBD- binding antibody or antigen-binding fragment thereof and a second RBD-binding antibody or antigen-binding fragment thereof, that are capable of binding the RBD of the spike protein in a partially distinct manner.
[0278] Additional embodiments for combination therapies
[0279] In some embodiments, the pharmaceutical compositions described herein include (i) a first antibody having binding affinity to SARS-CoV-2 S protein identified herein (e.g., any one of the antibodies set forth in Table 1) and (ii) one or more antibodies previously characterized as having binding affinity to SARS-CoV-2 S protein (e.g., previously characterized therapeutic antibody) such as antibodies approved by FDA for treatment of coronavirus infection. Examples of suitable antibodies previously characterized as having binding affinity to SARS-CoV-2 S protein include, but are not limited to casirivimab, imdevimab, bamlanivimab, etesevimab, and sotrovimab.
[0280] In some embodiments, the pharmaceutical compositions described herein include (i) a first antibody having binding affinity to SARS-CoV-2 S protein identified herein (e.g., any one of the antibodies set forth in Table 1) and (ii) one or more antibodies and/or small molecule entities having affinity for an immune pathway target. Non-limiting examples of suitable antibodies for an immune pathway target include interleukin antibodies, IL-1 pathway target antibodies and IL-6 pathway target antibodies.
[0281] IL-1 pathway target antibodies.
[0282] Non-limiting examples of IL-1 pathway target antibodies include Ilaris (canakinumab) (IL-1 inhibitor), IL ip-specifie antibodies SK48-E26, 47F11, and XOMA 052 (gevokizumab), IL- la-specific MABpl.
[0283] IL-6 pathway target antibodies :
[0284] Non-limiting examples of IL-6 pathway target antibodies include Actemra (tocilizumab) (blocks IL-6 receptor) and Sirukumab (anti-IL-6 antibody candidate).
[0285] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to an individual. The composition can be in liquid form or in a lyophilized or freeze-dried form and can include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. The scope of the present disclosure includes desiccated, e.g., freeze-dried, compositions comprising an anti- CoV-S antigen-binding polypeptides, e.g., antibody or antigen-binding fragment thereof (e.g., of Table 1), or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual or an animal (some non-limiting examples include a human, or a mammal). In some embodiments, the individual is a human.
[0286] The terms “administration” and “administering,” as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, oral, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.
[0287] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example, the pharmaceutical composition can be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition can be formulated for oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial administration. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.
[0288] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In some embodiments, the composition should be sterile and should be fluid to the extent that easy syringability exists. It can be stabilized under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0289] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
[0290] In some embodiments, the pharmaceutical composition of the disclosure further includes a further therapeutic agent. Non-limiting examples of further therapeutic agents include
(i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. Accordingly, in some embodiments, the pharmaceutical composition of the disclosure further includes a further therapeutic agent selected from the group consisting of: (i) an antiviral agent,
(ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. In some embodiments, the further therapeutic agent is a second antibody or antigen-binding fragment disclosed herein, e.g., of Table 1. In some embodiments, one, two, three, four, or more antibodies, or antigen-binding fragments thereof, of Table 1 can be used in combination.
[0291] In some embodiments, antibodies and antigen-binding fragments from different human donors can be combined. For example, some embodiments of the disclosure relate to a composition comprising two (or more) antibodies and antigen-binding fragments comprising variable domains from human subjects, wherein the two (or more) antibodies or antigen-binding fragments are derived from different subjects (e.g., two different human subjects). In some embodiments, antibody variable regions derived from human B cells cane be combined with a constant region not from those B cells to produce hybrid antibodies. In some embodiments, a composition of the disclosure can include a combination of an antibody or antigen-binding fragment thereof with variable domains derived from one donor and an antibody or antigen binding fragment thereof with variable domains derived from another donor.
[0292] In some embodiments, the one or more further therapeutic agents includes an antiviral drug or a vaccine. One of ordinary skill in the art will understand that the antiviral drug of the disclosure can include any anti -infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. In some embodiments, the antiviral drug includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, ribavirin, or interferon- alpha2b. Methods for treating or preventing virus (e.g., coronavirus) infection in a subject in need of said treatment or prevention by administering an antibody or antigen-binding fragment of Table 1 in association with a further therapeutic agent are part of the present disclosure.
[0293] For example, in some embodiments of the disclosure, the further therapeutic agent is a vaccine, e.g., a coronavirus vaccine. In some embodiments, a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.
METHODS OF THE DISCLOSURE
Methods for identifying antibodies with binding affinity to a target antisen, e.s.. a coronavirus spike protein (CoV-S)
[0294] In described in more detail below, one aspect of the disclosure relates to new approaches and methods for the identification and characterization of antigen-binding molecules, e.g., antibodies and antigen-binding fragments. In some embodiments, these methods are used to identify antigen binding molecules that are derived from B cells obtained from subjects who have been exposed to a coronavirus, by using single-cell immune profiling methodologies, so as to generate antibodies and antigen-binding fragments having a binding specificity for a coronavirus spike protein (CoV-S).
[0295] Advantages of the new approaches and methods disclosed herein are numerous. For example, as illustrated herein, workflows disclosed herein identified higher numbers of antibody hits that neutralize target antigen at greater potency, and within a much shorter timeframe than traditional discovery approaches. The antibody hits identified via workflows disclosed herein are likely to have lower developability burden than those identified using display methodologies. Furthermore, as illustrated herein, affinity and functional profiles of antibodies identified via workflows disclosed herein are typically superior or non-inferior to those of antibodies derived using slower and lower-throughput approaches. Accordingly, the workflows disclosed herein yield greater numbers of antibodies with superior properties as compared to traditional antibody discovery workflows. Such workflows are particularly advantageous in the face of rapidly changing disease landscapes where variants of concern evolve over time.
[0296] Some embodiments of the disclosure relate to methods for identifying an antibody having specific binding affinity (e.g., ability to bind, with varying degrees of specificity) for a target antigen relative to a non-target antigen, the methods including: (a) partitioning a sample comprising biological particles producing antigen binding molecules and a plurality of antigens, wherein the plurality of antigens includes a target antigen and a non-target antigen, and wherein each of the antigens include a reporter oligonucleotide, and wherein the sample comprises at least one biological particle bound to the target antigen, and wherein the partitioning provides a partition including (i) the biological particle bound to the target antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence; (c) identifying a sequence of at least one antigen binding molecule produced by the biological particle that has been bound to the target antigen; and d) assessing the binding affinity (e.g., ability to bind, with varying degrees of specificity) of the antigen-binding molecule to the target antigen; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the target antigen if the barcoded antibody specifically binds to the target antigen.
[0297] Some embodiments of the disclosure relates to methods for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the methods including: (a) contacting a plurality of B cells obtained from a subject who has been exposed to a coronavirus with a plurality of antigens, wherein the plurality of antigens includes a CoV-S antigen and a non-CoV-S antigen, and wherein each of the antigens include a reporter oligonucleotide, wherein the contacting provides a B cell bound to a CoV-S antigen; (b) partitioning the B cell bound to the CoV-S antigen in a partition of a plurality of partitions, wherein the partitioning provides a partition including (i) the B cell bound to the CoV-S antigen and (ii) a plurality of nucleic acid barcode molecules including a common barcode sequence; (c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the CoV-S antigen; and d) assessing the binding affinity of the barcoded antibody or antigen-binding fragment to a CoY-S protein; and e) identifying the isolated antibody antigen-binding fragment as an antibody having a binding specificity for the CoV-S protein if the barcoded antibody specifically binds to the CoV-S protein.
[0298] Non-limiting exemplary embodiments of the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein can include one or more of the following features. In some embodiments, the reporter oligonucleotide includes (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence. In some embodiments, the methods of the disclosure further include coupling a barcode moiety to the antibody or antigen binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment. In some embodiments, a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further includes a capture sequence configured to couple to the capture handle sequence of the reporter oligonucleotide and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further includes a capture sequence configured to couple to an mRNA analyte. In some embodiments, the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide is complementary to the capture handle sequence of the reporter oligonucleotide. In some embodiments, the capture sequence configured to couple to an mRNA analyte includes a polyT sequence. In some embodiments, the first and second nucleic acid barcode molecules each include a unique molecule identifier (UMI).
[0299] In some embodiments, the antibody or antigen-binding fragment has a binding specificity to an epitope on a domain of the CoV-S protein. In some embodiments, the domain of the CoV-S protein is in the SI domain (i.e., subunit. In some embodiments, the domain of the CoV-S protein is the S2 domain (i.e., subunit). In some embodiments, the antibody or antigen binding fragment has a binding affinity to a RBD or a NTD of the S 1 subunit. In some embodiments, the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein.
[0300] In some embodiments, the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 500 nM, for example, less than 400 nM, less than 300 nM, less than 200 nM, less than 150 nM, less than 120 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 5 nM, or less than 1 nM. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with a KD value lower than the binding affinity between a SARS-CoV-2 S protein and its receptor ACE2, which has been previously estimated to have a KD value of about 120 nM. Accordingly, in some embodiment of the disclosure, the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 120 nM.
[0301] In some embodiments, the antibody or antigen-binding fragment has a sub nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof. In some embodiments, the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 500 pM, for example, less than 100 pM, less than 50 pM, less than 10 pM, less than 5 pM, less than 1 pM, less than 0.5 pM. In some embodiments, the antibodies or antigen-binding fragments provided herein can bind to and/or neutralize the CoV-S protein of an alphacoronavirus, a betacoronavirus, a gammacoronavirus, and/or a deltacoronavirus. In certain embodiments, this binding and/or neutralization can be specific for a particular genus of coronavirus or for a particular subgroup of a genus. In some embodiments, the CoV-S protein is a spike protein of SARS-CoV-1, SARS-CoV-2, or MERS-CoV. In some embodiments, the subject is suspected of being infected with a coronavirus, has been infected with a coronavirus, has been vaccinated, or has been recovered from a coronavirus infection. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human.
[0302] In some embodiments, the antigens are each coupled to a fluorescent label identifying the antigens. In some embodiments, the methods for identifying an antibody having binding affinity for a CoV-S protein as described herein further include isolating and/or enriching the plurality of B cells prior to (b). In some embodiments, the enrichment further includes sorting of the B cells bound to the CoV-S antigen and/or non-CoV-S antigen based on detection of one or more of the fluorescent labels coupled to the antigens. In some embodiments, the CoV-S protein is coupled to a barcode moiety.
[0303] As discussed in greater detail below, cell separation techniques can be used to enrich for specific populations of cells of interest. Non-limiting examples of separation techniques useful for separating ( e.g ., sorting) one or more different types of cells can include, for example, centrifugation, filtration, microfluidic-based sorting, flow cytometry, fluorescence- activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy-activated cell sorting (BACS), or any other useful methods. [0304] In certain embodiments, the disclosure provides methods for identifying a B cell as expressing an antibody that has binding affinity for a CoV-S antigen or a non-CoV-S antigen, or for identifying an antibody that has binding affinity for a CoV-S antigen or a non-CoV-S antigen. In one embodiment, the method comprises contacting a plurality of B cells with a plurality of antigens. The plurality of antigens may include at least two antigens that are different from one another, e.g.. a first antigen and a second antigen, wherein the first antigen is a different type of antigen than the second antigen. For example, the plurality of antigens may include, without limitation, (i) antigens that are the same, (ii) antigens that are different, (iii) a CoV-S antigen and a control antigen ( e.g ., a non-CoV-S antigen) or (iv) a first CoV-S antigen and a second CoV-S antigen which are different types of CoV-S antigens. In another embodiment, the plurality of B cells is obtained from a subject (or obtained from a sample that was obtained from a subject) who has been exposed to a coronavirus.
[0305] In an additional embodiment, the method further comprises partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions comprises a B cell from said plurality of B cells, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In one embodiment, the B cell in said partition comprises (i) a surface-bound CoV-S antigen comprising a CoV-S reporter oligonucleotide having a CoV-S reporter sequence and (ii) a surface-bound control antigen having a control reporter oligonucleotide that comprises a control reporter sequence.
[0306] In one additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules using said CoV-S reporter oligonucleotide, said control reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules comprises a first barcoded nucleic acid molecule comprising said CoV-S reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further comprise a second barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof.
[0307] In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating a third barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the third barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof. Suitable methods, compositions, systems, and kits for single cell analysis of immune receptors and/or antigen binding are disclosed in US20180105808A1, US20180179590A1, US20190338353A1, and US20190367969A1.
[0308] In a further embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that has binding affinity for said CoV-S, or to identify an antibody that has binding affinity for a CoV-S antigen or a non-CoV-S antigen. In another embodiment, the method further comprises determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method comprises identifying an antibody expressed by said B cell as having binding affinity for a CoV-S antigen or a non-CoV-S antigen based on a determination of (a) a sequence comprising the CoY-S reporter sequence and the partition barcode sequence or complement thereof, and another sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof; and (b) a sequence comprising the CoV-S reporter sequence and the partition barcode sequence or complement thereof, another sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof; and an additional sequence corresponding to an immune receptor and the partition barcode sequence or complement thereof.
[0309] In other embodiments, the disclosure provides methods for identifying a B cell as expressing an antibody that is cross-reactive against more than one antigen, or for identifying an antibody that is cross-reactive against more than one antigen. In one embodiment, the method comprises contacting a plurality of B cells with a plurality of antigens. The plurality of antigens may include at least two antigens that are different from one another, e.g., a first antigen and a second antigen, wherein the first antigen is a different type of antigen than the second antigen. For example, the plurality of antigens may include, without limitation, (i) antigens that are different or (ii) a first antigen and a second antigen which are different types of antigens, e.g ., a non-CoV-S antigen. The plurality of antigens may further comprise a control antigen (e.g., an antigen that is unrelated to the first or second antigens). For example, where the first and second antigen are viral protein antigens, the control antigen is a non-viral protein antigen. In another embodiment, the plurality of B cells is obtained from a subject (or obtained from a sample that was obtained from a subject) who has been exposed to a pathogen ( e.g ., a virus, coronavirus).
[0310] In an additional embodiment, the method further comprises partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions comprises a B cell from said plurality of B cells, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In one embodiment, the B cell in said partition comprises (i) a first surface-bound antigen comprising a first reporter oligonucleotide having a first reporter sequence and (ii) a second surface-bound antigen comprising a second reporter oligonucleotide having a second reporter sequence. In one additional embodiment, the B cell in the partition further comprises a surface-bound control antigen having a control reporter oligonucleotide that comprises a control reporter sequence.
[0311] In one additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules using said first reporter oligonucleotide, said second reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules comprises a first barcoded nucleic acid molecule comprising said first reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further comprise a second barcoded nucleic acid molecule comprising said second reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In a further embodiment, the plurality of barcoded nucleic acid molecules may further comprise a third barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating an additional barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the additional barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof.
[0312] In a further embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that is cross-reactive against more than one antigen, or to identify an antibody that is cross-reactive against more than one antigen. In another embodiment, the method further comprises determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method comprises identifying an antibody expressed by said B cell as cross-reactive against more than one antigen based on a determination of a sequence comprising the first reporter sequence and the partition barcode sequence or complement thereof, and another sequence comprising the second reporter sequence and the partition barcode sequence or complement thereof. The determination may further comprise determination of (i) a sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof, and/or (ii) an additional sequence corresponding to an immune receptor and the partition barcode sequence or complement thereof.
[0313] In an additional embodiment, the method further comprises partitioning the plurality of B cells into a plurality of partitions, which can be a plurality of droplets in an emulsion or a plurality of wells. In a further embodiment, a partition of said plurality of partitions comprises a B cell from said plurality of B cells, and a plurality of nucleic acid barcode molecules comprising a partition barcode sequence. In one embodiment, the B cell in said partition comprises (i) a first surface-bound CoV-S antigen comprising a first CoV-S reporter oligonucleotide having a first CoV-S reporter sequence and (ii) a second surface-bound CoV-S antigen comprising a second CoV-S reporter oligonucleotide having a second CoV-S reporter sequence. In one additional embodiment, the B cell in the partition further comprises a surface- bound control antigen having a control reporter oligonucleotide that comprises a control reporter sequence.
[0314] In one additional embodiment, the method further comprises generating in said partition a plurality of barcoded nucleic acid molecules using said first CoV-S reporter oligonucleotide, said second CoV-S reporter oligonucleotide, and said plurality of nucleic acid barcode molecules. In other embodiments, the plurality of barcoded nucleic acid molecules comprises a first barcoded nucleic acid molecule comprising said first CoV-S reporter sequence or complement thereof and said partition barcode sequence or complement thereof. The plurality of barcoded nucleic acid molecules may further comprise a second barcoded nucleic acid molecule comprising said second CoV-S reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In a further embodiment, the plurality of barcoded nucleic acid molecules may further comprise a third barcoded nucleic acid molecule comprising said control reporter sequence or complement thereof and said partition barcode sequence or complement thereof. In other embodiments, the method further comprises obtaining immune receptor information from the plurality of B cells. In one embodiment, the method comprises generating an additional barcoded nucleic acid molecule using a nucleic acid molecule encoding for an immune receptor and said plurality of nucleic acid barcode molecules. The nucleic acid molecule encoding for an immune receptor may be a messenger ribonucleic acid (mRNA) molecule. In another embodiment, the additional barcoded nucleic acid molecule comprises an immune receptor nucleic acid sequence from the B cell or complement thereof and the partition barcode sequence or complement thereof.
[0315] In a further embodiment, the method further comprises determining the sequence of said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule to identify said B cell as expressing an antibody that is cross-reactive against more than one CoV-S antigen, or to identify an antibody that is cross-reactive against more than one CoV-S antigen. In another embodiment, the method further comprises determining the sequence of the third barcoded nucleic acid molecule. In some embodiments, the method comprises identifying an antibody expressed by said B cell as cross-reactive against more than one CoV-S antigen based on a determination of a sequence comprising the first CoY-S reporter sequence and the partition barcode sequence or complement thereof, and another sequence comprising the second CoV-S reporter sequence and the partition barcode sequence or complement thereof. The determination may further comprise determination of (i) a sequence comprising the control reporter sequence and the partition barcode sequence or complement thereof, and/or (ii) an additional sequence corresponding to an immune receptor and the partition barcode sequence or complement thereof.
[0316] In some embodiments, the method further comprises determining the binding affinity and/or binding specificity of the antigen binding molecule (e.g., antibody or antigen binding fragment) to a CoV-S antigen according to a BEAM-seq method, e.g., as described further herein. [0317] In certain embodiments, the partition-based compositions and methods use droplet- based partitions ( e.g ., droplets in an emulsion) or well-based partitions. In one embodiment, the plurality of partitions is a plurality of droplets (e.g., a plurality of droplets in an emulsion) or a plurality of wells. In other embodiments, the plurality of nucleic acid barcode molecules is coupled to a support. The support may be a bead, which is optionally a gel bead. In another embodiment, the plurality of nucleic acid barcode molecules is coupled to a support via a labile moiety. In other embodiments, the plurality of nucleic acid barcode molecules is releasably coupled to said support. The plurality of nucleic acid barcode molecules may be releasable from said support upon application of a stimulus. In one embodiment, the stimulus is selected from the group consisting of a thermal stimulus, an enzymatic stimulus, a photo stimulus, and a chemical stimulus. In another embodiment, the application of said stimulus results in one or more of (i) cleavage of a linkage between nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules and said bead, and (ii) degradation of said bead to release nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules from said bead. In one embodiment, the bead is provided in said partition, and wherein said nucleic acid barcode molecule is released from said bead within said partition.
Systems and methods for partitioning
[0318] In some aspects, such as those that have been described above, the methods provided herein include a step of partitioning, or include a step of generating barcoded nucleic acid molecules, or may include an additional processing step(s) This description sets forth examples, embodiments and characteristics of steps of the methods and of reagents useful in the methods or as may be provided in the partitions.
[0319] In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g., biological particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions.
[0320] In some embodiments disclosed herein, the partitioned particle is a labelled cell of B cell lineage, e.g., a plasma cell or a memory B cell, which expresses an antigen -binding molecule (e.g., an immune receptor, an antibody or a functional fragment thereof) on its surface. In other examples, the partitioned particle can be a labelled cell engineered to express an antibody. In some embodiments, the labelled cell of B-cell lineage is a B cell which expresses antigen-binding molecules ( e.g ., an immune receptors, antibodies or functional fragments thereof).
[0321] The term “partition,” as used herein, generally, refers to a space or volume that can be suitable to contain one or more cells, one or more species of features or compounds, or conduct one or more reactions. A partition can be a physical container, compartment, or vessel, such as a droplet, a flow cell, a reaction chamber, a reaction compartment, a tube, a well, or a microwell. In some embodiments, the compartments or partitions include partitions that are flowable within fluid streams. These partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core, or, in some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix. In some aspects, partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase). A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in detail in, e.g., U.S. Patent Application Publication No. 2010/010511.
[0322] In some embodiments, a partition herein includes a space or volume that can be suitable to contain one or more species or conduct one or more reactions. A partition can be a physical compartment, such as a droplet or well. The partition can be an isolated space or volume from another space or volume. The droplet can be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet can be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition can include one or more other (inner) partitions. In some cases, a partition can be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment can include a plurality of virtual compartments.
[0323] In some embodiments, the methods and systems described herein provide for the compartmentalization, depositing or partitioning of individual cells from a sample material containing cells after at least one labelling agent or reporter agent molecule has been bound to a cell surface feature of a cell, into discrete partitions, where each partition maintains separation of its own contents from the contents of other partitions. Identifiers including unique identifiers ( e.g ., UMI) and common or universal tags, e.g., barcodes, can be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments. Further, identifiers including unique identifiers and common or universal tags, e.g., barcodes, can be coupled to labelling agents and previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments. Identifiers including unique identifiers and common or universal tags, e.g., barcodes, can be delivered, for example on an oligonucleotide, to a partition via any suitable mechanism, for example by coupling the barcoded oligonucleotides to a bead. In some embodiments, the barcoded oligonucleotides are reversibly (e.g, releasably) coupled to a bead. The bead suitable for the compositions and methods of the disclosure can have different surface chemistries and/or physical volumes. In some embodiments, the bead includes a polymer gel. In some embodiments, the polymer gel is a polyacrylamide. Additional non-limiting examples of suitable beads include microparticles, nanoparticles, beads, and microbeads. The partition can be a droplet in an emulsion. A partition can include one or more particles. A partition can include one or more types of particles. For example, a partition of the present disclosure can include one or more biological particles, e.g, labelled engineered cells, B cells, or memory B cells, and/or macromolecular constituents thereof. A partition can include one or more gel beads. A partition can include one or more cell beads. A partition can include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition can include one or more reagents. Alternatively, a partition can be unoccupied. For example, a partition cannot comprise a bead. Unique identifiers, such as barcodes, can be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a bead, as described elsewhere herein. Microfluidic channel networks (e.g, on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.
[0324] The partitions can be flowable within fluid streams. The partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core. In some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials ( e.g ., expressed antibodies or antigen binding-fragments thereof) within its matrix (e.g., via a capture agent configured to couple to both the matrix and the expressed antibody or antigen binding-fragment thereof). The partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible. For example, the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase). In another example, the partitions can be droplets of a non-aqueous fluid within an aqueous phase. In some examples, the partitions can be provided in a water-in-oil emulsion or oil-in-water emulsion. A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in, for example, U.S. Patent Application Publication No. 2010/0105112.
[0325] In the case of droplets in an emulsion, allocating individual particles (e.g., labelled engineered cells) to discrete partitions can, in one non-limiting example, be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non- aqueous fluid, such that droplets are generated at the junction of the two streams. Fluid properties (e.g., fluid flow rates, fluid viscosities, etc.), particle properties (e.g, volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g, channel geometry, etc ), and other parameters can be adjusted to control the occupancy of the resulting partitions (e.g, number of biological particles per partition, number of beads per partition, etc.). For example, partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles. To generate single biological particle partitions, the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions can contain less than one biological particle per partition in order to ensure that those partitions that are occupied are primarily singly occupied. In some cases, partitions among a plurality of partitions can contain at most one biological particle (e.g, bead, DNA, cell, such as a labelled engineered cells, B cells or memory B cells, plasma cells, or cellular material). In some embodiments, the various parameters (e.g, fluid properties, particle properties, microfluidic architectures, etc.) can be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions. The flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions.
[0326] In some embodiments, the method further includes individually partitioning one or more single cells from a plurality of cells in a partition of a second plurality of partitions.
[0327] In some embodiments, at least one of the first and second plurality of partitions includes a microwell, a flow cell, a reaction chamber, a reaction compartment, or a droplet. In some embodiments, at least one of the first and second plurality of partitions includes individual droplets in emulsion. In some embodiments, the partitions of the first plurality and/or the second plurality of partition have the same reaction volume.
[0328] In the case of droplets in emulsion, allocating individual cells to discrete partitions can generally be accomplished by introducing a flowing stream of cells in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. By providing the aqueous cell-containing stream at a certain concentration of cells, the occupancy of the resulting partitions ( e.g ., number of cells per partition) can be controlled. For example, where single cell partitions are desired, the relative flow rates of the fluids can be selected such that, on average, the partitions contain less than one cell per partition, in order to ensure that those partitions that are occupied, are primarily singly occupied. In some embodiments, the relative flow rates of the fluids can be selected such that a majority of partitions are occupied, e.g., allowing for only a small percentage of unoccupied partitions. In some embodiments, the flows and channel architectures are controlled as to ensure a desired number of singly occupied partitions, less than a certain level of unoccupied partitions and less than a certain level of multiply occupied partitions.
[0329] In some embodiments, the methods described herein can be performed such that a majority of occupied partitions include no more than one cell per occupied partition. In some embodiments, the partitioning process is performed such that fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5%, fewer than 2%, or fewer than 1% the occupied partitions contain more than one cell. In some embodiments, fewer than 20% of the occupied partitions include more than one cell. In some embodiments, fewer than 10% of the occupied partitions include more than one cell per partition. In some embodiments, fewer than 5% of the occupied partitions include more than one cell per partition. In some embodiments, it is desirable to avoid the creation of excessive numbers of empty partitions. For example, from a cost perspective and/or efficiency perspective, it may be desirable to minimize the number of empty partitions. While this can be accomplished by providing sufficient numbers of cells into the partitioning zone, the Poissonian distribution can optionally be used to increase the number of partitions that include multiple cells. As such, in some embodiments described herein, the flow of one or more of the cells, or other fluids directed into the partitioning zone are performed such that no more than 50% of the generated partitions, no more than 25% of the generated partitions, or no more than 10% of the generated partitions are unoccupied. Further, in some aspects, these flows are controlled so as to present non-Poissonian distribution of single occupied partitions while providing lower levels of unoccupied partitions. Restated, in some aspects, the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in some embodiments, the use of the systems and methods described herein creates resulting partitions that have multiple occupancy rates of less than 25%, less than 20%, less than 15%), less than 10%, and in some embodiments, less than 5%, while having unoccupied partitions of less than 50%), less than 40%, less than 30%, less than 20%, less than 10%, and in some embodiments, less than 5%.
[0330] Although described in terms of providing substantially singly occupied partitions, above, in some embodiments, the methods as described herein include providing multiply occupied partitions, e.g., containing two, three, four or more cells and/or microcapsules (e.g., beads) comprising nucleic acid barcode molecules within a single partition.
[0331] In some embodiments, the reporter oligonucleotides contained within a partition are distinguishable from the reporter oligonucleotides contained within other partitions of the plurality of partitions.
[0332] In some embodiments, it may be desirable to incorporate multiple different barcode sequences within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known barcode sequences set can provide greater assurance of identification in the subsequent processing, e.g, by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.
Micro fluidic channel structures
[0333] Microfluidic channel networks (e.g, on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.
