ANTIBODIES THAT BIND SARS-COV-2 SPIKE PROTEIN
20240270826 · 2024-08-15
Assignee
Inventors
Cpc classification
G01N2469/10
PHYSICS
C07K16/1003
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K47/68
HUMAN NECESSITIES
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
Abstract
Described herein are antibodies that specifically recognize the SARS-CoV-2 spike (S) polypeptide, compositions comprising said antibodies, uses thereof, and methods employing said antibodies. Each antibody specifically recognizes the S1-RBD domain, S1-NTD domain, or S2 subunit of the SARS-CoV-2 spike polypeptide. Some antibodies are cross-reactive with variants of SARS-CoV-2 and other coronavirus spike polypeptides, such as SARS-CoV S, pangolin CoV S, bat SARS-like CoV S, and civet SARS-CoV S.
Claims
1. An isolated or purified antibody that specifically recognizes at least one coronavirus spike polypeptide, wherein the antibody comprises an antigen binding portion of an antibody heavy chain, wherein the antigen binding portion comprises a first complementarity determining region (CDR1), a second complementarity determining region (CDR2), and a third complementarity determining region (CDR3), and wherein CDR1, CDR2, and CDR3, respectively, comprise the amino acid sequence set forth in: SEQ ID NO: 1, SEQ ID NO: 45, and SEQ ID NO: 90; SEQ ID NO: 2, SEQ ID NO: 45, and SEQ ID NO: 91; SEQ ID NO: 1, SEQ ID NO: 46, and SEQ ID NO: 92; SEQ ID NO: 3, SEQ ID NO: 47, and SEQ ID NO: 93; SEQ ID NO: 4, SEQ ID NO: 48, and SEQ ID NO: 94; SEQ ID NO: 5, SEQ ID NO: 49, and SEQ ID NO: 95; SEQ ID NO: 6, SEQ ID NO: 50, and SEQ ID NO: 96; SEQ ID NO: 7, SEQ ID NO: 51, and SEQ ID NO: 97; SEQ ID NO: 8, SEQ ID NO: 52, and SEQ ID NO: 98; SEQ ID NO: 9, SEQ ID NO: 53, and SEQ ID NO: 99; SEQ ID NO: 10, SEQ ID NO: 54, and SEQ ID NO: 100; SEQ ID NO: 11, SEQ ID NO: 55, and SEQ ID NO: 101; SEQ ID NO: 12, SEQ ID NO: 56, and SEQ ID NO: 102; SEQ ID NO: 13, SEQ ID NO: 57, and SEQ ID NO: 103; SEQ ID NO: 14, SEQ ID NO: 58, and SEQ ID NO: 104; SEQ ID NO: 15, SEQ ID NO: 59, and SEQ ID NO: 105; SEQ ID NO: 16, SEQ ID NO: 60, and SEQ ID NO: 106; SEQ ID NO: 17, SEQ ID NO: 61, and SEQ ID NO: 107; SEQ ID NO: 18, SEQ ID NO: 62, and SEQ ID NO: 108; SEQ ID NO: 19, SEQ ID NO: 63, and SEQ ID NO: 109; SEQ ID NO: 20, SEQ ID NO: 64, and SEQ ID NO: 110; SEQ ID NO: 21, SEQ ID NO: 65, and SEQ ID NO: 111; SEQ ID NO: 22, SEQ ID NO: 66, and SEQ ID NO: 112; SEQ ID NO: 23, SEQ ID NO: 67, and SEQ ID NO: 113; SEQ ID NO: 24, SEQ ID NO: 68, and SEQ ID NO: 114; SEQ ID NO: 25, SEQ ID NO: 69, and SEQ ID NO: 115; SEQ ID NO: 26, SEQ ID NO: 70, and SEQ ID NO: 116; SEQ ID NO: 27, SEQ ID NO: 71, and SEQ ID NO: 117; SEQ ID NO: 28, SEQ ID NO: 72, and SEQ ID NO: 118; SEQ ID NO: 29, SEQ ID NO: 73, and SEQ ID NO: 119; SEQ ID NO: 30, SEQ ID NO: 74, and SEQ ID NO: 120; SEQ ID NO: 31, SEQ ID NO: 75, and SEQ ID NO: 121; SEQ ID NO: 32, SEQ ID NO: 76, and SEQ ID NO: 122; SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 123; SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 124; SEQ ID NO: 35, SEQ ID NO: 79, and SEQ ID NO: 125; SEQ ID NO: 36, SEQ ID NO: 80, and SEQ ID NO: 125; SEQ ID NO: 20, SEQ ID NO: 81, and SEQ ID NO: 126; SEQ ID NO: 20, SEQ ID NO: 81, and SEQ ID NO: 127; SEQ ID NO: 37, SEQ ID NO: 82, and SEQ ID NO: 128; SEQ ID NO: 38, SEQ ID NO: 83, and SEQ ID NO: 129; SEQ ID NO: 39, SEQ ID NO: 84, and SEQ ID NO: 130; SEQ ID NO: 40, SEQ ID NO: 85, and SEQ ID NO: 131; SEQ ID NO: 41, SEQ ID NO: 86, and SEQ ID NO: 132; SEQ ID NO: 42, SEQ ID NO: 87, and SEQ ID NO: 133; SEQ ID NO: 43, SEQ ID NO: 88, and SEQ ID NO: 134; or SEQ ID NO: 44, SEQ ID NO: 89, and SEQ ID NO: 135.
2. The antibody of claim 1, wherein the antibody is a neutralizing antibody and wherein CDR1, CDR2, and CDR3, respectively, comprise the amino acid sequence set forth in: SEQ ID NO: 11, SEQ ID NO: 55, and SEQ ID NO: 101; SEQ ID NO: 13, SEQ ID NO: 57, and SEQ ID NO: 103; SEQ ID NO: 19, SEQ ID NO: 63, and SEQ ID NO: 109; or SEQ ID NO: 21, SEQ ID NO: 65, and SEQ ID NO: 111.
3. The antibody of claim 1, wherein the antibody comprises the amino acid sequence set forth in SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, or SEQ ID NO: 186.
4. The antibody of claim 1, wherein the antibody comprises the amino acid sequence set forth in SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, or SEQ ID NO: 182, or an amino acid sequence having at least 75% sequence identity to the full length of the amino acid sequence set forth in SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, or SEQ ID NO: 182.
5. The antibody of claim 1, wherein the antibody is a single domain antibody.
6-7. (canceled)
8. The antibody of claim 1, wherein the antibody is in a multivalent display format.
9. The antibody of claim 8, wherein the antibody is linked to an Fc fragment.
10. (canceled)
11. A nucleic acid molecule encoding the antibody of claim 1.
12. A vector comprising the nucleic acid molecule of claim 11.
13. (canceled)
14. A host cell comprising the vector of claim 12.
15. A pharmaceutical composition comprising at least one antibody as defined in claim 1 and a pharmaceutically acceptable carrier and/or diluent.
