WO2022221314A1 - Compositions and methods involving severe acute respiratory syndrome coronavirus 2 receptor binding domain - Google Patents

Compositions and methods involving severe acute respiratory syndrome coronavirus 2 receptor binding domain Download PDF

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WO2022221314A1
WO2022221314A1 PCT/US2022/024471 US2022024471W WO2022221314A1 WO 2022221314 A1 WO2022221314 A1 WO 2022221314A1 US 2022024471 W US2022024471 W US 2022024471W WO 2022221314 A1 WO2022221314 A1 WO 2022221314A1
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sequence
seq
antibody
nos
identity
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PCT/US2022/024471
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French (fr)
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Daniel Kim
Mong-Shang LIN
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BioLegend, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5- 9; and
  • the disclosure features an expression vector comprising the nucleic acid described above.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Specificity can be determined using standard methods, e.g, solid-phase ELISA immunoassays (see, e.g, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22 and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:46. .
  • the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts.
  • a humanized antibody i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., PNAS USA , 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol ., 44:65-92 (1988); Verhoeyen et al., Science , 239:1534-1536 (1988); Padlan, Molec. Immun ., 28:489-498 (1991); Padlan, Molec.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more substantial changes may be introduced and the products may be screened. Examples of substitutions are listed below:
  • the sample barcode sequence is a particular barcode that can be unique relative to other barcodes.
  • Methods for conjugating or hybridizing an oligonucleotide label can be performed in a manner set forth in WO/2018/144813, WO/2017/018960, WO/2018/089438, WO/2014/182528, WO/2018/026873, WO/2021/188838.
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) can also be suitable. These examples are illustrative rather than limiting.
  • a solid support or substrate can be any physically separable solid to which an anti-SARS-CoV-2 RBD antibody can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles.
  • antibodies and methods for detecting SARS-CoV-2 RBD are provided herein.
  • the biological sample is a solid tissue, fluid, or cell.
  • the solid tissue may comprise solid tissue from one or more of adipose tissue, bladder, bone, brain breast cervix, endothelium, gallbladder, kidney, liver, lung, lymph, ovary, prostate, salivary gland, stomach, testis, thyroid, urethra, uterus, vagina, and vulva.
  • the fluid comprises one or more of amniotic fluid, bile, blood, breast milk, breast fluid, cerebrospinal fluid, lavage fluid, lymphatic fluid, mucous, plasma, saliva, semen, serum, spinal fluid, sputum, tears, umbilical cord blood, urine, and vaginal fluid.
  • an HCDR3 having the sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO:66), wherein Xi is Y or absent; X2 is Y, R, A, or absent; X3 is G or absent; X4 is S, D, L or A; X5 is S, R, L, M, or Y; Xe is Y or N; X7 is G, D, R, or absent; Xs is G, Y, or absent; X9 is Y, T, S, or absent; X10 is F or V; X11 is D or A; and X12 is V or Y; and b) a light chain variable region comprising:
  • an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS: 1-4;
  • Xi is D, N, or S;
  • X 2 is I or W; and
  • X 3 is L or H in HCDR1;
  • a method of diagnosing a disease or disorder comprising: a) isolating a sample from a subject; b) incubating the sample with the antibody of any one of embodiments 1 to 99, for a period of time sufficient to generate SARS-CoV-2 RBD:anti- SARS-CoV-2 RBD complexes; c) detecting the presence or absence of the SARS-COV-2 RBD:anti-SARS- COV-2 RBD complexes from the isolated tissue; and d) associating presence or abundance of SARS-COV-2 RBD with a location of interest of a tissue sample.

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Abstract

Compositions and methods comprising severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD) antibodies are provided.

Description

COMPOSITIONS AND METHODS INVOLVING SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 RECEPTOR-BINDING DOMAIN
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Application No. 63/175,758, filed April 16, 2021, entitled “COMPOSITIONS AND METHODS INVOLVING SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 RECEPTOR-BINDING DOMAIN”, the contents of which are hereby incorporated by reference in their entirety for all purposes.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “BLD018PCT”, created on April 8, 2022, which is 93,439 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND
[0003] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, non-segmented, positive sense RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2 has four main structural proteins including spike glycoprotein (S), small envelope glycoprotein (E), membrane glycoprotein (M), and nucleocapsid protein (N), and also several accessory proteins. The spike glycoprotein, a transmembrane protein found on the outer portion of the virus, facilitates binding of the enveloped viruses to angiotensin-converting enzyme 2 (ACE2), which is the functional receptor expressed on the cell surface of susceptible cells, e.g., cells in the lower respiratory tract. The spike glycoprotein is cleaved by the furin-like protease in the host cell into two subunits, SI and S2. The spike glycoprotein SI subunit contains a receptor-binding domain (RBD) that specifically recognizes ACE2.
[0004] There is a need to better understand the cell entry mechanism of SARS-CoV-2. Described herein are particular monoclonal antibodies to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), including anti-SARS-CoV-2 RBD antibodies, SARS-CoV-2 RBD-binding antibody fragments, derivatives, and variants of such antibodies and antibody fragments (including immunoconjugates, labeled antibodies, antigen-binding antibody fragments, and the like), diagnostic reagents that comprise such antibodies, containers and kits including such antibodies, and methods of making and using the same.
BRIEF SUMMARY
[0005] In one aspect, the disclosure features an isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody comprises: a) a heavy chain variable region comprising:
(i) a heavy chain complementary determining region 1 (HCDR1) having the sequence of X1YX2MX3 (SEQ ID NO:64), wherein Xi is D, E, N, or S; X2 is I, T, or W; and X3 is L or H;
(ii) an HCDR2 having the sequence of X1IX2PX3X4X5X6TX7X8NX9KFKX10 (SEQ ID NO:65), wherein Xi is N, G, M, or E; X2 is N or D; X3 is Y, N, or S; X4 is Y, N, or D; X5 is G, V, or S; Xe is S, D, E, or R; X7 is S, N, or R; Xs is Y or L; X9 is L, Q, or E; and X10 is G, D, or S;
(iii) an HCDR3 having the sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO:66), wherein Xi is Y or absent; X2 is Y, R, A, or absent; X3 is G or absent; X4 is
S, D, L or A; X5 is S, R, L, M, or Y; Xe is Y or N; X7 is G, D, R, or absent; Xs is G, Y, or absent; X9 is Y, T, S, or absent; X10 is F or V; X11 is D or A; and X12 is V or Y; and b) a light chain variable region comprising:
(iv) a light chain complementary determining region 1 (LCDR1) having the sequence of X1X2SX3X4X5X6X7X8X9X1X11X12X13X14X15 (SEQ ID NO:67), wherein Xi is S or R; X2 is A or S; X3 is S, E, K, Q, or T; X4 is S, E, or G; X5 is L or absent; Xe is V, L, or absent; X7 is D, H, A, or absent; Xx is N, S, V, or absent; X9 is Y, N, T, or absent; X10 is G, I,
T, or absent; X11 is L, I, N, or S; X12 is N, S, T, or G; X13 is F or Y; X14 is M, L, or A; and X15 is Y, N, or S;
(v) a LCDR2 having the sequence of X1X2X3X4X5X6X7 (SEQ ID NO:68), wherein Xi is L, A, R, or G; X2 is T, A, or M; X3 is S or N; X4 is N or T; X5 is L, Q, or R; Xe is A, G, or D; and X7 is S or P; and
(vi) a LCDR3 having the sequence of X1X2X3X4X5X6X7X8X9 (SEQ ID NO:69), wherein Xi is Q, M, L, or A; X2 is Q or L; X3 is W, S, H, or Y; X4 is S, K, L, A, or Y; Xs is T, E, or S; Xe is N, V, or Y; X7 is P or H; X8 is L, Y, or W; and X9 is T or V. [0006] In some embodiments, the antibody comprises:
(a) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 1- 4;
(b) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5- 9; and
(c) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:10-14.
[0007] In some embodiments, the antibody comprises:
(1) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:23-28;
(2) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:29-33; and
(3) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:34-38.
[0008] In some embodiments, the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 1- 4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5-
9;
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:10-14;
(4) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:23-28;
(5) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:29-33; and
(6) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:34-38.
[0009] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO:7, and a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36.
[0010] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO:7, and a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:28, a LCDR2 having the sequence of SEQ ID NO:33, and a LCDR3 having the sequence of SEQ ID NO:38.
[0011] In some embodiments, the heavy chain variable region comprises at least 90% identity to the sequence set forth in any one of SEQ ID NOS: 15-22. In some embodiments, the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO:22.
[0012] In some embodiments, the light chain variable region comprises at least 90% identity to a sequence set forth in any of SEQ ID NOS:39-46. In some embodiments, the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:45. In some embodiments, the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:46.
[0013] In some embodiments, the heavy chain variable region comprises at least 90% identity to the sequence set forth in any one of SEQ ID NOS: 15-22, and wherein the light chain variable region comprises at least 90% identity to the sequence set forth in any of SEQ ID NOS:39-46. In some embodiments, the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:22, and the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:45. In some embodiments, the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:22, and the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:46.
[0014] In some embodiments, the antibody comprises an antigen-binding fragment thereof. In some embodiments, the antibody comprises an Fc polypeptide having at least 90% identity to a sequence of SEQ ID NO:47.
[0015] In another aspect, the disclosure features an isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody competes binding to the SARS-CoV-2 RBD with an antibody described herein.
[0016] In some embodiments, the antibody binds to the same epitope as the antibody described herein.
[0017] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody comprises one or more human framework regions. In some embodiments, the antibody is conjugated to a detectable marker or label. In some embodiments, the antibody is non-diffusively immobilized on a solid support.
[0018] In another aspect, the disclosure features an isolated nucleic acid encoding the isolated antibody described herein.
[0019] In another aspect, the disclosure features an isolated nucleic acid comprising a nucleotide sequence that encodes a heavy chain variable domain comprising at least 90% sequence identity to the sequence set forth in any of SEQ ID NOS: 70-79 and 86. [0020] In another aspect, the disclosure features an isolated nucleic acid comprising a nucleotide sequence that encodes a light chain variable domain comprising at least 90% sequence identity to the sequence set forth in any of SEQ ID NOS: 80-85 and 87-93.
[0021] In another aspect, the disclosure features an expression vector comprising the nucleic acid described above.
[0022] In another aspect, the disclosure features an isolated host cell comprising the vector described above.
[0023] In another aspect, the disclosure features a pharmaceutical composition comprising the isolated antibody described herein and a pharmaceutically acceptable carrier.
[0024] In another aspect, the disclosure features a diagnostic reagent comprising the isolated antibody described herein.
[0025] In another aspect, the disclosure features a kit comprising the isolated antibody described herein or the diagnostic reagent described herein.
[0026] In another aspect, the disclosure features a method of detecting severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), comprising: a) contacting a sample with the antibody described herein, under conditions to bind said antibody to a SARS-CoV-2 RBD receptor on said sample, wherein the binding generates the production of a receptor/antibody complex; and b) detecting the presence of the receptor/antibody complexes, wherein the detecting comprises the presence or absence of the SARS-CoV-2 RBD receptor on said sample.
[0027] In another aspect, the disclosure features a method of treating or preventing a disease or disorder associated with severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD) in a subject, comprising: a) contacting a sample known or suspected to contain SARS-CoV-2 RBD with the antibody described herein; b) detecting the presence of complexes comprising SARS-CoV-2 RBD and the antibody; wherein the presence of the complexes indicates the presence of a disease or disorder; and c) administering to the subject the antibody described herein or antigen binding fragment thereof. [0028] In another aspect, the disclosure features a method of diagnosing a disease or disorder, comprising: a) isolating a sample from a subject; b) incubating the sample with the antibody described herein, for a period of time sufficient to generate SARS-CoV-2 RBD:anti- SARS-CoV-2 RBD complexes; c) detecting the presence or absence of the SARS-COV-2 RBD:anti-SARS- COV-2 RBD complexes from the isolated tissue; and d) associating presence or abundance of SARS-CoV-2 RBD with a location of interest of a tissue sample.
[0029] In some embodiments, the method is performed in vitro.
[0030] In some embodiments of the methods described herein, the detection comprises hybridization of a detectable moiety to the antibody. In some embodiments, the detectable moiety comprises an oligonucleotide. In some embodiments, the detectable moiety comprises a fluorescent label. In some embodiments of the methods described herein, the sample comprises a cell. In some embodiments, the sample comprises a tissue sample.
DETAILED DESCRIPTION
I Introduction
[0031] The inventors have discovered new antibodies that bind to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD). The RBD of SARS- CoV-2 is located on the SI subunit of the spike glycoprotein, which can be cleaved by the furin-like protease in the host cell into SI and S2 subunits. The disclosure provides antibodies and antigen-binding fragments thereof that can bind to SARS-CoV-2 RBD under physiological and/or in vitro conditions. Such antibodies and antigen-binding fragments thereof can be useful for laboratory/ research purposes ( e.g flow cytometry, ELISA, and/or Western blot), and can be used in treatment and/or prevention of various diseases or disorders through the delivery of pharmaceutical or other compositions that contain such antibodies.
II. Definitions
[0032] The term "antibody" as used herein includes antibody fragments that retain binding specificity. For example, there are a number of well characterized antibody fragments. Thus, for example, pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.
[0033] An antibody as described herein can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. In some embodiments, the antibody is IgG (e.g, IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, or IgE.
[0034] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
[0035] In an antibody, substitution variants have at least one amino acid residue removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework alterations are also contemplated. Examples of conservative substitutions are described above.
[0036] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a b-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) Non-polar: Norleucine, Met, Ala, Val, Leu, lie;
(2) Polar without charge: Cys, Ser, Thr, Asn, Gin;
(3) Acidic (negatively charged): Asp, Glu;
(4) Basic (positively charged): Lys, Arg;
(5) Residues that influence chain orientation: Gly, Pro; and
(6) Aromatic: Trp, Tyr, Phe, His.
Non-conservative substitutions are made by exchanging a member of one of these classes for another class.
[0037] One type of substitution that can be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there can be a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody. In some embodiments, the cysteine is canonical ( e.g ., involved in di sulfide bond formation). Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.
[0038] Antibodies include VH-VL dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker (e.g., Huston, el al. Proc. Nat. Acad. Sci. USA , 85:5879-5883, 1988). While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently. Alternatively, the antibody can be another fragment. Other fragments can also be generated, e.g, using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies can also include diantibodies and miniantibodies. Antibodies of the disclosure also include heavy chain dimers, such as antibodies from camelids. In some embodiments an antibody is dimeric. In other embodiments, the antibody may be in a monomeric form that has an active isotype. In some embodiments the antibody is in a multivalent form, e.g., a trivalent or tetravalent form.
[0039] As used herein, the terms “variable region” and “variable domain” refer to the portions of the light and heavy chains of an antibody that include amino acid sequences of complementary determining regions (CDRs, e.g., HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCDR3) and framework regions (FRs). The variable region for the heavy and light chains is commonly designated VH and VL, respectively. The variable region is included on Fab, F(ab’)2, Fv and scFv antibody fragments described herein, and involved in specific antigen recognition.