[0334] FIG. 5 shows an example of a microfluidic channel structure 500 for partitioning individual biological particles. The channel structure 500 can include channel segments 502,
504, 506 and 508 communicating at a channel junction 510. In operation, a first aqueous fluid 512 that includes suspended biological particles ( e.g ., cells, for example, labelled engineered cells, B cells, memory B cells, or plasma cells) 514 can be transported along channel segment 502 into junction 510, while a second fluid 516 that is immiscible with the aqueous fluid 512 is delivered to the junction 510 from each of channel segments 504 and 506 to create discrete droplets 518, 520 of the first aqueous fluid 512 flowing into channel segment 508, and flowing away from junction 510. The channel segment 508 can be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated can include an individual biological particle 514 (such as droplets 518). A discrete droplet generated can include more than one individual biological particle (e.g., labelled B cell such as memory B cell, or plasma cell) 514 (not shown in FIG. 5). A discrete droplet can contain no biological particle 514 (such as droplet 520). Each discrete partition can maintain separation of its own contents (e.g., individual biological particle 514) from the contents of other partitions.
[0335] The second fluid 516 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 518, 520. Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010/0105112.
[0336] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure 500 can have other geometries. For example, a microfluidic channel structure can have more than one channel junction. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles (e.g, biological particles, cell beads, and/or gel beads) that meet at a channel junction. Fluid can be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g, providing positive pressure), pumps (e.g, providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.
[0337] The generated droplets can include two subsets of droplets: (1) occupied droplets 518, containing one or more biological particles 514, e.g., labelled engineered cells, labelled B cells, memory B cells, or plasma cell, and (2) unoccupied droplets 520, not containing any biological particles 514. Occupied droplets 518 can include singly occupied droplets (having one biological particle, such as one labelled B cell, memory B cell, or plasma cell) and multiply occupied droplets (having more than one biological particle, such as multiple engineered cells, labelled B cells, memory B cells, or plasma cells). As described elsewhere herein, in some cases, the majority of occupied partitions can include no more than one biological particle, e.g., labelled engineered cells, labelled B cells, memory B cells, or plasma cells, per occupied partition and some of the generated partitions can be unoccupied (of any biological particle, or labelled engineered cell, labelled B cells, memory B cells, or plasma cells). In some cases, though, some of the occupied partitions can include more than one biological particle, e.g., labelled engineered cells, labelled B cells, or memory B cells. In some cases, the partitioning process can be controlled such that fewer than about 25% of the occupied partitions contain more than one biological particle, and in many cases, fewer than about 20% of the occupied partitions have more than one biological particle, while in some cases, fewer than about 10% or even fewer than about 5% of the occupied partitions include more than one biological particle per partition.
[0338] In some cases, it can be desirable to minimize the creation of excessive numbers of empty partitions, such as to reduce costs and/or increase efficiency. While this minimization can be achieved by providing a sufficient number of biological particles (e.g, biological particles, such as labelled engineered cells, labelled B cells, memory B cells, or plasma cells 514) at the partitioning junction 510, such as to ensure that at least one biological particle is encapsulated in a partition, the Poissonian distribution can expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied.
[0339] In some cases, the flow of one or more of the biological particles, such as labelled B cells, memory B cells, or plasma cells (e.g, in channel segment 502), or other fluids directed into the partitioning junction (e.g, in channel segments 504, 506) can be controlled such that, in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions. The above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, the use of the systems and methods described herein can create resulting partitions that have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having unoccupied partitions of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.
[0340] As will be appreciated, the above-described occupancy rates are also applicable to partitions that include both biological particles ( e.g ., labelled B cells or plasma cells) and additional reagents, including, but not limited to, microcapsules or beads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g., nucleic acid barcode molecules or barcoded oligonucleotides) (described in relation to FIGS. 5 and 6). The occupied partitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can include both a microcapsule (e.g, bead) comprising barcoded nucleic acid nucleic acid molecules (e.g, nucleic acid barcode molecules) and a biological particle.
[0341] FIG. 14 shows an example of a microfluidic channel structure 1400 for delivering barcode carrying beads to droplets. The channel structure 1400 can include channel segments 1401, 1402, 1404, 1406 and 1408 communicating at a channel junction 1410. In operation, the channel segment 1401 may transport an aqueous fluid 1412 that includes a plurality of beads 1414 (e.g, with nucleic acid molecules, e.g, nucleic acid barcode molecules or barcoded oligonucleotides, molecular tags) along the channel segment 1401 into junction 1410. The plurality of beads 1414 may be sourced from a suspension of beads. For example, the channel segment 1401 may be connected to a reservoir comprising an aqueous suspension of beads 1414. The channel segment 1402 may transport the aqueous fluid 1412 that includes a plurality of biological particles 1416 along the channel segment 1402 into junction 1410. The plurality of biological particles 1416 may be sourced from a suspension of biological particles. For example, the channel segment 1402 may be connected to a reservoir comprising an aqueous suspension of biological particles 1416. In some instances, the aqueous fluid 1412 in either the first channel segment 1401 or the second channel segment 1402, or in both segments, can include one or more reagents, as further described below. A second fluid 1418 that is immiscible with the aqueous fluid 1412 ( e.g ., oil) can be delivered to the junction 1410 from each of channel segments 1404 and 1406. Upon meeting of the aqueous fluid 1412 from each of channel segments 1401 and 1402 and the second fluid 1418 from each of channel segments 1404 and 1406 at the channel junction 1410, the aqueous fluid 1412 can be partitioned as discrete droplets 1420 in the second fluid 1418 and flow away from the junction 1410 along channel segment 1408. The channel segment 1408 may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 1408, where they may be harvested. As an alternative, the channel segments 1401 and 1402 may meet at another junction upstream of the junction 1410. At such junction, beads and biological particles may form a mixture that is directed along another channel to the junction 1410 to yield droplets 1420. The mixture may provide the beads and biological particles in an alternating fashion, such that, for example, a droplet comprises a single bead and a single biological particle.
[0342] In another aspect, in addition to or as an alternative to droplet based partitioning, biological particles (e.g., cells) can be encapsulated within a microcapsule that comprises an outer shell, layer or porous matrix in which is entrained one or more individual biological particles or small groups of biological particles. In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g, cells) may be encapsulated within a particulate material to form a “cell bead.” In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g, cells) may be comprised within a particulate material to form a “cell bead.”
[0343] The microcapsule or cell bead can include other reagents. Encapsulation of biological particles, e.g, labelled engineered cell, B cells, memory B cells, or plasma cells, can be performed by a variety of processes. Such processes can combine an aqueous fluid containing the biological particles with a polymeric precursor material that can be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. Such stimuli can include, for example, thermal stimuli (e.g, either heating or cooling), photo-stimuli (e.g, through photo-curing), chemical stimuli (e.g, through crosslinking, polymerization initiation of the precursor (e.g., through added initiators)), mechanical stimuli, or a combination thereof. [0344] Preparation ( e.g ., encapsulation) of microcapsules comprising biological particles, e.g., labelled engineered cells, B cells, memory B cells, or plasma cells, can be performed by a variety of methods. For example, air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form microcapsules or cell beads that include individual biological particles or small groups of biological particles (e.g., labelled B cells or plasma cells). Likewise, membrane-based encapsulation systems may be used to generate cell beads comprising encapsulated biological particles (e.g, B cells or plasma cells) as described herein. Microfluidic systems of the present disclosure, such as that shown in FIG. 5, may be readily used in encapsulating biological particles (e.g., cells) as described herein. Exemplary methods for encapsulating biological particles (e.g, cells) are also further described in U S. Patent Application Pub. No. US 2015/0376609 and PCT Pub. No. WO2018140966A1. In particular, and with reference to FIG. 5, the aqueous fluid 512 comprising (i) the biological particles (e.g, labelled B cells or plasma cells) 514 and (ii) the polymer precursor material (not shown) is flowed into channel junction 510, where it is partitioned into droplets 518, 520 through the flow of non-aqueous fluid 516. In the case of encapsulation methods, non-aqueous fluid 516 may also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule (e.g, bead) that includes the entrained biological particles. Examples of polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345.
[0345] For example, in the case where the polymer precursor material comprises a linear polymer material, such as a linear polyacrylamide, PEG, or other linear polymeric material, the activation agent can include a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets. Likewise, for polymer precursors that comprise polymerizable monomers, the activation agent can include a polymerization initiator. For example, in certain cases, where the polymer precursor comprises a mixture of acrylamide monomer with aN,N’- bis-(acryloyl)cystamine (BAC) comonomer, an agent such as tetraethylmethylenediamine (TEMED) can be provided within the second fluid streams 516 in channel segments 504 and 506, which can initiate the copolymerization of the acrylamide and BAC into a cross-linked polymer network, or hydrogel.
[0346] Upon contact of the second fluid stream 516 with the first fluid stream 512 at junction 510, during formation of droplets, the TEMED can diffuse from the second fluid 516 into the aqueous fluid 512 comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets 518, 520, resulting in the formation of gel ( e.g ., hydrogel) microcapsules or cell beads, as solid or semi-solid beads or particles entraining the cells (e.g., labelled B cells or plasma cells) 514. Although described in terms of polyacrylamide encapsulation, other “activatable” encapsulation compositions can also be employed in the context of the methods and compositions described herein. For example, formation of alginate droplets followed by exposure to divalent metal ions (e.g, Ca2+ ions), can be used as an encapsulation process using the described processes. Likewise, agarose droplets can also be transformed into capsules through temperature based gelling (e.g, upon cooling, etc.).
[0347] In some cases, encapsulated biological particles can be selectively releasable from the microcapsule or cell beads, such as through passage of time or upon application of a particular stimulus, that degrades the encapsulating material (e.g., microcapsule) sufficiently to allow the biological particles (e.g, labelled B cells or plasma cells), or its other contents to be released from the encapsulating material, such as into a partition (e.g., droplet). For example, in the case of the polyacrylamide polymer described above, degradation of the polymer can be accomplished through the introduction of an appropriate reducing agent, such as DTT or the like, to cleave disulfide bonds that cross-link the polymer matrix. See, for example, U.S. Patent Application Publication No. 2014/0378345.
[0348] The biological particle (e.g, labelled B cell, memory B cells, or plasma cell), can be subjected to other conditions sufficient to polymerize or gel the precursors. The conditions sufficient to polymerize or gel the precursors can include exposure to heating, cooling, electromagnetic radiation, and/or light. The conditions sufficient to polymerize or gel the precursors can include any conditions sufficient to polymerize or gel the precursors. Following polymerization or gelling, a polymer or gel can be formed around the biological particle (e.g, labelled B cell or plasma cell). The polymer or gel can be diffusively permeable to chemical or biochemical reagents. The polymer or gel can be diffusively impermeable to macromolecular constituents (e.g, secreted antibodies or antigen-binding fragments thereof) of the biological particle (e.g, labelled B cell, memory B cell, or plasma cell). In this manner, the polymer or gel can act to allow the biological particle (e.g, labelled B cell, memory B cell, or plasma cell) to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel. The polymer or gel can include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG- alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel can include any other polymer or gel.
[0349] The polymer or gel can be functionalized ( e.g ., coupled to a capture agent) to bind to targeted analytes (e.g., secreted antibodies or antigen-binding fragment thereof), such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel can be polymerized or gelled via a passive mechanism. The polymer or gel can be stable in alkaline conditions or at elevated temperature. The polymer or gel can have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel can be of a similar size to the bead. The polymer or gel can have a mechanical strength (e.g., tensile strength) similar to that of the bead. The polymer or gel can be of a lower density than an oil. The polymer or gel can be of a density that is roughly similar to that of a buffer. The polymer or gel can have a tunable pore size. The pore size can be chosen to, for instance, retain denatured nucleic acids. The pore size can be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The polymer or gel can be biocompatible. The polymer or gel can maintain or enhance cell viability. The polymer or gel can be biochemically compatible. The polymer or gel can be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.
[0350] The polymer can include poly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages. The preparation of the polymer can include a two-step reaction. In the first activation step, poly(acrylamide-co-acrylic acid) can be exposed to an acylating agent to convert carboxylic acids to esters. For instance, the poly(acryl amide-co-acrylic acid) can be exposed to 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). The polyacrylamide-co-acrylic acid can be exposed to other salts of 4-(4,6-dimethoxy-l,3,5-triazin-2- yl)-4-methylmorpholinium. In the second cross-linking step, the ester formed in the first step can be exposed to a disulfide crosslinking agent. For instance, the ester can be exposed to cystamine (2,2’-dithiobis(ethylamine)). Following the two steps, the biological particle can be surrounded by polyacrylamide strands linked together by disulfide bridges. In this manner, the biological particle can be encased inside of or comprise a gel or matrix (e.g., polymer matrix) to form a “cell bead.” A cell bead can contain biological particles ( e.g ., labelled B cell, memory B cell, or plasma cell) or macromolecular constituents (e.g., RNA, DNA, proteins, secreted antibodies or antigen-binding fragments thereof etc.) of biological particles. A cell bead can include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example, after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Systems and methods disclosed herein can be applicable to both (i) cell beads (and/or droplets or other partitions) containing biological particles and (ii) cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles.
[0351] Encapsulated biological particles (e.g., labelled B cell, memory B cell, or plasma cell) can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it can be desirable to allow biological particles (e.g, labelled B cell, memory B cell, or plasma cell) to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli (e.g., cytokines, antigens, etc.). In such cases, encapsulation can allow for longer incubation than partitioning in emulsion droplets, although in some cases, droplet partitioned biological particles can also be incubated for different periods of time, e.g., at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or more. The encapsulation of biological particles (e.g., labelled B cells, memory B cells, or plasma cells) can constitute the partitioning of the biological particles into which other reagents are co-partitioned. Alternatively or in addition, encapsulated biological particles can be readily deposited into other partitions (e.g., droplets) as described above.
Microwells
[0352] As described herein, one or more processes can be performed in a partition, which can be a well. The well can be a well of a plurality of wells of a substrate, such as a microwell of a microwell array or plate, or the well can be a microwell or microchamber of a device (e.g., microfluidic device) comprising a substrate The well can be a well of a well array or plate, or the well can be a well or chamber of a device (e.g., fluidic device). Accordingly, the wells or microwells can assume an “open” configuration, in which the wells or microwells are exposed to the environment ( e.g ., contain an open surface) and are accessible on one planar face of the substrate, or the wells or microwells can assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate. In some instances, the wells or microwells can be configured to toggle between “open” and “closed” configurations. For instance, an “open” microwell or set of microwells can be “closed” or “sealed” using a membrane (e.g., semi-permeable membrane), an oil (e.g., fluorinated oil to cover an aqueous solution), or a lid, as described elsewhere herein. The wells or microwells can be initially provided in a “closed” or “sealed” configuration, wherein they are not accessible on a planar surface of the substrate without an external force. For instance, the “closed” or “sealed” configuration can include a substrate such as a sealing film or foil that is puncturable or pierceable by pipette tip(s). Suitable materials for the substrate include, without limitation, polyester, polypropylene, polyethylene, vinyl, and aluminum foil.
[0353] In some embodiments, the well can have a volume of less than 1 milliliter (mL). For example, the well can be configured to hold a volume of at most 1000 microliters ( m L), at most 100 pL, at most 10 pL, at most 1 pL, at most 100 nanoliters (nL), at most 10 nL, at most 1 nL, at most 100 picoliters (pL), at most 10 (pL), or less. The well can be configured to hold a volume of about 1000 pL, about 100 pL, about 10 pL, about 1 pL, about 100 nL, about 10 nL, about 1 nL, about 100 pL, about 10 pL, etc. The well can be configured to hold a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 pL, at least 10 pL, at least 100 pL, at least 1000 pL, or more. The well can be configured to hold a volume in a range of volumes listed herein, for example, from about 5 nL to about 20 nL, from about 1 nL to about 100 nL, from about 500 pL to about 100 pL, etc. The well can be of a plurality of wells that have varying volumes and can be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.
[0354] In some instances, a microwell array or plate includes a single variety of microwells. In some instances, a microwell array or plate includes a variety of microwells. For instance, the microwell array or plate can include one or more types of microwells within a single microwell array or plate. The types of microwells can have different dimensions (e.g, length, width, diameter, depth, cross-sectional area, etc.), shapes (e.g, circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, etc.), aspect ratios, or other physical characteristics. The microwell array or plate can include any number of different types of microwells. For example, the microwell array or plate can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000 or more different types of microwells. A well can have any dimension ( e.g ., length, width, diameter, depth, cross-sectional area, volume, etc.), shape (e.g., circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, other polygonal, etc.), aspect ratios, or other physical characteristics described herein with respect to any well.
[0355] In certain instances, the microwell array or plate includes different types of microwells that are located adjacent to one another within the array or plate. For example, a microwell with one set of dimensions can be located adjacent to and in contact with another microwell with a different set of dimensions. Similarly, microwells of different geometries can be placed adjacent to or in contact with one another. The adjacent microwells can be configured to hold different articles; for example, one microwell can be used to contain a cell, cell bead, or other sample (e.g., cellular components, nucleic acid molecules, nucleic acid barcode molecules, etc.) while the adjacent microwell can be used to contain a microcapsule, droplet, bead, or other reagent. In some cases, the adjacent microwells can be configured to merge the contents held within, e.g., upon application of a stimulus, or spontaneously, upon contact of the articles in each microwell.
[0356] As is described elsewhere herein, a plurality of partitions can be used in the systems, compositions, and methods described herein. For example, any suitable number of partitions (e.g., wells or droplets) can be generated or otherwise provided. For example, in the case when wells are used, at least about 1,000 wells, at least about 5,000 wells, at least about 10,000 wells, at least about 50,000 wells, at least about 100,000 wells, at least about 500,000 wells, at least about 1,000,000 wells, at least about 5,000,000 wells at least about 10,000,000 wells, at least about 50,000,000 wells, at least about 100,000,000 wells, at least about 500,000,000 wells, at least about 1,000,000,000 wells, or more wells can be generated or otherwise provided. Moreover, the plurality of wells can include both unoccupied wells (e.g, empty wells) and occupied wells.
[0357] A well can include any of the reagents described herein, or combinations thereof. These reagents can include, for example, barcode molecules, enzymes, adapters, and combinations thereof. The reagents can be physically separated from a sample (for example, a cell, cell bead, or cellular components, e.g., proteins, nucleic acid molecules, etc.) that is placed in the well. This physical separation can be accomplished by containing the reagents within, or coupling to, a microcapsule or bead that is placed within a well. The physical separation can also be accomplished by dispensing the reagents in the well and overlaying the reagents with a layer that is, for example, dissolvable, meltable, or permeable prior to introducing the polynucleotide sample into the well. This layer can be, for example, an oil, wax, membrane (e.g., semi- permeable membrane), or the like. The well can be sealed at any point, for example, after addition of the microcapsule or bead, after addition of the reagents, or after addition of either of these components. The sealing of the well can be useful for a variety of purposes, including preventing escape of beads or loaded reagents from the well, permitting select delivery of certain reagents (e.g, via the use of a semi -permeable membrane), for storage of the well prior to or following further processing, etc.
[0358] Once sealed, the well may be subjected to conditions for further processing of a cell (or cells) in the well. For instance, reagents in the well may allow further processing of the cell, e.g., cell lysis, as further described herein. Alternatively, the well (or wells such as those of a well-based array) comprising the cell (or cells) may be subjected to freeze-thaw cycling to process the cell (or cells), e.g., cell lysis. The well containing the cell may be subjected to freezing temperatures (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, - 40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C, or -85°C). Freezing may be performed in a suitable manner, e.g., sub-zero freezer or a dry ice/ethanol bath. Following an initial freezing, the well (or wells) comprising the cell (or cells) may be subjected to freeze-thaw cycles to lyse the cell (or cells). In one embodiment, the initially frozen well (or wells) are thawed to a temperature above freezing (e.g., 4°C or above, 8°C or above, 12°C or above, 16°C or above, 20°C or above, room temperature, or 25°C or above). In another embodiment, the freezing is performed for less than 10 minutes (e.g., 5 minutes or 7 minutes) followed by thawing at room temperature for less than 10 minutes (e.g., 5 minutes or 7 minutes). This freeze-thaw cycle may be repeated a number of times, e.g., 2, 3, 4 or more times, to obtain lysis of the cell (or cells) in the well (or wells). In one embodiment, the freezing, thawing and/or freeze/thaw cycling is performed in the absence of a lysis buffer. Additional disclosure related to freeze-thaw cycling is provided in WO2019165181A1, which is incorporated herein by reference in its entirety.
[0359] A well can include free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with microcapsules, beads, or droplets. In some embodiments, any of the reagents described in this disclosure can be encapsulated in, or otherwise coupled to, a droplet or bead, with any chemicals, particles, and elements suitable for sample processing reactions involving biomolecules, such as, but not limited to, nucleic acid molecules and proteins. For example, a bead or droplet used in a sample preparation reaction for DNA sequencing can include one or more of the following reagents: enzymes, restriction enzymes ( e.g ., multiple cutters), ligase, polymerase, fluorophores, oligonucleotide barcodes, adapters, buffers, nucleotides (e.g., dNTPs, ddNTPs) and the like.
[0360] Additional examples of reagents include, but are not limited to: buffers, acidic solution, basic solution, temperature-sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions, magnesium chloride, sodium chloride, manganese, aqueous buffer, mild buffer, ionic buffer, inhibitor, enzyme, protein, polynucleotide, antibodies, saccharides, lipid, oil, salt, ion, detergents, ionic detergents, non-ionic detergents, oligonucleotides, nucleotides, deoxyribonucleotide triphosphates (dNTPs), dideoxyribonucleotide triphosphates (ddNTPs), DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, polymerase, ligase, restriction enzymes, proteases, nucleases, protease inhibitors, nuclease inhibitors, chelating agents, reducing agents, oxidizing agents, fluorophores, probes, chromophores, dyes, organics, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, pharmaceuticals, radioactive molecules, preservatives, antibiotics, aptamers, and pharmaceutical drug compounds. As described herein, one or more reagents in the well can be used to perform one or more reactions, including but not limited to: cell lysis, cell fixation, permeabilization, nucleic acid reactions, e.g., nucleic acid extension reactions, amplification, reverse transcription, transposase reactions (e.g., tagmentation), etc.
[0361] The wells disclosed herein can be provided as a part of a kit. For example, a kit can include instructions for use, a microwell array or device, and reagents (e.g, beads). The kit can include any useful reagents for performing the processes described herein, e.g., nucleic acid reactions, barcoding of nucleic acid molecules, sample processing (e.g., for cell lysis, fixation, and/or permeabilization). [0362] In some cases, a well includes a microcapsule, bead, or droplet that includes a set of reagents that has a similar attribute, for example, a set of enzymes, a set of minerals, a set of oligonucleotides, a mixture of different barcode molecules, a mixture of identical barcode molecules. In other cases, a microcapsule, bead, or droplet includes a heterogeneous mixture of reagents. In some cases, the heterogeneous mixture of reagents can include all components necessary to perform a reaction. In some cases, such mixture can include all components necessary to perform a reaction, except for 1, 2, 3, 4, 5, or more components necessary to perform a reaction. In some cases, such additional components are contained within, or otherwise coupled to, a different microcapsule, droplet, or bead, or within a solution within a partition ( e.g ., microwell) of the system.
[0363] A non-limiting example of a microwell array in accordance with some embodiments of the disclosure is schematically presented in FIG. 9. In this example, the array can be contained within a substrate 900. The substrate 900 includes a plurality of wells 902. The wells 902 can be of any size or shape, and the spacing between the wells, the number of wells per substrate, as well as the density of the wells on the substrate 900 can be modified, depending on the particular application. In one such example application, a sample molecule 906, which can include a cell or cellular components (e.g., nucleic acid molecules) is co-partitioned with a bead 904, which can include a nucleic acid barcode molecule coupled thereto. The wells 902 can be loaded using gravity or other loading technique (e.g., centrifugation, liquid handler, acoustic loading, optoelectronic, etc.). In some instances, at least one of the wells 902 contains a single sample molecule 906 (e.g., cell) and a single bead 904.
[0364] Reagents can be loaded into a well either sequentially or concurrently. In some cases, reagents are introduced to the device either before or after a particular operation. In some cases, reagents (which can be provided, in certain instances, in microcapsules, droplets, or beads) are introduced sequentially such that different reactions or operations occur at different steps.
The reagents (or microcapsules, droplets, or beads) can also be loaded at operations interspersed with a reaction or operation step. For example, microcapsules, droplets, or beads including reagents for fragmenting polynucleotides (e.g, restriction enzymes) and/or other enzymes (e.g, transposases, ligases, polymerases, etc.) can be loaded into the well or plurality of wells, followed by loading of microcapsules, droplets, or beads including reagents for attaching nucleic acid barcode molecules to a sample nucleic acid molecule. Reagents can be provided concurrently or sequentially with a sample, e.g., a cell or cellular components (e.g., organelles, proteins, nucleic acid molecules, carbohydrates, lipids, etc.). Accordingly, use of wells can be useful in performing multi-step operations or reactions.
[0365] FIG. 10 schematically shows an example workflow for processing nucleic acid molecules within a sample. A substrate 1000 including a plurality of microwells 1002 can be provided. A sample 1006 which can include a cell, cell bead, cellular components or analytes (e.g, proteins and/or nucleic acid molecules) can be co-partitioned, in a plurality of microwells 1002, with a plurality of beads 1004 including nucleic acid barcode molecules. During a partitioning process, the sample 1006 can be processed within the partition. For instance, in the case of live cells, the cell can be subjected to conditions sufficient to lyse the cells and release the analytes contained therein. In process 1020, the bead 1004 can be further processed. By way of example, processes 1020a and 1020b schematically illustrate different workflows, depending on the properties of the bead 1004.
[0366] In 1020a, the bead includes nucleic acid barcode molecules that are attached thereto, and sample nucleic acid molecules (e.g., RNA, DNA) can attach, e.g., via hybridization of ligation, to the nucleic acid barcode molecules. Such attachment can occur on the bead. In process 1030, the beads 1004 from multiple wells 1002 can be collected and pooled. Further processing can be performed in process 1040. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 1050, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g., in a plot.
[0367] In 1020b, the bead includes nucleic acid barcode molecules that are releasably attached thereto, as described below. The bead can degrade or otherwise release the nucleic acid barcode molecules into the well 1002; the nucleic acid barcode molecules can then be used to barcode nucleic acid molecules within the well 1002. Further processing can be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 1050, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g., in a plot.
[0368] As described elsewhere herein, the nucleic acid barcode molecules and other reagents can be contained within a microcapsule, bead, or droplet. These microcapsules, beads or droplets can be loaded into a partition (e.g., a microwell) before, after, or concurrently with the loading of a cell, such that each cell is contacted with a different microcapsule, bead, or droplet. This technique can be used to attach a unique nucleic acid barcode molecule to nucleic acid molecules obtained from each cell. Alternatively or in addition, the sample nucleic acid molecules can be attached to a support. For example, the partition (e.g., microwell) can include a bead which has coupled thereto a plurality of nucleic acid barcode molecules. The sample nucleic acid molecules, or derivatives thereof, can couple or attach to the nucleic acid barcode molecules attached on the support. The resulting barcoded nucleic acid molecules can then be removed from the partition, and in some instances, pooled and sequenced. In such cases, the nucleic acid barcode sequences can be used to trace the origin of the sample nucleic acid molecule. For example, polynucleotides with identical barcodes can be determined to originate from the same cell or partition, while polynucleotides with different barcodes can be determined to originate from different cells or partitions.