16. (canceled)
17. A composition comprising at least one antibody as defined in claim 1 linked to another molecule.
18. (canceled)
19. The composition of claim 17, wherein the other molecule is an ACE2 polypeptide or a fragment thereof.
20. A composition comprising at least one antibody as defined in claim 1 immobilized on a substrate.
21-26. (canceled)
27. A method for treating or preventing a coronavirus infection, the method comprising administering at least one antibody as defined claim 1 to a subject in need thereof.
28-30. (canceled)
31. A method for detecting the presence of a coronavirus or a coronavirus spike polypeptide or fragment thereof in a sample, the method comprising exposing the sample to at least one antibody as defined in claim 1 and assaying for specific binding between the at least one antibody and the sample, wherein specific binding indicates a presence of the at least one coronavirus or coronavirus spike polypeptide or fragment thereof in the sample.
32. A method for capturing a coronavirus or a coronavirus spike polypeptide or fragment thereof from a sample, the method comprising exposing the sample to the composition as defined in claim 20.
33. The method of claim 31, wherein the coronavirus is a coronavirus that specifically binds an ACE2 receptor or the coronavirus spike polypeptide is a coronavirus spike polypeptide that specifically binds an ACE2 receptor.
34. The method of claim 31, wherein the coronavirus is SARS-CoV-2 or SARS-CoV, or the coronavirus spike polypeptide or fragment thereof is a SARS-CoV-2 or SARS-CoV coronavirus spike polypeptide or fragment thereof.
35-44. (canceled)
45. An antibody cocktail composition comprising two or more of the antibodies of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0266] Throughout the present disclosure, including in the drawings, antibodies may be referred to by their full name, e.g. NRCoV2-1d, NRCoV2-02, NRCoV2-SR03, or NRCoV2-MRed02, or by an abbreviation in which the NRCoV2- portion of the antibody name is omitted, e.g. 1 d, 02, SR03, or MRed02. Further, RBD and S1-RBD are used interchangeably, as are NTD and S1-NTD.
[0267]
[0268]
[0269]
[0270]
[0271]
[0272]
[0273]
[0274]
[0275]
[0276]
[0277]
[0278]
[0279]
[0280]
[0281]
[0282]
[0283]
[0284]
[0285]
[0286]
[0287]
[0288]
[0289]
[0290]
[0291]
[0292]
[0293]
[0294]
[0295]
[0296]
[0297]
[0298]
DETAILED DESCRIPTION
[0299] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.
[0300] Terms defined below may have the meanings ascribed to them, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Definitions
[0301] The coronavirus spike polypeptide or coronavirus spike protein (S) is the major coronavirus surface protein, and is a glycosylated homotrimer that binds to a host cell receptor and mediates coronavirus entry into a host cell. The coronavirus may be SARS-CoV-2, SARS-CoV, or another coronavirus. SARS-CoV-2 may be used herein to refer to any strain or variant of the SARS-CoV-2 virus. Similarly, SARS-CoV may be used to refer to any strain or variant of the SARS-CoV virus. A SARS-CoV-2 variant is a strain of SARS-CoV-2 that comprises one or more mutations relative to the Wuhan strain of SARS-CoV-2. A variant may be, but need not be, a variant that has been designated as a variant of concern or a variant of interest by the World Health Organization.
[0302] As used herein, the term polypeptide refers to a molecule comprising two or more amino acid residues linked by peptide bonds. A polypeptide may have primary, secondary, and/or tertiary structure. A protein comprises at least one polypeptide and may have primary, secondary, tertiary, and/or quaternary structure. The terms polypeptide and protein are often used interchangeably, and a polypeptide may be comprised by a protein. For example, a protein may be a homo- or hetero-multimer that comprises two or more polypeptides, or a protein may comprise a single polypeptide. A polypeptide or protein may include one or more post-translational modifications, such as, but not limited to, glycosylation, phosphorylation, lipidation, S-nitrosylation, N-acetylation, or methylation.
[0303] As used herein, the term fragment, in the context of a polypeptide, refers to a portion of a polypeptide comprising a series of consecutive amino acid residues from a parent polypeptide. In a specific embodiment, the term fragment refers to an amino acid sequence of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive amino acid residues from a parent polypeptide. In embodiments, a fragment may comprise an epitope or binding domain from a parent polypeptide. In embodiments, a fragment may be a biologically active fragment that retains one or more functional characteristics of a parent polypeptide.
[0304] The term antibody, as used herein, refers to an antigen binding protein comprising at least a heavy chain variable region (V.sub.H) that binds a target epitope. The term antibody includes monoclonal antibodies comprising immunoglobulin heavy and light chain molecules, single heavy chain variable domain antibodies, and variants and derivatives thereof, including chimeric variants of monoclonal and single heavy chain variable domain antibodies. The antibody may be a naturally-occurring antibody, it may be obtained by manipulation of a naturally-occurring antibody, or it may be produced using recombinant methods. For example, an antibody may include, but is not limited to a Fv, single-chain Fv (scFv; a molecule consisting of V.sub.L and V.sub.H connected with a peptide linker), Fab, F(ab)2, single domain antibody (sdAb; an antibody composed of a single V.sub.L or V.sub.H), or a multivalent presentation of any of these. Antibodies such as those just described may require linker sequences, disulfide bonds, or other types of covalent bond to link different portions of the antibody. Those of skill in the art will be familiar with the requirements of the different types of antibodies and various approaches for their construction.
[0305] In a non-limiting example, the antibody may be a single domain antibody derived from a naturally-occurring source. Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed V.sub.HH. sdAbs have also been observed in shark and are termed VNAR (Nuttall et al, 2003). Other sdAbs may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term single domain antibody includes single domain antibodies directly isolated from V.sub.H, V.sub.HH, V.sub.L, or VNAR reservoir of any origin through phage display or other technologies, single domain antibodies derived from the aforementioned single domain antibodies, recombinantly produced single domain antibodies, as well as single domain antibodies generated through further modification of such single domain antibodies by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the disclosure are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the single domain antibody.
[0306] Single domain antibodies possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997). They can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of non-canonical disulfide bonds (Hussack et al, 2011; Kim et al, 2012), may also be brought to a single domain antibody.
[0307] A person of skill in the art would be well-acquainted with the structure of a single-domain antibody. A single domain antibody comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by one of skill in the art, not all CDRs may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDRs may contribute to binding and recognition of the antigen by a single domain antibody. The CDRs of the single domain antibody or variable domain are referred to herein as CDR1, CDR2, and CDR3, and numbered as defined by Lefranc et al., 2003.
[0308] As described herein, the amino acid sequence and structure of a heavy chain variable domain, including a V.sub.HH, can be considered-without however being limited thereto to be comprised of four framework regions or FR, which are referred to in the art and herein as Framework region 1 orFR1; as Framework region 2 orFR2; as Framework region 3 or FR3; and as Framework region 4 orFR4, respectively; which framework regions are interrupted by three complementarity determining regions or CDR's, which are referred to in the art as Complementarity Determining Region 1 or CDR1; as Complementarity Determining Region 2 or CDR2; and as Complementarity Determining Region 3 or CDR3, respectively. CDRs described in the present disclosure have been defined using the IMGT numbering system (Lefranc et al, 2003).