[0040] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDRl is the CDRl from the variable domain of the light chain of the antibody in which it is found.
[0041] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
[0042] The amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g, Rabat, North method (see, e.g, North et ak, J Mol Biol. 406(2):228-256, 2011), Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g, Johnson etal, supra ; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et ak, 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et ak, 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799- 817; Al-Lazikani et ah, J.Mol.Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et ak, IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol. , 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996).
[0043] As used herein, "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigen specificity; or with corresponding sequences from another species or from another antibody class or subclass.
[0044] As used herein, "humanized antibody" refers to an immunoglobulin molecule in CDRs from a donor antibody are grafted onto human framework sequences. Humanized antibodies may also comprise residues of donor origin in the framework sequences. The humanized antibody can also comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed using methods known in the art (e.g, Jones et al, Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-321 , 1988; Verhoeyen etal., Science 239:1534-1536, 1988); Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Patent No. 4,816,567), including techniques such as “superhumanizing" antibodies (Tan et al, J. Immunol. 169: 1119, 2002) and "resurfacing” (e.g, Staelens et al, Mol. Immunol. 43: 1243, 2006; and Roguska et al, Proc. Natl. Acad. Sci USA 91: 969, 1994).
[0045] The term “recombinant” when used with reference, e.g, to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. [0046] The terms “antigen,” “immunogen,” “antibody target,” “target analyte,” and like terms are used herein to refer to a molecule, compound, or complex that is recognized by an antibody, i.e., can be specifically bound by the antibody. The term can refer to any molecule that can be specifically recognized by an antibody, e.g., a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g, phosphorylated or glycosylated polypeptides, etc.). One of skill will understand that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody.
[0047] Antibodies bind to an “epitope” on an antigen. The epitope is the localized site on the antigen that is recognized and bound by the antibody. Epitopes can include a few amino acids or portions of a few amino acids, e.g, 5 or 6, or more, e.g, 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g, from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three- dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g, a discontinuous epitope). The same is true for other types of target molecules that form three-dimensional structures. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
[0048] A “label” or a “detectable moiety” is a diagnostic agent or component detectable by spectroscopic, radiological, photochemical, biochemical, immunochemical, chemical, or other physical means. Exemplary labels include radiolabels (e.g, U1ln, 99mTc, 131I, 67Ga) and other FDA-approved imaging agents. Additional labels include 32P, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g, by incorporating a radiolabel into the targeting agent. Any method known in the art for conjugating a nucleic acid or nanocarrier to the label may be employed, e.g, using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.
[0049] A “labeled” or “tagged” antibody or agent is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the antibody or agent may be detected by detecting the presence of the label bound to the antibody or agent.
[0050] Techniques for conjugating detectable and therapeutic agents to antibodies are well known (see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery"in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody- Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)).
[0051] The terms “specific for,” “specifically binds,” and like terms refer to a molecule (e.g, antibody or antibody fragment) that binds to a target with at least 2-fold greater affinity than non-target compounds, e.g, at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity. For example, an antibody that specifically binds a target (e.g, SARS-CoV-2 RBD) will typically bind the target with at least a 2-fold greater affinity than a non-target. Specificity can be determined using standard methods, e.g, solid-phase ELISA immunoassays (see, e.g, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
[0052] The term “binds” with respect to an antibody target (e.g, antigen, analyte, immune complex), typically indicates that an antibody binds a majority of the antibody targets in a pure population (assuming appropriate molar ratios). For example, an antibody that binds a given antibody target typically binds to at least 2/3 of the antibody targets in a solution (e.g, at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). One of skill will recognize that some variability will arise depending on the method and/or threshold of determining binding.
[0053] A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g, in the presence of a test compound, and compared to samples from known conditions, e.g, in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value or a range gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data ( e.g ., half-life) or therapeutic measures (e.g., comparison of benefit and/or side effects). Controls can be designed for in vitro applications. One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.
[0054] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 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 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g, NCBI web site ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 or more amino acids or nucleotides in length.
[0055] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0056] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art.
[0057] An algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0058] The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0059] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof ( e.g ., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
[0060] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms encompass to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
[0061] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0062] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. [0063] The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen-binding portion thereof, competes for binding with a second antibody, or an antigen-binding portion thereof, where binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross competition occurs ( e.g steric hindrance, conformational change, or binding to a common epitope, or portion thereof, and the like), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.
[0064] Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al, Methods in Enzymology 9:242-253 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al. , J. Immunol. 137:3614-3619 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel etal., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct biotin-avidin EIA (Cheung et al, Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al, Scand. J. Immunol. 32:77-82 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50 or 75%.
III. Antibodies that bind SARS-CoV-2 RBI) and antigen-binding fragments thereof
[0065] Antibodies (including antibody fragments) that specifically bind to SARS-CoV-2 RBD are provided. For example, particular monoclonal antibodies to SARS-CoV-2 RBD that provide superior target specificity, signal-to-noise ratios, and the like as compared to other reported anti-SARS-CoV-2 RBD antibodies, as well as antigen-binding fragments of such antibodies, are described herein. Also provided herein are methods for producing anti-SARS- CoV-2 RBD antibodies and methods for detecting SARS-CoV-2 RBD using such antibodies.
[0066] In some embodiments, the anti-SARS-CoV-2 RBD antibody is isolated ( e.g ., separated from a component of its natural environment (e.g., an animal, a biological sample)). In some embodiments, the anti-SARS-CoV-2 RBD antibody is a humanized antibody, or an antigen binding fragment thereof. In some embodiments, the anti-SARS-CoV-2 RBD antibody is a derivative of a humanized antibody that binds SARS-CoV-2 RBD. In some embodiments, the anti-SARS-CoV-2 RBD antibody binds SARS-CoV-2 RBD under laboratory conditions (e.g, binds SARS-CoV-2 RBD in vitro, binds SARS-CoV-2 RBD in a flow cytometry assay, binds SARS-CoV-2 RBD in an ELISA). In some embodiments, the anti-SARS-CoV-2 RBD antibody binds SARS-CoV-2 RBD under physiological conditions (e.g, binds SARS-CoV-2 RBD in a cell in a subject).
[0067] Generally, the anti-SARS-CoV-2 RBD antibodies provided herein comprise at least one immunoglobulin heavy chain variable region and at least one immunoglobulin light chain variable region. In some embodiments, an anti-SARS-CoV-2 RBD antibody described herein comprises two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions. Typically, each immunoglobulin heavy chain variable region of the anti-SARS-CoV-2 RBD antibody comprises first, second, and third heavy chain complementarity determining regions (CDRs; HCDR1, HCDR2, and HCDR3), and each immunoglobulin light chain variable region of the anti-SARS-CoV-2 RBD antibody comprises first, second, and third light chain CDRs (LCDR1, LCDR2, and LCDR3).
[0068] In some embodiments, the antibodies are antibody fragments such as Fab, F(ab’)2, Fv or scFv. The antibody fragments can be generated using any means known in the art including, chemical digestion (e.g, papain or pepsin) and recombinant methods. Methods for isolating and preparing recombinant nucleic acids are known to those skilled in the art (see, Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). The antibodies can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, and HeLa cells lines and myeloma cell lines.
[0069] In some embodiments, antibodies of the disclosure can comprise sequences of a heavy chain complementary determining region 1 (HCDR1), an HCDR2, an HCDR3, a light chain complementary determining region 1 (LCDR1), a LCDR2, a LCDR3, a heavy chain variable region (VH), and/or a light chain variable region (VL) as described in Table 1.
Table 1
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
[0070] The disclosure provides an isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody comprises: (1) a heavy chain complementary determining region 1 (HCDR1) having the sequence of X1YX2MX3 (SEQ ID NO:64), wherein Xi is D, E, N, or S; X2 is I, T, or W; and X3 is L or H; (2) an HCDR2 having the sequence of X1IX2PX3X4X5X6TX7X8NX9KFKX10 (SEQ ID NO:65), wherein Xi is N, G, M, or E; X2 is N or D; X3 is Y, N, or S; X4 is Y, N, or D; X5 is G, V, or S; Xe is S, D, E, or R; Xv is S, N, or R; X8 is Y or L; X9 is L, Q, or E; and X10 is G, D, or S; (3) an HCDR3 having the sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO:66), wherein Xi is Y or absent; X2 is Y, R, A, or absent; X3 is G or absent; X4 is S, D, L or A; X5 is S, R, L, M, or Y; Xe is Y or N; X7 is G, D, R, or absent; Xs is G, Y, or absent; X9 is Y, T, S, or absent; X10 is F or V; X11 is D or A; and X12 is V or Y; (4) a light chain complementary determining region 1 (LCDR1) having the sequence of X1X2SX3X4X5X6X7X8X9X1X11X12X13X14X15 (SEQ ID NO:67), wherein Xi is S or R; X2 is A or S; X3 is S, E, K, Q, or T; X4 is S, E, or G; X5 is L or absent; Xe is V, L, or absent; X7 is D, H, A, or absent; Xs is N, S, V, or absent; X9 is Y, N, T, or absent; X10 is G, I, T, or absent; Xu is L, I, N, or S; X12 is N, S, T, or G; X13 is F or Y; X14 is M, L, or A; and X15 is Y, N, or S; (5) a LCDR2 having the sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 68), wherein Xi is L, A, R, or G; X2 is T, A, or M; X3 is S or N; X4 is N or T; X5 is L, Q, or R; Xe is A, G, or D; and X7 is S or P; and (6) a LCDR3 having the sequence of X1X2X3X4X5X6X7X8X9 (SEQ ID NO:69), wherein Xi is Q, M, L, or A; X2 is Q or L; X3 is W, S, H, or Y; X is S, K, L, A, or Y; Xs is T, E, or S; Xe is N, V, or Y; X7 is P or H; X8 is L, Y, or W; and X9 is T or V.
[0071] In particular embodiments of the antibodies described herein, (1) Xi is D, N, or S; X2 is I or W; and X3 is L or H in HCDR1; (2) Xi is N or M; X2 is N or D; X3 is Y or S; X4 is Y or D; X5 is G or S; Cb is S or E; X7 is S or R; Xs is Y or L; X9 is L or Q; and X10 is G or D in HCDR2; (3) Xi is Y or absent; X2 is Y or R; X3 is G; X4 is S or L; X5 is S or L; Xe is Y or N; X7 is G or absent; Xs is G or absent; X9 is Y or absent; X10 is F; X11 is D; and X12 is V or Y; (4) Xi is S or R; X2 is A or S; X3 is S or T; X4 is S or G; X5 is absent; Cb is absent; X7 is A or absent; Xs is V or absent; X9 is T or absent; X10 is T or absent; X11 is L or S; X12 is N; X13 is F or Y; Xi4 is M or A; and X15 is Y or N in LCDR1 ; (5) Xi is L or G; X2 is T; X3 is S or N; X4 is N; Xs is L or R; Xe is A; and X7 is S or P in LCDR2; (6) Xi is Q or A; X2 is Q or L; X3 is W; X4 is S or Y; X5 is T or S; Xe is N; X7 is P; Xs is L or W; and X9 is T or V in LCDR3.
[0072] In some embodiments, an antibody of the disclosure comprises: (1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a variant thereof that has a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS: 1-4; (2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to a sequence of any one of SEQ ID NOS:5-9; (3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a variant thereof that has a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS: 10-14; (4) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a variant thereof that has a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS:23-28; (5) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a variant thereof that has a sequence having one substitution relative to a sequence of any one of SEQ ID NOS:29-33; and (6) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:34-38.
[0073] In some embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO: 1 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 1, an HCDR2 having the sequence of SEQ ID NO: 5 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO:5, and an HCDR3 having the sequence of SEQ ID NO: 10 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 10. In some embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:2 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:2, an HCDR2 having the sequence of SEQ ID NO:6 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO: 6, and an HCDR3 having the sequence of SEQ ID NO: 11 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 11. In some embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO: 3 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO: 7 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO:7, and an HCDR3 having the sequence of SEQ ID NO: 12 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 12. In some embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:3 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO: 7 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO: 7, and an HCDR3 having the sequence of SEQ ID NO: 13 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 13. In further embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO: 3 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO: 8 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO:8, and an HCDR3 having the sequence of SEQ ID NO: 14 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 14. In further embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:l or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:l, an HCDR2 having the sequence of SEQ ID NO: 9 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO: 9, and an HCDR3 having the sequence of SEQ ID NO: 10 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 10. In further embodiments, an antibody of the disclosure can comprise an HCDR1 having the sequence of SEQ ID NO:4 or a variant thereof that has a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:4, an HCDR2 having the sequence of SEQ ID NO: 7 or a variant thereof that has a sequence having one, two, or three amino acid substitutions relative to the sequence of SEQ ID NO:7, and an HCDR3 having the sequence of SEQ ID NO: 12 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 12.
[0074] An antibody of the disclosure can comprise a heavy chain variable region (VH) having an HCDR1, an HCDR2, and an HCDR3 as described herein. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:l, 5, and 10, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 15. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:2, 6, and 11, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 16. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 7, and 12, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 17. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 7, and 13, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 18. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 8, and 14, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 19. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:l, 9, and 10, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:20. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:l, 9, and 10, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:21. In certain embodiments, an antibody of the disclosure can comprise a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:4, 7, and 12, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:22.
[0075] In some embodiments, an antibody of the disclosure can comprise an LCDR1 having the sequence of SEQ ID NO:23 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:23, an LCDR2 having the sequence of SEQ ID NO:29 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:29, and an LCDR3 having the sequence of SEQ ID NO:34 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:34. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:24 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:24, a LCDR2 having the sequence of SEQ ID NO:30 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:30, and a LCDR3 having the sequence of SEQ ID NO:35 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:35. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:25 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:25, a LCDR2 having the sequence of SEQ ID NO: 31 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:36. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:26 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO:31 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:36. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:27 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:27, a LCDR2 having the sequence of SEQ ID NO:32 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:32, and a LCDR3 having the sequence of SEQ ID NO:37 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:37. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:23 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:23, a LCDR2 having the sequence of SEQ ID NO:29 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:29, and a LCDR3 having the sequence of SEQ ID NO:34 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:34. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:26 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO:31 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:36. In some embodiments, an antibody of the disclosure can comprise a LCDR1 having the sequence of SEQ ID NO:28 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:28, a LCDR2 having the sequence of SEQ ID NO:33 or a variant thereof that has a sequence having one amino acid substitutions relative to the sequence of SEQ ID NO:33, and a LCDR3 having the sequence of SEQ ID NO:38 or a variant thereof that has a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:38.