[0369] The samples or reagents can be loaded in the wells or microwells using a variety of approaches. For example, the samples (e.g, a cell, cell bead, or cellular component) or reagents (as described herein) can be loaded into the well or microwell using an external force, e.g., gravitational force, electrical force, magnetic force, or using mechanisms to drive the sample or reagents into the well, for example, via pressure-driven flow, centrifugation, optoelectronics, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc. In certain cases, a fluid handling system can be used to load the samples or reagents into the well. The loading of the samples or reagents can follow a Poissonian distribution or a non-Poissonian distribution, e.g., super Poisson or sub-Poisson. The geometry, spacing between wells, density, and size of the microwells can be modified to accommodate a useful sample or reagent distribution; for example, the size and spacing of the microwells can be adjusted such that the sample or reagents can be distributed in a super-Poissonian fashion.
[0370] In one non-limiting example, the microwell array or plate includes pairs of microwells, in which each pair of microwells is configured to hold a droplet ( e.g ., including a single cell) and a single bead (such as those described herein, which can, in some instances, also be encapsulated in a droplet). The droplet and the bead (or droplet containing the bead) can be loaded simultaneously or sequentially, and the droplet and the bead can be merged, e.g., upon contact of the droplet and the bead, or upon application of a stimulus (e.g., external force, agitation, heat, light, magnetic or electric force, etc.). In some cases, the loading of the droplet and the bead is super-Poissonian. In other examples of pairs of microwells, the wells are configured to hold two droplets including different reagents and/or samples, which are merged upon contact or upon application of a stimulus. In such instances, the droplet of one microwell of the pair can include reagents that can react with an agent in the droplet of the other microwell of the pair. For example, one droplet can include reagents that are configured to release the nucleic acid barcode molecules of a bead contained in another droplet, located in the adjacent microwell. Upon merging of the droplets, the nucleic acid barcode molecules can be released from the bead into the partition (e.g., the microwell or microwell pair that are in contact), and further processing can be performed (e.g., barcoding, nucleic acid reactions, etc.). In cases where intact or live cells are loaded in the microwells, one of the droplets can include lysis reagents for lysing the cell upon droplet merging.
[0371] In some embodiments, a microcapsule, droplet, or bead can be partitioned into a well. The droplets can be selected or subjected to pre-processing prior to loading into a well. For instance, the droplets can include cells, and only certain droplets, such as those containing a single cell (or at least one cell), can be selected for use in loading of the wells. Such a pre selection process can be useful in efficient loading of single cells, such as to obtain a non- Poissonian distribution, or to pre-filter cells for a selected characteristic prior to further partitioning in the wells. Additionally, the technique can be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.
[0372] In some embodiments, the wells can include nucleic acid barcode molecules attached thereto. The nucleic acid barcode molecules can be attached to a surface of the well (e.g, a wall of the well). The nucleic acid barcode molecules may be attached to a droplet or bead that has been partitioned into the well. The nucleic acid barcode molecule ( e.g ., a partition barcode sequence) of one well can differ from the nucleic acid barcode molecule of another well, which can permit identification of the contents contained with a single partition or well. In some embodiments, the nucleic acid barcode molecule can include a spatial barcode sequence that can identify a spatial coordinate of a well, such as within the well array or well plate. In some embodiments, the nucleic acid barcode molecule can include a unique molecular identifier for individual molecule identification. In some instances, the nucleic acid barcode molecules can be configured to attach to or capture a nucleic acid molecule from or within a sample or cell distributed in the well. For example, the nucleic acid barcode molecules can include a capture sequence that can be used to capture or hybridize to a nucleic acid molecule (e.g., RNA, DNA) from or within the sample. In some embodiments, the nucleic acid barcode molecules can be releasable from the microwell. In some instances, the nucleic acid barcode molecules may be releasable from the bead or droplet. For example, the nucleic acid barcode molecules can include a chemical cross-linker which can be cleaved upon application of a stimulus (e.g., photo-, magnetic, chemical, biological, stimulus). The released nucleic acid barcode molecules, which can be hybridized or configured to hybridize to a sample nucleic acid molecule, can be collected and pooled for further processing, which can include nucleic acid processing (e.g, amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In some instances nucleic acid barcode molecules attached to a bead in a well may be hybridized to sample nucleic acid molecules, and the bead with the sample nucleic acid molecules hybridized thereto may be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In such cases, the unique partition barcode sequences can be used to identify the cell or partition from which a nucleic acid molecule originated.
[0373] Characterization of samples within a well can be performed. Such characterization can include, in non-limiting examples, imaging of the sample (e.g, cell, cell bead, or cellular components) or derivatives thereof. Characterization techniques such as microscopy or imaging can be useful in measuring sample profiles in fixed spatial locations. For example, when cells are partitioned, optionally with beads, imaging of each microwell and the contents contained therein can provide useful information on cell doublet formation (e.g, frequency, spatial locations, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression level of a biomarker ( e.g ., a surface marker, a fluorescently labeled molecule therein, etc.), cell or bead loading rate, number of cell-bead pairs, etc. In some instances, imaging can be used to characterize live cells in the wells, including, but not limited to: dynamic live-cell tracking, cell cell interactions (when two or more cells are co-partitioned), cell proliferation, etc. Alternatively or in addition to, imaging can be used to characterize a quantity of amplification products in the well.
[0374] In operation, a well can be loaded with a sample and reagents, simultaneously or sequentially. When cells or cell beads are loaded, the well can be subjected to washing, e.g., to remove excess cells from the well, microwell array, or plate. Similarly, washing can be performed to remove excess beads or other reagents from the well, microwell array, or plate. In the instances where live cells are used, the cells can be lysed in the individual partitions to release the intracellular components or cellular analytes. Alternatively, the cells can be fixed or permeabilized in the individual partitions. The intracellular components or cellular analytes can couple to a support, e.g., on a surface of the microwell, on a solid support (e.g., bead), or they can be collected for further downstream processing. For example, after cell lysis, the intracellular components or cellular analytes can be transferred to individual droplets or other partitions for barcoding. Alternatively, or in addition, the intracellular components or cellular analytes (e.g., nucleic acid molecules) can couple to a bead including a nucleic acid barcode molecule; subsequently, the bead can be collected and further processed, e.g., subjected to nucleic acid reaction such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon can be further characterized, e.g., via sequencing. Alternatively, or in addition, the intracellular components or cellular analytes can be barcoded in the well (e.g., using a bead including nucleic acid barcode molecules that are releasable or on a surface of the microwell including nucleic acid barcode molecules). The barcoded nucleic acid molecules or analytes can be further processed in the well, or the barcoded nucleic acid molecules or analytes can be collected from the individual partitions and subjected to further processing outside the partition. Further processing can include nucleic acid processing (e.g., performing an amplification, extension) or characterization (e.g., fluorescence monitoring of amplified molecules, sequencing). At any convenient, suitable, and/or useful step, the well (or microwell array or plate) can be sealed (e.g., using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents. Beads
[0375] In some embodiments of the disclosure, a partition can include one or more unique identifiers, such as barcodes ( e.g ., a plurality of barcode nucleic acid molecules, also referred herein to as nucleic acid barcode molecules which can be, for example, a plurality of partition barcode sequences). Barcodes can be previously, subsequently, or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle (e.g., labelled B cell, memory B cell, or plasma cell). For example, barcodes can be injected into droplets previous to, subsequent to, or concurrently with droplet generation. In some embodiments, the delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle (e.g., labelled B cell, memory B cell, or plasma cell) to the particular partition. Barcodes can be delivered, for example on a nucleic acid molecule (e.g., a barcoded oligonucleotide, nucleic acid barcode molecule), to a partition via any suitable mechanism. In some embodiments, barcoded nucleic acid molecules, e.g., nucleic acid barcode molecules can be delivered to a partition via a microcapsule. A microcapsule, in some instances, can include a bead. Beads are described in further detail below.
[0376] In some embodiments, barcodes (e.g., barcoded nucleic acid molecules, nucleic acid barcode molecules can be initially associated with the microcapsule and then released from the microcapsule. In some embodiments, release of the barcoded nucleic acid molecules, e.g., nucleic acid barcode molecules can be passive (e.g., by diffusion out of the microcapsule). In addition or alternatively, release from the microcapsule can be upon application of a stimulus which allows the barcoded nucleic acid molecules to dissociate or to be released from the microcapsule. Such stimulus can disrupt the microcapsule, an interaction that couples the barcoded nucleic acid molecules to or within the microcapsule, or both. Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g, change in pH or use of a reducing agent), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof. Methods and systems for partitioning barcode carrying beads into droplets are provided in US. Patent Publication Nos. 2019/0367997 and 2019/0064173, and International Publication Nos. WO2020167862 and WO2020176882.
[0377] Beneficially, a discrete droplet partitioning a biological particle and a barcode carrying bead can effectively allow the attribution of the barcode to macromolecular constituents of the biological particle within the partition. The contents of a partition can remain discrete from the contents of other partitions.
[0378] In operation, the barcoded oligonucleotides can be released ( e.g ., in a partition), as described elsewhere herein. Alternatively, the nucleic acid molecules bound to the bead (e.g., gel bead) can be used to hybridize and capture analytes (e.g., one or more types of analytes) on the solid phase of the bead.
[0379] In some examples, beads, biological particles (e.g., labelled B cells memory B cells, or plasma cells) and droplets can flow along channels (e.g., the channels of a microfluidic device), in some cases at substantially regular flow profiles (e.g., at regular flow rates). Such regular flow profiles can permit a droplet to include a single bead and a single biological particle. Such regular flow profiles can permit the droplets to have an occupancy (e.g, droplets having beads and biological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flow profiles and devices that can be used to provide such regular flow profiles are provided in, for example, U.S. Patent Publication No. 2015/0292988.
[0380] A bead can be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead can be dissolvable, disruptable, and/or degradable. Degradable beads, as well as methods for degrading beads, are described in PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety. In some cases, any combination of stimuli, e.g., stimuli described in PCT Publication No. WO2014210353 and US Patent Application Pub. No. 2015/0376609, hereby incorporated by reference in its entirety, may trigger degradation of a bead. For example, a change in pH may enable a chemical agent (e.g., DTT) to become an effective reducing agent.
[0381] In some cases, a bead cannot be degradable. In some cases, the bead can be a gel bead. A gel bead can be a hydrogel bead. A gel bead can be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid bead can be a liposomal bead. Solid beads can include metals including iron oxide, gold, and silver. In some cases, the bead can be a silica bead. In some cases, the bead can be rigid. In other cases, the bead can be flexible and/or compressible.
[0382] A bead can be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.
[0383] Beads can be of uniform size or heterogeneous size. In some cases, the diameter of a bead can be at least about 10 nanometers (nm), 100 nm, 500 nm, 1 micrometer (mhi), 5mih, IOmih, 20mhi, 30mm, 40mhi, 50mm, 60mm, 70mm, 80mm, 90mhi, 100mm, 250mm, 500mm, 1mm, or greater. In some cases, a bead can have a diameter of less than about 10 nm, 100 nm, 500 nm, Imhi, 5mhi, IOmhi, 20mih, 30mih, 40mhi, 50mm, 60mm, 70mm, 80mm, 90mih, 100mm, 250mm, 500mm, 1mm, or less. In some cases, a bead can have a diameter in the range of about 40-75pm, 30-75pm, 20-75pm, 40-85mih, 40-95pm, 20-100pm, 10-100pm, l-100pm, 20-250pm, or 20- 500pm.
[0384] In certain aspects, beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide relatively consistent amounts of reagents within partitions, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency. In some embodiments, the beads described herein can have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.
[0385] A bead can include natural and/or synthetic materials. For example, a bead can include a natural polymer, a synthetic polymer or both natural and synthetic polymers. See, e.g., PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety. Beads can also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others.
[0386] In some embodiments, the bead can include covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, linear polymers), nucleic acid molecules (e.g., nucleic acid barcode molecules or barcoded oligonucleotides), primers, and other entities. In some embodiments, the covalent bonds can be carbon-carbon bonds, thioether bonds, or carbon- heteroatom bonds.
[0387] In some embodiments, a bead can include an acrydite moiety, which in certain aspects can be used to attach one or more nucleic acid molecules (e.g., barcode sequence, barcoded nucleic acid molecule, nucleic acid barcode molecule, barcoded oligonucleotide, primer, or other oligonucleotide) to the bead. Acrydite moieties, as well as their uses in attaching nucleic acid molecules to beads, are described in PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety.
[0388] For example, precursors (e.g., monomers, cross-linkers) that are polymerized to form a bead can include acrydite moieties, such that when a bead is generated, the bead also includes acrydite moieties. The acrydite moieties can be attached to a nucleic acid molecule ( e.g ., oligonucleotide such as nucleic acid barcode molecule), which can include a priming sequence (e.g., a primer for amplifying target nucleic acids, random primer, primer sequence for messenger RNA) and/or one or more barcode sequences. The one or more barcode sequences can include sequences that are the same for all nucleic acid molecules coupled to a given bead (e.g, nucleic acid barcode molecules coupled to a given bead) and/or sequences that are different across all nucleic acid molecules coupled to the given bead (e.g, nucleic acid barcode molecules coupled to a given bead). The nucleic acid molecule can be incorporated into the bead.
[0389] In some embodiments, the nucleic acid molecule (e.g, nucleic acid barcode molecule) can include a functional sequence, e.g, for use in downstream sequencing methodologies, for example, a functional sequence for attachment to a sequencing flow cell, such as, for example, a P5 sequence for Illumina® sequencing. In some cases, the nucleic acid barcode molecule or derivative thereof (e.g., oligonucleotide or polynucleotide generated from the nucleic acid barcode molecule) can include another functional sequence, such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid barcode molecule can include a barcode sequence. In some cases, the primer can further include a unique molecular identifier (UMI). In some cases, the primer can include an R1 primer sequence for Illumina sequencing. In some embodiments, the nucleic acid barcode molecule can include adapters for compatibility with other sequencing platforms. Non limiting examples of nucleic acid sequencing methods include Maxam-Gilbert sequencing and chain-termination methods, de novo sequencing methods including shotgun sequencing and bridge PCR, next-generation methods including Polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD™ sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, and SMRT® sequencing.
[0390] Other examples of methods for sequencing nucleic acids include, but are not limited to, DNA hybridization methods, restriction enzyme digestion methods, Sanger sequencing methods, ligation methods, and microarray methods. Additional examples of sequencing methods that can be used include targeted sequencing, single molecule real-time sequencing, exon sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, co-amplification at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, Solexa Genome Analyzer sequencing, MS-PET sequencing, and any combinations thereof.
[0391] Accordingly, a wide variety of different approaches, systems, and techniques for nucleic acid sequencing, including next-generation sequencing (NGS) methods, can be used to determine the nucleic acid sequences encoding the antibodies produced by the partitioned single cells. Generally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) ( e.g ., digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR), and/or isothermal amplification. In some embodiments, the nucleic acid barcode molecule can include adapters for compatibility with long read sequencing platforms such as the PacBio SMRT-seq platform and nanopore sequencing
[0392] In some embodiments, the nucleic acid molecule (e.g., nucleic acid barcode molecule) or derivative thereof (e.g., oligonucleotide or polynucleotide generated from the nucleic acid barcode molecule) can include another functional sequence, such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid barcode molecule can include a barcode sequence. In some cases, the nucleic acid barcode molecule or primer can further include a unique molecular identifier (UMI). In some cases, the primer can include an R1 primer sequence for Illumina sequencing. In some embodiments, the nucleic acid barcode molecule or primer can include an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g, oligonucleotides, polynucleotides, etc.) and uses thereof, as can be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and 2015/0376609.
[0393] FIG. 7 illustrates an example of a barcode carrying bead. A nucleic acid molecule ( e.g ., nucleic acid barcode molecule, barcoded nucleic acid molecule) 702, such as an oligonucleotide, can be coupled to a bead 704 by a releasable linkage 706, such as, for example, a disulfide linker. The same bead 704 can be coupled (e.g., via releasable linkage) to one or more other nucleic acid molecules (e.g., other nucleic acid barcode molecules) 718, 720. The nucleic acid molecule 702 can be or include a barcode. As noted elsewhere herein, the structure of the barcode can include a number of sequence elements. The nucleic acid molecule 702 can include a functional sequence 708 that can be used in subsequent processing. For example, the functional sequence 708 can include one or more of a sequencer specific flow cell attachment sequence (e.g., a P5 sequence for Illumina® sequencing systems) and a sequencing primer sequence (e.g., a R1 primer for Illumina® sequencing systems). The nucleic acid molecule 702 can include a barcode sequence 710 for use in barcoding the sample (e.g., DNA, RNA, protein, etc.). In some cases, the barcode sequence 710 can be bead- specific such that the barcode sequence 710 is common to all nucleic acid molecules (e.g., including nucleic acid molecule 702) coupled to the same bead 704. Alternatively or in addition, the barcode sequence 710 can be partition-specific such that the barcode sequence 710 is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition. The nucleic acid molecule 702 can include a specific priming sequence 712, such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence. The nucleic acid molecule 702 can include an anchoring sequence 714 to ensure that the specific priming sequence 712 hybridizes at the sequence end (e.g., of the mRNA). For example, the anchoring sequence 714 can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.
[0394] The nucleic acid molecule 702 can include a unique molecular identifying sequence 716 (e.g, unique molecular identifier (UMI)). In some cases, the unique molecular identifying sequence 716 can include from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence 716 can compress less than about 5 or more than about 8 nucleotides. The unique molecular identifying sequence 716 can be a unique sequence that varies across individual nucleic acid molecules (e.g., 702, 718, 720, etc.) coupled to a single bead (e.g, bead 704). In some cases, the unique molecular identifying sequence 716 can be a random sequence (e.g, such as a random N-mer sequence). For example, the UMI can provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA. As will be appreciated, although FIG. 7 shows three nucleic acid molecules 702, 718, 720 coupled to the surface of the bead 704, an individual bead can be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands, millions, or even billion of individual nucleic acid molecules. The respective barcodes for the individual nucleic acid molecules can include both common sequence segments or relatively common sequence segments ( e.g ., 708, 710, 712, etc.) and variable or unique sequence segments (e.g., 716) between different individual nucleic acid molecules coupled to the same bead.
[0395] In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can be co-partitioned along with a barcode bearing bead 704. The barcoded nucleic acid molecules 702, 718, 720 can be released from the bead 704 in the partition. By way of example, in the context of analyzing sample RNA, the poly-T segment (e.g., 712) of one of the released nucleic acid molecules (e.g., 702) can hybridize to the poly-A tail of a mRNA molecule. Reverse transcription can result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments 708, 710, 716 of the nucleic acid molecule 702. Because the nucleic acid barcode molecule 702 includes an anchoring sequence 714, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. Within any given partition, all of the cDNA transcripts of the individual mRNA molecules can include a common barcode sequence segment 710. However, the transcripts made from the different mRNA molecules within a given partition can vary at the unique molecular identifying sequence 712 segment (e.g., UMI segment). Beneficially, even following any subsequent amplification of the contents of a given partition, the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g., cell). As noted above, the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences can also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid barcode molecules bound to the bead (e.g., gel bead) can be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents. In such cases, further processing can be performed, in the partitions or outside the partitions ( e.g ., in bulk). For instance, the RNA molecules on the beads can be subjected to reverse transcription or other nucleic acid processing, additional adapter sequences can be added to the barcoded nucleic acid molecules, or other nucleic acid reactions (e.g., amplification, nucleic acid extension) can be performed. The beads or products thereof (e.g., barcoded nucleic acid molecules) can be collected from the partitions, and/or pooled together and subsequently subjected to clean up and further characterization (e.g., sequencing).
[0396] The operations described herein can be performed at any useful or suitable step. For instance, the beads including nucleic acid barcode molecules can be introduced into a partition (e.g, well or droplet) prior to, during, or following introduction of a sample into the partition. The nucleic acid molecules of a sample can be subjected to barcoding, which can occur on the bead (in cases where the nucleic acid molecules remain coupled to the bead) or following release of the nucleic acid barcode molecules into the partition. In cases where the nucleic acid molecules from the sample remain attached to the bead, the beads from various partitions can be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, and/or sequencing). In other instances, the processing can occur in the partition. For example, conditions sufficient for barcoding, adapter attachment, reverse transcription, or other nucleic acid processing operations can be provided in the partition and performed prior to clean up and sequencing.
[0397] In some instances, a bead can include a capture sequence or binding sequence configured to bind to a corresponding capture sequence or binding sequence. In some instances, a bead can include a plurality of different capture sequences or binding sequences configured to bind to different respective corresponding capture sequences or binding sequences. For example, a bead can include a first subset of one or more capture sequences each configured to bind to a first corresponding capture sequence, a second subset of one or more capture sequences each configured to bind to a second corresponding capture sequence, a third subset of one or more capture sequences each configured to bind to a third corresponding capture sequence, and etc. A bead can include any number of different capture sequences. In some instances, a bead can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences, respectively. Alternatively or in addition, a bead can include at most about 10, 9, 8, 7, 6, 5, 4, 3, or 2 different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences. In some instances, the different capture sequences or binding sequences can be configured to facilitate analysis of a same type of analyte. In some instances, the different capture sequences or binding sequences can be configured to facilitate analysis of different types of analytes (with the same bead). The capture sequence can be designed to attach to a corresponding capture sequence. Beneficially, such corresponding capture sequence can be introduced to, or otherwise induced in, a biological particle ( e.g ., cell, cell bead, etc.) for performing different assays in various formats (e.g., barcoded antibodies including the corresponding capture sequence, barcoded MHC dextramers including the corresponding capture sequence, barcoded guide RNA molecules including the corresponding capture sequence, etc.), such that the corresponding capture sequence can later interact with the capture sequence associated with the bead. In some instances, a capture sequence coupled to a bead (or other support) can be configured to attach to a linker molecule, such as a splint molecule, wherein the linker molecule is configured to couple the bead (or other support) to other molecules through the linker molecule, such as to one or more analytes or one or more other linker molecules.
[0398] FIG. 8 illustrates a non-limiting example of a barcode carrying bead in accordance with some embodiments of the disclosure. A nucleic acid barcode molecule 805, such as an oligonucleotide, can be coupled to a bead 804 by a releasable linkage 806, such as, for example, a disulfide linker. The nucleic acid barcode molecule 805 can include a first capture sequence 860. The same bead 804 can be coupled, e.g., via releasable linkage, to one or more other nucleic acid molecules 803, 807 including other capture sequences. The nucleic acid barcode molecule 805 can be or include a barcode sequence. As described elsewhere herein, the structure of the barcode sequence can include a number of sequence elements, such as a functional sequence 808 (e.g., flow cell attachment sequence, sequencing primer sequence, etc ), a barcode sequence 810 (e.g., bead-specific sequence common to bead, partition-specific sequence common to partition, etc.), and a unique molecular identifier 812 (e.g., unique sequence within different molecules attached to the bead), or partial sequences thereof. The capture sequence 860 can be configured to attach to a corresponding capture sequence 865 (e.g., capture handle). In some instances, the corresponding capture sequence 865 can be coupled to another molecule that can be an analyte or an intermediary carrier. For example, as illustrated in FIG. 8, the corresponding capture sequence 865 is coupled to a guide RNA molecule 862 including a target sequence 864, wherein the target sequence 864 is configured to attach to the analyte. Another oligonucleotide molecule
807 attached to the bead 804 includes a second capture sequence 880 which is configured to attach to a second corresponding capture sequence ( e.g ., capture handle) 885. As illustrated in FIG. 8, the second corresponding capture sequence 885 is coupled to an antibody 882. In some cases, the antibody 882 can have binding specificity to an analyte (e.g., surface protein). Alternatively, the antibody 882 cannot have binding specificity. Another oligonucleotide molecule 803 attached to the bead 804 includes a third capture sequence 870 which is configured to attach to a third corresponding capture sequence 875. As illustrated in FIG. 8, the third corresponding capture sequence (e.g., capture handle) 875 is coupled to a molecule 872. The molecule 872 may or may not be configured to target an analyte. The other oligonucleotide molecules 803, 807 can include the other sequences (e.g., functional sequence, barcode sequence, UMI, etc.) described with respect to oligonucleotide molecule 805. While a single oligonucleotide molecule including each capture sequence is illustrated in FIG. 8, it will be appreciated that, for each capture sequence, the bead can include a set of one or more oligonucleotide molecules each including the capture sequence. For example, the bead can include any number of sets of one or more different capture sequences. Alternatively or in addition, the bead 808 can include other capture sequences. Alternatively or in addition, the bead
808 can include fewer types of capture sequences (e.g., two capture sequences). Alternatively or in addition, the bead 808 can include oligonucleotide molecule(s) including a priming sequence, such as a specific priming sequence such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence, for example, to facilitate an assay for gene expression.
[0399] The generation of a barcoded sequence, see, e.g., FIG. 7, is described herein.
[0400] A bead injected or otherwise introduced into a partition can include releasably, cleavably, or reversibly attached barcodes (e.g., partition barcode sequences). A bead injected or otherwise introduced into a partition can include activatable barcodes. A bead injected or otherwise introduced into a partition can be degradable, disruptable, or dissolvable beads.
[0401] Barcodes (e.g., nucleic acid barcode molecules or barcoded oligonucleotides), can be releasably, cleavably or reversibly attached to the beads such that barcodes can be released or be releasable through cleavage of a linkage between the barcode containing nucleic acid molecule and the bead, or released through degradation of the underlying bead itself, allowing the barcodes to be accessed or be accessible by other reagents, or both. In non-limiting examples, cleavage can be achieved through reduction of di-sulfide bonds, use of restriction enzymes, photo-activated cleavage, or cleavage via other types of stimuli ( e.g ., chemical, thermal, pH, enzymatic, etc.) and/or reactions, such as described elsewhere herein. Releasable barcodes can sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode can be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.
[0402] As will be appreciated from the above disclosure, the degradation of a bead can refer to the dissociation of a bound (e.g., capture agent configured to couple to a secreted antibody or antigen-binding fragment thereof) or entrained species (e.g., labelled B cell, e.g., memory B cell, or plasma cell, or secreted antibody or antigen-binding fragment thereof) from a bead, both with and without structurally degrading the physical bead itself. For example, the degradation of the bead can involve cleavage of a cleavable linkage via one or more species and/or methods described elsewhere herein. In another example, entrained species can be released from beads through osmotic pressure differences due to, for example, changing chemical environments. See, e.g., PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety.
[0403] A degradable bead can be introduced into a partition, such as a droplet of an emulsion or a well, such that the bead degrades within the partition and any associated species (e.g., oligonucleotides) are released within the droplet when the appropriate stimulus is applied. The free species (e.g., oligonucleotides, nucleic acid molecules, nucleic acid barcode molecules) can interact with other reagents contained in the partition. See, e.g., PCT Publication No. WO2014210353, which is hereby incorporated by reference in its entirety.
[0404] Any suitable number of molecular tag molecules (e.g, primer, barcoded oligonucleotide) can be associated with a bead such that, upon release from the bead, the molecular tag molecules (e.g., primer, e.g., barcoded oligonucleotide) are present in the partition at a pre-defmed concentration. Such pre-defmed concentration can be selected to facilitate certain reactions for generating a sequencing library, e.g., amplification, within the partition. In some cases, the pre-defmed concentration of the primer can be limited by the process of producing nucleic acid barcode molecule (e.g., oligonucleotide, e.g., nucleic acid barcode molecule) bearing beads.
[0405] In some cases, beads can be non-covalently loaded with one or more reagents. The beads can be non-covalently loaded by, for instance, subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interiors of the beads, and subjecting the beads to conditions sufficient to de-swell the beads. The swelling of the beads can be accomplished, for instance, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and/or subjecting the beads to an electric field. The swelling of the beads can be accomplished by various swelling methods. The de-swelling of the beads can be accomplished, for instance, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or high temperatures, subjecting the beads to a lower or higher ion concentration, and/or removing an electric field. The de-swelling of the beads can be accomplished by various de-swelling methods. Transferring the beads can cause pores in the bead to shrink. The shrinking can then hinder reagents within the beads from diffusing out of the interiors of the beads. The hindrance can be due to steric interactions between the reagents and the interiors of the beads. The transfer can be accomplished microfluidically. For instance, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream. The swellability and/or pore size of the beads can be adjusted by changing the polymer composition of the bead.