[0309] The term binding as used herein in the context of binding between an antibody, such as a V.sub.HH, and a coronavirus spike protein epitope as a target, refers to the process of a non-covalent interaction between molecules. Preferably, said binding is specific. The terms specific or specificity or grammatical variations thereof refer to the number of different types of antigens or their epitopes to which a particular antibody such as a V.sub.HH can bind. The specificity of an antibody, also referred to as specific binding, can be determined based on affinity. A specific antibody preferably has a binding affinity (Kd) for its epitope of less than 10.sup.?7 M, preferably less than 10.sup.?8 M.
[0310] The term affinity, as used herein, refers to the strength of a binding reaction between a binding domain of an antibody and an epitope. It is the sum of the attractive and repulsive forces operating between the binding domain and the epitope. The term affinity, as used herein, refers to the equilibrium dissociation constant, Kd.
[0311] The term epitope or antigenic determinant, as used herein, refers to a part of an antigen that is recognized by an antibody. The term epitope includes linear epitopes and conformational epitopes. A linear epitope is an epitope that is recognized by an antibody based on its primary structure, and a stretch of contiguous amino acids is sufficient for binding. A conformational epitope is based on 3-D surface features and shape and/or tertiary structure of the antigen.
[0312] The term neutralizing antibody, as used herein, refers to an antibody that, when bound to an epitope, interferes with at least one of the steps leading to the release of a virus genome, such as a coronavirus genome, into a host cell.
[0313] The term subject, as used herein, refers to an animal that is susceptible to infection by a coronavirus. The subject may be an animal that is susceptible to infection by a coronavirus that binds an ACE2 receptor, such as SARS-CoV-2 or SARS-CoV. The subject may be a human or non-human animal. Preferably the subject is a human or non-human mammal. Correspondingly, the ACE2 receptor may be a human ACE2 receptor or an animal ACE2 receptor.
[0314] The term administering, as used herein, refers to the introduction into a subject of a therapeutic agent. Many administration routes are known in the art, and include, but are not limited to, parenteral (intravenous, intramuscular, and subcutaneous), oral, nasal, ocular, transmucosal (buccal, vaginal, and rectal), transdermal, and pulmonary administration.
[0315] The terms strong interaction and strong binding, as used herein, refer to the presence of salt bridges and cation-pi interactions between amino acid residues, as is known to the skilled person.
[0316] The terms weak interaction and weak binding, as used herein, refer to the presence of hydrogen bonds and non-bonded/hydrophobic interactions, as is known to the skilled person.
[0317] The term purified, as used herein, refers to a molecule, e.g. a polypeptide or protein that has been identified and substantially separated and/or recovered from the components of its natural environment. The term isolated antibody, as used herein, refers to an antibody that is substantially freed from other antibody molecules having different antigenic specificities. Further, a purified or isolated antibody may be substantially free of one or more other cellular and/or chemical substances. Absolute purity is not required for a molecule to be considered purified or isolated.
[0318] The term pharmaceutically acceptable, as used herein, means generally regarded as safe when administered to humans. Preferably, as used herein, the term pharmaceutically acceptable is approved by a federal or state government regulatory agency for use in animals, more preferably in humans. The term carrier means a diluent, adjuvant, excipient, or vehicle with which a compound is formulated and/or administered. Such pharmaceutical carriers can be water and sterile liquids, such as petroleum, animal, vegetable or synthetically derived oils such as peanut oil, soybean oil, mineral oil, sesame oil. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers for injectable solutions. Suitable pharmaceutical carriers are, for example, described in Remington (23.sup.rd edition), The Science and Practice of Pharmacy.
[0319] As used herein the term linked or linkage includes covalent and non-covalent linkage (bonding). As used herein, the term linker refers to a chemical group or molecule that can be used to join one molecule to another. An antibody may be linked to another molecule by a linker or an antibody may be directly linked (aka joined, fused, or bonded) to another molecule, without the use of a linker. Suitable linkers are known in the art and may be selected based on the chemical nature of the molecules being joined. Examples of linkers include peptide linkers and chemical cross-linkers. Peptide linkers may comprise a single amino acid residue or a plurality of amino acid residues. An antibody and a polypeptide may, for example, be linked by chemical conjugation, with or without the use of a linker, or produced as a fusion, for example by recombinant protein expression.
[0320] As used herein the term label refers to a molecule or compound that can be used to label a molecule, such as an antibody, to allow detection of the molecule. Suitable labels will be known to one skilled in the art and include, but are not limited to, radioisotopes; enzymes, such as horse radish peroxidase (HRP) or calf intestinal alkaline phosphate (AP); fluorophores; antigen binding fragments from cleaved antibodies (Fabs); and colloidal gold. Covalent linkage is commonly used to link a label to a molecule of interest, however, non-covalent linkage is also possible, for example, when the label is a Fab.
[0321] As used herein, the term nucleic acid molecule refers to any nucleic acid-containing molecule including, but not limited to, DNA, RNA, and DNA/RNA hybrids, in any form and/or conformation. The term encompasses nucleic acids that include any of the known base analogs of DNA and RNA. For example, single-stranded, double-stranded, nuclear, extranuclear, extracellular, and isolated nucleic acids are all contemplated.
[0322] As used herein, the term vector refers to a synthetic nucleotide sequence used for manipulation of genetic material, including but not limited to cloning, subcloning, sequencing, or introduction of exogenous genetic material into cells, tissues or organisms. It is understood by one skilled in the art that vectors may contain synthetic DNA sequences, naturally occurring DNA sequences, or both. Examples of commonly used vectors include plasmids, viral vectors, cosmids, and artificial chromosomes.
[0323] As used herein, the term regulatory sequence includes promoters, enhancers and other expression control elements, such as polyadenylation sequences, matrix attachment sites, insulator regions for expression of multiple genes on a single construct, ribosome entry/attachment sites, introns that are able to enhance expression, and silencers. Promoters may be cell-specific or tissue-specific to facilitate expression in a desired target.
[0324] When referring to two nucleotide sequences, one being a regulatory sequence, the term operably linked is used herein to mean that the two sequences are associated in a manner that allows the regulatory sequence to affect expression of the other nucleotide sequence. It is not required that the operably-linked sequences be directly adjacent to one another with no intervening sequence(s).
[0325] As used herein, the term host cell refers to a cell into which a nucleic acid molecule or vector may be introduced, for example to allow for replication of the nucleic acid molecule or vector by the host cell and/or to allow for expression of the nucleic acid molecule, or of a nucleic acid molecule comprised by the vector, by the host cell to produce a product of interest, such as an RNA or protein. In a specific embodiment, the nucleic acid molecule may encode an antibody as described herein, and introduction of the nucleic acid molecule into the host cell may allow the antibody to be expressed by the host cell. A host cell may be any suitable cell, such as a bacterial cell or eukaryotic cell. Commonly used host cells include E. coli, yeast, and mammalian cells, such as, but not limited to, Chinese hamster ovary (CHO) cells, mouse myeloma cells, and human embryonic kidney (HEK) cells.
[0326] The term treatment and variations thereof, such as treat or treating, as used herein, refer to the administration of a therapeutic molecule or composition to a subject to reduce or eliminate one or more symptoms of an illness or disease in the subject and/or to reduce the duration of the illness or disease in the subject.