[0076] An antibody of the disclosure can comprise a light chain variable region (VL) having a LCDR1, a LCDR2, and a LCDR3 as described herein. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 29, and 34, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:39. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:24, 30, and 35, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:40. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:25, 31, and 36, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:41. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:26, 31, and 36, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:42. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:27, 32, and 37, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:43. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 29, and 34, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:44. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:26, 31, and 36, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:45. In certain embodiments, an antibody of the disclosure can comprise a light chain variable region having a LCDR1, aLCDR2, and aLCDR3 of SEQ IDNOS:28, 33, and 38, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:46.
[0077] In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof described herein comprises: a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; and a CDRH3 comprising the sequence of SEQ ID NO: 12.
[0078] In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof described herein comprises: a CDRLl comprising the sequence of SEQ ID NO:26 or 28; a CDRL2 comprising the sequence of SEQ ID NO:31 or 33; and a CDRL3 comprising the sequence of SEQ ID NO:36 or 38.
[0079] In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof described herein comprises: a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; and a CDRH3 comprising the sequence of SEQ ID NO: 12; a CDRLl comprising the sequence of SEQ ID NO:26 or 28; a CDRL2 comprising the sequence of SEQ ID NO:31 or 33; and a CDRL3 comprising the sequence of SEQ ID NO:36 or 38.
[0080] In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof described herein comprises: a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; and a CDRH3 comprising the sequence of SEQ ID NO: 12; a CDRLl comprising the sequence of SEQ ID NO:26; a CDRL2 comprising the sequence of SEQ ID NO:31; and a CDRL3 comprising the sequence of SEQ ID NO:36.
[0081] In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof described herein comprises: a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; and a CDRH3 comprising the sequence of SEQ ID NO: 12; a CDRLl comprising the sequence of SEQ ID NO:28; a CDRL2 comprising the sequence of SEQ ID NO:33; and a CDRL3 comprising the sequence of SEQ ID NO:38.
[0082] An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein may comprise a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22. In some embodiments, any of the anti-SARS-CoV- 2 RBD antibody or antigen-binding fragment thereof provided herein may comprise a VH comprising the sequence of SEQ ID NO:22. In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; a CDRH3 comprising the sequence of SEQ ID NO: 12; and a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22.
[0083] An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein may comprise a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:45. In some embodiments, any of the anti-SARS-CoV- 2 RBD antibody or antigen-binding fragment thereof provided herein may comprise a VL comprising the sequence of SEQ ID NO:45. In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRLl comprising the sequence of SEQ ID NO:26; a CDRL2 comprising the sequence of SEQ ID NO:31; a CDRL3 comprising the sequence of SEQ ID NO:36; and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:45.
[0084] An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein may comprise a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:46. In some embodiments, any of the anti-SARS-CoV- 2 RBD antibody or antigen-binding fragment thereof provided herein may comprise a VL comprising the sequence of SEQ ID NO:46. In some embodiments, an anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRLl comprising the sequence of SEQ ID NO:28; a CDRL2 comprising the sequence of SEQ ID NO:33; a CDRL3 comprising the sequence of SEQ ID NO:38; and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:46.
[0085] An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22 and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:45. In some of any embodiments, any of the anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a VH comprising the sequence of SEQ ID NO:22 and a VL comprising a sequence the sequence of SEQ ID NO:45.
[0086] An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22 and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:46. . In some of any embodiments, any of the anti-SAR.S-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a VH comprising the sequence of SEQ ID NO:22 and a VL comprising a sequence the sequence of SEQ ID NO:46.
[0087] An anti-SAR.S-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; a CDRH3 comprising the sequence of SEQ ID NO: 12; and a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22; and a CDRL1 comprising the sequence of SEQ ID NO:26; a CDRL2 comprising the sequence of SEQ ID NO:31; a CDRL3 comprising the sequence of SEQ ID NO:36; and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:45. An anti-SAR.S-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; a CDRH3 comprising the sequence of SEQ ID NO: 12; and a VH comprising the sequence of SEQ ID NO:22; and a CDRL1 comprising the sequence of SEQ ID NO:26; a CDRL2 comprising the sequence of SEQ ID NO:31; a CDRL3 comprising the sequence of SEQ ID NO:36; and a VL comprising the sequence of SEQ ID NO:45. [0088] An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; a CDRH3 comprising the sequence of SEQ ID NO: 12; and a VH comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:22; and a CDRLl comprising the sequence of SEQ ID NO:28; a CDRL2 comprising the sequence of SEQ ID NO:33; a CDRL3 comprising the sequence of SEQ ID NO:38; and a VL comprising a sequence having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:46. An anti-SARS-CoV-2 RBD antibody or antigen-binding fragment thereof provided herein comprises a CDRH1 comprising the sequence of SEQ ID NO:4; a CDRH2 comprising the sequence of SEQ ID NO:7; a CDRH3 comprising the sequence of SEQ ID NO: 12; and a VH comprising the sequence of SEQ ID NO:22; and a CDRLl comprising the sequence of SEQ ID NO:28; a CDRL2 comprising the sequence of SEQ ID NO:33; a CDRL3 comprising the sequence of SEQ ID NO:38; and a VL comprising the sequence of SEQ ID NO:46.
AB1
[0089] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO: 1 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:l; (2) an HCDR2 having the sequence of SEQ ID NO:5 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:5; (3) an HCDR3 having the sequence of SEQ ID NO: 10 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 10; (4) a LCDR1 having the sequence of SEQ ID NO:23 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:29 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:29; and (6) a LCDR3 having the sequence of SEQ ID NO:34 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:34. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDRl having the sequence of SEQ ID NO:l; (2) an HCDR2 having the sequence of SEQ ID NO:5; (3) an HCDR3 having the sequence of SEQ ID NO: 10; (4) a LCDR1 having the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:29; and (6) a LCDR3 having the sequence of SEQ ID NO:34. [0090] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS: 1, 5, and 10, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 15, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 29, and 34, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:39. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0091] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48:
EIQLQQTGPELVKPGASVKISCKASGYSFTDYIMLWVKQSHGKSLEWIGNINPYYGST NYNLKFKGK ATLT VDKS S ST AYMQLN SLT SED S AVYY C ARYY GS S Y GGYFD VW GA GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKT HT CPPCP APELLGGP S VFLFPPKPKDTLMISRTPE VT C VVVD V SHEDPEVKFNW YVD GVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCK V SNK ALP APIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPP VLD SDGSFFL Y SKLT VDK SRWQQGNVF S C S VMHE ALHNH YT QK SL SL SPGK, and a light chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49:
Q A V VT QES ALTT SPGET VTLTCRS S T GA VTT SN Y ANW V QEKPDHLF T GLIGGTNNRA PGVP ARE SGSLIGDKAALTITGAQTEDEAIYF C ALWY SNHWVF GGGTKLTVLRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK D S T Y SL S STLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0092] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 15 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:39 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49. AB2
[0093] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:2 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:2; (2) an HCDR2 having the sequence of SEQ ID NO:6 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:6; (3) an HCDR3 having the sequence of SEQ ID NO: 11 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 11; (4) a LCDR1 having the sequence of SEQ ID NO:24 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:24; (5) a LCDR2 having the sequence of SEQ ID NO:30 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:30; and (6) a LCDR3 having the sequence of SEQ ID NO:35 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:35. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:2; (2) an HCDR2 having the sequence of SEQ ID NO:6; (3) an HCDR3 having the sequence of SEQ ID NO:l l; (4) aLCDRl having the sequence of SEQ ID NO:24; (5) a LCDR2 having the sequence of SEQ ID NO: 30; and (6) a LCDR3 having the sequence of SEQ ID NO:35.
[0094] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:2, 6, and 11, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 16, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:24, 30, and 35, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:40. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0095] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:50:
EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNNV DTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDRYDYTVDYWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCP APELLGGP S VFLFPPKPKDTLMISRTPE VT C VVVD V SHEDPEVKFNW YVDGVE VHNAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 51 :
DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVP KRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPWTFGGGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS L S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0096] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 16 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:50; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:40 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:51.
AB3
[0097] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO: 7 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 12 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 12; (4) a LCDR1 having the sequence of SEQ ID NO:25 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:25; (5) a LCDR2 having the sequence of SEQ ID NO:31 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:31; and (6) a LCDR3 having the sequence of SEQ ID NO:36 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:36. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 12; (4) a LCDR1 having the sequence of SEQ ID NO:25; (5) a LCDR2 having the sequence of SEQ ID NO:31; and (6) a LCDR3 having the sequence of SEQ ID NO:36. [0098] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 17, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:25, 31, and 36, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:41. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0099] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 52:
QVQLQQSGPQLVRPGASVKISCKASGYSFTSYWMHWVKQRPGQGLEWIGMIDPSDS ETRLN QKFKDK ATLT VDK S S S T A YMQL S SPT SED S A V Y Y C ARRGLLNFD YW GQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF P AVLQ S SGL Y SL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53:
QIVLT Q SP ALMS ASPGEK VTMT C S AS S SLNYMYW Y QQKPRS SPKPWI YLT SNL ASGV PARFSGSGSGTSYSLTINSMEAEDAATYYCQQWSTNPLTFGAGTKLELKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0100] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 17 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:41 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53. AB4
[0101] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO: 7 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 13 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 13; (4) a LCDR1 having the sequence of SEQ ID NO:26 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:26; (5) a LCDR2 having the sequence of SEQ ID NO:31 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:31; and (6) a LCDR3 having the sequence of SEQ ID NO:36 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:36. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 13; (4) a LCDR1 having the sequence of SEQ ID NO:26; (5) a LCDR2 having the sequence of SEQ ID NO:31; and (6) a LCDR3 having the sequence of SEQ ID NO:36.
[0102] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 7, and 13, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 18, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:26, 31, and 36, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:42. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0103] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 54:
QVQLQQSGPQLVRPGASVKISCKASGYSFTSYWMHWVKQRPGQGLEWIGMIDPSDS ETRLN QKFKDK ATLT VDK S S S T A YMQL S SPT SED S A V Y Y C ARRGLMNFD YW GQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKRVEPK S CDKTHT CP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 55:
QIVLT Q SP ALMS ASPGEK VTMT C S AS S SLNFMYW Y QQKPRS SPKPWI YLT SNL ASGV PARFSGSGSGTSYSLTINSMEAEDAATYYCQQWSTNPLTFGAGTKLELKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0104] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 18 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:54; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:42 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55.
AB5
[0105] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO: 8 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:8; (3) an HCDR3 having the sequence of SEQ ID NO: 14 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 14; (4) a LCDR1 having the sequence of SEQ ID NO:27 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:27; (5) a LCDR2 having the sequence of SEQ ID NO:32 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:32; and (6) a LCDR3 having the sequence of SEQ ID NO:37 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:37. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:3; (2) an HCDR2 having the sequence of SEQ ID NO:8; (3) an HCDR3 having the sequence of SEQ ID NO: 14; (4) a LCDR1 having the sequence of SEQ ID NO:27; (5) a LCDR2 having the sequence of SEQ ID NO:32; and (6) a LCDR3 having the sequence of SEQ ID NO:37. [0106] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:3, 8, and 14, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 19, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:27, 32, and 37, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:43. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0107] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56:
QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSN GRTNYNEKFKSKATLTVDKS S STAYMQLS SLTSEDS AVYY C AS AGAYYRY SF AYWG QGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQS SGLY SLS S VVTVPSS SLGTQT YICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCK V SNK ALP APIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPP VLD SDGSFFL Y SKLT VDK SRWQQGNVF S C S VMHE ALHNH YT QK SL SL SPGK, and a light chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:57:
DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQ GSGVPARF SGSGSGTDF SLNIHPMEEDDTAMYF CQQSKEVP YTF GGGTKLEIKRT VA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KD S T YSL S S TLTL SK AD YEKHK V Y ACE VTHQ GL S SP VTK SFNRGEC .
[0108] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:43 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:57. AB6
[0109] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO: 1 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:l; (2) an HCDR2 having the sequence of SEQ ID NO:9 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:9; (3) an HCDR3 having the sequence of SEQ ID NO: 10 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 10; (4) a LCDR1 having the sequence of SEQ ID NO:23 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:29 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:29; and (6) a LCDR3 having the sequence of SEQ ID NO:34 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:34. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDRl having the sequence of SEQ ID NO:l; (2) an HCDR2 having the sequence of SEQ ID NO:9; (3) an HCDR3 having the sequence of SEQ ID NO: 10; (4) a LCDR1 having the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:29; and (6) a LCDR3 having the sequence of SEQ ID NO:34.
[0110] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS: 1, 9, and 10, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:20, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 29, and 34, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:39. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0111] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 58:
EIQLQQTGPELVKPGASVKISCKASGYSFTDYIMLWVKQSHGKSLEWIGNINPYYGST S YNLKFKGK ATLTVDKS S ST AYMQLN SLT SED S AV YY C ARYY GS S Y GGYFD VWGA GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKRVEPKSCDKT HT CPPCP APELLGGP S VFLFPPKPKDTLMISRTPE VT C VVVD V SHEDPEVKFNW YVD GVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCK V SNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPP VLD SDGSFFL Y SKLT VDK SRWQQGNVF S C S VMHE ALHNH YT QK SL SL SPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49:
Q A V VT QES ALTT SPGET VTLTCRS S T GA VTT SN Y ANW V QEKPDHLF T GLIGGTNNRA PGVPARF SGSLIGDKAALTITGAQTEDEAIYF CALWY SNHWVF GGGTKLTVLRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK D S T Y SL S STLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0112] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:20 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:58; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:39 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49.
AB7
[0113] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO: 1 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: l; (2) an HCDR2 having the sequence of SEQ ID NO:9 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:9; (3) an HCDR3 having the sequence of SEQ ID NO: 10 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 10; (4) a LCDR1 having the sequence of SEQ ID NO:23 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:29 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:29; and (6) a LCDR3 having the sequence of SEQ ID NO:34 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:34. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDRl having the sequence of SEQ ID NO:l; (2) an HCDR2 having the sequence of SEQ ID NO:9; (3) an HCDR3 having the sequence of SEQ ID NO: 10; (4) a LCDR1 having the sequence of SEQ ID NO:23; (5) a LCDR2 having the sequence of SEQ ID NO:29; and (6) a LCDR3 having the sequence of SEQ ID NO:34. [0114] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS: 1, 9, and 10, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:21, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:23, 29, and 34, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:44. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0115] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:59:
EIQMQQTGPELVKPGASVKISCKASGYSFTDYIMLWVKQSHGKSLEWIGNINPYYGS T S YNLKFKGK ATLT VDKS S ST AYMQLN SLTSED S AVYY C ARYY GS S Y GGYFD VW G AGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQS SGLY SLS S VVTVPSS SLGTQT YICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCK V SNK ALP APIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPP VLD SDGSFFL Y SKLT VDK SRWQQGNVF S C S VMHE ALHNH YT QK SL SL SPGK, and a light chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 60:
QTVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRA PGVP ARE SGSLIGDKAALTITGAQTEDEAIYF C ALWY SNHWVF GGGTKLTVLRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK D S T Y SL S STLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0116] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:21 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:59; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:44 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:60. AB8
[0117] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:4 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:4; (2) an HCDR2 having the sequence of SEQ ID NO:7 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 12 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 12; (4) a LCDR1 having the sequence of SEQ ID NO:26 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:26; (5) a LCDR2 having the sequence of SEQ ID NO:31 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:31; and (6) a LCDR3 having the sequence of SEQ ID NO:36 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:36. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:4; (2) an HCDR2 having the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 12; (4) a LCDR1 having the sequence of SEQ ID NO:26; (5) a LCDR2 having the sequence of SEQ ID NO:31; and (6) a LCDR3 having the sequence of SEQ ID NO:36.