[0406] In some cases, an acrydite moiety linked to a precursor, another species linked to a precursor, or a precursor itself can include a labile bond, such as chemically, thermally, or photo sensitive bond e.g., disulfide bond, UV sensitive bond, or the like. Once acrydite moieties or other moieties including a labile bond are incorporated into a bead, the bead can also include the labile bond. The labile bond can be, for example, useful in reversibly linking (e.g., covalently linking) species (e.g, barcodes, primers, etc.) to a bead. In some cases, a thermally labile bond can include a nucleic acid hybridization based attachment, e.g., where an oligonucleotide is hybridized to a complementary sequence that is attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide, e.g., a barcode containing sequence, from the bead or microcapsule.
[0407] The addition of multiple types of labile bonds to a gel bead can result in the generation of a bead capable of responding to varied stimuli. Each type of labile bond can be sensitive to an associated stimulus ( e.g ., chemical stimulus, light, temperature, enzymatic, etc.) such that release of species attached to a bead via each labile bond can be controlled by the application of the appropriate stimulus. Such functionality can be useful in controlled release of species from a gel bead. In some cases, another species including a labile bond can be linked to a gel bead after gel bead formation via, for example, an activated functional group of the gel bead as described above. As will be appreciated, barcodes that are releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable through cleavage of a linkage between the barcode molecule and the bead, or that are released through degradation of the underlying bead itself, allowing the barcodes to be accessed or accessible by other reagents, or both.
[0408] The barcodes that are releasable as described herein can sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode can be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.
[0409] In addition to thermally cleavable bonds, disulfide bonds and UY sensitive bonds, other non-limiting examples of labile bonds that can be coupled to a precursor or bead include an ester linkage (e.g., cleavable with an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodium periodate), a Diels- Alder linkage (e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via a base), a silyl ether linkage (e.g, cleavable via an acid), a glycosidic linkage (e.g, cleavable via an amylase), a peptide linkage (e.g, cleavable via a protease), or a phosphodi ester linkage (e.g, cleavable via a nuclease (e.g, DNAase)). A bond can be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g, restriction endonucleases), as described further below.
[0410] Species can be encapsulated in beads (e.g, capture agent) during bead generation (e.g, during polymerization of precursors). Such species may or may not participate in polymerization. Such species can be entered into polymerization reaction mixtures such that generated beads include the species upon bead formation. In some cases, such species can be added to the gel beads after formation. Such species can include, for example, nucleic acid molecules (e.g, oligonucleotides, e.g. , nucleic acid barcode molecules), reagents for a nucleic acid amplification reaction (e.g, primers, polymerases, dNTPs, co-factors (e.g, ionic co-factors, buffers) including those described herein, reagents for enzymatic reactions ( e.g ., enzymes, co factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g., tagmentation) for one or more sequencing platforms (e.g., Nextera® for Illumina®). Such species can include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g, endonuclease), transposase, ligase, proteinase K, DNAse, etc. Such species can include one or more reagents described elsewhere herein (e.g., lysis agents, inhibitors, inactivating agents, chelating agents, stimulus). Trapping of such species can be controlled by the polymer network density generated during polymerization of precursors, control of ionic charge within the gel bead (e.g., via ionic species linked to polymerized species), or by the release of other species. Encapsulated species can be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the species from the bead. Alternatively or in addition, species can be partitioned in a partition (e.g., droplet) during or subsequent to partition formation. Such species can include, without limitation, the abovementioned species that can also be encapsulated in a bead.
[0411] Although FIG. 5 and FIG. 6 have been described in terms of providing substantially singly occupied partitions, above, in certain cases, it may be desirable to provide multiply occupied partitions, e.g., containing two, three, four or more cells and/or microcapsules (e.g., beads) including barcoded nucleic acid molecules, e.g., nucleic acid barcode molecules (e.g., oligonucleotides) within a single partition (e.g, multiomics method described elsewhere, herein). Accordingly, as noted above, the flow characteristics of the biological particle and/or bead containing fluids and partitioning fluids can be controlled to provide for such multiply occupied partitions. In particular, the flow parameters can be controlled to provide a given occupancy rate at greater than about 50% of the partitions, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher.
[0412] In some cases, additional microcapsules or beads can be used to deliver additional reagents to a partition. In such cases, it can be advantageous to introduce different beads into a common channel or droplet generation junction, from different bead sources (e.g, containing different associated reagents) through different channel inlets into such common channel or droplet generation junction (e.g, junction 210). In such cases, the flow and frequency of the different beads into the channel or junction can be controlled to provide for a certain ratio of microcapsules or beads from each source, while ensuring a given pairing or combination of such beads into a partition with a given number of biological particles ( e.g ., one biological particle and one bead per partition).
[0413] The partitions described herein can include small volumes, for example, less than about 10 microliters (pL), 5pL, lpL, 10 nanoliters (nL), 5 nL, 1 nL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.
[0414] For example, in the case of droplet based partitions, the droplets can have overall volumes that are less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, or less. Where co-partitioned with microcapsules, it will be appreciated that the sample fluid volume, e.g., including co-partitioned biological particles and/or beads, within the partitions can be less than about 90% of the above described volumes, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the above described volumes.
[0415] As is described elsewhere herein, partitioning species can generate a population or plurality of partitions. In such cases, any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions, or more partitions can be generated or otherwise provided. Moreover, the plurality of partitions can include both unoccupied partitions (e.g., empty partitions) and occupied partitions.
Reagents
[0416] In accordance with certain aspects, biological particles can be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition. See, e.g, U.S. Pat. Pub. 2018/0216162 (now U.S. Pat. 10,428,326), U.S. Pat. Pub. 2019/0100632 (now U S. Pat. 10,590,244), and U.S. Pat. Pub. 2019/0233878. Biological particles (e.g., cells, cell beads, cell nuclei, organelles, and the like) can be partitioned together with nucleic acid barcode molecules and the nucleic acid molecules of or derived from the biological particle (e.g, mRNA, cDNA, gDNA, etc.,) can be barcoded as described elsewhere herein. In some embodiments, biological particles are co-partitioned with barcode carrying beads (e.g., gel beads) and the nucleic acid molecules of or derived from the biological particle are barcoded as described elsewhere herein. In such cases, the lysis agents can be contacted with the biological particle suspension concurrently with, or immediately prior to, the introduction of the biological particles into the partitioning junction/droplet generation zone (e.g., junction 210), such as through an additional channel or channels upstream of the channel junction. In accordance with other aspects, additionally or alternatively, biological particles can be partitioned along with other reagents, as will be described further below.
[0417] Beneficially, when lysis reagents and biological particles are co-partitioned, the lysis reagents can facilitate the release of the contents of the biological particles within the partition. The contents released in a partition can remain discrete from the contents of other partitions.
[0418] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structures can have other geometries and/or configurations. For example, a microfluidic channel structure can have more than two channel junctions. For example, a microfluidic channel structure can have 2, 3, 4, 5 channel segments or more each carrying the same or different types of beads, reagents, and/or biological particles that meet at a channel junction. Fluid flow in each channel segment can be controlled to control the partitioning of the different elements into droplets. Fluid can be directed flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can include compressors (e.g, providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.
[0419] Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g, gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, MO), as well as other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be co-partitioned with the biological particles to cause the release of the biological particle’s contents into the partitions. For example, in some cases, surfactant-based lysis solutions can be used to lyse cells ( e.g ., labelled engineered cells, B cells, memory B cells, or plasma cells), although these can be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions. In some cases, lysis solutions can include non ionic surfactants such as, for example, Triton X-100 and Tween 20. In some cases, lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of biological particles that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.
[0420] Alternatively or in addition to the lysis agents co-partitioned with the biological particles (e.g., labelled engineered cells, B cells, memory B cells, or plasma cells) described above, other reagents can also be co-partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. In addition, in the case of encapsulated biological particles (e.g., labelled engineered cells, B cells, memory B cells, or plasma cells, or cell beads comprising labelled engineered cells, B cells, or plasma cells), the biological particles can be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned microcapsule or cell bead. For example, in some cases, a chemical stimulus can be co-partitioned along with an encapsulated biological particle to allow for the degradation of the encapsulating material or microcapsule and release of the cell or its contents into the larger partition. In some cases, this stimulus can be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g, nucleic acid barcode molecules or oligonucleotides, e.g, barcoded oligonucleotides) from their respective microcapsule (e.g, bead). In alternative aspects, this can be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules (e.g, nucleic acid barcode molecules or barcoded oligonucleotides) into the same partition. [0421] Additional reagents can also be co-partitioned with the biological particles (e.g., labelled engineered cells, B cells, memory B cells, or plasma cells), such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments. Other enzymes can be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching oligonucleotides can include a hybridization region and a template region. Template switching oligonucleotides are further described in PCT Pub. No. WO2018119447, which is hereby incorporated by reference in its entirety.
[0422] Once the contents of the cells (e.g., labelled B cells, memory B cells, or plasma cells) are released into their respective partitions, the macromolecular components (e.g, macromolecular constituents of biological particles, such as RNA, DNA, proteins, or secreted antibodies or antigen-binding fragments thereof) contained therein can be further processed within the partitions. In accordance with the methods and systems described herein, the macromolecular component contents of individual biological particles (e.g, labelled B cells, memory B cells, or plasma cells) can be provided with unique identifiers such that, upon characterization of those macromolecular components they can be attributed as having been derived from the same biological particle or particles. The ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles. Unique identifiers, e.g., in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles.
[0423] In some aspects, this is performed by co-partitioning the individual biological particle (e.g., labelled B cell, memory B cell, or plasma cell) or groups of biological particles (e.g, labelled B cells, memory B cells, or plasma cells) with the unique identifiers, such as described above (with reference to FIGS. 5 and 6). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules (e.g, oligonucleotides) that include nucleic acid barcode sequences that can be attached to or otherwise associated with the nucleic acid contents of individual biological particle, or to other components of the biological particle, and particularly to fragments of those nucleic acids. The nucleic acid molecules are partitioned such that as between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences can be present.
[0424] The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g, oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be completely contiguous, i.e ., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by one or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at least about 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
[0425] The co-partitioned nucleic acid molecules can also include other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles ( e.g labelled B cells, memory B cells, or plasma cells). These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides can also be employed, including, e.g., coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g., droplets within microfluidic systems.
[0426] In an example, microcapsules, such as beads are provided that each include large numbers of the above described barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) releasably attached to the beads, where all of the nucleic acid molecules attached to a particular bead will include the same nucleic acid sequence, but where a large number of diverse barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, e.g., including polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid molecules into the partitions, as they are capable of carrying large numbers of nucleic acid molecules, and can be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences, or more. Additionally, each bead can be provided with large numbers of nucleic acid ( e.g oligonucleotide) molecules attached. In particular, the number of molecules of nucleic acid molecules including the barcode sequence on an individual bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules, or more. Nucleic acid molecules of a given bead can include identical (or common) sequences, different sequences, or a combination of both. Nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. Nucleic acid molecules of a given set can include identical barcode sequences. The identical barcode sequences can be different from barcode sequences of nucleic acid molecules of another set.
[0427] Moreover, when the population of beads is partitioned, the resulting population of partitions can also include a diverse barcode library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Additionally, each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules.
[0428] In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known set of barcode sequences can provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.
[0429] The nucleic acid molecules (e.g., oligonucleotides) are releasable from the beads upon the application of a particular stimulus to the beads. In some cases, the stimulus can be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the nucleic acid molecules. In other cases, a thermal stimulus can be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the nucleic acid molecules from the beads. In still other cases, a chemical stimulus can be used that cleaves a linkage of the nucleic acid molecules to the beads, or otherwise results in release of the nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrices described above for encapsulation of biological particles, and can be degraded for release of the attached nucleic acid barcode molecules through exposure to a reducing agent, such as DTT.
Systems and methods for controlled partitioning
[0430] In some aspects, provided are systems and methods for controlled partitioning. Droplet size can be controlled by adjusting certain geometric features in channel architecture (e.g, microfluidics channel architecture). For example, an expansion angle, width, and/or length of a channel can be adjusted to control droplet size. FIG. 6 shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. A channel structure 600 can include a channel segment 602 communicating at a channel junction 606 (or intersection) with a reservoir 604. The reservoir 604 can be a chamber. Any reference to “reservoir,” as used herein, can also refer to a “chamber.” In operation, an aqueous fluid 608 that includes suspended beads 612 can be transported along the channel segment 602 into the junction 606 to meet a second fluid 610 that is immiscible with the aqueous fluid 608 in the reservoir 604 to create droplets 616, 618 of the aqueous fluid 608 flowing into the reservoir 604. At the junction 606 where the aqueous fluid 608 and the second fluid 610 meet, droplets can form based on factors such as the hydrodynamic forces at the junction 606, flow rates of the two fluids 608, 610, fluid properties, and certain geometric parameters (e.g., w, ho, a, etc.) of the channel structure 600. A plurality of droplets can be collected in the reservoir 604 by continuously injecting the aqueous fluid 608 from the channel segment 602 through the junction 606. A discrete droplet generated can include a bead ( e.g ., as in occupied droplets 616). Alternatively, a discrete droplet generated can include more than one bead. Alternatively, a discrete droplet generated cannot include any beads (e.g., as in unoccupied droplet 618). In some instances, a discrete droplet generated can contain one or more biological particles, as described elsewhere herein. In some instances, a discrete droplet generated can include one or more reagents, as described elsewhere herein.
[0431] In some instances, the aqueous fluid 608 can have a substantially uniform concentration or frequency of beads 612. The beads 612 can be introduced into the channel segment 602 from a separate channel (not shown in FIG. 6). The frequency of beads 612 in the channel segment 602 can be controlled by controlling the frequency in which the beads 612 are introduced into the channel segment 602 and/or the relative flow rates of the fluids in the channel segment 602 and the separate channel. In some instances, the beads can be introduced into the channel segment 602 from a plurality of different channels, and the frequency controlled accordingly.
[0432] In some instances, the aqueous fluid 608 in the channel segment 602 can include biological particles (e.g., described with reference to FIG. 5). In some instances, the aqueous fluid 608 can have a substantially uniform concentration or frequency of biological particles. As with the beads, the biological particles (e.g, labelled B engineered cells, memory B cells, or plasma cells) can be introduced into the channel segment 602 from a separate channel. The frequency or concentration of the biological particles in the aqueous fluid 608 in the channel segment 602 can be controlled by controlling the frequency in which the biological particles are introduced into the channel segment 602 and/or the relative flow rates of the fluids in the channel segment 602 and the separate channel. In some instances, the biological particles can be introduced into the channel segment 602 from a plurality of different channels, and the frequency controlled accordingly. In some instances, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment 202. The first separate channel introducing the beads can be upstream or downstream of the second separate channel introducing the biological particles.
[0433] The second fluid 610 can include an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets. [0434] In some instances, the second fluid 610 cannot be subjected to and/or directed to any flow in or out of the reservoir 604. For example, the second fluid 610 can be substantially stationary in the reservoir 604. In some instances, the second fluid 610 can be subjected to flow within the reservoir 604, but not in or out of the reservoir 604, such as via application of pressure to the reservoir 604 and/or as affected by the incoming flow of the aqueous fluid 608 at the junction 606. Alternatively, the second fluid 610 can be subjected and/or directed to flow in or out of the reservoir 604. For example, the reservoir 604 can be a channel directing the second fluid 610 from upstream to downstream, transporting the generated droplets.
[0435] The channel structure 600 at or near the junction 606 can have certain geometric features that at least partly determine the sizes of the droplets formed by the channel structure 600. The channel segment 602 can have a height, ho and width, w, at or near the junction 606. By way of example, the channel segment 602 can include a rectangular cross-section that leads to a reservoir 604 having a wider cross-section (such as in width or diameter). Alternatively, the cross-section of the channel segment 602 can be other shapes, such as a circular shape, trapezoidal shape, polygonal shape, or any other shapes. The top and bottom walls of the reservoir 604 at or near the junction 606 can be inclined at an expansion angle, a. The expansion angle, a, allows the tongue (portion of the aqueous fluid 608 leaving channel segment 602 at junction 606 and entering the reservoir 604 before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. Droplet size can decrease with increasing expansion angle. The resulting droplet radius, Rd, can be predicted by the following equation for the aforementioned geometric parameters of ho, w, and or
Rd * 0.44
Figure imgf000149_0001
[0436] Systems and methods for controlled partitioning are described further in W02019040637, which is hereby incorporated by reference in its entirety.
[0437] The methods and systems described herein can be used to greatly increase the efficiency of single cell applications and/or other applications receiving droplet-based input.
[0438] Subsequent operations that can be performed can include generation of amplification products, purification ( e.g ., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations can occur in bulk (e.g., outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. Additional reagents that can be co-partitioned along with the barcode bearing bead can include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA from cells. Alternatively, rRNA removal agents can be applied during additional processing operations. The configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing and/or sequence the 5’ end of a polynucleotide sequence. The amplification products, for example, first amplification products and/or second amplification products, can be subject to sequencing for sequence analysis. In some cases, amplification can be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method.
[0439] A variety of applications require the evaluation of the presence and quantification of different biological particle or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like.
[0440] Partitions including a barcode bead (e.g., a gel bead) associated with barcode molecules and a bead encapsulating cellular constituents (e.g, a cell bead) such as cellular nucleic acids can be useful in constituent analysis as is described in U.S. Patent Publication No. 2018/0216162.
Sample and cell processing
[0441] A sample can be derived from any useful source including any subject, such as a human subject. A sample can include material (e.g., one or more cells) from one or more different sources, such as one or more different subjects. Multiple samples, such as multiple samples from a single subject (e.g., multiple samples obtained in the same or different manners from the same or different bodily locations, and/or obtained at the same or different times (e.g., seconds, minutes, hours, days, weeks, months, or years apparat)), or multiple samples from different subjects, can be obtained for analysis as described herein. For example, a first sample can be obtained from a subject at a first time and a second sample can be obtained from the subject at a second time later than the first time. The first time can be before a subject undergoes a treatment regimen or procedure (e.g., to address a disease or condition), and the second time can be during or after the subject undergoes the treatment regimen or procedure. In another example, a first sample can be obtained from a first bodily location or system of a subject (e.g., using a first collection technique) and a second sample can be obtained from a second bodily location or system of the subject ( e.g ., using a second collection technique), which second bodily location or system can be different than the first bodily location or system. In another example, multiple samples can be obtained from a subject at a same time from the same or different bodily locations. Different samples, such as different samples collected from different bodily locations of a same subject, at different times, from multiple different subjects, and/or using different collection techniques, can undergo the same or different processing (e.g., as described herein). For example, a first sample can undergo a first processing protocol and a second sample can undergo a second processing protocol.
[0442] A sample can be a biological sample, such as a cell sample (e.g., as described herein). A sample can include one or more biological particles, such as one or more cells and/or cellular constituents, such as one or more cell nuclei. For example, a sample can include a plurality of cells and/or cellular constituents. Components (e.g., cells or cellular constituents, such as cell nuclei) of a sample can be of a single type or a plurality of different types. For example, cells of a sample can include one or more different types of blood cells.
[0443] A biological sample can include a plurality of cells having different dimensions and features. In some cases, processing of the biological sample, such as cell separation and sorting (e.g., as described herein), can affect the distribution of dimensions and cellular features included in the sample by depleting cells having certain features and dimensions and/or isolating cells having certain features and dimensions.
[0444] A sample may undergo one or more processes in preparation for analysis (e.g., as described herein), including, but not limited to, filtration, selective precipitation, purification, centrifugation, permeabilization, isolation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In an example, a filtration process can include the use of microfluidics (e.g., to separate biological particles of different sizes, types, charges, or other features).
[0445] In an example, a sample including one or more cells can be processed to separate the one or more cells from other materials in the sample (e.g., using centrifugation and/or another process). In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort groups of cells and/or cellular constituents, such as to separate and/or sort cells and/or cellular constituents of different types. Examples of cell separation include, but are not limited to, separation of white blood cells or immune cells from other blood cells and components, separation of circulating tumor cells from blood, and separation of bacteria from bodily cells and/or environmental materials. A separation process can include a positive selection process ( e.g ., targeting of a cell type of interest for retention for subsequent downstream analysis, such as by use of a monoclonal antibody that targets a surface marker of the cell type of interest), a negative selection process (e.g., removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g., removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).
[0446] Separation of one or more different types of cells can include, for example, centrifugation, filtration, microfluidic-based sorting, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy-activated cell sorting (BACS), or any other useful method. For example, a flow cytometry method can be used to detect cells and/or cellular constituents based on a parameter such as a size, morphology, or protein expression. Flow cytometry-based cell sorting can include injecting a sample into a sheath fluid that conveys the cells and/or cellular constituents of the sample into a measurement region one at a time. In the measurement region, a light source such as a laser can interrogate the cells and/or cellular constituents and scattered light and/or fluorescence can be detected and converted into digital signals. A nozzle system (e.g, a vibrating nozzle system) can be used to generate droplets (e.g, aqueous droplets) including individual cells and/or cellular constituents. Droplets including cells and/or cellular constituents of interest (e.g., as determined via optical detection) can be labeled with an electric charge (e.g, using an electrical charging ring), which charge can be used to separate such droplets from droplets including other cells and/or cellular constituents. For example, FACS can include labeling cells and/or cellular constituents with fluorescent markers (e.g, using internal and/or external biomarkers). Cells and/or cellular constituents can then be measured and identified one by one and sorted based on the emitted fluorescence of the marker or absence thereof. MACS can use micro- or nano-scale magnetic particles to bind to cells and/or cellular constituents (e.g, via an antibody interaction with cell surface markers) to facilitate magnetic isolation of cells and/or cellular constituents of interest from other components of a sample (e.g, using a column-based analysis). BACS can use microbubbles (e.g, glass microbubbles) labeled with antibodies to target cells of interest. Cells and/or cellular components coupled to microbubbles can float to a surface of a solution, thereby separating target cells and/or cellular components from other components of a sample. Cell separation techniques can be used to enrich for populations of cells of interest ( e.g ., prior to partitioning, as described herein). For example, a sample including a plurality of cells including a plurality of cells of a given type can be subjected to a positive separation process. The plurality of cells of the given type can be labeled with a fluorescent marker (e.g., based on an expressed cell surface marker or another marker) and subjected to a FACS process to separate these cells from other cells of the plurality of cells. The selected cells can then be subjected to subsequent partition-based analysis (e.g., as described herein) or other downstream analysis. The fluorescent marker can be removed prior to such analysis or can be retained. The fluorescent marker can include an identifying feature, such as a nucleic acid barcode sequence and/or unique molecular identifier.
[0447] In another example, a first sample including a first plurality of cells including a first plurality of cells of a given type (e.g., immune cells expressing a particular marker or combination of markers) and a second sample including a second plurality of cells including a second plurality of cells of the given type can be subjected to a positive separation process. The first and second samples can be collected from the same or different subjects, at the same or different types, from the same or different bodily locations or systems, using the same or different collection techniques. For example, the first sample can be from a first subject and the second sample can be from a second subject different than the first subject. The first plurality of cells of the first sample can be provided a first plurality of fluorescent markers configured to label the first plurality of cells of the given type. The second plurality of cells of the second sample can be provided a second plurality of fluorescent markers configured to label the second plurality of cells of the given type. The first plurality of fluorescent markers can include a first identifying feature, such as a first barcode, while the second plurality of fluorescent markers can include a second identifying feature, such as a second barcode, that is different than the first identifying feature. The first plurality of fluorescent markers and the second plurality of fluorescent markers can fluoresce at the same intensities and over the same range of wavelengths upon excitation with a same excitation source (e.g., light source, such as a laser). The first and second samples can then be combined and subjected to a FACS process to separate cells of the given type from other cells based on the first plurality of fluorescent markers labeling the first plurality of cells of the given type and the second plurality of fluorescent markers labeling the second plurality of cells of the given type. Alternatively, the first and second samples can undergo separate FACS processes and the positively selected cells of the given type from the first sample and the positively selected cells of the given type from the second sample can then be combined for subsequent analysis. The encoded identifying features of the different fluorescent markers can be used to identify cells originating from the first sample and cells originating from the second sample. For example, the first and second identifying features can be configured to interact ( e.g ., in partitions, as described herein) with nucleic acid barcode molecules (e.g., as described herein) to generate barcoded nucleic acid products detectable using, e.g., nucleic acid sequencing.
Multiplexing methods
[0448] In some embodiments of the disclosure, steps (a) and (b) of the methods described herein are performed in multiplex format. For example, in some embodiments, step (a) of the methods disclosed herein can include individually partitioning additional single cells (e.g., B cells) of the plurality of cells in additional partitions of the plurality of partitions, and step (b) can further include determining all or a part of the nucleic acid sequences encoding antibodies or antigen-binding fragments thereof produced by the additional cells (e.g., B cells).
[0449] Accordingly, in some embodiments, the present disclosure provides methods and systems for multiplexing, and otherwise increasing throughput of samples for analysis. For example, a single or integrated process workflow may permit the processing, identification, and/or analysis of more or multiple analytes, more or multiple types of analytes, and/or more or multiple types of analyte characterizations. For example, in the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more cells or cell features can be used to characterize cells and/or cell features. In some instances, cell features include cell surface features. Cell surface features can include, but are not limited to, a receptor, an antigen or antigen fragment (e.g, an antigen or antigen fragment that binds to an antigen-binding molecule located on a cell surface), a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen- presenting complex, a major histocompatibility complex, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features can include intracellular analytes, such as proteins, protein modifications ( e.g ., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof. A labelling agent can include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), an antigen, an antigen fragment, a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a Darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide can include a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) can have a first reporter oligonucleotide coupled thereto, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) can have a different reporter oligonucleotide coupled thereto. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969.
[0450] In a particular example, a library of potential cell feature labelling agents can be provided, where the respective cell feature labelling agents are associated with nucleic acid reporter molecules, such that a different reporter oligonucleotide sequence is associated with each labelling agent capable of binding to a specific cell feature. In other aspects, different members of the library can be characterized by the presence of a different oligonucleotide sequence label. For example, an antibody capable of binding to a first protein can have associated with it a first reporter oligonucleotide sequence, while an antibody, (which may be the same antibody), capable of binding to a second protein can have a different, (or additional if the same antibody), reporter oligonucleotide sequence(s) associated with it. The presence of the particular oligonucleotide sequence(s) can be indicative of the presence of a particular antibody or cell feature which can be recognized or bound by the particular antibody.
[0451] Labelling agents capable of binding to or otherwise coupling to one or more cells can be used to characterize a cell as belonging to a particular set of cells. For example, labeling agents can be used to label a sample of cells, e.g., to provide a sample index. For other example, labelling agents can be used to label a group of cells belonging to a particular experimental condition. In this way, a group of cells can be labeled as different from another group of cells. In an example, a first group of cells can originate from a first sample and a second group of cells can originate from a second sample. Labelling agents can allow the first group and second group to have a different labeling agent (or reporter oligonucleotide associated with the labeling agent). This can, for example, facilitate multiplexing, where cells of the first group and cells of the second group can be labeled separately and then pooled together for downstream analysis. The downstream detection of a label can indicate analytes as belonging to a particular group.