[0327] The term prevention and variations thereof, such as prevent or preventing, as used herein, refer to the prophylactic administration of a therapeutic molecule or composition to a subject to prevent the occurrence of, or to reduce the severity of, an illness or disease in the subject.
[0328] The term sample as used herein, refers to a sample in which a coronavirus presence is suspected or expected. For example, the sample may be a biological sample from a subject, such as, but not limited to, blood or a fraction thereof, saliva, cellular material, urine, or feces; a sample from a bioreactor; or an environmental sample.
[0329] The term sequence identity as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. % identity=(number of identical overlapping positions/total number of positions)?100). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
[0330] As used herein the singular forms a, an, and the include plural references unless the context clearly dictates otherwise.
[0331] The phrase and/or, as used herein, should be understood to mean either or both of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with and/or should be construed in the same fashion, i.e., one or more of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the and/or clause, whether related or unrelated to those elements specifically identified.
[0332] As used herein in the specification and in the claims, or should be understood to have the same meaning as and/or as defined above. For example, when separating items in a list, or or and/or shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as only one of or exactly one of or, when used in the claims, consisting of will refer to the inclusion of exactly one element of a number or list of elements. In general, the term or as used herein shall only be interpreted as indicating exclusive alternatives (i.e., one or the other but not both) when preceded by terms of exclusivity, such as either, one of, only one of, or exactly one of.
[0333] As used herein, all transitional phrases such as comprising, including, carrying, having, containing, involving, holding, composed of, and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases consisting of and consisting essentially of shall be closed or semi-closed transitional phrases, respectively.
[0334] As used herein, the phrase at least one, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase at least one refers, whether related or unrelated to those elements specifically identified.
Description
[0335] The present disclosure relates to SARS-CoV-2 spike protein-specific antibodies and uses thereof. Provided are isolated or purified antibodies comprising complementarity determining region (CDR) 1, CDR2, and CDR3 sequences as outlined in Table 6. The antibodies described herein recognize a variety of spike protein epitopes in different subunit and domains of the coronavirus spike protein, specifically S2, the N-terminal domain of S1 (S1-NTD), and the receptor binding domain of S1 (S1-RBD). Within these subunits/domains, antibodies described herein recognize several different epitopes. Because of this epitopic diversity, antibodies described herein may be used in combination, for example for combination therapy, or as bispecific or multi-specific antibodies.
[0336] An antibody as described herein comprises an antigen binding portion of an antibody heavy chain, wherein the antigen binding portion comprises a first complementarity determining region (CDR1), a second complementarity determining region (CDR2), and a third complementarity determining region (CDR3), and wherein CDR1, CDR2, and CDR3, respectively, comprise the amino acid sequence set forth in: SEQ ID NO: 1, SEQ ID NO: 45, and SEQ ID NO: 90; SEQ ID NO: 2, SEQ ID NO: 45, and SEQ ID NO: 91; SEQ ID NO: 1, SEQ ID NO: 46, and SEQ ID NO: 92; SEQ ID NO: 3, SEQ ID NO: 47, and SEQ ID NO: 93; SEQ ID NO: 4, SEQ ID NO: 48, and SEQ ID NO: 94; SEQ ID NO: 5, SEQ ID NO: 49, and SEQ ID NO: 95; SEQ ID NO: 6, SEQ ID NO: 50, and SEQ ID NO: 96; SEQ ID NO: 7, SEQ ID NO: 51, and SEQ ID NO: 97; SEQ ID NO: 8, SEQ ID NO: 52, and SEQ ID NO: 98; SEQ ID NO: 9, SEQ ID NO: 53, and SEQ ID NO: 99; SEQ ID NO: 10, SEQ ID NO: 54, and SEQ ID NO: 100; SEQ ID NO: 11, SEQ ID NO: 55, and SEQ ID NO: 101; SEQ ID NO: 12, SEQ ID NO: 56, and SEQ ID NO: 102; SEQ ID NO: 13, SEQ ID NO: 57, and SEQ ID NO: 103; SEQ ID NO: 14, SEQ ID NO: 58, and SEQ ID NO: 104; SEQ ID NO: 15, SEQ ID NO: 59, and SEQ ID NO: 105; SEQ ID NO: 16, SEQ ID NO: 60, and SEQ ID NO: 106; SEQ ID NO: 17, SEQ ID NO: 61, and SEQ ID NO: 107; SEQ ID NO: 18, SEQ ID NO: 62, and SEQ ID NO: 108; SEQ ID NO: 19, SEQ ID NO: 63, and SEQ ID NO: 109; SEQ ID NO: 20, SEQ ID NO: 64, and SEQ ID NO: 110; SEQ ID NO: 21, SEQ ID NO: 65, and SEQ ID NO: 111; SEQ ID NO: 22, SEQ ID NO: 66, and SEQ ID NO: 112; SEQ ID NO: 23, SEQ ID NO: 67, and SEQ ID NO: 113; SEQ ID NO: 24, SEQ ID NO: 68, and SEQ ID NO: 114; SEQ ID NO: 25, SEQ ID NO: 69, and SEQ ID NO: 115; SEQ ID NO: 26, SEQ ID NO: 70, and SEQ ID NO: 116; SEQ ID NO: 27, SEQ ID NO: 71, and SEQ ID NO: 117; SEQ ID NO: 28, SEQ ID NO: 72, and SEQ ID NO: 118; SEQ ID NO: 29, SEQ ID NO: 73, and SEQ ID NO: 119; SEQ ID NO: 30, SEQ ID NO: 74, and SEQ ID NO: 120; SEQ ID NO: 31, SEQ ID NO: 75, and SEQ ID NO: 121; SEQ ID NO: 32, SEQ ID NO: 76, and SEQ ID NO: 122; SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 123; SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 124; SEQ ID NO: 35, SEQ ID NO: 79, and SEQ ID NO: 125; SEQ ID NO: 36, SEQ ID NO: 80, and SEQ ID NO: 125; SEQ ID NO: 20, SEQ ID NO: 81, and SEQ ID NO: 126; SEQ ID NO: 20, SEQ ID NO: 81, and SEQ ID NO: 127; SEQ ID NO: 37, SEQ ID NO: 82, and SEQ ID NO: 128; SEQ ID NO: 38, SEQ ID NO: 83, and SEQ ID NO: 129; SEQ ID NO: 39, SEQ ID NO: 84, and SEQ ID NO: 130; SEQ ID NO: 40, SEQ ID NO: 85, and SEQ ID NO: 131; SEQ ID NO: 41, SEQ ID NO: 86, and SEQ ID NO: 132; SEQ ID NO: 42, SEQ ID NO: 87, and SEQ ID NO: 133; SEQ ID NO: 43, SEQ ID NO: 88, and SEQ ID NO: 134; or SEQ ID NO: 44, SEQ ID NO: 89, and SEQ ID NO: 135. In an embodiment, the antibody comprises the amino acid sequence set forth in SEQ ID NO: 183, 184, 185, or 186.