[0118] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:4, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:22, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:26, 31, and 36, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:45. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0119] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:61 :
QVQLQQSGPQLVRPGASVKISCKASGYSFSNYWMHWVKQRPGQGLEWIGMIDPSDS ETRLN QKFKDK ATLT VDK S S S T A YMQL S SPT SED S A V Y Y C ARRGLLNFD YW GQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF P AVLQ S SGL Y SL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 62:
QIVLT Q SP ALMS ASPGEK VTMT C S AS S SLNFMYW Y QQKPRS SPKPWI YLT SNL ASGV PARE S GS GS GT S Y SLTIN CMEAED A AT Y Y C QQ W S TNPLTF GAGTKLELKRT V AAP S V FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0120] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:22 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:61; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:45 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:62.
AB9
[0121] In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:4 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:4; (2) an HCDR2 having the sequence of SEQ ID NO:7 or a sequence having one, two or three amino acid substitutions relative to the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 12 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO: 12; (4) a LCDR1 having the sequence of SEQ ID NO:28 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:28; (5) a LCDR2 having the sequence of SEQ ID NO:33 or a sequence having one amino acid substitution relative to the sequence of SEQ ID NO:33; and (6) a LCDR3 having the sequence of SEQ ID NO:38 or a sequence having one or two amino acid substitutions relative to the sequence of SEQ ID NO:38. In particular embodiments, an antibody of the disclosure can comprise: (1) an HCDR1 having the sequence of SEQ ID NO:4; (2) an HCDR2 having the sequence of SEQ ID NO:7; (3) an HCDR3 having the sequence of SEQ ID NO: 12; (4) a LCDR1 having the sequence of SEQ ID NO:28; (5) a LCDR2 having the sequence of SEQ ID NO:33; and (6) a LCDR3 having the sequence of SEQ ID NO:38. [0122] In some embodiments, the antibody can comprise (1) a heavy chain variable region having an HCDR1, an HCDR2, and an HCDR3 of SEQ ID NOS:4, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:22, and (2) a light chain variable region having a LCDR1, a LCDR2, and a LCDR3 of SEQ ID NOS:28, 33, and 38, respectively, and at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:46. Such an antibody can be an IgGl, IgG2, IgG3, or IgG4.
[0123] In certain embodiments, the antibody comprises a heavy chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:61 :
QVQLQQSGPQLVRPGASVKISCKASGYSFSNYWMHWVKQRPGQGLEWIGMIDPSDS ETRLN QKFKDK ATLT VDK S S S T A YMQL S SPT SED S A V Y Y C ARRGLLNFD YW GQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF P AVLQ S SGL Y SL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:63:
DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSN L ASGVPDRF SGS GS GT AF TLRI SRVE AED V GV Y Y CMQHLE YP YTF GGGTKLEIKRT V AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKD S T Y SL S STLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[0124] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:22 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:61; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:46 and a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:63. Nucleic acid sequences
[0125] Provided below are examples of VH nucleotide (NT) sequences related to the antibodies described herein.
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
[0126] Provided below are examples of VL nucleotide (NT) sequences related to the antibodies described herein.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
IV. Fc polypeptide
[0127] An anti-SARS-CoV-2 RBD antibody provided herein can comprise a fragment crystallizable region (Fc region), also referred to as an Fc polypeptide herein. An Fc polypeptide is part of each of the two heavy chains in the antibody and can interact with certain cell surface receptors and certain components of the complement system. An Fc polypeptide typically includes the CH2 domain and the CH3 domain, which are immunoglobulin constant region domain polypeptides. In some embodiments, the Fc polypeptide in an antibody described herein can be a wild-type Fc polypeptide, e.g., a human IgGl Fc polypeptide. In certain embodiments, an antibody described herein can comprise a wild-type Fc polypeptide having the sequence of SEQ ID NO:47:
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQP REPQ VYTLPP SRDELTKN Q V SLTCL VKGF YP SDI AVEWESNGQPENNYKTTPP VLD S DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. In other embodiments, an antibody described herein can comprise a variant of the wild-type Fc polypeptide that has at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to the sequence of a wild-type Fc polypeptide (e.g, SEQ ID NO:47) and at least one amino acid substitution relative to the sequence of a wild-type Fc polypeptide (e.g, SEQ ID NO:47).
[0128] In some embodiments, an Fc polypeptide includes one or more modifications (e.g, one or more amino acid substitutions, insertions, or deletions relative to a comparable wild- type Fc region). Antibodies comprising modified Fc polypeptides typically have altered phenotypes relative to antibodies comprising wild-type Fc polypeptides. For example, antibodies comprising modified Fc polypeptides can have altered serum half-life, altered stability, altered susceptibility to cellular enzymes, and/or altered effector function ( e.g ., as assayed in an NK-dependent or macrophage-dependent assay).
[0129] In some embodiments, an Fc polypeptide in an antibody described herein can include amino acid substitutions that modulate effector function. In certain embodiments, an Fc polypeptide in an antibody described herein can include amino acid substitutions that reduce or eliminate effector function. Illustrative Fc polypeptide amino acid substitutions that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 4 and 5 (position numbering relative to the sequence of SEQ ID NO:47) (see, e.g, Lund et al., J Immunol. 147(8):2657-62, 1991). For example, in some embodiments, one or both Fc polypeptides in an antibody described herein can comprise L4A and L5A substitutions.
[0130] Additional Fc polypeptide amino acid substitutions that modulate an effector function include, e.g, substitution at position 99 (position numbering relative to the sequence of SEQ ID NO:47). For example, in some embodiments, one or both Fc polypeptides in an antibody described herein can comprise P99G substitution. In certain embodiments, one or both Fc polypeptides in an antibody described herein can have L4A, L5A, and P99G substitutions.
[0131] In some embodiments, an Fc polypeptide includes one or more modifications that alter (relative to a wild-type Fc polypeptide) the Ratio of Affinities of the modified Fc polypeptide to an activating FcyR (such as FcyRIIA or FcyRIIIA) relative to an inhibiting FcyR (such as FcyRIIB):
Wild-Type to Variant Change m Affinity to FcyR .
Ratio of Affinities — -
Figure imgf000054_0001
Wild-Type to Variant Change in Affinity to FcyR
Figure imgf000054_0002
[0132] Where a modified Fc polypeptide has a Ratio of Affinities greater than 1, an anti-SARS-CoV-2 RBD antibody herein may have particular use in providing a therapeutic or prophylactic treatment of a disease, disorder, or infection, or the amelioration of a symptom thereof, where an enhanced efficacy of effector cell function (e.g, ADCC) mediated by FcyR is desired, e.g, cancer or infectious disease. Where a modified Fc region has a Ratio of Affinities less than 1, an anti-SARS-CoV-2 RBD antibody herein may have particular use in providing a therapeutic or prophylactic treatment of a disease or disorder, or the amelioration of a symptom thereof, where a decreased efficacy of effector cell function mediated by FcyR is desired, e.g, autoimmune or inflammatory disorders. Table 2 lists examples of single, double, triple, quadruple, and quintuple amino acid substitutions in an Fc polypeptide that provide a Ratio of Affinities greater than 1 or less than 1 (see e.g., PCT Publication Nos. WO 04/063351; WO 06/088494; WO 07/024249; WO 06/113665; WO 07/021841; WO 07/106707; WO 2008/140603). Amino acid positions are numbered according to EU numbering scheme.
Table 2
Figure imgf000055_0001
Figure imgf000056_0001
V Antibodies that competitively bind with an anti-SARS-CoV-2 RBI) antibody
[0133] Also provided herein are anti-SARS-CoV-2 RBD antibodies that competitively bind, or are capable of competitively binding ( e.g ., competitor antibodies), with one or more anti- SARS-CoV-2 Spike glycoprotein SI antibodies described herein. In certain instances, an antibody (i.e., competitor antibody) may be considered to compete for binding to SARS-CoV- 2 RBD when the competitor binds to the same general region of SARS-CoV-2 RBD as an anti-SARS-CoV-2 RBD antibody described herein. In certain instances, an antibody (i.e., competitor antibody) may be considered to compete for binding to SARS-CoV-2 RBD when the competitor binds to the exact same region of SARS-CoV-2 RBD as an anti-SARS-CoV-2 RBD antibody described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)). In certain instances, an antibody (i.e., competitor antibody) may be considered capable of competing for binding to SARS-CoV-2 RBD when the competitor binds to the same general region of SARS-CoV-2 RBD as an anti-SARS-CoV- 2 RBD antibody described herein (i.e., extracellular region or leucine-rich binding domain) under suitable assay conditions. In certain instances, an antibody (i.e., competitor agent) may be considered capable of competing for binding to SARS-CoV-2 RBD when the competitor binds to the exact same region of SARS-CoV-2 RBD as an anti-SARS-CoV-2 RBD antibody described herein ( e.g ., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)) under suitable assay conditions.
[0134] In certain instances, an antibody (i.e., competitor antibody) may be considered to compete for binding to SARS-CoV-2 RBD when the competitor blocks the binding of one or more anti-SARS-CoV-2 RBD antibodies described herein to SARS-CoV-2 RBD, for example, under suitable assay conditions. Whether a competitor blocks the binding of one or more anti- SARS-CoV-2 RBD antibodies described herein to SARS-CoV-2 RBD may be determined using a suitable competition assay or blocking assay, such as, for example, a blocking assay as described in herein. A competitor antibody may block binding of one or more anti-SARS- CoV-2 RBD antibodies described herein to SARS-CoV-2 RBD in a competition or blocking assay by 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%), and conversely, one or more anti-SARS-CoV-2 RBD antibodies described herein may block binding of the competitor antibody to SARS-CoV-2 RBD in a competition or blocking assay by about 50% or more (e.g, e.g, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%).
[0135] In certain instances, an antibody (i.e., competitor antibody) may be considered to compete for binding to SARS-CoV-2 RBD when the competitor binds to SARS-CoV-2 RBD with a similar affinity as one or more anti-SARS-CoV-2 RBD antibodies described herein, for example, under suitable assay conditions. In some embodiments, an antibody (i.e., competitor antibody) is considered to compete for binding to SARS-CoV-2 RBD when the competitor binds to SARS-CoV-2 RBD with an affinity that is at least about 50% (e.g, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the affinity of one or more anti-SARS-CoV-2 RBD antibodies described herein.
[0136] Also provided herein are anti-SARS-CoV-2 RBD antibodies that bind to, or are capable of binding to, the same epitope as one or more anti-SARS-CoV-2 RBD antibodies described herein. In particular, provided herein are anti-SARS-CoV-2 RBD antibodies that compete with one or more anti-SARS-CoV-2 RBD antibodies described herein for binding to the same epitope (e.g, same peptide (linear epitope) or same surface amino acids (conformational epitope)) on SARS-CoV-2 RBD. Such antibodies that bind the same epitope may be referred to as epitope competitors. VI. Polyclonal and monoclonal antibodies
[0137] Polyclonal antibodies may be raised in animals (vertebrate or invertebrates, including mammals, birds and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein or other carrier that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or R1N=C=NR, where R and R1 are different alkyl groups. Non-protein carriers (e.g, colloidal gold) also may be used for antibody production.
[0138] Animals can be immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g, 100 pg or 5 pg of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with one-fifth to one- tenth of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Often, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
[0139] Monoclonal antibodies may be made using a hybridoma, e.g, the hybridoma method first described by Kohler et ah, Nature, 256:495 (1975), or may be made by other methods such as recombinant DNA methods (see, e.g, U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see, e.g, Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). [0140] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as SP-2 or X63- Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et ah, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0141] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation, by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA), or by flow cytometric analysis of cells expressing the membrane antigen. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et ah, Anal. Biochem., 107:220 (1980).
[0142] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
[0143] DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Alternatively, cDNA may be prepared from mRNA and the cDNA then subjected to DNA sequencing. The hybridoma cells serve as a preferred source of such genomic DNA or RNA for cDNA preparation. Once isolated, the DNA may be placed into expression vectors, which are well known in the art, and which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
VII. Humanization and amino acid variants
[0144] General methods for humanization of antibodies are described, for example, in U.S. Patent Nos. 5861155, 6479284, 6407213, 6639055, 6500931, 5530101, 5585089, 5693761, 5693762, 6180370, 5714350, 6350861, 5777085, 5834597, 5882644, 5932448, 6013256, 6129914, 6210671 , 6329511 , 5225539, 6548640, and 5624821. In certain embodiments, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly where these improve the binding affinity or other biological properties ( e.g ., half- life) of the antibody.
[0145] In some embodiments, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., PNAS USA , 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol ., 44:65-92 (1988); Verhoeyen et al., Science , 239:1534-1536 (1988); Padlan, Molec. Immun ., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994). Techniques for humanizing antibodies are well known in the art and are described in e.g, U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87702671; EP Patent Application 0173494; Jones et al. (1986 ) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534. Humanized antibodies are further described in, e.g, Winter and Milstein (1991) Nature 349:293. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells. The CDRs for producing the immunoglobulins of the present disclosure can be similarly derived from monoclonal antibodies capable of specifically binding to SARS-CoV-2 RBD.
[0146] Amino acid sequence variants of the anti-SARS-CoV-2 RBD antibody can be prepared by introducing appropriate nucleotide changes into the anti-SARS-CoV-2 RBD antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-SARS-CoV-2 RBD antibodies for the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post- translational processes of the humanized or variant anti-SARS-CoV-2 RBD antibody, such as changing the number or position of glycosylation sites.
[0147] One method for identification of certain residues or regions of the anti-SARS-CoV-2 RBD antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis,” as described by, e.g., Cunningham and Wells, Science , 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g, charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably Ala or poly-Ala) to affect the interaction of the amino acids with SARS-CoV-2 RBD antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-SARS-CoV-2 RBD antibody variants are screened for the desired activity. Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants include the fusion of an enzyme or a polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody.