[0452] For example, a reporter oligonucleotide can be linked to an antibody or an epitope binding fragment thereof, and labeling a cell can include subjecting the antibody-linked barcode molecule or the epitope binding fragment-linked barcode molecule to conditions suitable for binding the antibody to a molecule present on a surface of the cell. The binding affinity between the antibody or the epitope binding fragment thereof and the molecule present on the surface can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule. For example, the binding affinity can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule during various sample processing steps, such as partitioning and/or nucleic acid amplification or extension. A dissociation constant (Kd) between the antibody or an epitope binding fragment thereof and the molecule to which it binds can be less than about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 900 hM, 800 hM, 700 hM, 600 hM, 500 hM, 400 hM, 300 hM, 200 hM, 100 hM, 90 hM, 80 hM, 70 hM, 60 hM, 50 hM, 40 hM, 30 hM, 20 hM, 10 hM, 9 hM, 8 hM, 7 hM, 6 hM, 5 hM, 4 hM, 3 hM, 2 hM, 1 hM, 900 rM, 800 rM, 700 rM, 600 rM, 500 rM, 400 rM, 300 rM, 200 rM, 100 rM, 90 rM, 80 rM, 70 rM, 60 rM, 50 rM, 40 rM, 30 rM, 20 rM, 10 rM, 9 rM, 8 rM, 7 rM, 6 rM, 5 rM, 4 rM, 3 rM, 2 rM, or 1 rM. For example, the dissociation constant can be less than about 10 mM. In some embodiments, the antibody or antigen-binding fragment thereof has a desired dissociation rate constant (koff), such that the antibody or antigen binding-fragment thereof remains bound to the target antigen or antigen fragment during various sample processing steps.
[0453] In another example, a reporter oligonucleotide can be coupled to a cell-penetrating peptide (CPP), and labeling cells can include delivering the CPP coupled reporter oligonucleotide into a biological particle. Labeling biological particles can include delivering the CPP conjugated oligonucleotide into a cell and/or cell bead by the cell-penetrating peptide. A CPP that can be used in the methods provided herein can include at least one non-functional cysteine residue, which can be either free or derivatized to form a disulfide link with an oligonucleotide that has been modified for such linkage. Non-limiting examples of CPPs that can be used in embodiments herein include penetratin, transportan, pi si, TAT(48-60), pVEC, MTS, and MAP. Cell-penetrating peptides useful in the methods provided herein can have the capability of inducing cell penetration for at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of cells of a cell population. The CPP can be an arginine- rich peptide transporter. The CPP can be Penetratin or the Tat peptide. In another example, a reporter oligonucleotide can be coupled to a fluorophore or dye, and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the surface of the cell. In some instances, fluorophores can interact strongly with lipid bilayers and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or is inserted into a membrane of the cell. In some cases, the fluorophore is a water-soluble, organic fluorophore. In some instances, the fluorophore is Alexa 532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, Sulfo-Cy3 maleimide, Alexa 546 carboxylic acid/succinimidyl ester, Atto 550 maleimide, Cy3 carboxylic acid/succinimidyl ester, Cy3B carboxylic acid/succinimidyl ester, Atto 565 biotin, Sulforhodamine B, Alexa 594 maleimide, Texas Red maleimide, Alexa 633 maleimide, Abberior STAR 635P azide, Atto 647N maleimide, Atto 647 SE, or Sulfo-Cy5 maleimide. See, e.g., Hughes L D, et al. PLoS One. 2014 Feb. 4; 9(2):e87649, for a description of organic fluorophores.
[0454] A reporter oligonucleotide can be coupled to a lipophilic molecule, and labeling cells can include delivering the nucleic acid barcode molecule to a membrane of a cell or a nuclear membrane by the lipophilic molecule. Lipophilic molecules can associate with and/or insert into lipid membranes such as cell membranes and nuclear membranes. In some cases, the insertion can be reversible. In some cases, the association between the lipophilic molecule and the cell or nuclear membrane can be such that the membrane retains the lipophilic molecule {e.g., and associated components, such as nucleic acid barcode molecules, thereof) during subsequent processing {e.g., partitioning, cell permeabilization, amplification, pooling, etc.). The reporter nucleotide can enter into the intracellular space and/or a cell nucleus. In some embodiments, a reporter oligonucleotide coupled to a lipophilic molecule will remain associated with and/or inserted into lipid membrane (as described herein) via the lipophilic molecule until lysis of the cell occurs, e.g., inside a partition. Exemplary embodiments of lipophilic molecules coupled to reporter oligonucleotides are described in PCT/US2018/064600.
[0455] A reporter oligonucleotide can be part of a nucleic acid molecule including any number of functional sequences, as described elsewhere herein, such as a target capture sequence, a random primer sequence, and the like, and coupled to another nucleic acid molecule that is, or is derived from, the analyte.
[0456] Prior to partitioning, the cells can be incubated with the library of labelling agents, that can be labelling agents to a broad panel of different cell features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides. Unbound labelling agents can be washed from the cells, and the cells can then be co-partitioned (e.g., into droplets or wells) along with partition-specific barcode oligonucleotides (e.g., attached to a support, such as a bead or gel bead) as described elsewhere herein. As a result, the partitions can include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides.
[0457] In other instances, e.g., to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature can have a first plurality of the labelling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide. For example, the first plurality of the labeling agent and second plurality of the labeling agent can interact with different cells, cell populations or samples, allowing a particular report oligonucleotide to indicate a particular cell population (or cell or sample) and cell feature. In this way, different samples or groups can be independently processed and subsequently combined together for pooled analysis (e.g, partition-based barcoding as described elsewhere herein). See, e.g, U S. Pat. Pub. 20190323088.
[0458] In some embodiments, to facilitate sample multiplexing, individual samples can be stained with lipid tags, such as cholesterol-modified oligonucleotides (CMOs, see, e.g, FIG. 11), anti-calcium channel antibodies, or anti-ACTB antibodies. Non-limiting examples of anti calcium channel antibodies include anti-KCNN4 antibodies, anti-BK channel beta 3 antibodies, anti-alB calcium channel antibodies, and anti-CACNAlA antibodies. Examples of anti-ACTB antibodies suitable for the methods of the disclosure include, but are not limited to, mAbGEa, ACTN05, AC-15, 15G5A11/E2, BA3R, and HHF35. [0459] As described elsewhere herein, libraries of labelling agents can be associated with a particular cell feature as well as be used to identify analytes as originating from a particular cell population, or sample. Cell populations can be incubated with a plurality of libraries such that a cell or cells include multiple labelling agents. For example, a cell can include coupled thereto a lipophilic labeling agent and an antibody. The lipophilic labeling agent can indicate that the cell is a member of a particular cell sample, whereas the antibody can indicate that the cell includes a particular analyte. In this manner, the reporter oligonucleotides and labelling agents can allow multi-analyte, multiplexed analyses to be performed.
[0460] In some instances, these reporter oligonucleotides can include nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The use of oligonucleotides as the reporter can provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using sequencing or array technologies.
[0461] Attachment (coupling) of the reporter oligonucleotides to the labelling agents can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, reporter oligonucleotides can be covalently attached to a portion of a labelling agent (such a protein, e.g., an antigen or antigen fragment, an antibody or antibody fragment) , e.g., via a linker, using chemical conjugation techniques (e.g, Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g, using biotinylated antibodies (or biotinylated antigens, or biotinylated antigen fragments) and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin, an streptavidin linker in monomeric or multimeric form (e.g, tetramic form of streptavidin). Those of skill in the art will recognize that a streptavidin monomer encompasses streptavidin molecules with 1 biotin binding site, while a streptavidin multimer encompasses strepatavidin molecules with more than 1 biotin binding site. For example, a streptavidin tetramer has 4 biotin binding sites. However, a skilled artisan will also recognize that in a streptavidin tetramer does not necessarily 4 streptavidins complexed together. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, el ah, “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552. Furthermore, click reaction chemistry such as 5’ Azide oligos and Alkyne-NHS for click chemistry, 4’-Amino oligos for HyNic-4B chemistry, a Methyl tetrazine-PEG5-NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, strain-promoted alkyne-azide cycloaddition (SPAAC), or the like, can be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art can be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide including a barcode sequence that identifies the label agent. For instance, the labelling agent can be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that includes a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide can be attached to the labeling agent through a labile bond (e.g, chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein can include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).
[0462] In some cases, the labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a monomer. In some cases, the labelling agent is presented as a multimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a dimer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a trimer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a tetramer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a pentamer. In some cases, a labelling agent (e.g, an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a hexamer. In some cases, a labelling agent ( e.g ., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a heptamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as an octamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a nonamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a decamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a 10+-mer.
[0463] In some cases, the labelling agent can include a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to an oligonucleotide that is complementary to a sequence of the reporter oligonucleotide, and the oligonucleotide can be allowed to hybridize to the reporter oligonucleotide.
[0464] FIG. 11 describes exemplary labelling agents (1110, 1120, 1130) including reporter oligonucleotides (1140) attached thereto. Labelling agent 1110 (e.g., any of the labelling agents described herein) is attached (either directly, e.g., covalently attached, or indirectly) to reporter oligonucleotide 1140. Reporter oligonucleotide 1140 can include barcode sequence 1142 that identifies labelling agent 1110. Reporter oligonucleotide 1240 can also include one or more functional sequences 1143 that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, or a sequencing primer or primer biding sequence (such as an Rl, R2, or partial Rl or R2 sequence).
[0465] Referring to FIG. 11, in some instances, reporter oligonucleotide 1140 conjugated to a labelling agent (e.g., 1110, 1120, 1130) includes a functional sequence 1141, a reporter barcode sequence 1142 that identifies the labelling agent (e.g., 1110, 1120, 1130), and reporter capture handle 1143. Reporter capture handle sequence 1143 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule (not shown), such as those described elsewhere herein ( e.g ., FIGS. 7, 8, 12 and 13). In some instances, nucleic acid barcode molecule is attached to a support (e.g., a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1190 can be attached to the support via a releasable linkage (e.g., including a labile bond), such as those described elsewhere herein (e.g., FIGS. 7, 8, 12 and 13). In some instances, reporter oligonucleotide 1140 includes one or more additional functional sequences, such as those described above.
[0466] In some instances, the labelling agent 1110 is a protein or polypeptide (e.g., an antigen or prospective antigen) including reporter oligonucleotide 1140. Reporter oligonucleotide 1140 includes reporter barcode sequence 1142 that identifies polypeptide 1110 and can be used to infer the presence of an analyte, e.g., a binding partner of polypeptide 1110 (i.e., a molecule or compound to which polypeptide 1110 can bind). In some instances, the labelling agent 1110 is a lipophilic moiety (e.g., cholesterol) including reporter oligonucleotide 1140, where the lipophilic moiety is selected such that labelling agent 1110 integrates into a membrane of a cell or nucleus. Reporter oligonucleotide 1140 includes reporter barcode sequence 1142 that identifies lipophilic moiety 1110 which in some instances is used to tag cells (e.g., groups of cells, cell samples, etc.) and can be used for multiplex analyses as described elsewhere herein. In some instances, the labelling agent is an antibody 1120 (or an epitope binding fragment thereof) including reporter oligonucleotide 1140. Reporter oligonucleotide 1140 includes reporter barcode sequence 1142 that identifies antibody 1120 and can be used to infer the presence of, e.g., a target of antibody 1120 (i.e., a molecule or compound to which antibody 1120 binds). In other embodiments, labelling agent 1130 includes an MHC molecule 1131 including peptide 1132 and reporter oligonucleotide 1140 that identifies peptide 1132. In some instances, the MHC molecule is coupled to a support 1133. In some instances, support 1133 can be a polypeptide, such as streptavidin, or a polysaccharide, such as dextran. In some instances, reporter oligonucleotide 1140 can be directly or indirectly coupled to MHC labelling agent 1130 in any suitable manner. For example, reporter oligonucleotide 1140 can be coupled to MHC molecule 1131, support 1133, or peptide 1132. In some embodiments, labelling agent 1130 includes a plurality of MHC molecules, (e.g. is an MHC multimer, which can be coupled to a support (e.g., 1133)). There are many possible configurations of Class I and/or Class II MHC multimers that can be utilized with the compositions, methods, and systems disclosed herein, e.g., MHC tetramers, MHC pentamers (MHC assembled via a coiled-coil domain, e.g., Pro5® MHC Class I Pentamers, (Prolmmune, Ltd.), MHC octamers, MHC dodecamers, MHC decorated dextran molecules (e.g., MHC Dextramer® (Immudex)), etc. For a description of exemplary labelling agents, including antibody and MHC -based labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429 and U.S. Pat. Pub. 20190367969.
[0467] Exemplary barcode molecules attached to a support (e.g., a bead) is shown in FIG. 12. In some embodiments, analysis of multiple analytes (e.g., RNA and one or more analytes using labelling agents described herein) can include nucleic acid barcode molecules as generally depicted in FIG. 12. In some embodiments, nucleic acid barcode molecules 1210 and 1220 are attached to support 1230 via a releasable linkage 1240 (e.g., including a labile bond) as described elsewhere herein. Nucleic acid barcode molecule 1210 can include functional sequence 1211, barcode sequence 1212 and capture sequence 1213. Nucleic acid barcode molecule 1220 can include adapter sequence 1221, barcode sequence 1212, and adapter sequence 1223, wherein adapter sequence 1223 includes a different sequence than adapter sequence 1213. In some instances, adapter 1211 and adapter 1221 include the same sequence. In some instances, adapter 1211 and adapter 1221 include different sequences. Although support 1230 is shown including nucleic acid barcode molecules 1210 and 1220, any suitable number of barcode molecules including common barcode sequence 1212 are contemplated herein. For example, in some embodiments, support 1230 further includes nucleic acid barcode molecule 1250. Nucleic acid barcode molecule 1350 can include adapter sequence 1251, barcode sequence 1212 and adapter sequence 1253, wherein adapter sequence 1253 includes a different sequence than adapter sequence 1313 and 1223. In some instances, nucleic acid barcode molecules (e.g., 1210, 1220, 1250) include one or more additional functional sequences, such as a UMI or other sequences described herein. The nucleic acid barcode molecules 1210, 1220 or 1250 can interact with analytes as described elsewhere herein, for example, as depicted in FIGS. 13A-13C.
[0468] Referring to FIG. 13A, in an instance where cells are labelled with labeling agents, capture sequence 1323 can be complementary to an adapter sequence of a reporter oligonucleotide. Cells can be contacted with one or more reporter oligonucleotide 1320 conjugated labelling agents 1310 (e.g., polypeptide such as an antigen, antibody, or others described elsewhere herein). In some cases, the cells can be further processed prior to barcoding. For example, such processing steps can include one or more washing and/or cell sorting steps. In some instances, a cell that is bound to labelling agent 1310 which is conjugated to reporter oligonucleotide 1320 and support 1330 ( e.g ., ahead, such as a gel bead) including nucleic acid barcode molecule 1390 is partitioned into a partition amongst a plurality of partitions (e.g., a droplet of a droplet emulsion or a well of a microwell array). In some instances, the partition includes at most a single cell bound to labelling agent 1310. In some instances, reporter oligonucleotide 1320 conjugated to labelling agent 1310 (e.g., polypeptide such as an antigen, an antibody, pMHC molecule such as an MHC multimer, etc.) includes a first adapter sequence 1311 (e.g., a primer sequence), a barcode sequence 1312 that identifies the labelling agent 1310 (e.g, the polypeptide such as an antigen, antibody, or peptide of a pMHC molecule or complex), and a capture handle sequence 1313. Capture handle sequence 1313 can be configured to hybridize to a complementary sequence, such as capture sequence 1323 present on a nucleic acid barcode molecule 1390 (e.g, partition-specific barcode molecule). In some instances, reporter oligonucleotide 1320 includes one or more additional functional sequences, such as those described elsewhere herein.
[0469] Barcoded nucleic acid molecules can be generated (e.g, via a nucleic acid reaction, such as nucleic acid extension, reverse transcription, or ligation) from the constructs described in FIGS. 13A-13C. For example, capture handle sequence 1313 can then be hybridized to complementary capture sequence 1323 to generate (e.g, via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule including cell barcode (e.g, common barcode or partition-specific barcode) sequence 1322 (or a reverse complement thereof) and reporter barcode sequence 1312 (or a reverse complement thereof). In some embodiments, the nucleic acid barcode molecule 1390 (e.g, partition-specific barcode molecule) further includes a UMI. Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g, to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g, U.S. Pat. Pub. 2018/0105808. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform.
[0470] In some instances, analysis of multiple analytes (e.g, nucleic acids and one or more analytes using labelling agents described herein) can be performed. For example, the workflow can include a workflow as generally depicted in any of FIGS. 13A-13C, or a combination of workflows for an individual analyte, as described elsewhere herein. For example, by using a combination of the workflows as generally depicted in FIGS. 13A-13C, multiple analytes can be analyzed.
[0471] In some instances, analysis of an analyte ( e.g . a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.) includes a workflow as generally depicted in FIG. 13A. A nucleic acid barcode molecule 1390 can be co-partitioned with the one or more analytes. In some instances, nucleic acid barcode molecule 1390 is attached to a support 1330 (e.g., a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1390 can be attached to support 1330 via a releasable linkage 1340 (e.g., including a labile bond), such as those described elsewhere herein. Nucleic acid barcode molecule 1390 can include a functional sequence 1321 and optionally include other additional sequences, for example, a barcode sequence 1322 (e.g., common barcode, partition-specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence. The nucleic acid barcode molecule 1390 can include a capture sequence 1323 that can be complementary to another nucleic acid sequence, such that it can hybridize to a particular sequence.
[0472] For example, capture sequence 1323 can include a poly-T sequence and can be used to hybridize to mRNA. Referring to FIG. 13C, in some embodiments, nucleic acid barcode molecule 1390 includes capture sequence 1323 complementary to a sequence of RNA molecule 1360 from a cell. In some instances, capture sequence 1323 includes a sequence specific for an RNA molecule. Capture sequence 1323 can include a known or targeted sequence or a random sequence. In some instances, a nucleic acid extension reaction can be performed, thereby generating a barcoded nucleic acid product including capture sequence 1323, the functional sequence 1321, UMI and/or barcode sequence 1322, any other functional sequence, and a sequence corresponding to the RNA molecule 1360.
[0473] In another example, capture sequence 1323 can be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte. For example, referring to FIG. 13B, in some embodiments, primer 1350 includes a sequence complementary to a sequence of nucleic acid molecule 1360 (such as an RNA encoding for a BCR sequence) from a biological particle. In some instances, primer 1350 includes one or more sequences 1351 that are not complementary to RNA molecule 1360. Sequence 1351 can be a functional sequence as described elsewhere herein, for example, an adapter sequence, a sequencing primer sequence, or a sequence the facilitates coupling to a flow cell of a sequencer. In some instances, primer 1350 includes a poly-T sequence. In some instances, primer 1350 includes a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 1350 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Primer 1350 is hybridized to nucleic acid molecule 1360 and complementary molecule 1370 is generated. For example, complementary molecule 1370 can be cDNA generated in a reverse transcription reaction. In some instances, an additional sequence can be appended to complementary molecule 1370. For example, the reverse transcriptase enzyme can be selected such that several non-templated bases 1380 ( e.g ., a poly-C sequence) are appended to the cDNA. In another example, a terminal transferase can also be used to append the additional sequence. Nucleic acid barcode molecule 1390 includes a sequence 1324 complementary to the non- templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 1390 to generate a barcoded nucleic acid molecule including cell (e.g., partition specific) barcode sequence 1322 (or a reverse complement thereof) and a sequence of complementary molecule 1370 (or a portion thereof). In some instances, capture sequence 1323 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Capture sequence 1323 is hybridized to nucleic acid molecule 1360 and a complementary molecule 1370 is generated. For example, complementary molecule 1370 can be generated in a reverse transcription reaction generating a barcoded nucleic acid molecule including cell barcode (e.g., common barcode or partition-specific barcode) sequence 1322 (or a reverse complement thereof) and a sequence of complementary molecule 1370 (or a portion thereof). Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in International Patent Application WO2018/075693, U.S. Patent Publication No. 2018/0105808, U.S. Patent Publication No. 2015/0376609, filed June 26, 2015, and U.S. Patent Publication No. 2019/0367969.
[0474] In some embodiments, biological particles (e.g., cells, nuclei) from a plurality of samples (e.g., a plurality of subjects) can be pooled, sequenced, and demultiplexed by identifying mutational profiles associated with individual samples and mapping sequence data from single biological particles to their source based on their mutational profile. See, e.g., Xu J. el al, Genome Biology Vol. 20, 290 (2019); Huang Y. et al., Genome Biology Vol. 20, 273 (2019); and Heaton et al, Nature Methods volume 17, pages 615-620(2020), which are hereby incorporated by reference in their entirety.
[0475] Gene expression data can reflect the underlying genome and mutations and structural variants therein. As a result, the variation inherent in the captured and sequenced RNA molecules can be used to identify genotypes de novo or used to assign molecules to genotypes that were known a priori. In some embodiments, allelic variation that is present due to haplotypic states (including linkage disequilibrium of the human leucocyte antigen loci (HLA), immune receptor loci ( e.g ., BCR), and other highly polymorphic regions of the genome), can also be used for demultiplexing. Expressed B cell receptors can be used to infer germline alleles from unrelated individuals, which information may be used for demultiplexing.
Methods for detecting SARS-CoV-2 S protein and/or SARS-CoV-2
[0476] As discussed above, one aspect of the present disclosure relates to methods for detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, the methods including contacting an antibody or antigen-binding fragment as disclosed herein with a biological sample from an individual infected with or suspected of being infected with SARS-CoV-2. For example, the anti- SARS-CoV-2 S antibodies and antigen-binding fragments of the present disclosure can be used to detect and/or measure SARS-CoV in a sample, e.g., for diagnostic purposes.
[0477] In some embodiments, the methods include (i) contacting an antibody or antigen binding fragment as disclosed herein with a biological sample from an individual infected with or suspected of being infected with SARS-CoV-2, (ii) detecting the formation of an antigen- antibody complex between the antibody or antigen-binding fragment and a SARS-CoV-2 S protein present in the biological sample. The formation of the antigen-antibody complex can be detected by one or more techniques known in the art, such as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IF A), dot blot or western blot. In some embodiments, the formation of the antigen-antibody complex can be detected by ELISA, dot blot or western blot.
[0478] For example, the anti-CoV-S antigen-binding polypeptides, e.g., antibodies or antigen-binding fragments thereof of the present disclosure (e.g., of Table 1), can be used to detect and/or measure CoV-S in a sample. Exemplary assays for CoV-S include, but are not limited to, contacting a biological sample with an anti-CoV-S antigen-binding polypeptide ( e.g ., antibody or antigen-binding fragment thereof) of the disclosure, wherein the anti-CoV-S antigen binding polypeptide is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from biological samples. The presence of an anti-CoV-S antigen-binding polypeptide complexed with CoV-S indicates the presence of CoV-S in the sample. Alternatively, an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, b- galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CoV-S in a sample include neutralization assays, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA), and fluorescence-activated cell sorting (FACS). Thus, the present disclosure includes a method for detecting the presence of spike protein polypeptide in a sample comprising contacting the sample with an anti-CoV-S antigen binding polypeptide and detecting the presence of a CoV-S/anti-CoV-S antigen-binding polypeptide wherein the presence of the complex indicates the presence of CoV-S.
[0479] In principle, there are no particular restrictions in regard to the types of biological samples suitable for use in the methods described herein. For example, samples that can be suitably used in SARS-CoV diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a subject, which contains detectable quantities of either SARS-CoV spike protein, or fragments thereof, under normal or pathological conditions. In some embodiments, the biological sample includes sputum, bronchoalveolar lavage, pleural effusion, tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, circulating tumor cells, circulating nucleic acids, bone marrow, or any combination thereof. In some embodiments, the biological sample includes cells or tissue. For example, the biological sample can be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. In some embodiments, the biological sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. In some embodiments, the biological sample can be a skin sample. In some embodiments, the biological sample can be a cheek swab. In some embodiments, the biological sample includes whole blood and blood components.
[0480] Generally, levels of SARS-CoV spike protein in a particular sample obtained from a healthy subject ( e.g ., a subject not afflicted with a disease associated with SARS-CoV) are measured to initially establish a baseline, or standard, level of SARS-CoV. This baseline level of MERS-CoV can then be compared against the levels of SARS-CoV measured in samples obtained from individuals suspected of having a SARS-CoV -associated condition, or symptoms associated with such condition.
[0481] The antibodies and antigen-binding fragments specific for SARS-CoV spike protein may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In some embodiments, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.
Methods of treatment
[0482] As discussed above, one aspect of the disclosure relate to methods for treating or preventing viral infection (e.g., reducing the likelihood of a viral infection such as coronavirus infection) by administering a composition comprising therapeutically effective amount of an anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment, (e.g., of Table 1) to a subject in need of such treatment or prevention. In a related aspect, some embodiments of the disclosure relate methods for reducing binding of SARS-Co-2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject, the method including administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment as disclosed herein. In some embodiments, the composition includes at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven antibodies or antigen-binding fragments as disclosed herein. In some embodiments, the composition includes (a) a first antibody or antigen-binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to a full- length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein); (b) a first antibody or antigen-binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to a NTD of a SARS-CoV-2 S protein; or (c) a first antibody or antigen-binding fragment having a binding affinity to a NTD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SAR.S-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein).
[0483] The treatment methods of the disclosure involve administering an anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment of the present disclosure (e.g., of Table 1), to a subject having one or more signs or symptoms of a disease or infection, e.g., viral infection, for which the antigen-binding polypeptide is effective when administered to the subject at an effective or therapeutically effective amount or dose. An effective or therapeutically effective dose of anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment (e.g., of Table 1), for treating or preventing a viral infection refers to the amount of the antibody or fragment sufficient to alleviate one or more signs and/or symptoms of the infection in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms. Health conditions and symptoms associated with SARS-CoV-2 infection include respiratory tract infections, often in the lower respiratory tract. Accordingly, some embodiments of the disclosure relate to methods of for reducing one or more signs or symptoms associated with coronavirus infection, such as high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe cases. In some embodiments, a sign or symptom of a coronavirus infection in a subject is survival or proliferation of virus in the body of the subject, e.g, as determined by viral titer assay (e.g, coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay). Other signs and symptoms of viral infection include, but are not limited to fever or feeling feverish/chills, cough, sore throat, runny or stuffy nose, sneezing, muscle or body aches, headaches, fatigue (tiredness), vomiting, diarrhea, respiratory tract infection, chest discomfort, shortness of breath, bronchitis, and pneumonia.
[0484] The present disclosure also encompasses prophylactically administering an anti- CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment thereof of the present disclosure (e.g, of Table 1), to a subject who is at risk of viral infection so as to prevent such infection (e.g, reducing the likelihood of a viral infection). Passive antibody-based immunoprophylaxis has proven an effective strategy for preventing subject from viral infection. The preventive methods of the disclosure involve administering a composition comprising an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment of the present disclosure (e.g, of Table 1), to a subject to inhibit the manifestation of a disease or infection ( e.g ., viral infection) in the body of a subject, for which the antigen-binding polypeptide is effective when administered to the subject at an effective or therapeutically effective amount or dose.
[0485] The dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In some embodiments, an effective or therapeutically effective dose of antibody or antigen-binding fragment thereof of the present disclosure, for treating or preventing viral infection, e.g. , in an adult human subject, is about 0.01 to about 200 mg/kg, e.g., up to about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, or about 175 mg/kg. In some embodiments, the dosage is up to about 10.8 or 11 grams (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or about 11 grams). Depending on the severity of the infection, the frequency and the duration of the treatment can be adjusted. In some embodiments, the antibody or antigen binding fragment of the present disclosure can be administered at an initial dose, followed by one or more secondary doses. In some embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
[0486] In some embodiments, the administered composition reduces the likelihood of a coronavirus infection by at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject that has not been administered with the composition.
[0487] In some embodiments, the administered composition reduces binding of SARS-Co- 2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject by at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject that has not been administered with the composition.
[0488] In a related aspect, some embodiments of the disclosure relate methods for reducing binding of SARS-Co-2V S protein to a cell of a subject and/or reducing SARS-CoV-2 entry into a cell of a subject, the method including administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment as disclosed herein.