[0337] In an embodiment, an antibody as described herein comprises the amino acid sequence set forth in SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, or SEQ ID NO: 182. In another embodiment, the antibody comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the full length of the amino acid sequence set forth in SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, and/or SEQ ID NO: 182 and comprises CDR1, CDR2, and CDR3 sequences that, respectively, comprise the amino acid sequence set forth in: SEQ ID NO: 1, SEQ ID NO: 45, and SEQ ID NO: 90; SEQ ID NO: 2, SEQ ID NO: 45, and SEQ ID NO: 91; SEQ ID NO: 1, SEQ ID NO: 46, and SEQ ID NO: 92; SEQ ID NO: 3, SEQ ID NO: 47, and SEQ ID NO: 93; SEQ ID NO: 4, SEQ ID NO: 48, and SEQ ID NO: 94; SEQ ID NO: 5, SEQ ID NO: 49, and SEQ ID NO: 95; SEQ ID NO: 6, SEQ ID NO: 50, and SEQ ID NO: 96; SEQ ID NO: 7, SEQ ID NO: 51, and SEQ ID NO: 97; SEQ ID NO: 8, SEQ ID NO: 52, and SEQ ID NO: 98; SEQ ID NO: 9, SEQ ID NO: 53, and SEQ ID NO: 99; SEQ ID NO: 10, SEQ ID NO: 54, and SEQ ID NO: 100; SEQ ID NO: 11, SEQ ID NO: 55, and SEQ ID NO: 101; SEQ ID NO: 12, SEQ ID NO: 56, and SEQ ID NO: 102; SEQ ID NO: 13, SEQ ID NO: 57, and SEQ ID NO: 103; SEQ ID NO: 14, SEQ ID NO: 58, and SEQ ID NO: 104; SEQ ID NO: 15, SEQ ID NO: 59, and SEQ ID NO: 105; SEQ ID NO: 16, SEQ ID NO: 60, and SEQ ID NO: 106; SEQ ID NO: 17, SEQ ID NO: 61, and SEQ ID NO: 107; SEQ ID NO: 18, SEQ ID NO: 62, and SEQ ID NO: 108; SEQ ID NO: 19, SEQ ID NO: 63, and SEQ ID NO: 109; SEQ ID NO: 20, SEQ ID NO: 64, and SEQ ID NO: 110; SEQ ID NO: 21, SEQ ID NO: 65, and SEQ ID NO: 111; SEQ ID NO: 22, SEQ ID NO: 66, and SEQ ID NO: 112; SEQ ID NO: 23, SEQ ID NO: 67, and SEQ ID NO: 113; SEQ ID NO: 24, SEQ ID NO: 68, and SEQ ID NO: 114; SEQ ID NO: 25, SEQ ID NO: 69, and SEQ ID NO: 115; SEQ ID NO: 26, SEQ ID NO: 70, and SEQ ID NO: 116; SEQ ID NO: 27, SEQ ID NO: 71, and SEQ ID NO: 117; SEQ ID NO: 28, SEQ ID NO: 72, and SEQ ID NO: 118; SEQ ID NO: 29, SEQ ID NO: 73, and SEQ ID NO: 119; SEQ ID NO: 30, SEQ ID NO: 74, and SEQ ID NO: 120; SEQ ID NO: 31, SEQ ID NO: 75, and SEQ ID NO: 121; SEQ ID NO: 32, SEQ ID NO: 76, and SEQ ID NO: 122; SEQ ID NO: 33, SEQ ID NO: 77, and SEQ ID NO: 123; SEQ ID NO: 34, SEQ ID NO: 78, and SEQ ID NO: 124; SEQ ID NO: 35, SEQ ID NO: 79, and SEQ ID NO: 125; SEQ ID NO: 36, SEQ ID NO: 80, and SEQ ID NO: 125; SEQ ID NO: 20, SEQ ID NO: 81, and SEQ ID NO: 126; SEQ ID NO: 20, SEQ ID NO: 81, and SEQ ID NO: 127; SEQ ID NO: 37, SEQ ID NO: 82, and SEQ ID NO: 128; SEQ ID NO: 38, SEQ ID NO: 83, and SEQ ID NO: 129; SEQ ID NO: 39, SEQ ID NO: 84, and SEQ ID NO: 130; SEQ ID NO: 40, SEQ ID NO: 85, and SEQ ID NO: 131; SEQ ID NO: 41, SEQ ID NO: 86, and SEQ ID NO: 132; SEQ ID NO: 42, SEQ ID NO: 87, and SEQ ID NO: 133; SEQ ID NO: 43, SEQ ID NO: 88, and SEQ ID NO: 134; or SEQ ID NO: 44, SEQ ID NO: 89, and SEQ ID NO: 135.
[0338] Another embodiment is a nucleic acid molecule encoding an antibody as described herein. A further embodiment is a vector comprising the nucleic acid molecule. Optionally, the nucleic acid molecule may be operably linked to at least one promoter and/or regulatory element to enable expression in a host cell. A further embodiment is a host cell comprising the nucleic acid or vector.
[0339] An antibody as described herein may be comprised within a composition. For example, the antibody may be comprised within a pharmaceutical composition that comprises a pharmaceutically acceptable carrier and/or diluent, the antibody may be linked to another molecule, or the antibody may be immobilized on a substrate. In an embodiment, the pharmaceutical composition may be for delivery by inhalation or nebulization.
[0340] Antibodies and compositions as described herein may be used, or for use, to treat or prevent a coronavirus infection, including an infection caused by at least one coronavirus that specifically binds an ACE2 receptor. Antibodies as described herein may also be used in the manufacture of a medicament for prevention or treatment of a coronavirus infection. In a specific embodiment, the at least one coronavirus is SARS-CoV-2 and/or SARS-CoV. Further provided is a method for prevention or treatment of a coronavirus infection comprising administering an antibody or composition as described herein to a subject in need thereof. In an embodiment, the administration is by inhalation or nebulization.
[0341] Antibodies and compositions as described herein may also be used, or for use, to detect, quantify, and/or capture a coronavirus, a coronavirus spike polypeptide or a coronavirus spike polypeptide fragment. Further provided are methods for detecting, quantifying, and/or capturing a coronavirus, a coronavirus spike polypeptide or a coronavirus spike polypeptide fragment using an antibody or composition as described herein. In an embodiment, the coronavirus or spike polypeptide is a coronavirus or spike polypeptide that specifically binds an ACE2 receptor. The ACE2 receptor may be a human ACE2 receptor or an animal ACE2 receptor. In a specific embodiment, the coronavirus is SARS-CoV-2 or SARS-CoV, or the spike polypeptide or fragment thereof is from SARS-CoV-2 or SARS-CoV
[0342] Several of the antibodies described herein have the characteristics of neutralizing antibodies, and some have been demonstrated to be cross-reactive with the spike protein of other coronaviruses, such as SARS-CoV and related coronaviruses that infect bats, pangolin, and civet, suggesting that antibodies described herein may be useful for binding the spike protein of more than one coronavirus; including coronaviruses that bind an ACE2 receptor, such as SARS-CoV-2 and SARS-CoV. Antibodies described herein have also been demonstrated to bind various SARS-CoV-2 spike protein variants, such as the Wuhan-Hu-1 variant that was first identified in China; the B.1.1.7 variant that was first identified in the United Kingdom (also referred to herein as the UK variant, or the Alpha variant); the B.1.352 variant that was first identified in South Africa (also referred to herein as the South Africa variant, or the Beta variant), the B.1.617.1 variant that was first detected in India (also referred to herein as Kappa); the B.1.617.2 variant that was first detected in India (also referred to herein as Delta); and the B.1.1.529 variant that was first detected in South Africa (also referred to herein as Omicron).