[0148] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more substantial changes may be introduced and the products may be screened. Examples of substitutions are listed below:
Ala (A): Val; Leu; He; Val
Arg (R): Lys; Gin; Asn; Lys
Asn (N): Gin; His; Asp, Lys; Gin; Arg
Asp (D): Glu; Asn
Cys (C): Ser; Ala
Gin (Q): Asn; Glu
Glu (E): Asp; Gin
Gly (G): Ala
His (H): Asn; Gin; Lys; Arg lie (I): Leu; Val; Met; Ala; Leu; Phe; Norleucine
Leu (L): Norleucine; He; Val; He; Met; Ala; Phe
Lys (K): Arg; Gin; Asn
Met (M): Leu; Phe; He
Phe (F): Leu; Val; He; Ala; Tyr
Pro (P): Ala
Ser (S): Thr
Thr (T): Ser
Trp (W): Tyr; Phe
Tyr (Y): Trp; Phe; Thr; Ser
Val (V): He; Leu; Met; Phe; Ala; Norleucine
[0149] Substantial modifications in the biological properties of an antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side- chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;
(2) neutral hydrophilic: Cys, Ser, Thr;
(3) acidic: Asp, Glu;
(4) basic: Asn, Gin, His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; and
(6) aromatic: Trp, Tyr, Phe
[0150] Non-conservative substitutions will entail exchanging a member of one of the above classes for another class.
[0151] Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
[0152] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites ( e.g ., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated can be displayed in the monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage- displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. [0153] Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one of more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked and/or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O- linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to the antibody can be accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
VIII. Other modifications
[0154] Other modifications of an anti-SARS-CoV-2 RBD antibody are contemplated. For example, technology herein also pertains to immunoconjugates comprising an anti-SARS- CoV-2 RBD antibody described herein conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug. Such conjugates are sometimes referred to as “antibody-drug conjugates” or “ADC.” Conjugates can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2, 4-dinitrobenzene).
[0155] Anti-SARS-CoV-2 RBD antibodies (e.g., anti-SARS-CoV-2 RBD antibodies) disclosed herein may be formulated as immunoliposomes. Liposomes containing an antibody are prepared by methods know in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. For example, liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an antibody provided herein can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. Another active ingredient is optionally contained within the liposome.
[0156] Enzymes or other polypeptides can be covalently bound to an anti-SARS-CoV-2 RBD antibody by techniques well known in the art such as the use of the heterobifunctional cross-linking reagents discussed above. In some embodiments, fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984)).
[0157] In certain embodiments, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase penetration of target tissues and cells, for example. In such instances, it may be desirable to modify the antibody fragment in order to increase its serum half-life. This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g, by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g, by DNA or peptide synthesis; see, e.g, W096/32478 published Oct. 17, 1996).
[0158] In some embodiments, a modification can optionally be introduced into the antibodies (e.g, within the polypeptide chain or at either the N- or C-terminal), e.g, to extend in vivo half-life, such as PEGylation or incorporation of long-chain polyethylene glycol polymers (PEG). Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the polypeptides, for example, to prevent rapid filtration into the urine. In some embodiments, a lysine residue in the sequence is conjugated to PEG directly or through a linker. Such linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site- specifically attached to a PEG chain containing, for example, a maleimide function. Methods for incorporating PEG or long chain polymers of PEG are known in the art (described, for example, in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, M. J., et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which are incorporated herein by reference.
[0159] In some embodiments, any of the antibodies or antigen fragments thereof disclosed herein are conjugated or hybridized to an oligonucleotide label. In some embodiments, the oligonucleotide label includes a sample barcode sequence, a binding site for a primer and an anchor. In some embodiments, the oligonucleotide label can be conjugated or hybridized to any of the detectable markers or labels disclosed herein. In some embodiments, the oligonucleotide label is a polymeric sequence. In some embodiments, the terms “oligonucleotide” and “polynucleotide” are used interchangeably to refer to a single-stranded multimer of nucleotides from about 2 to about 500 nucleotides in length. In some embodiments, any of the oligonucleotide labels described herein can be synthetic, made enzymatically (e.g., via polymerization), or using a “split-pool” method. In some embodiments, any of the oligonucleotide labels described herein can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some embodiments, any of the oligonucleotide labels described herein can include a combination of both deoxyribonucleotide monomers and ribonucleotide monomers in the oligonucleotide (e.g., random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). In some embodiments, the oligonucleotide label can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400-500 nucleotides in length. In some embodiments, any of the oligonucleotide labels described herein can include one or more functional moieties that are attached (e.g., covalently or non-covalently) to another structure. In some embodiments, any of the oligonucleotide labels described herein can include one or more detectable labels (e.g., a radioisotope or fluorophore). In some embodiments, the anchor is a defined polymer, e.g., a polynucleotide or oligonucleotide sequence, which is designed to hybridize to a complementary oligonucleotide sequence. In some embodimentsthe anchor is designed for the purpose of generating a double stranded construct oligonucleotide sequence. In some embodiments, the anchor is positioned at the 3’ end of the construct oligonucleotide sequence. In other embodiments, the anchor is positioned at the 5’ end of the construct oligonucleotide sequence. Each anchor is specific for its intended complementary sequence.
[0160] In some embodiments, the sample barcode sequence is a polymer, e.g., a polynucleotide, which when it is a functional element, is specific for a single ligand. In some embodiments, the sample barcode sequence can be used for identifying a particular cell or substrate, e.g., Drop-seq microbead. In some embodiments, the sample barcode sequence can be formed of a defined sequence of DNA, RNA, modified bases or combinations of these bases, as well as any other polymer defined above. In some embodiments, the sample barcode sequence is about 2 to 4 monomeric components, e.g., nucleotide bases, in length. In other embodiments, the barcode is at least about 1 to 100 monomeric components, e.g., nucleotides, in length. Thus in various embodiments, the barcode is formed of a sequence of at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In some embodiments, the sample barcode sequence is a particular barcode that can be unique relative to other barcodes.
[0161] In some of any embodiments, the sample barcode sequences can have a variety of different formats. For example, sample barcode sequences can include polynucleotide barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. A sample barcode sequence can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. A sample barcode sequences can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Sample barcode sequences can allow for identification and/or quantification of individual sequencing-reads (e.g., a barcode can be or can include a unique molecular identifier or “UMI”).
[0162] Sample barcode sequences can spatially-resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”). In some embodiments, a barcode includes both a UMI and a spatial barcode. In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode sequences. [0163] In some embodiments, the binding site for a primer is a functional component of the oligonucleotide which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for amplification of the oligonucleotide. The binding site for a primer can be formed of polymers of DNA, RNA, PNA, modified bases or combinations of these bases, or polyamides, etc. In some embodiments, the binding site for a primer is about 10 of such monomeric components, e.g., nucleotide bases, in length. In other embodiments, the binding site for a primer is at least about 5 to 100 monomeric components, e.g., nucleotides, in length. Thus in various embodiments, the binding site for a primer is formed of a sequence of at least
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81
82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In certain embodiments, the binding site for a primer can be a generic sequence suitable as an annealing site for a variety of amplification technologies. Amplification technologies include, but are not limited to, DNA-polymerase based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA) and polymerization by any number of DNA polymerases (for example, T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase) and RNA-polymerase based amplification systems (such as T7-, T3-, and SP6-RNA- polymerase amplification), nucleic acid sequence based amplification (NASBA), self- sustained sequence replication (3 SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase dependent amplification (I), ramification amplification method and RNA-seq. Methods for conjugating or hybridizing an oligonucleotide label can be performed in a manner set forth in WO/2018/144813, WO/2016/018960, WO/2018/089438, WO/2014/182528, WO/2018/026873, WO/2021/188838.
[0164] In some embodiments, a modification can optionally be introduced into the antibodies (e.g., within the polypeptide chain or at either the N- or C-terminal), e.g, to extend in vivo half- life, such as PEGylation or incorporation of long-chain polyethylene glycol polymers (PEG). Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the polypeptides, for example, to prevent rapid filtration into the urine. In some embodiments, a lysine residue in the sequence is conjugated to PEG directly or through a linker. Such linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site- specifically attached to a PEG chain containing, for example, a maleimide function. Methods for incorporating PEG or long chain polymers of PEG are known in the art (described, for example, in Veronese, F. M., et ah, Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et ah, Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, M. J., et ah, Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which are incorporated herein by reference.
[0165] Covalent modifications of an anti-SARS-CoV-2 RBD antibody are also included within the scope of this technology. For example, modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of an anti-SARS-CoV-2 RBD antibody. Other types of covalent modifications of an antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Example covalent modifications of polypeptides are described in Ci.S. Pat. No. 5,534,615, specifically incorporated herein by reference. A preferred type of covalent modification of the antibody comprises linking the antibody to one of a variety of non-proteinaceous polymers, e.g, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in, e.g, U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
IX. Nucleic acids, vectors, host cells, and recombinant methods
[0166] The disclosure also provides isolated nucleic acids encoding an anti-SARS-CoV-2 RBD antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody. A nucleic acid herein may include one or more subsequences, each referred to as a polynucleotide.
[0167] Provided herein are nucleic acids (e.g, isolated nucleic acids) comprising a nucleotide sequence that encodes an anti-SARS-CoV-2 RBD antibody, or fragment thereof. In some embodiments, a nucleic acid encodes an immunoglobulin heavy chain variable domain of an anti-SARS-CoV-2 RBD antibody provided herein. In some embodiments, a nucleic acid encodes an immunoglobulin light chain variable domain of an anti-SARS-CoV-2 RBD antibody provided herein. In some embodiments, a nucleic acid encodes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an anti- SARS-CoV-2 RBD antibody provided herein. In some embodiments, a nucleic acid comprises a nucleotide sequence that encodes an amino acid sequence of any one of SEQ ID NOS: 1-46.
[0168] Provided herein is an isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain of any of the anti-SARS-CoV-2 RBD antibodies or antigen-binding fragments thereof described herein. In some of any embodiments, the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOS:70-79 and 86.
[0169] Provided herein is an isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin light chain variable domain of any of the anti-SARS-CoV-2 RBD antibodies or antigen-binding fragments thereof described herein. In some of any embodiments, the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 80-85 and 87-93.
[0170] Provided herein is an isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of any of the anti-SARS-CoV-2 RBD antibodies or antigen-binding fragments thereof described herein.
[0171] In some of any embodiments, the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in any of SEQ ID NOS:70-79 and 86; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in any of SEQ ID NOS: 80-85 and 87-93.
[0172] In some of any embodiments, any of the nucleic acids provided herein can comprise a signal sequence. In some of any embodiments, any of the nucleic acids described herein do not comprise a signal sequence.
[0173] For recombinant production of an anti-SARS-CoV-2 RBD antibody, a nucleic acid encoding the anti-SARS-CoV-2 RBD antibody may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In certain instances, an anti-SARS-CoV-2 RBD antibody may be produced by homologous recombination. DNA encoding an anti-SARS-CoV-2 RBD antibody can be readily isolated and sequenced using conventional procedures ( e.g by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, and origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
[0174] Suitable host cells for cloning or expressing DNA in vectors herein can be prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia , e.g., E. coli , Enterohacter , Erwinia, Klebsiella , Proteus , Salmonella , e.g., Salmonella typhimurium , Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as . subtilis and . licheniformis , Pseudomonas such as P. aeruginosa , and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) can also be suitable. These examples are illustrative rather than limiting.
[0175] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-SARS-CoV-2 RBD antibody-encoding vectors. Saccharomyces cerevisiae , or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. A number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe ; Kluyveromyces hosts such as, e.g., K. lactis , K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans , and K. marxianus yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida ; Trichoderma reesia (EP 244,234); Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalism and filamentous fungi such as, e.g., Neurospora , Penicillium , Tolypocladium , and Aspergillus hosts such as A. nidulans and A. niger.
[0176] Suitable host cells for the expression of anti-SARS-CoV-2 RBD antibodies can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk moth) have been identified. A variety of viral strains for transfection are publicly available, e.g, the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present technology, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts. [0177] Suitable host cells for the expression of anti-SARS-CoV-2 RBD antibodies also may include vertebrate cells ( e.g mammalian cells). Vertebrate cells may be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BEK, ATCC CCL 10); Chinese hamster ovary cells/- DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et ak, Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0178] Host cells may be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Host cells used to produce antibodies provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham etak, Meth. Enz. 58:44 (1979), Barnes et ak, Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. [0179] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
[0180] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human g3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
[0181] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between, e.g., about 2.5-4.5, and may be performed at low salt concentrations (e.g, from about 0-0.2 5M salt). X Pharmaceutical formulations, dosing, and routes of administration
[0182] The present disclosure provides anti-SARS-CoV-2 RBD antibodies and related compositions, which may be useful for elimination of SARS-CoV-2 RBD-expressing pathogens from the body, for example, and for identification and quantification of the number of SARS-CoV-2 RBD-expressing pathogens in biological samples, for example.
[0183] Anti-SARS-CoV-2 RBD antibodies may be formulated in a pharmaceutical composition that is useful for a variety of purposes, including the treatment of diseases or disorders. Pharmaceutical compositions comprising one or more anti-SARS-CoV-2 RBD antibodies may be administered using a pharmaceutical device to a patient in need thereof, and according to one embodiment of the technology, kits are provided that include such devices. Such devices and kits may be designed for routine administration, including self administration, of the pharmaceutical compositions herein.
[0184] Therapeutic formulations of an antibody may be prepared for storage by mixing the agent or antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues ) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes ( e.g Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLEIRONICSTM, or polyethylene glycol (PEG).
[0185] Formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
[0186] Formulations for in vivo administration generally are sterile. This may be accomplished for instance by filtration through sterile filtration membranes, for example.
[0187] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated agents/antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thiol-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
[0188] For therapeutic applications, anti-SARS-CoV-2 RBD antibodies provided herein are administered to a mammal, e.g, a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. For the prevention or treatment of disease, the appropriate dosage of agent or antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventative or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.
[0189] Depending on the type and severity of the disease, about 1 pg/kg to about 50 mg/kg (e.g, 0.1-20 mg/kg) of antibody may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about 1 pg/kg to about 20 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using the antibody to determine SAR.S- CoV-2 RBD levels in bodily fluids or tissues may be used in order to optimize patient exposure to the therapeutic antibody.
[0190] In some embodiments, a composition comprising an anti-SARS-CoV-2 RBD antibody herein can be administered as a monotherapy, and in some embodiments, the composition comprising the anti-SARS-CoV-2 RBD antibody can be administered as part of a combination therapy. In some cases, the effectiveness of the antibody in preventing or treating diseases may be improved by administering the antibody serially or in combination with another drug that is effective for those purposes, such as a chemotherapeutic drug for treatment of cancer or a microbial infection. In other cases, the anti-SARS-CoV-2 RBD antibody may serve to enhance or sensitize cells to chemotherapeutic treatment, thus permitting efficacy at lower doses and with lower toxicity. Certain combination therapies include, in addition to administration of the composition comprising an antibody that reduces the number of SAR.S- CoV-2 RBD-expressing cells, delivering a second therapeutic regimen selected from the group consisting of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing. Such other agents may be present in the composition being administered or may be administered separately. Also, the anti-SARS-CoV-2 RBD antibody may be suitably administered serially or in combination with the other agent or modality, e.g, chemotherapeutic drug or radiation for treatment of cancer, infection, and the like, or an immunosuppressive drug. XI. Research and diagnostic
[0191] Also provided herein are diagnostic reagents comprising an anti-SARS-CoV-2 RBD antibody described herein. For example, anti-SARS-CoV-2 RBD antibodies provided herein may be used to detect and/or purify SARS-CoV-2 RBD, e.g., from bodily fluid(s) or tissues. Also provided herein are methods for detecting SARS-CoV-2 RBD. For example, a method may comprise contacting a sample (e.g, a biological sample known or suspected to contain SARS-CoV-2 RBD) with an anti-SARS-CoV-2 RBD antibody provided herein, and, if the sample contains SARS-CoV-2 RBD, detecting SARS-CoV-2 RBD: anti-SARS-CoV-2 RBD antibody complexes. Also provided herein are reagents comprising an anti-SARS-CoV-2 RBD antibody described herein and methods for detecting SARS-CoV-2 RBD for research purposes.