[0489] Non-limiting exemplary embodiments of the methods as described herein can include one or more of the following features. In some embodiments, the antibody or antigen binding fragment is administered in combination with a SARS-Co-2V S protein conjugated to a therapeutic agent of the disclosure. In some embodiments, the subject is administered one or more further therapeutic agents.
[0490] In some embodiments, the one or more further therapeutic agents is selected from the group consisting of: (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds TMPRSS2, and (iv) an antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. In some embodiments, the further therapeutic agent is a second antibody or antigen-binding fragment disclosed herein, e.g., of Table 1. In some embodiments, one, two, three, four, or more antibodies, or antigen-binding fragments thereof, of Table 1 can be administered in combination (e.g., concurrently or sequentially).
[0491] In some embodiments, the one or more further therapeutic agents includes an antiviral drug or a vaccine. One of ordinary skill in the art will understand that the antiviral drug of the disclosure can include any anti -infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. In some embodiments, the antiviral drug includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, ribavirin, or interferon- alpha2b. Methods for treating or preventing virus (e.g, coronavirus) infection in a subject in need of said treatment or prevention by administering an antibody or antigen-binding fragment of Table 1 in association with a further therapeutic agent are part of the present disclosure.
[0492] For example, in some embodiments of the disclosure, the further therapeutic agent is a vaccine, e.g, a coronavirus vaccine. In some embodiments, a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.
[0493] In some embodiments, the therapeutic composition is formulated to be compatible with its intended route of administration. For example, the antibodies and antigen-binding fragments of the disclosure may be given orally or by inhalation, but it is more likely that they will be administered through a parenteral route. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), and transmucosal administration. In some embodiments, the antibody or antigen-binding fragment is administered to the subject subcutaneously, intravenously, and/or intramuscularly.
[0494] Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and/or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide ( e.g ., to a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0495] Dosage, toxicity and therapeutic efficacy of such subject antibodies and antigen binding fragments of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are generally suitable. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
[0496] For example, the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
Combination therapies
[0497] As discussed above, according to some specific examples, the methods of the disclosure include administering to a subject one or more additional therapies in combination with an anti-CoV-S protein antibody or antigen-binding fragment as disclosed herein.
[0498] Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. Accordingly, the additional therapies can be administered before, after, or at the same time as a pharmaceutical composition comprising an anti-CoV-S protein antibody or antigen-binding fragment of the disclosure. Administration “in combination with” also includes the continuous or simultaneous administration of an anti-CoV-S antibody or antigen-binding fragment and a second therapy.
[0499] For example, when the pharmaceutical composition containing the anti-CoV-S antibody or antigen-binding fragment of the disclosure is administered “before,” the additional therapy can be administered for about 72 hours, about 60 hours, or about 48 hours, to the pharmaceutical composition containing the anti-CoV-S antibody or antigen-binding fragment thereof. About 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before. When the pharmaceutical composition containing the anti-CoV-S antibody or antigen-binding fragment is administered “after,” the additional therapy can be administered for about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after.
[0500] The combination therapy may include an anti-CoV-S antibody or antigen-binding fragment of the disclosure and any additional therapeutic agent that can be advantageously combined with an anti-CoV-S antibody or antigen-binding fragment of the disclosure.
[0501] For example, a second or third therapeutic agent may be used to help reduce viral load in the lungs, such as an antiviral agent ( e.g ., ribavirin). Antibodies can also be used in combination with other therapies as described above, including vaccines specific to CoV, secondary antibodies specific to CoV, antiviral agents, anti-inflammatory agents, antibodies specifically binds the serine protease TMPRSS2 of a target cell, and additional antibodies or antigen-binding fragment thereof that specifically bind to CoV-S protein. In some embodiments, the second and/or third therapeutic agent include an antibody approved by FDA for treatment of coronavirus infection such as, for example, casirivimab, imdevimab, bamlanivimab, etesevimab, and sotrovimab. Accordingly, in some embodiments, the second and third therapeutic agent are independently selected from the group consisting of casirivimab, imdevimab, bamlanivimab, etesevimab, and sotrovimab. In some embodiments, the second and/or third therapeutic agent includes an antibody and/or small molecule entity having affinity for an immune pathway target. Non-limiting examples of suitable antibodies for an immune pathway target are disclosed herein.
[0502] In some embodiments, the first therapeutic agent is a first antibody or antigen binding fragment belonging to bin 1 or bin 2 described in Example 14, and the second therapeutic agent is a second antibody or antigen-binding fragment belonging to bin 3, bin 4, or bin 3/4 described in Example 14. In some embodiments, the first therapeutic agent is a first antibody or antigen-binding fragment belonging to bin 1 described in Example 14, and the second therapeutic agent is a second antibody or antigen-binding fragment belonging to bin 3, bin 4, or bin 3/4 described in Example 14. In some embodiments, the first therapeutic agent is a first antibody or antigen-binding fragment belonging to bin 2 described in Example 14, and the second therapeutic agent is a second antibody or antigen-binding fragment belonging to bin bin 3, bin 4, or bin 3/4 described in Example 14. In some embodiments, the first therapeutic agent is a first antibody or antigen-binding fragment belonging to bin 3 described in Example 14, and the second therapeutic agent is a second antibody or antigen-binding fragment belonging to bin 4 described in Example 14. In some embodiments, the first therapeutic agent is a first antibody or antigen-binding fragment belonging to bin 3 described in Example 14, and the second therapeutic agent is a second antibody or antigen-binding fragment belonging to bin 3/4 described in Example 14. In some embodiments, the first therapeutic agent is a first antibody or antigen-binding fragment belonging to bin 4 described in Example 14, and the second therapeutic agent is a second antibody or antigen-binding fragment belonging to bin 3/4 described in Example 14.
[0503] As noted above, in some embodiments of the disclosure, the subject may be a non human animal, and the antigen-binding polypeptides ( e.g ., antibodies and antigen-binding fragments) discussed herein may be used in a veterinary context to treat and/or prevent disease associated with coronavirus in the non-human animals (e.g., cats, dogs, pigs, cows, horses, goats, rabbits, sheep, etc.). KITS
[0504] Further provided herein are kits for the practice of a method described herein. In some embodiments, provided herein are kits for identification of antibodies and antigen-binding fragments having binding affinity for a CoV-S. Such kits can include: (a) a plurality of CoV-S antigens and non-CoV-S antigens, and wherein each of the antigens comprise a reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and (b) instructions for performing a method of identifying an antibody or antigen-binding fragment having binding affinity for CoV-S as described herein.
[0505] Also provided, in some embodiments of the disclosure, are kits for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject. A kit can include instructions for use thereof and one or more of the antibodies or antigen-binding fragments thereof, recombinant nucleic acids, recombinant cells, and pharmaceutical compositions as described and provided herein. For example, some embodiments of the disclosure provide kits that include one or more of the antibodies described herein and/or antigen-binding fragments thereof, and instructions for use. In some embodiments, provided herein are kits that include one or more recombinant nucleic acids, recombinant cells, and pharmaceutical compositions as described herein and instructions for use thereof.
[0506] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. Accordingly, in some embodiments of the disclosure, the kit includes an anti-CoV-S antigen binding polypeptide, e.g., an antibody or antigen-binding fragment thereof as disclosed herein ( e.g ., of Table 1), or a pharmaceutical composition thereof in one container {e.g., in a sterile glass or plastic vial, a chromatography column, hollow bore needle, or a syringe cylinder) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial, a chromatography column, hollow bore needle, or a syringe cylinder).
[0507] In another embodiment, the kit includes a combination of the compositions described herein, including an anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment thereof as disclosed herein (e.g., of Table 1), or pharmaceutical composition thereof in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.
[0508] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device ( e.g ., an injection device or catheter) for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above containing the anti-CoV-S antigen-binding polypeptide, e.g., antibody or antigen-binding fragment thereof of the present disclosure (e.g., of Table 1).
[0509] In some embodiments, a kit can further include instructions for using the components of the kit to practice a method described herein. For example, the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and intellectual property information.
[0510] The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g, via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
[0511] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0512] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
[0513] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.
[0514] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.
EXAMPLES
EXAMPLE 1 Biological samples
[0515] Sample procurement: The experiments described in the below Examples were performed with peripheral blood mononuclear cells (PBMCs) collected from convalescent human survivors of natural infection with SARS-2. Specifically, Donor 531 PBMCs were purchased from Cellero (~112m/vial product, Cat. # 1146-4785JY20) and used in these experiments.
[0516] Sample background/timeline: The donor tested positive via nasopharyngeal swab while presenting asymptomatic/presymptomatic on Day 0. Hospitalization was not required. The donor tested negative for SARS-2 on Day 23. Plasma and apheresis sample collection were performed on Day 104. The donor is also seropositive for cytomegalovirus, a ubiquitous human herpesvirus.
EXAMPLE 2
Enrichment of B cells
[0517] A vial of frozen PBMCs was thawed for 1-2 min in a water bath, then transferred into 8-10 mL of 10% Fetal Bovine Serum (FBS) in PBS, and centrifuged for 5 min at 350g. The cell pellet was washed three times by resuspending in 0.04% Bovine Serum Albumin (BSA) in PBS and centrifuging at RT at 350g for 5 min each wash, with the final pellet resuspended to a concentration of ~20 million cells per mL in a total volume of 5 mL (~100 million cells total). B cells were enriched using the B Cell Isolation Kit II (human; MACS™ Miltenyi) according to manufacturer’s instructions, and approximately 50 million cells were applied to each of two LS columns designed for positive selection of cells. The effluent was concentrated and prepared for cell labeling.
EXAMPLE 3
Antigen sourcing preparation and conjugation [0518] Biotinylated antigens were sourced from suppliers as follows:
[0519] 1) Biotinylated trimerized S (SARS-2) was sourced from ACRO Biosystems, catalog # SPN-C82E9-25 (https://www.acrobiosystems.com/P3345-Biotinylated-SARS-CoV-2- S-protein-HisAvitag™-Superstable-trimer-%28MALS-verified%29.html). This protein carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using Avitag™ technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.
[0520] 2) Biotinylated trimerized S D614G (SARS-2), from ACRO Biosystems, catalog
# SPN-C82E3-25 (https://www.acrobiosystems.com/P343 l-Biotinylated-SARS-CoV-2-S- protein-%28D614G%29-HisAvitag™-Super-stable-trimer-%28MALS-verified%29.html). This protein contains D614G mutation, which has become increasingly common in SARS-CoV-2 viruses from around the world. This protein also carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using Avitag™ technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.
[0521] 3) Biotinylated Human Serum Albumin (HSA) HSA-H82E3, from Sapphire
Figure imgf000179_0001
[0522] Biotinylated antigens were each solubilized per manufacturer’s instructions. In each case, they were thawed and dissolved in sterile deionized water for 30-60 minutes at room temperature with occasional gentle mixing for a final concentration of 100 microgram/mL (for HSA) or 200 microgram/mL for both of the trimerized S antigens.
[0523] Solubilized antigens were each conjugated with, e.g., allowed to form a complex with (or bind to) one of the following Total SeqC reagents, supplied by BioLegend, which each contain a unique barcoded DNA oligonucleotide supplied by the vendor as follows:
[0524] 1) TotalSeq-C0951 PE Streptavidin was conjugated to biotinylated trimerized S glycoprotein (SARS-2).
[0525] 2) TotalSeq-C0952 PE Streptavidin was conjugated to biotinylated human serum albumin.
[0526] 3) TotalSeq-C0956 APC Streptavidin was conjugated to biotinylated trimerized S
D614G glycoprotein (SARS-2).
[0527] 4) TotalSeq-C0957 APC Streptavidin with biotinylated human serum albumin.
[0528] Briefly, each TotalSeq-C barcoded streptavidin PE or APC reagent was diluted to 0.1 mg/mL and then mixed with biotinylated antigen at a 5X molar excess of antigen to streptavidin, based on a fixed amount of 0.5
Figure imgf000180_0001
PE-SA. One fifth of the streptavidin-oligo PE or
APC conjugate was added to the antigen every 20 minutes at 4°C. The reaction was then quenched with 5 mΐ 4mM biotin (Pierce, Thermo Fisher) for 30 minutes for a total probe volume of 20 pL. The final conjugated antigen probes (streptavidin-antigen complexes) were then immediately used for cell labeling at a dilution of 1:50.
EXAMPLE 4 Cell labeling
[0529] This Example describes experiments performed to stain B cells with a number of barcoded antibodies and conjugated antigens. In these experiments, approximately 4.4 million enriched B cells were first resuspended in labeling buffer (1% BSA in PBS) and performed Fc blocking for 10 minutes on ice using Human TruStain FcX (BioLegend).
[0530] Next, cells were stained with the following cocktail of antibodies, antigens and dyes: CD19 PE-Cy7 (clone SJ25C1, BD Pharmingen) for discrimination of CD19+ cells by using fluorescence-activated cell sorting (FACS).
[0531] Barcoded Antibodies for lOx Single Cell Immune profiling, which included the following TotalSeq-C oligo barcoded antibodies:
[0532] - TotalSeq-C0389 anti -human CD38.
[0533] - TotalSeq-C0154 anti -human CD27.
[0534] - TotalSeq-C0189 anti -human CD24.
[0535] - TotalSeq-C0384 anti -human IgD.
[0536] - TotalSeq-COlOO anti -human CD20. [0537] - TotalSeq-C0050 anti -human CD 19 (clone HIB19, to distinguish it from the flow clone).
[0538] - TotalSeq-C0049 anti-human CD3E.
[0539] - TotalSeq-C0045 anti-human CD4.
[0540] - TotalSeq-C0046 anti-human CD8A.
[0541] - TotalSeq-C0051 anti-human CD 14.
[0542] - TotalSeq-C0083 anti -human CD 16.
[0543] - TotalSeq-C0090 mouse IgGl K isotype control.
[0544] - TotalSeq-C0091 mouse IgG2a K isotype control.
[0545] - TotalSeq-C0092 mouse IgG2b K isotype control.
[0546] Final conjugated antigens:
[0547] - TotalSeq-C0951 PE trimerized S (SARS-2).
[0548] - TotalSeq-C0952 PE Human Serum Albumin.
[0549] - TotalSeq-C0956 APC trimerized S D614G (SARS-2).
[0550] - TotalSeq-C0957 APC Human Serum Albumin.
[0551] - 7AAD for live/dead cell discrimination.
[0552] Cells were stained in labeling buffer (1% BSA in PBS) in the dark for 30 minutes on ice, then cells were washed 3 times with 2 mL of cold labeling buffer at 350*g for 5 minutes at 4°C, resuspended in cold labeling buffer and a 1:200 addition of live/dead cell discriminating agent 7AAD for 10 minutes on ice in the dark, then washed one more time with labeling buffer at 350*g for 5 minutes at 4°C, then resuspended in labeling buffer and loaded into a Sony MA900 Cell Sorter using a 70 microM sorting chip.
EXAMPLE 5
Antigen-specific enrichment via FACS
[0553] Cells were initially gated on being single, live (7AADnegative) and PE-Cy7-CD19+ and then sorted on their PE and/or APC status directly into master mixed and water based on one of four criteria:
[0554] 1) PE+, representing trimerized S (SARS-2) antigen† and/or HSA+ control antigen cells (gate Q1 in FIG. 1; 2,430 cells);
[0555] 2) APC+, representing trimerized S D614G (SARS-2) antigen and/or HSA control antigen cells (gate Q3 in FIG. 1; 728 cells); [0556] 3) Dual PE+ and APC+, representing a combination of trimerized S (SARS-2) antigen†, trimerized S D614G (SARS-2) antigen† and/or HSA control antigen-positive cells (gate Q2 in FIG. 1; 828 cells);
[0557] 4) PE and APC negative cells, representing cells not binding either SARS-2 antigen or control HSA antigen (gate Q4 in FIG. 1; 5,000 cells).
[0558] In FIG. 1, the Y axis represents PE (representing trimerized S (SARS-2) antigen† and/or HSA† control antigen cells) signal. The X axis represents APC trimerized S (SARS-2) D614G antigen† and/or HSA† control antigen cells. The numbers adjacent to each gate name represent the fraction of events of the parent population (single, live, CD19† cells) for that gate. FACS data were analyzed with FlowJo.
[0559] The resulting volume was adjusted with additional water to match the recommended volume and target for loading with the 10x 5’V2 Single Cell Immune Profiling kit. FACS data were analyzed using FlowJo. Standard gene expression, V(D)J, and barcoded antigen libraries were constructed using the lO 5Ύ2 Single Cell Immune Profiling kit per manufacturer's instructions. Additional information in this regard can be found at “support.10xgenomics.com/permalink/getting-started-immune-profiling-feature-barcoding.”
EXAMPLE 6 Sequencing analysis
[0560] The libraries resulting from the experiments described in Example 6 above were sequenced on a NovaSeq 3 using a NovaSeq S4200 cycles 2020 vl .5 kit, targeting using read 28, 10, 10, and 90 cycles targeting 20,000, 30,000, or 6000 reads per cell for gene expression, barcoded antigen, or Ig libraries, respectively. Sequence analysis (described further herein, see, e.g., Example 7) identified a total of 239 antibodies. The binding affinity of 80 exemplary antibodies to a trimerized wild-type SARS-CoV-2 spike protein (SEQ ID NO: 1484) and a SARS-CoV-2 spike protein variant with D614G substitution (SEQ ID NO: 1485) is summarized in Table 3 below. In these experiments, the binding affinity of an antigen-binding molecule (e.g, antibody) to a target antigen (wild-type S protein or a variant thereof) was determined based on quantity/numbers of unique molecular identifiers (UMIs) associated with each of the antigen binding molecules bound to the target antigen. Generally, the higher target antigen UMI counts were used as a predictor of higher binding affinity. When an antibody was found “fluorophore reactive” or “biotin-reactive” then it was categorized as a non-specific antibody, even if it had non-zero target antigen UMI counts. As shown in Table 3 below, all of the identified antibodies displayed high target antigen counts and low non-target antigen counts. As such, they were predicted to have specific binding affinity for the target antigen and were distinguishable from non-specific binders.
Table 3: Binding affinity of 80 exemplary antibodies. The integer values displayed in the table below represent antigen UMI counts for each of the individual on-target (Wild-type S, or D416G mutant (Mutant S)) and off-target (human serum albumin control/HSA 1, human serum albumin control/HSA 2) antigens.
Figure imgf000183_0001
Figure imgf000184_0001
EXAMPLE 7
Statistical analysis
[0561] Binding antibodies with a maximum spike antigen count greater than 40 UMIs (as summarized in Table 3 above) were selected for further analysis using lOx Genomics “Enclone” (available at https://bit.ly/enclone), which is a computational tool developed for clonal grouping to study the adaptive immune system. In this computational tool, the lOx Genomics Chromium Single Cell V(D)J data containing B cell receptor (BCR) and T cell receptor (TCR) RNA sequences are provided as input data to Enclone. Based on the input, Enclone finds and organizes cells arising from the same progenitors into groups ( e.g ., clonotype families) and compactly displays each clonotype along with its salient features, including mutated amino acids.
Antibodies in the dataset were classified into 3 categories, as listed below, via a process termed “barcode-enabled antigen mapping by sequencing” (BEAM-seq):
[0562] Category 1. (e.g., Fluorophore-reactive): Antibodies are classified into this category if the mix of antigens includes target and non-target antigens linked to different fluorophores, and counts are detected for target and non-target antigen linked to fluorophore 1 but not fluorophore 2, which indicates that the antibodies bind to the fluorophore and not the target antigen. In this particular Example, antibodies were classified into this category if counts were detected for only one spike protein and the corresponding albumin labeled with the same fluorophore.
[0563] Category 2. (e.g., Biotin-reactive, streptavidin-reactive, or polyreactive): Antibodies are classified into this category if the mix of antigens includes target and non-target antigens linked to different fluorophores, and counts are detected for target and non-target antigen linked to both fluorophores, which indicates that the antibodies does not bind the antigen but instead binds to a core component of the reagent (e.g., streptavidin, biotin) or is polyreactive (e.g., sticky and non-specific). In this particular Example, antibodies were classified into this category (e.g., classified as biotin-reactive, streptavidin-reactive, or polyreactive, if counts were detected for both spike proteins (trimerized wild-type S and trimerized S D614G) and both albumins (PE-HSA and APC-HSA).
[0564] Category 3. (e.g., Candidate SARS-2-reactive antibodies): Antibodies are classified into this category if counts are detected for target antigen but absent or at lower levels for non-target antigen, which indicates that the antibodies specifically binds the target antigen and has affinity for the target antigen. In this particular Example, antibodies were classified into this category ( e.g ., classified as candidate SARS-2-reactive antibodies, if counts were considerably higher for one or both spike proteins relative to the albumins; most antibodies bound the wild-type spike protein and the common population variant D614G. Category 3 antibodies are disclosed herein (see, e.g., Table 1, Table 3).
[0565] In a dataset where there is considerable enrichment for genuine antigen-binding cells, the binding affinity of an antigen-binding molecule (e.g., antibody or antigen-binding fragment) to a target antigen (such as S protein) were determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen bound to each cell. BEAM scores are approximately normally distributed, increase exponentially as target antigen-binding relative to expressed antibody and control antigen increases, are correlated with generation probability of the HCDR3 junction, e.g., following the known general relationship of somatic hypermutation (SHM) and increasing affinity, and also reveal that class switching increases predicted relative affinity in concordance with the literature (FIGS. 2 and 3). BEAM scores are also generally higher within sublineages that contain more daughter antibodies than narrow sublineages (representative example shown in FIG. 4).
EXAMPLE 8
Antibody synthesis cloning expression and purification [0566] Variable heavy chain and light chain domains of anti-SARS-CoV-2 antibodies were reformatted to IgGl and synthesized and cloned into mammalian expression vector pTwist CMV BG WPRE Neo utilizing the Twist Bioscience eCommerce portal. Light chain variable domains were reformatted into kappa and lambda frameworks accordingly. Clonal genes were delivered as purified plasmid DNA ready for transient transfection in human embryonic kidney (HEK) Expi293 cells (Thermo Scientific). Cultures in a volume of 1.2 ml were grown to four days, harvested and purified using Protein A resin (PhyNexus) on the Hamilton Microlab STAR platform into 43 mM Citrate, 148 mM HEPES, pH 6.
EXAMPLE 9
Further characterization of binding affinity [0567] This Example describes the results of experiments performed to further characterize binding affinity of select antibodies described herein. [0568] Antibodies with high predicted binding affinity based on antigen UMI count profiles were selected for further screening and analysis.
[0569] Subsequently, surface plasmon resonance (SPR) analyses were performed on the selected antibodies by using a Carterra LSA SPR biosensor equipped with a HC30M chip at 25°C in HBS-TE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20). Antibodies were diluted to 5 pg/ml in sodium acetate buffer, pH 4.5, and amine-coupled to the sensor chip by EDC/NHS activation, followed by ethanolamine HC1 quenching. Increasing concentrations of ligand were flowed over the sensor chip in HBSTE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20) with 0.5 mg/ml BSA with 5 minute association and 15 minute dissociation. Following each injection cycle the surface was regenerated with 2x 30 second injections of IgG elution buffer (Thermo). The following antigens and catalog #s from Aero Biosystems were used for serial analysis at the specified concentration ranges:
[0570] (1) SARS-CoV-2 S protein, His Tag, Super stable trimer (MALS & NS-EM verified), SPN-C52H9; 0 - 100 nM.
[0571] (2) SARS-CoV-2 S protein (D614G), His Tag, Super stable trimer (MALS verified), SPN-C52H3; 0 - 100 nM.
[0572] (3) SARS-CoV-2 (COVID-19) S protein RBD (triple mutant K417N, E484K,
N501Y), His Tag (MALS verified), SPD-C52Hp; 0 - 500 nM.
[0573] (4) SARS-CoV-2 (COVID-19) SI protein NTD, His Tag, S1D-C52H6; 0 - 500 nM.
[0574] (5) SARS-CoV-2 (COVID-19) S2 protein, His Tag, S2N-C52H5; 0 - 500 nM.
[0575] (6) MERS SI protein, His Tag, S1N-M52H5; 0 - 500 nM.
[0576] (7) HCoV-HKUl (isolate N5) S 1 protein, His Tag, SIN-V52H6; 0 - 500 nM.
[0577] Traces were analyzed and fit using Carterra's Kinetics Tool software, fit to a 1:1 receptor-ligand binding model.
[0578] Table 4 below provides a summary of the binding affinity of the exemplary antibodies to the following antigens: (1) a trimerized wild-type SARS-CoV-2 S protein (SEQ ID NO: 1483), (2) a SARS-CoV-2 S protein variant with D614G substitution (SEQ ID NO: 1484). For comparative analysis, also included in this study were the four following FDA-approved therapeutic antibodies previously reported to bind SARS-CoV-2 S protein: (1) imdevimab (REGN-COV2), (2) bamlanivimab (Eli Lilly / AbCellera), (3) etesevimab (Eli Lilly / AbCellera), and sotrovimab (Vir / GlaxoSmithKline). It was observed that the majority of antibodies tested in this study could bind to both wild-type S protein and D614G mutant in picomolar and nanomolar range. Remarkably, several antibodies described herein were found to have binding affinities as good as or superior to FDA-approved antibodies or antibodies in late clinical development. See also, FIGS. 16A-16B
TABLE 4: Binding affinity of three exemplary antibodies.
Figure imgf000188_0001
[0579] An UpSet plot was generated (see, e.g., FIG. 23) wherein antibodies are binned into antigen bins based on two rounds of SPR binding affinity data. For an antibody to be placed into a bin a detectable kinetic fit at all concentrations of antigen was required from at least one of the SPR experiments described in Examples 9 and 12, or orthogonal neutralization data. The results described in FIG. 23 illustrate that the BEAM-seq process described in the present disclosure allows for rapid identification of many antibodies with broad and robust binding affinity against several coronavirus S antigens, including several variants of concern (VoC), e.g.,
, beta, gamma, and kappa, as well as HKU1 (which is a different coronavirus).
[0580] Remarkably, several antibodies were found to be pan-coronavirus antibodies that recognizes a conserved epitope in the S 1 subunit and bind with high affinity to the S 1 subunit of a new human coronavirus strain HCoV-HKUl (see, e.g. , TXG-0085, TXG-0112, TXG-0136, TXG-0150, TXG-0192, TXG-0227, TXG-0228, TXG-0229, and TXG-0230 in Table 5). It was observed that several antibodies tested in this experiment could bind to human coronavirus strain HCoV-HKUl in low to mid nanomolar range. Accordingly, without being bound to any particular theory, these antibodies could be particularly useful in therapeutic combination against SARS-CoV-2 and other coronaviruses and in combination with RBD-binding, NTD-binding, or non-Sl binding therapeutic antibodies.
[0581] In addition, as shown in Table 5, several antibodies (e.g., TXG-0072, TXG-0137, TXG-0173, TXG-0174, and TXG-0230) were found to bind N-terminal domain of SARS-CoV-2 S protein with high affinity. Accordingly, these antibodies could be particularly useful in therapeutic combination against SARS-2 with RBD-binding and non-Sl binding therapeutic antibodies.
TABLE 5: Binding affinity of thirteen (13) exemplary antibodies to the N-terminal domain of SARS-CoV-2 S protein or the SI subunit of HCoV-HKUl. ND: Not determined.
Figure imgf000189_0001
EXAMPLE 10
Identification of antibodies with desired kinetic profiles [0582] Further analyses were performed using SPR binding curves to identify antibodies with particularly low K0rr constants. As shown in FIGS. 17A-17D and Table 6 below, it was observed that antibodies could be stratified into two major classes: optimal binding kinetics and less optimal binding kinetics. For example, it was found that one could identify antibodies with visibly longer half-lives have a K0rr lower than 4e-4, with an additional subclass of antibodies that have exceptionally long half lives and a K0h lower than le-4 (see, e.g., Table 6 and FIGS. 17A-17D). Antibody half-life values and mean-life values reported in Table 6 were calculated by using formula ln(2)/K0ff and formula 1/Koff, respectively.