[0343] Antibodies described herein may be linked to another molecule or substrate. For example, they may be linked to a detectable label to allow detection, quantification, and/or visualization; they may be linked to a molecule that extends antibody half-life, such as polyethylene glycol (PEG), Ig Fc, serum albumin, serum-albumin-specific antibody, serum-albumin-specific peptide, or Fc-specific peptides, proteins or antibodies; they may be linked to a therapeutic molecule; they may be immobilized onto a substrate, such as a plastic surface, a magnetic bead or a protein sheet or bead; and/or they may be linked to a polypeptide. In a specific embodiment, antibodies described herein may be linked to an ACE2 polypeptide or a fragment thereof.
[0344] Antibodies described herein may also be employed in various formats and combinations. For example, antibodies described herein may be monoparatropic or multiparatropic (including biparatropic), or monospecific or multispecific (including bispecific). Antibodies described herein may be in a monovalent format or in a multivalent format (including a bivalent format). Antibodies described herein that are specific for the same or different epitopes, or for the same or different spike protein subunit or domains, may be linked, for example to produce antibodies with different affinities and/or specificities. Further, antibodies described herein may be linked to one or more other antibodies or antibody fragments. In addition, antibodies described herein may be used individually or in combination. A combination may comprise any two or more antibodies described herein, or it may comprise at least one antibody described herein and another antibody. In some embodiments, the antibodies are V.sub.HH antibodies or V.sub.HH-Fc antibodies.
[0345] Antibodies described herein may be useful for a variety of applications. For example, they may be useful for detecting the presence of a coronavirus or a coronavirus spike polypeptide or fragment thereof, for capturing a coronavirus or a coronavirus spike polypeptide or fragment thereof, for quantifying the amount of a coronavirus or a coronavirus spike polypeptide or fragment thereof in a sample; for treatment or prevention of a coronavirus infection; for diagnosing a coronavirus infection; for monitoring the production of a coronavirus spike protein or fragment thereof, for purification of a coronavirus spike protein or fragment thereof, for detecting the level of expression of a coronavirus spike protein or fragment thereof, and/or for quantifying the amount of a coronavirus. Antibodies described herein have been shown to be stable against aerosolization, indicating that they may be suitable for delivery to the lungs by inhalation or nebulization. Further, cross-reactive antibodies may have general applicability for the treatment, prevention, detection, quantification or capture of coronaviruses, in addition to SARS-CoV-2, or coronavirus spike polypeptides or fragments thereof from coronaviruses in addition to SARS-CoV-2. In specific embodiments, cross-reactive antibodies may be used to bind coronaviruses or coronavirus spike polypeptides that bind an ACE2 receptor, including fragments of such coronavirus spike polypeptides.
[0346] Antibodies described herein may be classified based on the spike protein subunit or domain to which they bind. Nine antibodies were generated that bind to the S1-NTD domain, 24 antibodies were generated that bind to the S1-RBD domain, and 14 antibodies were generated that bind to the S2 subunit (see Tables 5 and 6). Neutralization assays, as described in the Examples, identified antibodies with neutralizing properties within each of these three groups. To the inventors' knowledge, this is the first known observation of single domain antibodies neutralizing the SARS-CoV-2 virus by targeting a non-S1-RBD region of S, i.e., S1-NTD or S2.
[0347] Within the three groups of antibodies identified above, further classification is possible based on epitope specificity, which was determined by epitope binning experiments (see Example 7). Preliminary results showed that antibodies binding to S1-NTD may be grouped into three epitope bins; antibodies binding S1-RBD may be grouped into six epitope bins, with some overlap between bins; and antibodies binding S2 may be grouped into five epitope bins (
[0348] Antibodies described herein may also be classified based on their pattern of cross-reactivity with different coronavirus spike proteins and/or spike protein variants, as shown in
[0349] As demonstrated in the Examples, several of the antibodies described herein have substantially increased binding affinity in comparison to a benchmark V.sub.HH spike protein antibody, V.sub.HH-72 (Wrapp et al., 2020). Further, many of the antibodies described herein are demonstrated to outperform V.sub.HH-72 in neutralization assays, and some are demonstrated to be more broadly neutralizing than V.sub.HH-72. Additionally, some antibodies described herein are demonstrated to be more broadly cross-reactive than V.sub.HH-72.
[0350] The antibodies described in the following examples may be modified, while still retaining antigen specificity. For example, changes may be introduced into the amino acid sequence of the framework regions, or the antibodies may be humanized. The antibodies may also be linked to other molecule(s). Antibodies and compositions resulting from such modifications are contemplated and encompassed by the present disclosure.
EXAMPLES
[0351] The following non-limiting examples are illustrative of the present disclosure and/or outline studies conducted pertaining to the present disclosure.
[0352] Several coronavirus spike protein fragments (spike protein antigens) were used in the Examples described below. Table 1 provides a list of spike protein fragments used in these studies.