[0192] Anti-SARS-CoV-2 RBD antibodies, for example, may be useful in diagnostic assays for SARS-CoV-2 RBD, e.g, detecting its presence in specific cells, tissues, or bodily fluids. Such diagnostic methods may be useful in diagnosis, e.g, of a hyperproliferative disease or disorder. Thus clinical diagnostic uses as well as research uses are comprehended herein. In some embodiments, an anti-SAR.S-CoV-2 RBD antibody comprises a detectable marker or label. In some embodiments, an anti-SAR.S-CoV-2 RBD antibody is conjugated to a detectable marker or label. For example, for research and diagnostic applications, an anti-SAR.S-CoV-2 RBD antibody may be labeled with a detectable moiety. Numerous labels are available which are generally grouped into the following categories:
(a) Radioisotopes, such as 35S, 14C, 1251, 3H, and 13 IF The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et ak, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation counting.
(b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, Texas Red and Brilliant VioletTM are available. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a flow cytometer, imaging microscope or fluorimeter.
(c) Various enzyme-substrate labels are available. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemilluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases ( e.g ., firefly luciferase and bacterial luciferase), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et ah, Methods for the Preparation of Enzyme- Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).
[0193] Examples of enzyme-substrate combinations include, for example:
(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, where the hydrogen peroxidase oxidizes a dye precursor (e.g, orthophenyl ene diamine (OPD) or 3, 3', 5,5'- tetramethyl benzidine hydrochloride (TMB));
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and
(iii) b-D-galactosidase (b-D-Gal) with a chromogenic substrate (e.g, p-nitrophenyl^-D- galactosidase) or fluorogenic substrate 4-methylumbelliferyl^-D-galactosidase.
[0194] In certain instances, the label is indirectly conjugated with the agent or antibody. The skilled artisan will be aware of various techniques for achieving this. For example, an antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g, digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g, anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.
[0195] In some embodiments, and anti-SARS-CoV-2 RBD antibody need not be labeled, and the presence thereof can be detected, e.g, using a labeled antibody which binds to an anti- SARS-CoV-2 RBD antibody. [0196] In some embodiments, an anti-SARS-CoV-2 RBD antibody herein is immobilized on a solid support or substrate. In some embodiments, an anti-SARS-CoV-2 RBD antibody herein is non-diffusively immobilized on a solid support ( e.g ., the anti-SARS-CoV-2 RBD antibody does not detach from the solid support). A solid support or substrate can be any physically separable solid to which an anti-SARS-CoV-2 RBD antibody can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports also can include, for example, chips, columns, optical fibers, wipes, filters (e.g, flat surface filters), one or more capillaries, glass and modified or functionalized glass (e.g, controlled-pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semi conductive materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethanes, TEFLON™, polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF), and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon, silica gel, and modified silicon, Sephadex®, Sepharose®, carbon, metals (e.g, steel, gold, silver, aluminum, silicon and copper), inorganic glasses, conducting polymers (including polymers such as polypyrole and polyindole); micro or nanostructured surfaces such as nucleic acid tiling arrays, nanotube, nanowire, or nanoparticulate decorated surfaces; or porous surfaces or gels such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibrous or stranded polymers. In some embodiments, the solid support or substrate may be coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Beads and/or particles may be free or in connection with one another (e.g, sintered). In some embodiments, a solid support or substrate can be a collection of particles. In some embodiments, the particles can comprise silica, and the silica may comprise silica dioxide. In some embodiments the silica can be porous, and in certain embodiments the silica can be non- porous. In some embodiments, the particles further comprise an agent that confers a paramagnetic property to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments the agent is a metal oxide, (e.g, iron or iron oxides, where the iron oxide contains a mixture of Fe2+ and Fe3+). An anti-SARS-CoV-2 RBD antibody may be linked to a solid support by covalent bonds or by non-covalent interactions and may be linked to a solid support directly or indirectly ( e.g ., via an intermediary agent such as a spacer molecule or biotin).
[0197] Antibodies provided herein may be employed in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147- 158 (CRC Press, Inc. 1987). Flow cytometry and mass cytometry assays generally involve the use of a single primary antibody to specifically identify the presence of the target molecule expressed on the surface of a dispersed suspension of individual cells. The dispersed cells are typically obtained from a biological fluid sample, e.g, blood, but may also be obtained from a dispersion of single cells prepared from a solid tissue sample such as spleen or tumor biopsy. The primary antibody may be directly conjugated with a detectable moiety, e.g, a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass cytometry. Alternatively, the primary antibody may be unlabeled or labeled with an undetectable tag such as biotin, and the primary antibody is then detected by a detectably labeled secondary antibody that specifically recognizes the primary antibody itself or the tag on the primary antibody. The labeled cells are then analyzed in an instrument capable of single cell detection, e.g, flow cytometer, mass cytometer, fluorescence microscope or brightfield light microscope, to identify those individual cells in the dispersed population or tissue sample that express the target recognized by the primary antibody. Detailed description of the technological basis and practical application of flow cytometry principles may be found in, e.g, Shapiro, Practical Flow Cytometry, 4th Edition, Wiley, 2003.
[0198] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein that is detected. In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. In a cell ELISA, the target cell population may be attached to the solid support using antibodies first attached to the support and that recognize different cell surface proteins. These first antibodies capture the cells to the support. SARS-CoV-2 Spike glycoprotein SI on the surface of the cells can then be detected by adding anti-SARS-CoV-2 RBD antibody to the captured cells and detecting the amount of SARS-CoV-2 RBD antibody attached to the cells. In certain instances, fixed and permeabilized cells may be used, an in such instances, surface SARS-CoV-2 Spike glycoprotein SI and intracellular SARS-CoV-2 Spike glycoprotein SI may be detected.
[0199] For immunohistochemistry, the blood or tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
[0200] The antibodies herein also may be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionuclide (such as 11 lln, 99Tc, 14C, 1311, 1251, 3H, 32P, or 35S) so that the bound target molecule can be localized using immunoscintillography.
XII Detection of SARS-Co V-2 RBD
[0201] Provided herein are antibodies and methods for detecting SARS-CoV-2 RBD. In some embodiments, antibodies and methods are provided for detecting SARS-CoV-2 RBD in a biological sample. In some embodiments, the biological sample is a solid tissue, fluid, or cell. The solid tissue may comprise solid tissue from one or more of adipose tissue, bladder, bone, brain breast cervix, endothelium, gallbladder, kidney, liver, lung, lymph, ovary, prostate, salivary gland, stomach, testis, thyroid, urethra, uterus, vagina, and vulva. In some embodiments, the fluid comprises one or more of amniotic fluid, bile, blood, breast milk, breast fluid, cerebrospinal fluid, lavage fluid, lymphatic fluid, mucous, plasma, saliva, semen, serum, spinal fluid, sputum, tears, umbilical cord blood, urine, and vaginal fluid.
[0202] In some of any embodiments, any of the antibodies or antigen binding fragments thereof provided herein can be used in the characterization of single cells by measurement of gene-expression levels and cellular proteins. Among such known single cell sequencing platforms suitable for integration with the antibodies or antigen binding fragments thereof described herein is the Drop-seq method, including, but not limited to, microfluidic, plate- based, or microwell, Seq-Well™ method and adaptations of the basic protocol, and InDrop™ method.
[0203] In another embodiment, a single cell sequencing platform suitable for integration with the antibodies or antigen binding fragments thereof described herein is lOx genomics single cell 3' solution or single cell V(D)J solution, either run on Chromium controller, or dedicated Chromium single cell controller. Other suitable sequencing methods include Wafergen iCell8™ method, Microwell-seq method, Fluidigm Cl™ method and equivalent single cell products. Still other known sequencing protocols useful with the antibodies or antigen binding fragments thereof described herein include BD Resolve™ single cell analysis platform and ddSeq (from Illumina® Bio-Rad® SureCell™ WTA 3' Library Prep Kit for the ddSEQ™ System, 2017, Pub. No. 1070-2016-014-B, Illumina Inc., Bio-Rad Laboratories, Inc.). In still other embodiment, the antibodies or antigen binding fragments thereof described herein are useful with combinatorial indexing based approaches (sci-RNA-seq™ method or SPLiT-seq™ method) and Spatial Transcriptomics, or comparable spatially resolved sequencing approaches. The methods and compositions described herein can also be used as an added layer of information on standard index sorting (FACS) and mRNA-sequencing-based approaches.
[0204] In some of any embodiments, any of the antibodies or antigen binding fragments thereof described herein can be used to detect the presence, absence or amount of the various nucleic acids, proteins, targets, oligonucleotides, amplification products and barcodes described herein.
[0205] In some of any embodiments, the detection comprises hybridization of a detectable moiety to the antibody or antigen binding fragment thereof. In some of any embodiments, the sample is contacted with a second antibody. In some of any embodiments, the second antibody is an antibody comprising a detectable moiety. In some of any embodiments, the detectable moiety comprises an oligonucleotide. In some of any embodiments, the detectable moiety comprises a fluorescent label. In some of any embodiments, the measurement comprises sequencing. In some of any embodiments, the detectable moiety comprises immunofluorescence. In some of any embodiments, the sample is a formalin-fixed paraffin- embedded sample. In some of any embodiments, the sample comprises a cell. In some of any embodiments, the sample comprises a tissue sample
XIII. Kits incorporating anti-SARS-CoV-2 RBI) antibodies
[0206] An anti-SARS-CoV-2 RBD antibody herein may be provided in a kit, for example, a packaged combination of reagents in predetermined amounts with instructions for use ( e.g ., instructions for performing a diagnostic assay; instructions for performing a laboratory assay). In some embodiments, the kit is a diagnostic kit configured to detect SARS-CoV-2 RBD in a sample (e.g., a biological sample). Where the anti-SARS-CoV-2 RBD antibody is labeled with a fluorophore, the kit may include an identical isotype negative control irrelevant antibody to control for non-specific binding of the anti-SARS-CoV-2 RBD antibody. Where the anti- SARS-CoV-2 RBD antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme ( e.g ., substrate precursor which provides the detectable chromophore or fluorophore). Additional additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer), and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. In certain instances, reagents may be provided as dry powders (e.g, lyophilized powder), including excipients that on dissolution will provide a reagent solution having the appropriate concentration.
EXAMPLES
Example 1 — Blocking assay
[0207] This example describes a blocking assay performed using anti-SARS-CoV-2 RBD antibodies.
[0208] SARS-CoV-2 S protein RBD-Fc chimera (BioLegend, cat. # 793104/06) was coated at 1.0 pg/mL, 100 pL/well in PBS on the 96-well plate overnight at 4°C. After washing the plate and blocking the coated plate with 1%BSA/PBS at 37°C for 1 hour. Anti-SARS-CoV-2 S Protein RBD antibodies (listed in Table 3 below) with starting final concentration of 30 pg/mL (1:3 serial dilutions; only the starting final concentration of AB2 was 27.5 pg/mL), 100 pL/well was added to coated plate. Subsequently, the plate was incubated on a shaker at room temperature for 1 hour. The 25 pL/well (final concentration was 0.5 pg/mL) of recombinant human ACE2 (BioLegend, cat. # 792002/04/06/08) was added to serial dilutions of antibody. The plate was incubated on a shaker for 5 hours, and then incubated overnight at 4°C. Next day, the plate was washed and then 100 pL of HRP anti-His Tag Antibody (BioLegend, cat. # 652503/04) at 1:1000 dilutions in the assay buffer was added to the plate. The plate was incubated on a shaker for 1 hour. After washing the plate 2 times, 100 pL/well of the mixture of color substrate TMB A+B (BioLegend, cat. # 421101) was added. Finally, 50 pL stop solution (BioLegend, cat. # 423001) was added to the plate and data were acquired at 450 nm.
[0209] To calculate the percentage of blocking, percentage original OD 450 nm was calculated by dividing the OD 450 nm of samples blocked with AB2, AB3, AB4, AB5, AB6, AB7, AB8, or AB9, by the OD 450 nm without blocking. This value was subtracted from 100 to get a blocking percentage. The formula is shown below:
[OD 450 nm blocking]
100 - ( * 100)
[OD 450 nm no blocking] [0210] For the blocking assay, AB2, AB3, AB4, AB8, and AB9 used in the blocking assay significantly blocked the binding of 0.5 pg/mL recombinant human ACE2 to 1 pg/mL immobilized SARS-CoV-2 S protein RBD-Fc chimera by a blocking percentage ranging from 70% to 100%. Overall, the blocking percentages of AB2, AB4, AB8 and AB9 are higher than 98%. Tables 3 below presents OD 450 nm and % blocking results.
Table 3
Figure imgf000084_0001
EXEMPLARY EMBODIMENTS
[0211] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:
1. An isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody comprises: a) a heavy chain variable region comprising:
(i) a heavy chain complementary determining region 1 (HCDR1) having the sequence of X1YX2MX3 (SEQ ID NO:64), wherein Xi is D, E, N, or S; X2 is I, T, or W; and X3 is L or H;
(ii) an HCDR2 having the sequence of X1IX2PX3X4X5X6TX7X8NX9KFKX10 (SEQ ID NO:65), wherein Xi is N, G, M, or E; X2 is N or D; X3 is Y, N, or S; X4 is Y, N, or D; X5 is G, V, or S; Xe is S, D, E, or R; X7 is S, N, or R; Xs is Y or L; X9 is L, Q, or E; and X10 is G, D, or S;
(iii) an HCDR3 having the sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO:66), wherein Xi is Y or absent; X2 is Y, R, A, or absent; X3 is G or absent; X4 is S, D, L or A; X5 is S, R, L, M, or Y; Xe is Y or N; X7 is G, D, R, or absent; Xs is G, Y, or absent; X9 is Y, T, S, or absent; X10 is F or V; X11 is D or A; and X12 is V or Y; and b) a light chain variable region comprising:
(iv) a light chain complementary determining region 1 (LCDR1) having the sequence of X1X2SX3X4X5X6X7X8X9X1X11X12X13X14X15 (SEQ ID NO:67), wherein Xi is S or R; X2 is A or S; X3 is S, E, K, Q, or T; X4 is S, E, or G; X5 is L or absent; Xe is V, L, or absent; X7 is D, H, A, or absent; Xx is N, S, V, or absent; X9 is Y, N, T, or absent; X10 is G, I,
T, or absent; X11 is L, I, N, or S; X12 is N, S, T, or G; X13 is F or Y; X14 is M, L, or A; and X15 is Y, N, or S;
(v) a LCDR2 having the sequence of X1X2X3X4X5X6X7 (SEQ ID NO:68), wherein Xi is L, A, R, or G; X2 is T, A, or M; X3 is S or N; X4 is N or T; X5 is L, Q, or R; Xe is A, G, or D; and X7 is S or P; and
(vi) a LCDR3 having the sequence of X1X2X3X4X5X6X7X8X9 (SEQ ID NO:69), wherein Xi is Q, M, L, or A; X2 is Q or L; X3 is W, S, H, or Y; X4 is S, K, L, A, or Y; Xs is T, E, or S; Xe is N, V, or Y; X7 is P or H; X8 is L, Y, or W; and X9 is T or V.