[0583] A smaller number of antibodies have less optimal binding kinetics due to their higher K0ff constants, which produce a less ideal KD even given their acceptable Kon constants (see, e.g., Table 6 and FIGS. 17A-17D). For comparative analysis, etesevimab and sotrovimab were also included in this study. TABLE 6: Half-lives of two exemplary antibodies. CTRL-0007: Etesevimab. CTRL-0008:
Sotrovimab.
Figure imgf000190_0002
EXAMPLE 11
Functional characterization of antibodies
[0584] To further characterize the antibodies and antigen-binding fragments described in Examples 1-10 above, ligand-blocking assays are performed using GFP+ reporter cells expressing ACE2 and dose competition of pre-fusion D614G spike protein in dual -fluorescent format, where the antigens being competed are in tetrameric format. In these experiments, a relative KD value for each mAb can be generated.
[0585] In addition, to determine whether the antibodies and antigen-binding fragments of the disclosure have a neutralizing activity ( e.g ., antagonistic activity) against SARS-CoV-2, e.g. , able to bind to and neutralize the activity of SARS-CoV-S, additional live virus or pseudovirus neutralization assays are performed using these mAbs in a dose-dependent manner to generate an IC50 of neutralization activity. In some experiments, a neutralization activity IC50 value for each antibody can be determined in a quantitative focus reduction neutralization test (FRNT) described previously by Zost etal. (Nature, 584:443-449, 2020). In some experiments, neutralization assays are used to determine infectivity of SARS-CoV-2 S protein-containing virus-like particles. In these experiments, a neutralizing or antagonistic CoV-S antibody or antigen-binding fragment can be identified based on its ability to inhibit an activity of CoV-S to any detectable degree, e.g., inhibits or reduces the ability of CoV-S protein to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or mediate viral reproduction in a host cell.
EXAMPLE 12
Additional surface plasmon resonance analysis
Figure imgf000190_0001
[0586] This Example describes the results of an additional round of experiments performed to further characterize binding affinity of select antibodies described herein.
[0587] A new lot of all 239 antibodies identified in Example 6 were synthesized, cloned, expressed, and purified according to the methods described in Example 8.
[0588] A second SPR experiment was performed under the same experimental settings (flow times, antigen concentration, coupling method, buffer, etc.) with one set of measurements per antibody (in comparison to the triplicate measurements completed as part of the first SPR experiment). Trimeric forms of the SARS-CoV-2 Wuhan entry strain (WT), beta, gamma, and kappa pre-fusion spike, SARS-CoV-2 NTD, HcoV-HKUl spike trimer, and human serum albumin were used as antigens to assess the affinity and reactivity of each antibody. The antigens used in these experiments were purchased from ACROBiosystems (His-tagged wild-type SARS- CoV-2: Cat# SPN-C52H9; His-tagged SARS-CoV-2 gamma variant: Cat# SPN-C52Hg; His- tagged SARS-CoV-2 kappa variant: Cat# SPN-C52Hr;. His-tagged SARS-CoV-2 beta variant: Cat# SPN-C52Hk; His-tagged SARS-CoV-2 NTD: Cat# SPN-C52H6; and His-tagged HcoV- HKU1 (isolate N5) spike trimer: Cat# SPN-C52H5). In addition, His-tagged human serum albumin (HSA) was also purchased from ACROBiosystems (Cat# HSA-H5220). Mutations identified in the beta, gamma, and kappa variants are as follows.
[0589] Beta variant: L18F, D80A, D215G, 242-244del, R246I, K417N, E484K, N501Y, D614G, A701V.
[0590] Gamma variant: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F.
[0591] Kappa variant: T95I, G142D, E154K, L452R, E484Q, D614G, P681R, and Q1071H.
[0592] It should be noted that the delta and kappa variants share two mutations E484Q and L452R. They were identified in India’s second COVID-19 wave, and have been reported to share significant similarity, presumably due to the fact that they are from the same lineage.
[0593] As shown in Table 7 below, it was observed that several antibodies described herein demonstrated high binding affinity to various spike variants of interrest (VoC), e.g., beta, gamma, and kappa variants. For comparative analysis, also included in this study were several control antibodies (denoted as CTRL) that had been previously described as having binding affinity for SARS-CoV-2 S protein. It was observed that several antibodies tested in this experiment could bind to one or more spike variants in low to mid nanomolar range. In addition, several antibodies were found to bind beta, gamma, and/or kappa variants with binding affinities as good as or superior to FDA-approved antibodies or antibodies in late clinical development.
TABLE 7: Binding affinity of exemplary antibodies to beta, gamma, and kappa spike variants. A total of 53 control antibodies (CTRL) were included in these experiments, including: CTRL- 0004: Casirivimab; CTRL-0005: Imdevimab; CTRL-0006: Bamlanivimab; CTRL-0007: Etesevimab; CTRL-0008: Sotrovimab; and CTRL-0009: Tixagevimab; ND: not determined.
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
EXAMPLE 13
Neutralization assays
[0594] All 239 antibodies identified in Example 6 above were screened in addition to 49 control antibodies of known SARS-2 and other viral binding. Assays were performed with a clinical isolate of the SARS-CoV-2 B.1 lineage (MEX-BC2/2020). This virus carries the D614G mutation in the spike protein (full sequence available at the GISAID/EpiCoV database ID: EPI_ISL_747242). The screen was performed with a microneutralization assay that utilizes prevention of the virus-induced cytopathic effect (CPE) in Vero E6 cells. All antibodies (i.e., test-items) were provided at varying concentrations (0.06 to 0.23mg/mL), and they were stored at 4°C until use. The screen was performed in ten different experiments performed in ten days, each one assessing the activity of approximately 30 Abs in parallel. All plates included a positive control — plasma from a convalescent patient who had also received the first dose of the Pfizer/BioNTech mRNA vaccine (BNT162b2). Plasma was collected 21 days after vaccination.
[0595] For this screen, Vero E6 cells were used to evaluate the neutralization activity of the antibody test-items against a replication competent SARS-CoV-2 virus. Antibodies were pre incubated first with the virus for 1 hour at 37°C before addition to cells. Following pre incubation of Ab/virus samples, Vero E6 cells were challenged with the mixture. After addition to cells, antibodies were present in the cell culture for the duration of the infection (96 hours), at which time a “ Neutral Red” uptake assay was performed to determine the extent of the virus- induced CPE. Prevention of the CPE was used as a surrogate marker to determine the neutralization activity of the test-items against SARS-CoV-2.
[0596] Eight dilutions of the antibodies were tested in duplicates for the neutralization assay using a five- fold dilution scheme starting at l,000ng/mL. Representative raw data from neutralization assay is shown in FIGS. 19 and 20. When possible, IC50 values of the antibodies displaying neutralizing activity were determined using GraphPad Prism software. Plasma control was assessed on each plate using singlet data-points (8 two-fold dilutions throughout 1 :20480). Representative neutralization curves (IC50) for control antibodies and antibody test- items are shown in FIGS. 21 and 22.
Data analysis of CPE-based neutralization assay [0597] The average absorbance at 540nm (A540) observed in infected cells in the presence of vehicle alone was calculated first, and then subtracted from all samples to determine the inhibition of the virus induced CPE. Data points were then normalized to the average A540 signal observed in uninfected cells (“mock”) after subtraction of the absorbance signal observed in infected cells.
[0598] In the neutral red CPE-based neutralization assay, uninfected cells remained viable and uptake the dye at higher levels than non-viable cells. In the absence of antibodies, the virus-induced CPE leads to cell death in infected cells and lowers the A540 signal (this value equals 0% neutralization). By contrast, incubation with neutralizing antibodies prevents the virus induced CPE and leads to absorbance levels similar to those observed in uninfected cells. Full recovery of cell viability in infected cells represents 100% neutralization of the virus. Each plate assessed 3 antibodies in triplicates (rows A-C and F-H) or duplicates (rows D and E). However, data analysis avoided samples located in rows A and H to minimize “edge effects.” Therefore, all antibodies were evaluated in duplicates. Uninfected cells and infected cells in the absence of antibodies were analyzed using six replica data-points of each. Control neutralizing plasma was run in singlet data-points (1:160 or 1:320 to 1:20480).
[0599] Every plate was analyzed during a QC step before data was selected for analysis. QC included signal to background values greater than 2.5, and percentage CV in uninfected lower than 20 (CV<20%). All plates passed QC and there was no need to perform repeats. In some instances, data-points identified as outliers may have been removed, or they were exchanged by an additional data-point of the extra row not used (the latter only for antibodies in A-C or F-H). However, these actions were rarely needed, and overall variation of the screen was excellent and within the ranges typically seen in the neutralization studies described herein.
Control inhibitors and quality controls in live SARS-CoV-2 assay
[0600] Quality controls for the infectivity assays were performed on every plate to determine: i) signal to background (S/B) values; ii) inhibition by plasma with neutralizing activity against SARS-CoV-2, and; iii) variation of the assay, as measured by the coefficient of variation (C.V.) of all data points. All controls worked as anticipated for the assay, and variation was within typical ranges seen in vendor laboratories.
[0601] The average of all C.V. (of all duplicate data-points) in each plate was below 10% (average CV 7.1% for the 10 plates, whereas the variation of uninfected controls (“mock”), which were repeated six times on each plate, was below 5% (average 3.2%) (see, e.g., Table 8). The ratio of signal-to-background (S/B) for the neutralization assays, estimated by dividing the average signal in uninfected cells (A540nm) by the average signal in infected cells (vehicle alone), was 4.1 -fold for the ten representative plates. When comparing the signal in uninfected cells to the signal in “no-cells” background wells, the S/B ratio of the assay was greater than 10 (data not shown). S B values and variation for each of the plates are available in the accompanying excel file summary.
[0602] To evaluate the neutralization activity of 288 Abs against SARS-CoV-2, the clinical isolate (MEX- BC2/2020) carrying a D614G mutation in the viral spike proteinwas used. Full sequence of this isolate is available at the GISAID/EpiCoV database with the identifier EPI_ISL_747242.
[0603] A CPE-based neutralization assay was performed by infecting Yero E6 cells in the presence or absence of antibodies. Infection of cells leads to significant cytopathic effect and cell death after 4 days of infection. In this screen, reduction of the virus CPE in the presence of antibodies was used as a surrogate marker to determine the neutralization activity of the tested items.
[0604] Vero E6 cells were maintained in DMEM with 10% fetal bovine serum (FBS), referred herein as DMEM10. Twenty-four hours after cell seeding, test samples were submitted to serial dilutions with DMEM with 2% FBS (DMEM2) in a different plate. Then, virus diluted in DMEM2 or DMEM2 alone was pre-incubated with antibody test-items for 1 hour at 37°C in a humidified incubator. Following incubation, media was removed from cells, and then cells were challenged with the SARS-CoV-2 / antibody pre-incubated mix. The amount of viral inoculum was previously titrated to result in a linear response inhibited by antibodies with known neutralizing activity against SARS-CoV-2. Cell culture media with the virus inoculum was not removed after virus adsorption, and antibodies and virus were maintained in the media for the duration of the assay (96 hours). After this period, the extent of cell viability was monitored with the neutral red (NR) uptake assay.
[0605] The virus-induced CPE was routinely monitored under the microscope after 3 days of infection, and after 4 days, cells were stained with neutral red to monitor cell viability. Viable cells incorporate neutral red in their lysosomes. The uptake of neutral red relies on the ability of live cells to maintain the pH inside the lysosomes lower than in the cytoplasm, a process that requires ATP. Inside the lysosome, the dye becomes charged and is retained. After a 3-h incubation with neutral red (0.017%), the extra dye is washed away, and the neutral red is extracted from lysosomes by incubating cells for 15 minutes with a solution containing 50% ethanol and 1% acetic acid. The amount of neutral red is estimated by measuring absorbance at 540nm in a plate reader. The general procedure followed to determine the anti-SARS-CoV-2 activity of antibody test-items is summarized in FIG. 18.
[0606] In these experiments, antibodies were evaluated in duplicates using five-fold serial dilutions starting at 1 pg/mL. Controls included uninfected cells (“mock-infected”), and infected cells treated with vehicle alone. Some cells were treated with a positive control plasma derived from a convalescent patient who was also administered the first dose of Pfizer / BioNTech mRNA vaccine (BNT162b2). Plasma was collected 21 days after the vaccine injection.
Results
[0607] Of the antibodies tested against SARS-CoV-2 (lineage B.l, carrying the D614G mutation), approximately 40% of all antibodies displayed measurable neutralization activity. Exemplary antibodies that displayed measurable neutralization activity include TXG-0049, TXG-0051, TXG-0068, TXG-0072, TXG-0098, TXG-0108, TXG-0115, TXG-0136, TXG-0137, TXG-0140, TXG-0147, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0208, TXG-0213, and TXG-0226. Table 8 below provides a summary of neutralization activity of six (6) exemplary antibodies as determined by IC50 in live SARS-CoV-2 assays.
TABLE 8: Neutralization activity of exemplary antibodies as determined in testing against SARS-CoV-2 (lineage B.l, carrying the D614G mutation).
Figure imgf000200_0001
Figure imgf000201_0001
[0608] Excluding the 29 control antibodies, at least two (2) of the hits displayed IC50 values below 100 ng/mL, including TXG-140 (40 ng/mL), and TXG-154 (22 ng/mL).
[0609] IC50 values varied from single digit ng/mL (9 ng/mL) to approximately 1.0 pg/mL. A precise IC50 value could not be generated for some of the Abs tested. Antibodies failing to neutralize the virus at any of the concentrations tested were given an IC50 value of greater than 1 pg/mL (> 1 pg/mL). A summary of the antibodies’ neutralization potency is also shown in FIG. 26. [0610] In these experiments, positive plasma controls (CS478 pi_vac_pfl, plasma of Pfizer vaccine) were run on every plate to determine the intra-plate and inter-day variation. The average NT50 value and standard deviation of all plates was 2,183 ± 551(CV=25.2%), with lower variation when plates in the same day were evaluated. Of note, NT50 values for the plasma control were generated with dose-response curves using singlet data-points (as compared to duplicates), and that may have increased the variation observed in these controls.
[0611] Additionally, an UpSet plot of antibodies identified as having neutralization activity against live SARS-COV-2 was generated (see, e.g., FIG. 24), wherein the antibodies are binned into antigen bins as described in FIG. 23. The data described in this figure illustrates that the BEAM-seq process described in the present disclosure allows for rapid identification of many antibodies with broad and robust neutralizing activity against several SARS-CoV-2 S variants of concern (VoC), e.g., beta, gamma, and kappa, as well as HKU1 (which is a different coronavirus).
[0612] In addition, UpSet plots of potently (IC50 <= 1000 ng / ml) neutralizing antibodies retrieved from the BEAM-seq workflow described herein were also generated (see, e.g., FIGS. 25A and 25B). FIG. 25A is an Upset plot of the potently neutralizing antibodies selected from 239 antibodies identified in Example 6. FIG. 25B is an Upset plot of the potently neutralizing antibodies selected from the antibodies of Table 3. In these UpSet plots, rows represent the binding of these neutralizing antibodies to pre-fusion spike trimers from major SARS-CoV-2 variants of concern, and the endemic HKU1 coronavirus spike protein as well as the SARS-CoV-2 N terminal domain. It was also observed that the antibodies identified in these experiments were diverse in their VH, VL, and isotype/subclass (see Table 3). These antibodies were found to use 18 diverse VH genes and 46 unique VH :VL pairings. Taken together, the data described in these figures illustrate that the BEAM-seq process described in the present disclosure allows for rapid identification of many antibodies with potent and broad neutralizing activity against several SARS-CoV-2 S variants of concern (VoC).
EXAMPLE 14 Epitope binning assays
[0613] This Example describes the results of experiments performed to assess binding characteristics of select antibodies described herein.
Methodoloev
[0614] 1) Antigens:
[0615] a) Pre-fusion trimerized spike protein from SARS-CoV-2 USA-WA1/2020 isolate, ACRO Biosystems.
[0616] b) Pre-fusion trimerized spike protein from SARS-CoV-2 delta variant,
ACRO Biosystems.
[0617] c) SARS-CoV-2 NTD, ACRO Biosystems.
[0618] 2) Surface Plasmon Resonance tSPR) techniques:
[0619] a) Competition/sandwich.
[0620] b) Bidirectional/premix.
[0621] Epitope binning experiments were performed in a premix format using a Carterra LSA SPR biosensor equipped with a HC30M chip at 25°C in HBS-TE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20). First, antibodies were amine-coupled to the sensor chip by EDC/NHS activation, followed by ethanolamine HC1 quenching. Antibodies and SARS-CoV-2 prefusion stabilized S trimers were combined and incubated for 1 hour in HBS-TE with 0.5 mg/ml BSA at 120 nM and 7.5 nM, respectively. This constituted a 5-molar excess relative to the S trimer antigens, accounting for three (3) binding sites for each molecule. Each premix sample was injected over the immobilized antibodies to determine blocking, partial blocking, or non -blocking activity. The sensor chip was regenerated between injections with Pierce IgG Elution Buffer (Thermo Fisher Scientific).
[0622] Data were analyzed using Carterra’ s Epitope Tool software. Briefly, blocking assignments were determined relative to the binding responses for S trimer alone (normalized to 1); premixes giving binding responses less than 0.5 were determined to be blocking, 0.5-0.7 were intermediate blocking, and above 0.7 were not blocking. Heat maps representing the competition results were generated where red, yellow, and green cells represent blocked, intermediate, and not blocked analyte/ligand pairs, respectively. A summary of binding characteristics of exemplary antibodies described herein is presented in FIGS. 27-28 and Table 9 below.
TABLE 9: Epitope binning of exemplary antibodies as determined in testing against a pre fusion trimerized spike protein from SARS-CoV-2 USA-WA1/2020 isolate (WA1) and/or SARS-CoV-2 delta variant (Delta) by using SPR competition assay and bidirectional assay. A number of control antibodies (CTRL) were included in these experiments, including: CTRL- 0004: Casirivimab; CTRL-0005 (Imdevimab), CTRL-0006: Bamlanivimab; CTRL-0007: (Etesevimab), CTRL-0008: Sotrovimab; and CTRL-0009: Tixagevimab. NA: not applicable. Other: antibodies capable of binding to an epitope different that the antibodies in any other of the bins identified in the same column of Table 8.
Figure imgf000204_0001
Data interpretation
[0623] In these experiments, antibodies that share an epitope bin are antibodies which compete for binding to the same epitopes in a dose-dependent manner.
[0624] As shown in the table above and further described below, the discovered antibodies group into five prominent epitope bins. Furthermore, a number of the discovered antibodies group into unique bins outside of the five prominent bins.
A. NTD targeting antibodies
[0625] As shown in Table 9, five (5) antibodies tested in these epitope binning experiments grouped into WA1 trimer bins 1 or 2. These likely represent antibodies that target NTD of the spike protein from SARS-CoV-2 USA-WA1/2020 isolate (WA1), based on SPR data indicating that antibodies that group into bins 1 and 2 exhibit high (nM) affinity for the N- terminal domain (NTD) of SARS-CoV-2 S protein and the observation that they do not compete for binding with the antibodies in WA1 trimer bins 3, 4, or 3/4 (which include the FDA- authorized antibodies which have been shown to target RBD epitopes). All of these 5 antibodies, were found to display measurable neutralization activity as determined by IC50 in live SARS- CoV-2 assays. Examples of antibodies that target NTD of the WA1 isolate and have measurable neutralization activity in live SARS-CoV-2 assays include TXG-0072, TXG-0136, TXG-0137, TXG-0173, and TXG-0174. Examples of antibodies that target NTD of the WA1 isolate and potently neutralize live SARS-CoV-2 include XG-0173, and TXG-0174.
[0626] As shown in Table 9, three (3) antibodies tested in these epitope binning experiments grouped into delta trimer bin 2 or bin 1/2. These likely represent antibodies that target NTD of the spike protein from SARS-CoV-2 delta variant, based on SPR data indicating that antibodies that group into these bins exhibit high (nM) affinity for the N-terminal domain (NTD) of SARS-CoV-2 S protein and the observation that they do not compete for binding with the antibodies in delta trimer bins 3, 4, or 3/4 (which include the FDA-authorized antibodies which have been shown to target RBD epitopes). All of these 3 antibodies were found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target NTD of the WA1 isolate and have measurable neutralization activity in live SARS-CoV-2 include TXG-0072, TXG-0137, and TXG-0174. Examples of antibodies that target NTD of the WA1 isolate and potently neutralize live SARS-CoV-2 include TXG-0174.
[0627] B. RBD targeting antibodies
[0628] As shown in Table 9, four (4) antibodies tested in these epitope binning experiments grouped into WA1 trimer bins 3 or bin 3/4. These likely represent antibodies targeting primarily RBD of the spike protein from SARS-CoV-2 USA-WA1/2020 isolate (WA1), based on the observation that they compete for binding with FDA-authorized antibodies which have been shown to target RBD epitopes. All of these 4 antibodies were found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target primarily RBD of the WA1 isolate and potently neutralize live SARS- CoV-2 include TXG-0115, TXG-0140, TXG-0153, and TXG-0154.
[0629] As shown in Table 9, one (1) antibody tested in these epitope binning experiments grouped into WA1 trimer bin 3, along with CTRL-0008 (sotrovimab). This antibody likely represents antibodies that target a WA1 RBD epitope that is at least partially distinctive from those targeted by bin 3/4, e.g., a distinctive RBD epitope from those targeted by the tested FDA- approved antibodies save for sotrovimab. This antibody (TXG-0153) was found to display measurable neutralization activity as determined by IC50 in live SARS-CoV-2 assays. This antibody (TXG-0153) is also an example of antibodies in WA1 trimer bin 3 and which potently neutralize live SARS-CoV-2.
[0630] As shown in Table 9, three (3) antibodies tested in these epitope binning experiments grouped into WA1 trimer bin 3/4, along with CTRL-0005 (imdevimab) and CTRL- 0006 (bamlanivimab). These likely represent antibodies that target a WA1 RBD epitope that is at least partially distinctive from those targeted by bin 3. All of these 3 antibodies were found to display potent neutralization activity as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies in WA1 trimer bin 4 and which potently neutralize live SARS-CoV-2 include TXG-0115, TXG-0140, and TXG-0154.
[0631] Two (2) antibodies tested in these epitope binning experiments grouped into delta trimer bin 3/4, along with CTRL-0005 (imdevimab). These likely represent antibodies targeting primarily RBD of the spike protein from SARS-CoV-2 delta variant, based on the observation that they compete for binding with FDA-authorized antibodies which have been shown to target RBD epitopes. Both of these antibodies (TXG-0140 and TXG-0154) were found to potently neutralize live SARS-CoV-2 as determined by IC50 in live SARS-CoV-2 assays. Examples of antibodies that target primarily RBD of the SARS-CoV-2 delta variant and potently neutralize live SARS-CoV-2 include TXG-0140 and TXG-0154.
[0632] C. Other epitopes
[0633] Seven (7) antibodies tested in these epitope binning experiments grouped into WA1 trimer bin “Other”. These likely represent antibodies that target epitopes that are distinctive from the epitopes targeted by any other of the WA1 trimer bins. Exemplary antibodies in these categories include TXG-0085, TXG-0112, TXG-0192, TXG-0227, TXG- 0228, TXG-0229, and TXG-0230.
[0634] Four (4) antibodies tested in these epitope binning experiments grouped into delta trimer bin 5. These likely represent antibodies that target a distinct delta variant epitope from other binned groups, e.g ., distinct from any of the tested FDA approved antibodies. Exemplary antibodies in this category include: TXG-0115, TXG-0136, TXG-0192, and TXG-0230. Of these, one antibody displays measurable neutralization activity as determined by live SARS- CoV-2 assays. This antibody (TXG-0115) is an example of antibodies in delta trimer bin 5 that can neutralize live SARS-CoV-2.
[0635] Six (6) antibodies tested in these epitope binning experiments grouped into delta trimer bins “Other”, along with CTRL-0006 (bamlanivimab). These likely represent antibodies that target epitopes that are distinctive from the epitopes targeted by any other of the delta trimer bins. Exemplary antibodies in these categories include TXG-0085, TXG-0112, TXG-0173, TXG-0227, TXG-0228, and TXG-0229. Of these six antibodies, one antibody (TXG-0173) exhibits potent neutralization activity againts live SARS-CoV-2.
[0636] Antibodies that group into different epitope bins can advantageously be used in a therapeutic antibody cocktail or combination therapy regimen. For example, a neutralizing antibody from bin A can thus be combined with a neutralizing antibody from bin B effectively as the two antibodies do not bind in the same location. Examples of such complementary bins that may be advantageously used in a combination therapy or antibody cocktail include:
[0637] NTD and RBD targeting combination: a. Antibody 1 : an antibody from bin 1 or bin 2, both of which represent NTD- binding antibodies b. Antibody 2: an antibody from bin 3, 4, or 3/4, which represent antibodies targeting primarily RBD
[0638] RBD distinctive targeting combination: a. Antibody 1 : an antibody from bin 3 (sotrovimab-like antibodies) b. Antibody 2: an antibody from bin 4
[0639] RBD partially distinctive targeting combination: a. Antibody 1 : an antibody from bin 3 or 4 b. Antibody 2: an antibody from bin 3/4 which partially competes with an antibody from bin 3 or 4
[0640] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. An isolated antibody, or an antigen-binding fragment thereof, that binds specifically to a spike (S) protein of SARS-CoV-2, wherein the antibody is selected from the group consisting of TXG-0153, TXG-0115, TXG-140, and TXG-154, wherein: a. the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0153 have the amino acid sequences as set forth in SEQ ID NOS: 88, 168, 248, 328, 408, and 488, respectively; b. the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0115 have the amino acid sequences as set forth in SEQ ID NOS: 72, 152, 232, 312, 392, and 472, respectively; c. the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0140 have the amino acid sequences as set forth in SEQ ID NOS: 83, 163, 243, 323, 403, and 483, respectively; and d. the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) of TXG-0154 have the amino acid sequences as set forth in SEQ ID NOS: 89, 169, 249, 329, 409, and 489, respectively.
2. An isolated antibody, or an antigen-binding fragment thereof, that binds specifically to a spike (S) protein of SARS-CoV-2, and wherein the antibody or fragment thereof shares an epitope bin with an antibody or antigen-binding fragment thereof that binds to amino acids 332- 337, 339-340, 343-346, 354, 356-361, 440-441, and 509 of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.
3. An isolated antibody, or an antigen-binding fragment thereof, that binds specifically to a spike (S) protein of SARS-CoV-2, wherein the antibody or antigen-binding fragment comprises: a) a heavy chain complementary determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3; b) a HCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 4-6; and c) a HCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 7-9.
4. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 1, 4, and 7, respectively.
5. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in in SEQ ID NOS: 2, 5, and 8, respectively.
6. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 3, 6, and 9, respectively.
7. An isolated antibody, or an antigen-binding fragment thereof, that binds specifically to a spike (S) protein of SARS-CoV-2, wherein the antibody or antigen-binding fragment comprises: a) a light chain complementary determining region 1 (LCDR1) comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 10-12; b) a LCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 13-15; and c) a LCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 16-18.
8. The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen binding fragment comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively.
9. The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen binding fragment comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 11, 14, and 17, respectively.
10. The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen binding fragment comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 12, 15, and 18, respectively.
11. An isolated antibody, or an antigen-binding fragment thereof, that binds specifically to a spike (S) protein of SARS-CoV-2, wherein the antibody or antigen-binding fragment comprises: a) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3; b) a HCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 4-6; c) a HCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 7-9; d) a LCDR1 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 10-12; e) a LCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 13-15; and f) a LCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 16-18.