TABLE-US-00001 TABLE 1 Coronavirus spike protein fragments used for library selection, binding and epitope study experiments Reference describing Accession expression & Description number Source Tag purification S1 (aa16-685) QHD43416.1.sup.b National Research FLAG, 6xHis Akache et al., (Wuhan) Council of 2021 Canada (NRC) S1 (aa16-685) QHD43416.1.sup.b ACROBiosystems AviTag, 6xHis na (Wuhan) S1 (aa16-685) QHD43416.1.sup.b ACROBiosystems Human IgG1 na (Wuhan) Fc NTD (aa16-305) QHD43416.1.sup.b NRC FLAG, 6xHis Akache et al., (Wuhan) 2021 RBD/SD1 (aa319-591) QHD43416.1.sup.b NRC Human IgG1 Wrapp et al., (Wuhan) Fc 2020 RBD/SD1 (aa319-591) QHD43416.1.sup.b NRC 6xHis Wrapp et al., (Wuhan) 2020 RBD_short (aa331-521) QHD43416.1.sup.b NRC 6xHis Akache et al., (Wuhan) 2021 RBD (aa319-541) QHD43416.1.sup.b NRC FLAG-6xHis Akache et al., (Wuhan) (N-term), E5 2021 (C-term) RBD_? (aa319-541) QHD43416.1.sup.b NRC FLAG, 6xHis Colwill et al., (B.1.1.7) 2021 RBD_? (aa319-541) QHD43416.1.sup.b NRC FLAG, 6xHis Colwill et al., (B.1.351) 2021 RBD_Wuhan QHD43416.1.sup.b ACROBiosystems AviTag, 6xHis na (aa319-541) RBD_SARS-CoV AAP13442.1.sup.b NRC FLAG, 6xHis Sulea et al., (aa306-527) 2022 S2 (aa686-1208) QHD43416.1.sup.b NRC FLAG, 6xHis Akache et al., (Wuhan) 2021 Swine deltaCoV S.sup.a AIH06857.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Avian_IBV S.sup.a AAP92675.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Pangolin CoV S.sup.a QIA48632.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Hedgehog CoV QGA70702.0.sup.b NRC FLAG-Dual Galipeau et al., HKU31 S.sup.a Strep-6xHis 2021 Bat CoV HKU9 S.sup.a YP_001039971.1.sup.c NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Bat SARS like CoV- AGZ48828.1.sup.b NRC FLAG-Dual Galipeau et al., WIVI S.sup.a Strep-6xHis 2021 Bat 229E-related CoV APD51507.1.sup.b NRC FLAG-Dual Galipeau et al., S.sup.a Strep-6xHis 2021 Bat CoV 512 S.sup.a YP_001351684.1.sup.c NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Bat SARS like CoV.sup.a ATO98157.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Civet SARS-CoV.sup.1 S.sup.a AAU04646.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Human MERS-CoV S.sup.a AGH58717.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Human CoV-NL63 S APF29071.1.sup.b Sino Biological 6xHis na Human CoV-OC43 S.sup.a AGT51431.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Human CoV-HKU1 S Q0ZME7.1.sup.b Sino Biological 6xHis na Human CoV-229E S.sup.a AAK32191.1.sup.b Sino Biological 6xHis na Human SARS-CoV S.sup.a AAP13442.1.sup.b NRC FLAG-Dual Galipeau et al., Strep-6xHis 2021 Human SARS-CoV- QHD43416.1.sup.b NRC FLAG, 6xHis Colwill et al., 2_Wuhan S.sup.a (SmT1) 2021 Human SARS-CoV- NRC FLAG, 6xHis Galipeau et al., 2_? (B.1.351) S.sup.a 2021 Human SARS-CoV- NRC FLAG, 6xHis Galipeau et al., 2_? (B.1.1.7) S.sup.a 2021 Human SARS-CoV- NRC none Galipeau et al., 2_? (P.1) S.sup.a 2021 Human SARS-CoV- NRC FLAG, 6xHis Stuible et al., 2_? (B.1.617.2) S.sup.a 2021 Human SARS-CoV- NRC FLAG, 6xHis Stuible et al., 2_?(B.1.617.1) S.sup.a 2021 Human SARS-CoV- NRC FLAG, 6xHis Sulea et al., 2_Omicron 2022 (B.1.1.529) S.sup.a Human ACE2 Q9BYF1-1.sup.d ACROBiosystems Human IgG1 na (aa18-740) Fc Human ACE2 Q9BYF1-1.sup.d NRC 6xHis Wrapp et al., (aa18-615) 2020 .sup.aProteins are C-terminally fused to the resistin trimerization domain. .sup.bGenBank; .sup.cNCBI; .sup.dUniProt. na, not applicable
Example 1: Antigen Validation
Introduction
[0353] Prior to use in library selection (panning) experiments, four SARS-CoV-2 spike protein antigens (S, S1, S1-RBD, and S2, as described in Table 1) were validated for structural integrity and functionality in adsorbed/captured states on microtiter wells by standard ELISA. Unless stated otherwise, all spike protein fragments used in the following Examples were produced as described in Stuible et al., 2021.
Materials and Methods
Binding to Cognate Human Angiotensin Converting Enzyme (ACE2) Receptor
[0354] ELISA was performed to determine if spike proteins were able to bind to human ACE2 when passively adsorbed (S, S1, S1-RBD and S2) or captured (S1, S1-RBD) on microtiter wells. For passive adsorption, wells of NUNC? Immulon 4 HBX MaxiSorp? microtiter plates (Thermo Fisher, Cat #3855) were coated with 50 ng of SARS-CoV-2 spike proteins (S, S1, S2, S1-RBD) in 100 ?L PBS overnight at 4? C. Following removal of protein solutions and three washes with PBST (PBS supplemented with 0.05% [v/v] Tween? 20), wells were blocked with PBSC (1% [w/v] casein [SIGMA, Cat #E3414] in PBS) at room temperature for 1 h. For capturing, in vivo biotinylated fragments harboring the AviTag? (AviTag-S1, AviTag-S1-RBD) were diluted in PBS and added at 50 ng/well (100 ?L) to pre-blocked Streptavidin Coated High Capacity Strip wells (Thermo Fisher, Cat #15501). After 1 h incubation at room temperature, wells were washed five times with PBST and incubated for an additional hour with 100 ?L/well of 2-fold serially diluted ACE2-Fc (human ACE2 fused to human IgG1 Fc domain; ACROBiosystems, Cat #AC2-H5257) in PBSTC (PBS/0.2% casein/0.1% Tween? 20). Wells were washed five times and incubated for 1 h with 1 ?g/mL HRP-conjugated goat anti-human IgG (SIGMA, Cat #A0170). Wells were washed 10 times and incubated with 100 ?L peroxidase substrate solution (SeraCare, Cat #50-76-00) at room temperature for 15 min. Reactions were stopped by adding 50 ?L 1 M H.sub.2SO.sub.4 to wells, and absorbance were subsequently measured at 450 nm using a Multiskan? FC photometer (Thermo Fisher).
Binding to Cognate Anti-Spike Protein Polyclonal Antibody
[0355] The four spike antigens were passively adsorbed as described above. After blocking with PBSC, wells were emptied, washed five times and incubated at room temperature for 1 h with 100 ?L of 1 ?g/mL anti-SARS-CoV-2 spike rabbit polyclonal antibody (Sino Biologicals, Cat #40589-T62) in PBSCT. Following 10 washes with PBST, wells were incubated with 100 ?L 1/2500 dilution (320 ng/mL) of goat anti-rabbit:HRP (Jackson Immunoresearch, Cat #111-035-144) in PBSCT for 1 h at room temperature. After 1 h incubation and final five washes with PBST, the peroxidase activity was determined as described above.
Results and Discussion
[0356] The passively adsorbed spike fragments, S, S1, S1-RBD, as well as the streptavidin-captured fragments, AviTag-S1-RBD and AviTag-S1, were found to bind to ACE2 with similarly high affinities (EC.sub.50=0.10-0.32 nM;
TABLE-US-00002 TABLE 2 Binding affinity (EC.sub.50) of passively absorbed spike fragments and streptavidin-captured spike fragments to ACE2 Antigen S S1 S2 S1-RBD AviTag-S1-RBD AviTag-S1 EC.sub.50 0.26 0.28 nb 0.32 0.10 0.28 (nM) nb indicates lack or no binding.
TABLE-US-00003 TABLE 3 Binding affinity (EC.sub.50) of passively absorbed spike fragments to a polyclonal antibody known to be specific for SARS-CoV-2 spike protein Antigen S S1 S2 S1-RBD EC.sub.50 (nM) 0.34 0.59 0.6 0.65
Example 2: Llama Immunization and Serum Analyses
Introduction
[0357] As described below, two llamas were immunized with SARS-CoV-2 S or S/S1-RBD to trigger the generation of a diverse pool of antibodies targeting manifold sites over the surface of S, and targeting the S1-RBD sub-domain of S which is used by the virus to start the process of host cell infection through interaction with the ACE2 receptor. Llama sera were assessed by ELISAs for generation of immune responses against SARS-CoV-2 spike proteins, and by flow cytometry surrogate neutralization assays for generation of neutralizing antibodies.