2. The isolated antibody of embodiment 1, wherein:
(1) Xi is D, N, or S; X2 is I or W; and X3 is L or H in HCDR1;
(2) Xi is N or M; X2 is N or D; X3 is Y or S; X4 is Y or D; X5 is G or S; Xe is S or E; X7 is S or R; Xs is Y or L; X9 is L or Q; and X10 is G or D in HCDR2; and
(3) Xi is Y or absent; X2 is Y or R; X3 is G; X4 is S or L; X5 is S or L; Xe is Y or N; X7 is G or absent; Xs is G or absent; X9 is Y or absent; X10 is F; X11 is D; and X12 is V or Y in HCDR3.
3. The antibody of embodiment 1 or 2, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS: 1-4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having one, two, or three amino acid substitutions relative to a sequence of any one of SEQ ID NOS:5-9; and
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS: 10-14. 4. The antibody of any one of embodiments 1 to 3, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 1- 4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5- 9; and
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:10-14.
5. The antibody of any one of embodiments 1 to 4, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9; and
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14.
6. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO: 1, an HCDR2 having the sequence of SEQ ID NO:5, and an HCDR3 having the sequence of SEQ ID NO: 10.
7. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO:2, an HCDR2 having the sequence of SEQ ID NO:6, and an HCDR3 having the sequence of SEQ ID NO: 11.
8. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO:7, and an HCDR3 having the sequence of SEQ ID NO: 12. 9. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO:7, and an HCDR3 having the sequence of SEQ ID NO: 13.
10. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:3, a HCDR2 having the sequence of SEQ ID NO:8, and a HCDR3 having the sequence of SEQ ID NO: 14.
11. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 1, a HCDR2 having the sequence of SEQ ID NO:9, and a HCDR3 having the sequence of SEQ ID NO: 10.
12. The isolated antibody of any one of embodiments 1 to 5, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO:7, and a HCDR3 having the sequence of SEQ ID NO: 12.
13. The isolated antibody of embodiment 1, wherein:
(a) Xi is S or R; X2 is A or S; X3 is S or T; X4 is S or G; X5 is absent; Xe is absent; X7 is A or absent; Xs is V or absent; X9 is T or absent; X10 is T or absent; X11 is L or S; X12 is N; X13 is F or Y; X14 is M or A; and X15 is Y or N in LCDR1;
(b) Xi is L or G; X2 is T; X3 is S or N; X4 is N; X5 is L or R; Xe is A; and X7 is S or P in LCDR2;
(c) Xi is Q or A; X2 is Q or L; X3 is W; X4 is S or Y; X5 is T or S; Xe is N; X7 is P; X8 is L or W; and X9 is T or V in LCDR3.
14. The antibody of embodiment 1 or 13, wherein the antibody comprises:
(1) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS:23-28;
(2) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS:29-33; and
(3) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS:34-38. 15. The antibody of any one of embodiments 1, 13, and 14, wherein the antibody comprises:
(1) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:23-28;
(2) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:29-33; and
(3) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:34-38.
16. The antibody of any one of embodiments 1 and 13 to 15, wherein the antibody comprises:
(1) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28;
(2) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33; and
(3) a LCDR3 having a sequence of any one of SEQ ID NOS: 34-38.
17. The isolated antibody of any one of embodiments 1 and 13 to 16, wherein the antibody comprises a LCDR1 having the sequence of SEQ ID NO:23, a LCDR2 having the sequence of SEQ ID NO: 29, and a LCDR3 having the sequence of SEQ ID NO:34.
18. The isolated antibody of any one of embodiments 1 and 13 to 16, wherein the antibody comprises a LCDR1 having the sequence of SEQ ID NO:24, a LCDR2 having the sequence of SEQ ID NO: 30, and a LCDR3 having the sequence of SEQ ID NO:35.
19. The isolated antibody of any one of embodiments 1 and 13 to 16, wherein the antibody comprises a LCDR1 having the sequence of SEQ ID NO:25, a LCDR2 having the sequence of SEQ ID NO: 31, and a LCDR3 having the sequence of SEQ ID NO:36. 20. The isolated antibody of any one of embodiments 1 and 13 to 16, wherein the antibody comprises a LCDR1 having the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO: 31, and a LCDR3 having the sequence of SEQ ID NO:36.
21. The isolated antibody of any one of embodiments 1 and 13 to 16, wherein the antibody comprises a LCDR1 having the sequence of SEQ ID NO:27, a LCDR2 having the sequence of SEQ ID NO: 32, and a LCDR3 having the sequence of SEQ ID NO:37.
22. The isolated antibody of any one of embodiments 1 and 13 to 16, wherein the antibody comprises a LCDR1 having the sequence of SEQ ID NO:28, a LCDR2 having the sequence of SEQ ID NO:33, and a LCDR3 having the sequence of SEQ ID NO:38.
23. The isolated antibody of embodiment 1, wherein:
(1) Xi is D, N, or S; X2 is I or W; and X3 is L or H in HCDR1;
(2) Xi is N or M; X2 is N or D; X3 is Y or S; X4 is Y or D; X5 is G or S; Xe is S or E; X7 is S or R; Xs is Y or L; X9 is L or Q; and X10 is G or D in HCDR2;
(3) Xi is Y or absent; X2 is Y or R; X3 is G; X4 is S or L; X5 is S or L; Xe is Y or N; X7 is G or absent; Xs is G or absent; X9 is Y or absent; X10 is F; X11 is D; and Xi2 is V or Y in HCDR3;
(4) Xi is S or R; X2 is A or S; X3 is S or T; X4 is S or G; X5 is absent; Xe is absent; X7 is A or absent; Xs is V or absent; X9 is T or absent; X10 is T or absent; X11 is L or S; Xi2 is N; Xi3 is F or Y; Xi4 is M or A; and X15 is Y or N in LCDR1;
(5) Xi is L or G; X2 is T; X3 is S or N; X4 is N; X5 is L or R; Xe is A; and X7 is S or P in LCDR2;
(6) Xi is Q or A; X2 is Q or L; X3 is W; X4 is S or Y; X5 is T or S; Xe is N; X7 is P; X8 is L or W; and X9 is T or V in LCDR3.
24. The antibody of embodiment 1 or 23, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS: 1-4; (2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having one, two, or three amino acid substitutions relative to a sequence of any one of SEQ ID NOS:5-9;
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS: 10-14;
(4) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS:23-28;
(5) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOS:29-33; and
(6) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having one or two amino acid substitutions relative to a sequence of any one of SEQ ID NOS:34-38.
25. The antibody of any one of embodiments 1, 23, and 24, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 1- 4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5- 9;
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:10-14;
(4) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:23-28; (5) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:29-33; and
(6) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:34-38.
26. The antibody of any one of embodiments 1 and 23 to 25, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS:5-9;
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14;
(4) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28;
(5) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33; and
(6) a LCDR3 having a sequence of any one of SEQ ID NOS: 34-38.
27. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO: 1, an HCDR2 having the sequence of SEQ ID NO:5, and an HCDR3 having the sequence of SEQ ID NO: 10, a LCDR1 having the sequence of SEQ ID NO:23, a LCDR2 having the sequence of SEQ ID NO:29, and a LCDR3 having the sequence of SEQ ID NO:34.
28. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO:2, an HCDR2 having the sequence of SEQ ID NO: 6, and an HCDR3 having the sequence of SEQ ID NO: 11, a LCDR1 having the sequence of SEQ ID NO:24, a LCDR2 having the sequence of SEQ ID NO:30, and a LCDR3 having the sequence of SEQ ID NO:35.
29. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO: 7, and an HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:25, a LCDR2 having the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36. 30. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises an HCDR1 having the sequence of SEQ ID NO:3, an HCDR2 having the sequence of SEQ ID NO: 7, and an HCDR3 having the sequence of SEQ ID NO: 13, a LCDR1 having the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36.
31. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:3, a HCDR2 having the sequence of SEQ ID NO:8, and a HCDR3 having the sequence of SEQ ID NO: 14, a LCDR1 having the sequence of SEQ ID NO:27, a LCDR2 having the sequence of SEQ ID NO:32, and a LCDR3 having the sequence of SEQ ID NO:37.
32. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 1, a HCDR2 having the sequence of SEQ ID NO: 9, and a HCDR3 having the sequence of SEQ ID NO: 10, a LCDR1 having the sequence of SEQ ID NO:23, a LCDR2 having the sequence of SEQ ID NO:29, and a LCDR3 having the sequence of SEQ ID NO:34.
33. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO: 7, and a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36.
34. The isolated antibody of any one of embodiments 1 and 23 to 26, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO: 7, and a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:28, a LCDR2 having the sequence of SEQ ID NO:33, and a LCDR3 having the sequence of SEQ ID NO:38.
35. The isolated antibody of any one of embodiments 1 to 34, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in any one of SEQ ID NOS: 15-22. 36. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO: 15.
37. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO: 16.
38. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO: 17.
39. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO: 18.
40. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO: 19.
41. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO:20.
42. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO:21.
43. The isolated antibody of embodiment 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO:22.
44. The isolated antibody of any one of embodiments 1 to 34, wherein the heavy chain variable region comprises the sequence of any one of SEQ ID NOS: 15-22.
45. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15.
46. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 16.
47. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 17.
48. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 18. 49. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 19.
50. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:20.
51. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:21.
52. The isolated antibody of embodiment 44, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:22.
53. The isolated antibody of any one of embodiments 1 to 52, wherein the light chain variable region comprises at least 90% identity to a sequence set forth in any of SEQ ID NOS:39-46.
54. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:39.
55. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:40.
56. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:41.
57. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:42.
58. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:43.
59. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:44.
60. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:45. 61. The isolated antibody of embodiment 53, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:46.
62. The isolated antibody of any one of embodiments 1 to 52, wherein the light chain variable region comprises the sequence set forth in any of SEQ ID NOS:39-46.
63. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:39.
64. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:40.
65. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:41.
66. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:42.
67. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:43.
68. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:44.
69. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:45.
70. The isolated antibody of embodiment 62, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:46.
71. The isolated antibody of any one of embodiments 1 to 70, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in any one of SEQ ID NOS: 15-22, and wherein the light chain variable region comprises at least 90% identity to the sequence set forth in any of SEQ ID NOS:39-46.
72. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO: 15, and wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:39.
73. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO: 16, and wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:40.
74. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO: 17, and wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:41.
75. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO: 18, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:42.
76. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO: 19, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:43.
77. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:20, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:39.
78. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:21, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:44.
79. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:22, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:45.
80. The isolated antibody of any one of embodiments 1 to 71, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:22, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:46.
81. The isolated antibody of any one of embodiments 1 to 70, wherein the heavy chain variable region comprises the sequence set forth in any one of SEQ ID NOS: 15- 22, and wherein the light chain variable region comprises the sequence set forth in any of SEQ ID NOS:39-46.
82. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:39.
83. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 16, and wherein the light chain variable region the sequence set forth in SEQ ID NO:40.
84. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 17, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:41.
85. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 18, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:42.
86. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 19, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:43.
87. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:20, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:39. 88. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in ID NO:21, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:44.
89. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in ID NO:22, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:45.
90. The isolated antibody of any one of embodiments 1 to 81, wherein the heavy chain variable region comprises the sequence set forth in ID NO:22, and wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:46.
91. The isolated antibody of any one of embodiments 1 to 90, wherein the antibody comprises an antigen-binding fragment thereof.
92. The isolated antibody of any one of embodiments 1 to 91, wherein the antibody comprises an Fc polypeptide having at least 90% identity to a sequence of SEQ ID NO:47.
93. An isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody competes binding to the SARS-CoV-2 RBD with an antibody of any one of embodiments 1 to 92.
94. The isolated antibody of embodiment 93, wherein the antibody binds to the same epitope as the antibody of any one of embodiments 1 to 92.
95. The isolated antibody of any one of embodiments 1 to 94, wherein the antibody is a monoclonal antibody.
96. The isolated antibody of any one of embodiments 1 to 95, wherein the antibody is a humanized antibody.
97. The isolated antibody of any one of embodiments 1 to 96, wherein the antibody comprises one or more human framework regions. 98. The isolated antibody of any one of embodiments 1 to 97, wherein the antibody is conjugated to a detectable marker or label.
99. The isolated antibody of any one of embodiments 1 to 98, wherein the antibody is non-diffusively immobilized on a solid support.
100. An isolated nucleic acid encoding the isolated antibody of any one of embodiments 1 to 99.
101. An isolated nucleic acid comprising a nucleotide sequence that encodes a heavy chain variable region comprising at least 90% sequence identity to the sequence set forth in any of SEQ ID NOS: 70-79 and 86.
102. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 70.
103. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 71.
104. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 72.
105. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 73.
106. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 74.
107. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 75. 108. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 76.
109. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 77.
110. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 78.
111. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 79.
112. The isolated nucleic acid of embodiment 101, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 86.
113. An isolated nucleic acid comprising a nucleotide sequence that encodes a heavy chain variable region comprising the sequence set forth in any of SEQ ID NOS: 70-79 and 86.
114. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 70.
115. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 71.
116. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 72.
117. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 73. 118. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 74.
119. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 75.
120. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 76.
121. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 77.
122. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region the sequence set forth in SEQ ID NO: 78.
123. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 79.
124. The isolated nucleic acid of embodiment 113, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 86.
125. An isolated nucleic acid comprising a nucleotide sequence that encodes a light chain variable region comprising at least 90% sequence identity to the sequence set forth in any of SEQ ID NOS: 80-85 and 87-93.
126. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 80.
127. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 81.
128. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 82. 129. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 83.
130. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 84.
131. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 85.
132. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 87.
133. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 88.
134. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 89.
135. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 90.
136. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID NO: 91.
137. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 92. 138. The isolated nucleic acid of embodiment 125, wherein the light chain variable region comprises at least 90% sequence identity to the sequence set forth in SEQ ID
NO: 93.