12. The antibody or antigen-binding fragment of claim 11, wherein the antibody or antigen binding fragment comprises: a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS: 1, 4, and 7, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS: 10, 13, and 16, respectively.
13. The antibody or antigen-binding fragment of claim 11, wherein the antibody or antigen binding fragment comprises: a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS:
2, 5, and 8, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS:
11, 14, and 17, respectively
14. The antibody or antigen-binding fragment of claim 11, wherein the antibody or antigen binding fragment comprises: a) the amino acid sequences of HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOS:
3, 6, and 9, respectively; and b) the amino acid sequences of LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOS:
12, 15, and 18, respectively.
15. The antibody or antigen-binding fragment of any one of claims 1 to 14, wherein the HCDR1 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122.
16. The antibody or antigen-binding fragment of any one of claims 1 to 15, wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-3 and SEQ ID NOS: 43-122, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid.
17. The antibody or antigen-binding fragment of claim 15, wherein the HCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122.
18. The antibody or antigen-binding fragment of any one of of claims 1 to 17, wherein the HCDR2 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-202.
19. The antibody or antigen-binding fragment of any one of claims of claims lto 18, wherein the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 4-6 and SEQ ID NOS: 123-202, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid.
20. The antibody or antigen-binding fragment of claim 18, wherein the HCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-202.
21. The antibody or antigen-binding fragment of any one of claims 1 to 20, wherein the HCDR3 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282.
22. The antibody or antigen-binding fragment of any one of of claims 1 to 21, wherein the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-9 and SEQ ID NOS: 203-282, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.
23. The antibody or antigen-binding fragment of claim 21, wherein the HCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282.
24. The antibody or antigen-binding fragment of any one of claims 1 to 23, wherein the LCDR1 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362.
25. The antibody or antigen-binding fragment of any one of claims 1 to 24, wherein the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 10-12 and SEQ ID NOS: 283-362, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.
26. The antibody or antigen-binding fragment of claim 24, wherein the LCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362.
27. The antibody or antigen-binding fragment of any one of claims 1 to 26, wherein the LCDR2 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442.
28. The antibody or antigen-binding fragment of any one of of claims 1 to 27, wherein the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-15 and SEQ ID NOS: 363-442, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid.
29. The antibody or antigen-binding fragment of claim 25, wherein the LCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442.
30. The antibody or antigen-binding fragment of any one of claims 1 to 29, wherein the LCDR3 amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522.
31. The antibody or antigen-binding fragment of any one of claims 1 to 30, wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-15 and SEQ ID NOS: 443-522, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid.
32. The antibody or antigen-binding fragment of claim 30, wherein the LCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522.
33. The antibody or antigen-binding fragment of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment comprises: a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-122; b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-202; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-282; d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-362; e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-442; and f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-522.
34. The antibody or antigen-binding fragment of claim 33, wherein the antibody or antigen binding fragment comprises: a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-45; b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 123-125; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 203-205; d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 283-285; e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 363-365; and f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 443-445.
35. The antibody or antigen-binding fragment of claim 33, wherein the antibody or antigen binding fragment comprises: a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 46-47; b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 126-127; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 206-207; d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 286-287; e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 366-367; and f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 446-447.
36. The antibody or antigen-binding fragment of claim 33, wherein the antibody or antigen binding fragment comprises: a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 117-120; b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 197-200; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 277-280; d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 357-360; e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 437-440; and f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 517-520.
37. The antibody or antigen-binding fragment of any one of claims 1 to 33, wherein the antibody or antigen-binding fragment comprises a framework region having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 523-1162.
38. The antibody or antigen-binding fragment of any one of claims 1 to 34, wherein the antibody or antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: a) TXG-0021 , TXG-0022, and TXG-0023; b) TXG-0027 and TXG-0028; or c) TXG-0227, TXG-0228, TXG-0229, and TXG-0230.
39. The antibody or antigen-binding fragment of any one of claims 1 to 38, wherein the antibody or antigen-binding fragment comprises:
(a) a heavy chain framework region 1 (HFWR1) comprising an amino acid sequence selected from the group consisting of SEQ ED NOS: 523-602;
(b) a heavy chain framework region 2 (HFWR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 603-682,
(c) a heavy chain framework region 3 (HFWR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 683-762; and
(d) a heavy chain framework region 4 (E1FWR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 763-842.
40. The antibody or antigen-binding fragment of any one of claims 1 to 39, wherein
41. The antibody or antigen-binding fragment of any one of claims 1 to 40, wherein the antibody or antigen-binding fragment comprises the HCDR1 , HCDR2, ITCDR3, LCDR1, LCDR2, and LCDR3 of an antibody of Table 1.
42. The antibody or antigen-binding fragment of claim 41, further comprising the heavy chain framework regions HFWR1, 1TFWR2, E1FWR3, and HFWR4 of the same antibody or antigen binding fragment.
43. The antibody or antigen-binding fragment of any one of claims 39 to 42, further comprising the light chain framework regions LFWR1, LFWR2, LFWR3, and LFWR4 of the same antibody or antigen-binding fragment.
44. The antibody or antigen-binding fragment of any one of claims 1 to 43, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163-1322.
45. The antibody or antigen-binding fragment of claim 44, wherein the HCVR comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163-1322.
46. The antibody or antigen-binding fragment of any one of claims lto 45, wherein the antibody or antigen-binding fragment comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482.
47. The antibody or antigen-binding fragment of claim 46, wherein the LCVR comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323-1482.
48. The antibody or antigen-binding fragment of any one of claims 1 to 47, wherein the antibody or antigen-binding fragment comprises: a) a HCVR comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163- 1322; and b) a LCVR comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323- 1482.
49. The antibody or antigen-binding fragment of any one of claims 1 to 48, wherein the antibody or antigen-binding fragment comprises a HCVR and a LCVR which respectively are 90% identical to the HCVR and LCVR of an antibody of Table 1.
50. The antibody or antigen-binding fragment of any one of claims 1 to 48, wherein the antibody or antigen-binding fragment comprises a HCVR and a LCVR of an antibody of Table 1.
51. The antibody or antigen-binding fragment of any one of claims 1 to 50, wherein the antibody or antigen-binding fragment is selected from Table 1.
52. The antibody or antigen-binding fragment of any one of claims 1 to 51, further comprising a constant region.
53. The antibody or antigen-binding fragment of claim 52, wherein the constant region is an IgA, IgD, IgE, IgG, or IgM heavy chain constant region.
54. The antibody or antigen-binding fragment of claim 52, wherein the constant region is a kappa type or lambda type light chain constant region.
55. The antibody or antigen-binding fragment of any one of claims 1 to 54, wherein the antibody is a human antibody.
56. The antibody or antigen-binding fragment of any one of claims 1 to 54, wherein the antibody is a humanized antibody or a chimeric antibody.
57. The antibody or antigen-binding fragment of any one of claims 1 to 56, wherein the antibody is a monoclonal antibody.
58. The antibody or antigen-binding fragment of any one of claims 1 to 57, which is a single chain antibody fragment (scFv), a Fab, a Fab', a Fab'-SH, a F(ab')2, or a Fv fragment.
59. The antibody or antigen-binding fragment of any one of claims 1 to 58, wherein the antibody or antigen-binding fragment has a binding affinity to an epitope in a subunit of the SARS-CoV-2 S protein.
60. The antibody or antigen-binding fragment of claim 59, wherein the subunit of the S protein of SARS-CoV-2 is the SI subunit.
61. The antibody or antigen-binding fragment of claim 60, wherein the antibody or antigen binding fragment has a binding affinity to a receptor binding domain (RBD) or a N-terminal domain (NTD) of the S 1 subunit.
62. The antibody or antigen-binding fragment of claim 59, wherein the subunit of the S protein of SARS-CoV-2 is the S2 subunit.
63. The antibody or antigen-binding fragment of any one of claims 1 to 62, wherein the SARS- CoV-2 S protein comprises one or more amino acid substitutions.
64. The antibody or antigen-binding fragment of claim 63, wherein the SARS-CoV-2 S protein comprises one or more amino acid substitutions at a position selected from the group consisting of K417, L452, K417, E484, E484, N501, and D614.
65. The antibody or antigen-binding fragment of claim 64, wherein the SARS-CoV-2 S protein comprises one or more amino acid substitutions selected from the group consisting of K417T, K417N, L452R, E484K, E484Q, N501Y, and D614G.
66. The antibody or antigen-binding fragment of claim 65, wherein the SARS-CoV-2 S protein comprises amino acid substitutions K417N, E484K, andN501Y.
67. The antibody or antigen-binding fragment of any one of claims 64 to 66, wherein the SARS-CoV-2 S protein comprises D614G substitution.
68. The antibody or antigen-binding fragment of any one of claims 1 to 67, wherein the antibody or antigen-binding fragment has binding affinity for a trimeric form of the SARS-CoV- S protein.
69. The antibody or antigen-binding fragment of any one of claims 1 to 67, wherein the antibody or antigen-binding fragment has binding affinity for a pre-fusion trimeric form of the SARS-CoV-S protein.
70. The antibody or antigen-binding fragment of any one of claims 1 to 67, wherein the antibody or antigen-binding fragment has binding affinity for a stabilized prefusion spike protein in monomeric or multimeric form.
71. The antibody or antigen-binding fragment of any one of claims 1 to 67, wherein the antibody or antigen-binding fragment has binding affinity for a non-prefusion spike protein in monomeric or multimeric form.
72. The antibody or antigen-binding fragment of any one of claims 1 to 67, wherein the antibody or antigen-binding fragment has binding affinity for a non-S2P-stabilized pre-fusion spike protein in monomeric or multimeric form.
73. The antibody or antigen-binding fragment of any one of claims 59 to 72, wherein the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 500 nM, less than 120 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 5 nM, less than 1 nM, or less than 1 pM.
74. The antibody or antigen-binding fragment of claim 73, wherein the antibody or antigen binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM.
75. The antibody or antigen-binding fragment of claim 74, wherein the antibody or antigen binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, TXG-0230.
76. The antibody or antigen-binding fragment of any one of claims 74 to 75, wherein the antibody or antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, TXG-0230.
77. The antibody or antigen-binding fragment of any one of claims 74 to 76, wherein the antibody or antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the following group of antibodies: TXG-0022, TXG-0023, TXG-0028, TXG-0049, TXG-0056, TXG-0062, TXG-0068, TXG-0072, TXG-0085, TXG-0089, TXG-0098, TXG-0112, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0152, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0192, TXG-0194, TXG-0195, TXG-0211, TXG-0213, TXG-0215, TXG-0217, TXG-0218, TXG-0226, TXG-0227, TXG-0228, TXG-0230.
78. The antibody or antigen-binding fragment of any one of claims 73 to 77, wherein the antibody or antigen-binding fragment has a sub-nanomolar binding affinity for a SARS-CoV-2 S protein, a fragment thereof, or a multimeric form thereof.
79. The antibody or antigen-binding fragment of claim 78, wherein the antibody or antigen binding fragment has a binding affinity with a KD value of less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM.
80. The antibody or antigen-binding fragment of claim 78, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0028, TXG-0049, TXG-0056, TXG-0072, TXG-0089, TXG-0113, TXG-0115, TXG-0118, TXG-0121, TXG-0136, TXG-0137, TXG-0138, TXG-0140, TXG-0145, TXG-0147, TXG-0153, TXG-0154, TXG-0165, TXG-0173, TXG-0174, TXG-0182, TXG-0194, and TXG-0217.
81. The antibody or antigen-binding fragment of any one of claims 1 to 80, wherein the antibody or antigen-binding fragment as a sub-nanomolar binding affinity for HCOV and/or for a SARS-CoV-2 S variant selected from the group consisting of beta, gamma, delta, and kappa.
82. The antibody or antigen-binding fragment of any one of claims 1 to 81, wherein the antibody or antigen-binding fragment further has a binding affinity to the N-terminal domain of (NTD) of the S 1 subunit.
83. The antibody or antigen-binding fragment of claim 81, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0115 and TXG-0154.
84. The antibody or antigen-binding fragment of claim 81, wherein the antibody or antigen binding fragment is TXG-0140.
85. The antibody or antigen-binding fragment of claim 81, wherein the antibody or antigen binding fragment is TXG-0153.
86. The antibody or antigen-binding fragment of claim 81, wherein the antibody or antigen binding fragment is TXG-0173.
87. The antibody or antigen-binding fragment of claim 81, wherein the antibody or antigen binding fragment is TXG-0174.
88. The antibody or antigen-binding fragment of any one of claims 1 to 87, wherein the antibody or antigen-binding fragment wherein the antibody has a neutralizing activity against SARS-CoV-2.
89. The antibody or antigen-binding fragment of claim 88, wherein the antibody or antigen binding fragment neutralizes at least 50% of 200 times the tissue culture infectious dose (200xTCID50) of the coronavirus.
90. The antibody or antigen-binding fragment of any one of claims 88 to 89, wherein the antibody has a neutralizing activity against SARS-CoV-2 with an IC50 value below 1 pg/mL. below 200 ng/mL, or below 40 ng/mL.
91. The antibody or antigen-binding fragment of claim 90, wherein the antibody has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 200 ng/mL to 1,000 ng mL.
92. The antibody or antigen-binding fragment of claim 90, wherein the antibody has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 40 ng/mL to 200 ng/mL.
93. The antibody or antigen-binding fragment of claim 90, wherein the antibody has a neutralizing activity against SARS-CoV-2 with an IC50 value ranging from 8 ng/mL to 40 ng mL.
94. The antibody or antigen-binding fragment of claim 91, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0153, TXG-0173, and TXG- 0174.
95. The antibody or antigen-binding fragment of claim 92, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0115 and TXG-0140.]
96. The antibody or antigen-binding fragment of claim 90, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0140 and TXG-0154.
97. The antibody or antigen-binding fragment of claim 93, wherein the antibody wherein the antibody or antigen-binding fragment is TXG-0154.
98. The antibody or antigen-binding fragment of any one of claims 1 to 97, wherein the isolated antibody or antigen-binding fragment thereof is a recombinant antibody or antigen binding fragment thereof.
99. The antibody or antigen-binding fragment of claim 88, wherein the antibody or antigen binding-fragment has a binding affinity to the N-terminal domain of (NTD) of a SARS-CoV-2 S protein and potently neutralizes live SARS-CoV-2.
100. The antibody or antigen-binding fragment of claim 99, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0072, TXG-0136, TXG-0137, TXG-0173, and TXG-0174.
101. The antibody or antigen-binding fragment of claim 100, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0173, and TXG-0174.
102. The antibody or antigen-binding fragment of claim 99, wherein the antibody or antigen binding fragment has a binding affinity to the N-terminal domain of (NTD) of an S protein from a SARS-CoV-2 delta variant.
103. The antibody or antigen-binding fragment of claim 102, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0072, TXG-0137, and TXG- 0174.
104. The antibody or antigen-binding fragment of claim 103, wherein the antibody or antigen binding fragment is TXG-0174.
105. The antibody or antigen-binding fragment of claim 88, wherein the antibody or antigen binding fragment has a binding affinity primarily to the RBD of a SARS-CoV-2 S protein and potently neutralizes live SARS-CoV-2.
106. The antibody or antigen-binding fragment of claim 105, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0115, TXG-0140, TXG-0153, and TXG-0154
107. The antibody or antigen-binding fragment of claim 105, wherein the antibody or antigen binding fragment is TXG-0153.
108. The antibody or antigen-binding fragment of claim 105, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0115, TXG-0140, and TXG- 0154.
109. The antibody or antigen-binding fragment of claim 105, wherein the antibody or antigen binding has a binding affinity primarily to the RBD of an S protein from a SARS-CoV-2 delta variant.
110. The antibody or antigen-binding fragment of claim 109, wherein the antibody or antigen binding fragment is selected from the group consisting of TXG-0140 and TXG-0154.
111. The antibody or antigen-binding fragment of any one of claims 88 to 110, wherein the antibody or antigen-binding fragment has a binding affinity for a SARS-CoV-2 S protein and is selected from the group consisting of TXG-0085, TXG-0112, TXG-0192, TXG-0227, TXG- 0228, TXG-0229, and TXG-0230.
112. The antibody or antigen-binding fragment of any one of claims 88 to 111, wherein the antibody or antigen-binding fragment has a binding affinity for an S protein of a SARS-CoV-2 delta variant and is selected from the group consisting of TXG-0115, TXG-0136, TXG-0192, and TXG-0230.
113. The antibody or antigen-binding fragment of claim 112, wherein the antibody or antigen binding fragment has a neutralizing activity against live SARS-CoV-2.
114. The antibody or antigen-binding fragment of claim 113, wherein the antibody or antigen binding fragment is TXG-0115.
115. The antibody or antigen-binding fragment of any one of claims 88 to 114, wherein the antibody or antigen-binding fragment has a binding affinity for an S protein from a SARS-CoV-2 delta variant and is selected from the group consisting of TXG-0085, TXG-0112, TXG-0173, TXG-0227, TXG-0228, and TXG-0229.
116. The antibody or antigen-binding fragment of claim 115, wherein the antibody or antigen binding fragment potently neutralizes live SARS-CoV-2.
117. The antibody or antigen-binding fragment of claim 116, wherein the antibody or antigen binding fragment is TXG-0173.
118. A recombinant nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 117.
119. The recombinant nucleic acid of claim 118, wherein the nucleic acid comprises a nucleic acid sequence encoding a HCVR comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1163- 1322.
120. The recombinant nucleic acid of claim 119, wherein nucleic acid comprises a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1487-1886.
121. The recombinant nucleic acid of claim 118, wherein the nucleic acid comprises a nucleic acid sequence encoding a LCVR comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1323- 1482.
122. The recombinant nucleic acid of claim 121, wherein the nucleic acid comprises a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1887-2286.
123. The recombinant nucleic acid of any one of claims 118 to 122, wherein the nucleic acid comprises a first nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1487-1886; and a second nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1887-2286.
124. A vector comprising the recombinant nucleic acid of any one of claims 118 to 123.
125. The vector of claim 124, wherein the vector is an expression vector.
126. The vector of any one of claims 124 to 125, wherein the vector is a plasmid vector or a viral vector.
127. A recombinant cell comprising: a) an antibody or antigen-binding fragment according to any one of claims 1 to 117; or b) a recombinant nucleic acid according to any one of claims 118 to 123; or c) a vector of any one of claims 124 to 126.
128. The recombinant cell of claim 127, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell.
129. A transgenic animal comprising a vector according to any one of claims 124 to 126.
130. The transgenic animal of claim 129, wherein the transgenic animal is a non-human animal.
131. The transgenic animal of any one of claims 129 to 130, wherein the transgenic animal produces the antibody or antigen-binding fragment according to any one of claims 1 to 117.
132. A method for producing an antibody or antigen-binding fragment thereof, comprising (i) rearing the transgenic animal of any one of claims 129-131 or (ii) culturing the recombinant cell of any one of claims 127-128 under conditions such that the antibody or antigen-binding fragment is produced.
133. The method of claim 132, further comprising isolating the produced antibody or antigen binding fragment from (i) the transgenic animal or (ii) recombinant cell and/or the medium in which the recombinant cell is cultured.
134. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 117, and a pharmaceutically acceptable carrier or diluent.
135. The pharmaceutical composition of claim 134, comprising:
(a) a first antibody or antigen-binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein); b) a first antibody or antigen-binding fragment having a binding affinity to a RBD and a second antibody or antigen-binding fragment having a binding affinity to aNTD of a SARS-CoV- 2 S protein; or c) a first antibody or antigen-binding fragment having a binding affinity to a NTD and a second antibody or antigen-binding fragment having a binding affinity to a full-length SARS-CoV-2 S protein (e.g, to the SI subunit of the full-length SARS-CoV-2 S protein)
136. The pharmaceutical composition of any one of claims 134 to 135, wherein the composition is a sterile composition.
137. The pharmaceutical composition of any one of claims 134 to 135, wherein the composition is formulated as a vaccine.
138. The pharmaceutical composition of any one of claims 134 to 137, wherein the composition further comprises an adjuvant.
139. The pharmaceutical composition of claim 134, further comprising a second therapeutic agent.
140. The pharmaceutical composition of claim 139, wherein the second therapeutic agent is selected from the group consisting of: (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein.
141. A method for treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the method comprising administering to the subject a composition comprising therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 117.
142. A method for reducing binding of SARS-Co-2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 117.
143. The method of any one of claims 141 to 142, wherein the antibody or antigen-binding fragment is administered in combination with a SARS-Co-2V S protein conjugated to a therapeutic agent.
144. The method of any one of claims 141 to 143, wherein the subject is administered one or more further therapeutic agents.
145. The method of claim 144, wherein the one or more further therapeutic agents comprises an antiviral drug or a vaccine.
146. The method of claim 144, wherein the one or more further therapeutic agents is selected from the group consisting of: (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds TMPRSS2, and (iv) an antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein.
147. The method of any one of claims 141 to 146, wherein the antibody or antigen-binding fragment is administered to the subject subcutaneously, intravenously, and/or intramuscularly.
148. A method for detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, the method comprising contacting an antibody or antigen-binding fragment of any one of claims 1 to 117 with a biological sample from an individual infected with or suspected of being infected with SARS-CoV-2.
149. A method for identifying an antibody having binding affinity for a coronavirus spike protein (CoV-S), the method comprising: a) contacting a plurality of B cells obtained from a subject who has been exposed to a coronavirus with a plurality of antigens, wherein the plurality of antigens comprises a CoV-S antigen and a non-CoY-S antigen, and wherein each of the antigens comprise a reporter oligonucleotide, wherein the contacting provides a B cell bound to a CoV-S antigen; b) partitioning the B cell bound to the CoV-S antigen in a partition of a plurality of partitions, wherein the partitioning provides a partition comprising (i) the B cell bound to the CoV- S antigen and (ii) a plurality of nucleic acid barcode molecules comprising a common barcode sequence; c) identifying a sequence of at least one antibody or antigen-binding fragment produced by the B cell that has been bound to the CoV-S antigen; and d) assessing the binding affinity of the antibody or antigen-binding fragment to a CoV-S protein; and e) identifying the antibody or antigen-binding fragment as having a binding specificity for the CoV-S protein if the antibody or antigen-binding fragment specifically binds to the CoV-S protein.
150. The method of claim 149, wherein the reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence.
151. The method of any one of claims 149 to 150, further comprising coupling a barcode moiety to the antibody or antigen-binding fragment produced by the B cell to generate a barcoded antibody or antigen-binding fragment.
152. The method of any one of claims 149 to 151, wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence of the reporter oligonucleotide and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further comprises a capture sequence configured to couple to an mRNA analyte.
153. The method of claim 152, wherein the capture sequence is configured to couple to the capture handle sequence of the reporter oligonucleotide is complementary to the capture handle sequence of the reporter oligonucleotide.
154. The method of claim 152, wherein the capture sequence configured to couple to an mRNA analyte comprises a polyT sequence.
155. The method of any one of claims 149 to 154, wherein the first and second nucleic acid barcode molecules each comprise a unique molecule identifier (UMI).
156. The method of any one of claims 149 to 155, wherein the antibody or antigen-binding fragment has a binding specificity to an epitope on a subunit of the CoV-S protein.
157. The method of claim 156, wherein the subunit of the CoV-S protein is the SI subunit.
158. The method of claim 156, wherein the domain of the CoV-S protein is the S2 domain.
159. The method of claim 157, wherein the antibody or antigen-binding fragment has a binding affinity to a RBD or a NTD of the S 1 subunit.
160. The method of any one of claims 149 to 159, wherein the antibody or antigen-binding fragment has binding affinity for a trimeric form of the CoV-S protein.
161. The method of any one of claims 149 to 159, wherein the antibody or antigen-binding fragment has binding affinity for pre-fusion trimeric form of the CoV-S protein.
162. The method of any one of claims 149 to 159, wherein the antibody or antigen-binding fragment has binding affinity for a stabilized pre-fusion spike protein in monomeric or multimeric form.
163. The method of any one of claims 149 to 159, wherein the antibody or antigen-binding fragment has binding affinity for a non-prefusion spike protein in monomeric or multimeric form.
164. The method of any one of claims 149 to 159, wherein the antibody or antigen-binding fragment has binding affinity for a non-S2P-stabilized pre-fusion spike protein in monomeric or multimeric form.
165. The method of any one of claims 149 to 164, wherein the antibody or antigen-binding fragment a binding affinity with an equilibrium dissociation constant (KD) value of less than 500 nM, less than 120 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 5 nM, less than 1 nM, or less than 1 pM.
166. The method of claim 165, wherein the antibody or antigen-binding fragment has a binding affinity with an equilibrium dissociation constant (KD) value of less than 120 nM.
167. The method of any one of claims 165 to 166, wherein the antibody or antigen-binding fragment has a sub-nanomolar binding affinity for a CoV-S protein, a fragment thereof, or a multimeric form thereof.
168. The method of claim 167, wherein the antibody or antigen-binding fragment has a binding affinity with a KD value of less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM.
169. The method of any one of claims 165 to 168, wherein the antibody or antigen-binding fragment as a neutralizing activity against a CoV-S protein.
170. The method of any one of claims 149 to 169, wherein the CoV-S protein is a spike protein of SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
171. The method of any one of claims 149 to 170, wherein the subject is suspected of being infected with a coronavirus, has been infected with a coronavirus, has been vaccinated, or has been recovered from a coronavirus infection.
172. The method of any one of claims 149 to 171, wherein the subject is a mammalian subject.
173. The method of claim 172, wherein the mammalian subject is a human.
174. The method of any one of claims 149 to 173, wherein the antigens are each coupled to a fluorescent label identifying the antigens.
175. The method of any one of claims 149 to 174, further comprising isolating and/or enriching the plurality of single B cells prior to (b).
176. The method of claim 175, wherein the enriching further comprises sorting of the B cells bound to the CoV-S antigen and/or non-CoV-S antigen based on detection of one or more of the fluorescent labels coupled to the antigens.
177. The method of any one of claims 149 to 176, wherein the CoV-S protein is coupled to a barcode moiety.
178. The method of any one of claims 149 to 177, further comprising purifying/isolating the antibody or antigen-binding fragment that has been identified as having a binding specificity for the CoV-S protein.
179. The method of any one of claims 149 to 178, wherein assessing the binding affinity of the barcoded antibody to the CoV-S protein comprises:
180. An isolated antibody identified by a method according to any one of claims 149 to 179.
181. A kit for identifying an antibody or antigen-binding fragment having binding affinity for a coronavirus spike protein (CoY-S), the kit comprising:
(a) a plurality of CoV-S antigens and non-CoV-S antigens, and wherein each of the antigens comprise a reporter oligonucleotide comprising (i) a reporter sequence that identifies the antigen and (ii) a capture handle sequence; and
(b) instructions for performing the method of any one of claims 148 to 179.
182. A kit for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the kit comprising instructions for use thereof and one or more of the following: a) an antibody or antigen-binding fragment of any one of claims lto 117; b) a recombinant nucleic acid according to any one of claims 118 to 123; or a vector of any one of claims 124-126; and c) a recombinant cell according to any one of claims 127-128.
183. A kit for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the kit comprising instructions for use thereof and a pharmaceutical composition of any one of claims 134 to 140.
184. A kit for (i) for producing an antibody or antigen-binding fragment thereof, (ii) detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, or (iii) treating, preventing, or ameliorating a health condition associated with SARS-CoV-2 infection in a subject, the kit comprising instructions for use thereof and one or more of the following: a) an antibody or antigen-binding fragment of any one of claims 1 to 117; b) a pharmaceutical composition of any one of claims 134 to 140; c) a recombinant nucleic acid according to any one of claims 118 to 123; or a vector of any one of claims 124-126; and d) a recombinant cell according to any one of claims 127-128.
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