139. An isolated nucleic acid comprising a nucleotide sequence that encodes a light chain variable region comprising the sequence set forth in any of SEQ ID NOS: 80-85 and 87-93.
140. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 80.
141. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 81.
142. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 82.
143. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 83.
144. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 84.
145. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 85.
146. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 87.
147. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 88.
148. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 89.
149. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 90. 150. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 91.
151. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 92.
152. The isolated nucleic acid of embodiment 139, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 93.
153. An expression vector comprising the nucleic acid of any one of embodiments 100 to 152.
154. An isolated host cell comprising the expression vector of embodiment
153.
155. A pharmaceutical composition comprising the isolated antibody of any one of embodiment 1 to 99 and a pharmaceutically acceptable carrier.
156. A diagnostic reagent comprising the isolated antibody of any one of embodiments 1 to 99.
157. A kit comprising the isolated antibody of any one of embodiments 1 to 99 or the diagnostic reagent of embodiment 156.
158. A method of detecting severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), comprising contacting a sample known or suspected to contain SARS-CoV-2 RBD with an antibody of any one of embodiments 1 to 99.
159. A method of detecting severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), comprising a) contacting a sample with the antibody of any one of embodiments 1 to 99, under conditions to bind said antibody to a SARS-CoV-2 RBD receptor on said sample, wherein the binding generates the production of a receptor/antibody complex; and b) detecting the presence of the receptor/antibody complexes, wherein the detecting comprises the presence or absence of the SARS-CoV-2 RBD receptor on said sample. 160. A method of treating or preventing a disease or disorder associated with severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV- 2 RBD) in a subject, comprising: a) contacting a sample known or suspected to contain SARS-CoV-2 RBD with the antibody of any one of embodiments 1 to 99; b) detecting the presence of complexes comprising SARS-CoV-2 RBD and the antibody; wherein the presence of the complexes indicates the presence of a disease or disorder; and c) administering to the subject the antibody or antigen binding fragment thereof of any one of embodiments 1 to 99.
161. A method of diagnosing a disease or disorder, comprising: a) isolating a sample from a subject; b) incubating the sample with the antibody of any one of embodiments 1 to 99, for a period of time sufficient to generate SARS-CoV-2 RBD:anti- SARS-CoV-2 RBD complexes; c) detecting the presence or absence of the SARS-COV-2 RBD:anti-SARS- COV-2 RBD complexes from the isolated tissue; and d) associating presence or abundance of SARS-COV-2 RBD with a location of interest of a tissue sample.
162. The method of embodiment 161, wherein the increase of SARS-CoV-2 RBD over a control level in the location of interest of the tissue sample is indicative of a disease or disorder in a subject.
163. The method of any one of embodiments 159 to 162, wherein the method is performed in vitro.
164. The method of any one of embodiments 159 to 162, wherein the method is performed in vivo.
165. The method of any one of embodiments 159 to 164, wherein the detection comprises intracellular detection. 166. The method of any one of embodiments 159 to 165, wherein the detection comprises detection on the surface of a cell.
167. The method of any one of embodiments 159 to 166, wherein the detection comprises hybridization of a detectable moiety to the antibody.
168. The method of any one of embodiments 159 to 167, wherein the sample is contacted with a second antibody.
169. The method of any one of embodiments 159 to 168, wherein the second antibody is an antibody comprising a detectable moiety.
170. The method of any one of embodiments 159 to 169, wherein the detectable moiety comprises an oligonucleotide.
171. The method of any one of embodiments 159 to 170, wherein the detectable moiety comprises a fluorescent label.
172. The method of any one of embodiments 159 to 171, wherein the measurement comprises sequencing.
173. The method of any one of embodiments 159 to 172, wherein the detectable moiety comprises immunofluorescence.
174. The method of any one of embodiments 159 to 173, wherein sample is a formalin-fixed paraffin-embedded sample.
175. The method of any one of embodiments 159 to 174, wherein the sample comprises a cell.
176. The method of any one of embodiments 159 to 175, wherein the sample comprises a tissue sample.
177. The method of any one of embodiments 159 to 176, wherein the sample comprises immune cells.
178. The method of embodiment 177, wherein the immune cells are selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).
179. The method of any one of embodiments 159 to 178, wherein the sample comprises a tissue or cells associated with a disease or disorder.
180. The method of any one of embodiments 159 to 179, wherein the disease or disorder is a cancer, an autoimmune disorder, an inflammatory disorder, or an infection.
181. The method of embodiment 179 or 180, wherein the disease or disorder is chosen fro non-viral cancers, virus-associated cancers, cancers associated with HBV infection, cancers associated with Epstein-Barr virus (EBV) infection, cancers associated with polyomavirus infection, erythema nodosum leprosum (ENL), autoimmune diseases, autoimmune inflammation, autoimmune thyroid diseases, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's Disease, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemias, myelodysplastie syndromes, solid phase cancer, herpes viral infections, and/or rejection of transplanted tissues or organs.
182. The antibody or antigen binding fragment thereof of any one of embodiments 1 99, for use in a method of associating presence or abundance of SARS-CoV- 2 RBD with a location of interest of a tissue sample.
183. The antibody or antigen binding fragment thereof of any one of embodiments 1 to 99, for use in a method of detecting SARS-CoV-2 RBD in a tissue sample.
184. The antibody or antigen binding fragment thereof of embodiment 182 or 183, wherein the method comprises generating a nucleic acid molecule comprising all or a portion of the sequence of the oligonucleotide or a complement thereof.
185. The antibody or antigen binding fragment thereof of any one of embodiments 1 to 99, for use in the construction of a protein library. 186. The antibody or antigen binding fragment thereof of embodiment 185, wherein the construction of a protein library comprises sequencing.
187. The antibody or antigen binding fragment thereof of embodiment 185, wherein the construction of a protein library comprises the use of flow cytometry.
[0212] The above examples are provided to illustrate the disclosure but not to limit its scope. Other variants of the disclosure will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, internet sources, patents, patent applications, and accession numbers cited herein are hereby incorporated by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:
1. An isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody comprises: a) a heavy chain variable region comprising:
(i) a heavy chain complementary determining region 1 (HCDR1) having the sequence of XIYX2MX3 (SEQ ID NO:64), wherein Xi is D, E, N, or S; X2 is I, T, or W; and X3 is L or H;
(ii) an HCDR2 having the sequence of X1IX2PX3X4X5X6TX7X8NX9KFKX10 (SEQ ID NO:65), wherein Xi is N, G, M, or E; X2 is N or D; X3 is Y, N, or S; X4 is Y, N, or D; X5 is G, V, or S; Cb is S, D, E, or R; X7 is S, N, or R; X8 is Y or L; X9 is L, Q, or E; and X10 is G, D, or S;
(iii) an HCDR3 having the sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO:66), wherein Xi is Y or absent; X2 is Y, R, A, or absent; X3 is G or absent; X4 is S, D, L or A; X5 is S, R, L, M, or Y; Cb is Y or N; X7 is G, D, R, or absent; X8 is G, Y, or absent; X9 is Y, T, S, or absent; X10 is F or V; Xn is D or A; and X12 is V or Y; and b) a light chain variable region comprising:
(iv) a light chain complementary determining region 1 (LCDR1) having the sequence of X1X2SX3X4X5X6X7X8X9X1X11X12X13X14X15 (SEQ ID NO:67), wherein Xi is S or R; X2 is A or S; X3 is S, E, K, Q, or T; X4 is S, E, or G; X5 is L or absent; Cb is V, L, or absent; X7 is D, H, A, or absent; X8 is N, S, V, or absent; X9 is Y, N, T, or absent; X10 is G, I, T, or absent; Xn is L, I, N, or S; X12 is N, S, T, or G; X13 is F or Y; X14 is M, L, or A; and X15 is Y, N, or S;
(v) a LCDR2 having the sequence of X1X2X3X4X5X6X7 (SEQ ID NO:68), wherein Xi is L, A, R, or G; X2 is T, A, or M; X3 is S or N; X4 is N or T; X5 is L, Q, or R; Cb is A, G, or D; and X7 is S or P; and
(vi) a LCDR3 having the sequence of X1X2X3X4X5X6X7X8X9 (SEQ ID NO:69), wherein Xi is Q, M, L, or A; X2 is Q or L; X3 is W, S, H, or Y; X4 is S, K, L, A, or Y; X5 is T, E, or S; X6 is N, V, or Y; X7 is P or H; X8 is L, Y, or W; and X9 is T or V.
2. The antibody of claim 1, wherein the antibody comprises:
(a) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 1-4;
(b) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5-9; and (c) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 10-14.
3. The antibody of claim 1 or 2, wherein the antibody comprises:
(1) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:23-28;
(2) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:29-33; and
(3) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:34-38.
4. The antibody of any of claims 1 to 3, wherein the antibody comprises:
(1) an HCDR1 having a sequence of any one of SEQ ID NOS: 1-4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 1-4;
(2) an HCDR2 having a sequence of any one of SEQ ID NOS: 5-9 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:5-9;
(3) an HCDR3 having a sequence of any one of SEQ ID NOS: 10-14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 10-14;
(4) a LCDR1 having a sequence of any one of SEQ ID NOS:23-28 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:23-28;
(5) a LCDR2 having a sequence of any one of SEQ ID NOS:29-33 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:29-33; and
(6) a LCDR3 having a sequence of any one of SEQ ID NOS:34-38 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS:34-38.
5. The isolated antibody of any one of claims 1 to 4, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO:7, and a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:26, a LCDR2 having the sequence of SEQ ID NO:31, and a LCDR3 having the sequence of SEQ ID NO:36.
6. The isolated antibody of any one of claims 1 to 4, wherein the antibody comprises a HCDR1 having the sequence of SEQ ID NO:4, a HCDR2 having the sequence of SEQ ID NO:7, and a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO:28, a LCDR2 having the sequence of SEQ ID NO:33, and a LCDR3 having the sequence of SEQ ID NO:38.
7. The isolated antibody of any one of claims 1 to 6, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in any one of SEQ ID NOS: 15-22.
8. The isolated antibody of any of claims 1 to 7, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO:22.
9. The isolated antibody of any one of claims 1 to 8, wherein the light chain variable region comprises at least 90% identity to a sequence set forth in any of SEQ ID NOS:39-46.
10. The isolated antibody of any of claims 1 to 9, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:45.
11. The isolated antibody of any of claims 1 to 9, wherein the light chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:46.
12. The isolated antibody of any one of claims 1 to 11, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in any one of SEQ ID NOS: 15-22, and wherein the light chain variable region comprises at least 90% identity to the sequence set forth in any of SEQ ID NOS:39-46.
13. The isolated antibody of any one of claims 1 to 12, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:22, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:45.
14. The isolated antibody of any one of claims 1 to 12, wherein the heavy chain variable region comprises at least 90% identity to the sequence set forth in SEQ ID NO:22, and wherein the light chain variable region comprises at least 90% identity to a sequence set forth in SEQ ID NO:46.
15. The isolated antibody of any of claims 1 to 14, wherein the antibody comprises an antigen-binding fragment thereof.
16. The isolated antibody of any one of claims 1 to 15, wherein the antibody comprises an Fc polypeptide having at least 90% identity to a sequence of SEQ ID NO:47.
17. An isolated antibody that specifically binds to severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), wherein the isolated antibody competes binding to the SARS-CoV-2 RBD with an antibody of any one of claims 1 to 16.
18. The isolated antibody of claim 17, wherein the antibody binds to the same epitope as the antibody of any one of claims 1 to 16.
19. The isolated antibody of any one of claims 1 to 18, wherein the antibody is a monoclonal antibody.
20. The isolated antibody of any one of claims 1 to 19, wherein the antibody is a humanized antibody.
21. The isolated antibody of any one of claims 1 to 20, wherein the antibody comprises one or more human framework regions.
22. The isolated antibody of any one of claims 1 to 21, wherein the antibody is conjugated to a detectable marker or label.
23. The isolated antibody of any one of claims 1 to 22, wherein the antibody is non-diffusively immobilized on a solid support.
24. An isolated nucleic acid encoding the isolated antibody of any one of claims
1 to 23.
25. An isolated nucleic acid comprising a nucleotide sequence that encodes a heavy chain variable domain comprising at least 90% sequence identity to the sequence set forth in any of SEQ ID NOS: 70-79 and 86.
I l l
26. An isolated nucleic acid comprising a nucleotide sequence that encodes a light chain variable domain comprising at least 90% sequence identity to the sequence set forth in any of SEQ ID NOS: 80-85 and 87-93.
27. An expression vector comprising the nucleic acid of any of claims 24 to 26.
28. An isolated host cell comprising the vector of claim 27.
29. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 23 and a pharmaceutically acceptable carrier.
30. A diagnostic reagent comprising the isolated antibody of any one of claims 1 to 23.
31. A kit comprising the isolated antibody of any one of claims 1 to 23 or the diagnostic reagent of claim 30.
32. A method of detecting severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), comprising a) contacting a sample with the antibody of any of claims 1 to 23, under conditions to bind said antibody to a SARS-CoV-2 RBD receptor on said sample, wherein the binding generates the production of a receptor/antibody complex; and b) detecting the presence of the receptor/antibody complexes, wherein the detecting comprises the presence or absence of the SAR.S-CoV-2 RBD receptor on said sample.
33. A method of treating or preventing a disease or disorder associated with severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SAR.S-C0V- 2 RBD) in a subject, comprising: a) contacting a sample known or suspected to contain SARS-CoV-2 RBD with the antibody of any of claims 1 to 23, b) detecting the presence of complexes comprising SAR.S-CoV-2 RBD and the antibody; wherein the presence of the complexes indicates the presence of a disease or disorder; and c) administering to the subject the antibody or antigen binding fragment thereof of any of claims 1 to 23.
34. A method of diagnosing a disease or disorder, comprising: a) isolating a sample from a subject; b) incubating the sample with the antibody of any of claims 1 to 23, for a period of time sufficient to generate SARS-CoV-2 RBD:anti- SARS-CoV-2 RBD complexes; c) detecting the presence or absence of the SARS-COV-2 RBD:anti-SARS- COV-2 RBD complexes from the isolated tissue; and d) associating presence or abundance of SARS-CoV-2 RBD with a location of interest of a tissue sample.
35. The method of any of claims 32 to 34, wherein the method is performed in vitro.
36. The method of any of claims 32 to 35, wherein the detection comprises hybridization of a detectable moiety to the antibody.
37. The method of claim 36, wherein the detectable moiety comprises an oligonucleotide.
38. The method of claim 36 or 37, wherein the detectable moiety comprises a fluorescent label.
39. The method of any of claims 32 to 38, wherein the sample comprises a cell.
40. The method of any of claims 32 to 38, wherein the sample comprises a tissue sample.
PCT/US2022/024471 2021-04-16 2022-04-12 Compositions and methods involving severe acute respiratory syndrome coronavirus 2 receptor binding domain WO2022221314A1 (en)

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