Materials and Methods
Llama Immunization
[0358] Immunizations were performed at Cedarlane Laboratories (Burlington, ON, Canada) and essentially as described (Hussack et al., 2011). One llama (Eva Green) was immunized with 100 ?g of S in 500 ?L PBS combined with 500 ?L of Freund's complete adjuvant on day 0, followed by immunization with 70 ?g of S1-RBD (ACROBiosystems, Cat #SPD-552H6) in Freund's incomplete adjuvant on each of days 7, 14, and 21. Bleeds were taken at days 0, 21, and 28. A second llama (Maple Red) was immunized with 100 ?g of S in 500 ?L PBS combined with 500 ?L of Freund's complete adjuvant on day 0, followed by immunization with 100 ?g of S mixed with Freund's incomplete adjuvant on day 7, and immunization with 50 ?g of S mixed with Freund's incomplete adjuvant on each of days 14 and 21.
Serum ELISA
[0359] Llama sera were tested for antigen-specific immune response by ELISA essentially as described (Hussack et al., 2011; Henry et al., 2016). Briefly, dilutions of sera in PBST were added to wells pre-coated with S, S1, S2 or S1-RBD. Negative antigen control wells were pre-coated with casein (100 ?L of 1% v/w) or recombinant human dipeptidase 1 ectodomain, DPEP1 (50 ng/well; Sino Biological, Cat #13543-H08H). Following 1 h incubation at room temperature, wells were washed 10 times with PBST and incubated with HRP-conjugated polyclonal goat anti-llama IgG heavy and light chain antibody (Bethyl, Cat #A160-100P) for 1 h at room temperature. After 10 washes, the peroxidase activity was determined as described above.
Serum Surrogate Neutralization Assay by Flow Cytometry
[0360] Trimeric SARS-CoV-2 S was chemically biotinylated using EZ-Link? NHS-LC-LC-Biotin following manufacturer's instructions (Thermo Fisher, Cat #21343). Vero E6 cells (ATCC, Cat #CRL-1586) were maintained according to ATCC protocols. Briefly, cells were grown to confluency in DMEM medium (Thermo Fisher, Cat #11965084) supplemented with 10% heat inactivated FBS (Thermo Fisher, Cat #10438034) and 2 mM Glutamax? (Thermofisher, Cat #35050061) at 37? C. in a humidified 5% CO.sub.2 atmosphere in T75 flasks. For flow cytometry experiments, cells were harvested by Accutase? (Thermo Fisher, Cat #A111050) treatment, washed once by centrifugation, and resuspended at 1?10.sup.6 cells/mL in PBSB (PBS containing 1% BSA) and 0.05% [v/v] sodium azide [SIGMA, Cat #52002]). Cells were kept on ice until use. To determine the presence of neutralizing antibodies in the immune sera of llamas, 400 ng of chemically biotinylated trimeric SARS-CoV-2 S was mixed with 5?10.sup.4 Vero E6 cells in the presence of 2-fold dilutions of sera (pre immune, day 21 and day 28 serum) in a final volume of 150 ?L. Following 1 h of incubation on ice, cells were washed twice with PBSB by centrifugation for 5 min at 1200 rpm and then incubated for an additional hour with 50 ?L of Streptavidin, R-Phycoerythrin Conjugate (SAPE, ThermoFisher, Cat #S866) at 250 ng/mL diluted in PBSB. After a final wash, cells were resuspended in 100 ?L PBSB and data were acquired on a CytoFLEX? S flow cytometer (Beckman Coulter, Brea, CA) and analyzed by FlowJo? software (FlowJo LLC, v10.6.2, Ashland, OR). Percent inhibition (neutralization) was calculated according to the following formula: % inhibition=100?[1?(F.sub.n?F.sub.min)/(F.sub.max?F.sub.min)], where, F.sub.n is the measured fluorescence at any given competitor serum dilution, F.sub.min is the baseline fluorescence measured in the presence of cells and SAPE only, and F.sub.max is the maximum fluorescence, measured in the absence of competitor serum.
Results and Discussion
[0361] The results of ELISAs performed with pre-immune (day 0) and immune (day 21 and 28) sera demonstrate that both llamas generated a strong immune response against target immunogens S, S1, S2 and S1-RBD (
TABLE-US-00004 TABLE 4 ELISA results summarizing day 0, 21 and 28 binding serum titers (EC.sub.50s) of Eva Green and Maple Red llamas against spike protein fragments S, S1, S2 and S1-RBD Binding serum titer, EC.sub.50 (reciprocal serum dilution) Llama Day S S1 S2 S1-RBD Eva Green 0 21 1.0 ? 10.sup.6 1.8 ? 10.sup.6 0.3 ? 10.sup.6 2.2 ? 10.sup.6 28 1.2 ? 10.sup.6 1.8 ? 10.sup.6 0.4 ? 10.sup.6 2.0 ? 10.sup.6 Maple Red 0 21 0.2 ? 10.sup.6 0.3 ? 10.sup.6 0.1 ? 10.sup.6 0.2 ? 10.sup.6 28 0.3 ? 10.sup.6 0.3 ? 10.sup.6 0.2 ? 10.sup.6 0.3 ? 10.sup.6 Dashes indicate lack of binding.
TABLE-US-00005 TABLE 5 Flow cytometry-based surrogate virus neutralization assay results summarizing day 21 and 28 inhibition serum titers (IC.sub.50s) of Eva Green and Maple Red llamas using spike protein S as surrogate for the virus Inhibition serum titer, IC.sub.50 (reciprocal serum dilution) Llama Day 21 Day 28 Eva Green 3300 6200 Maple Red <200 <400
Example 3: Phage Display Library Construction, Selection and Screening
Introduction
[0362] Two libraries (Eva Green and Maple Red) were constructed and subjected to selection against spike protein fragments. Selection and screening efforts were aimed at isolating not only S1-RBD binders, but also S1-NTD and S2 binders, as recent findings indicate that in addition to S1-RBD binders, S1-NTD and S2 binders could also be neutralizing (Rogers et al., 2020; Ravichandran et al., 2020). To this end, two libraries were generated and were selected under six different conditions to maximize the number and epitopic diversity of hits against S1-RBD, S1-NTD and S2. After two rounds of selection, monoclonal phages ELISA and DNA sequencing were performed to identify antigen-specific hits.
Materials and Methods
Phage Display Library Construction
[0363] On day 28, 100 mL of blood from each of the two llamas was drawn and peripheral blood mononuclear cells (PBMCs) were purified by Ficoll? gradient at Cedarlane Laboratories (Burlington, ON, Canada). Two independent phage-displayed V.sub.H/V.sub.HH libraries were constructed from ?5?10.sup.7 PBMCs as described previously (Henry et al., 2016; Rossotti et al., 2015; Henry et al., 2015). Total RNA was extracted from PBMCs using TRIzol? Plus RNA Purification Kit (Thermo Fisher, Cat #12183555) following manufacturer's instructions and used to reverse transcribe cDNA with SuperScript? IV VILO? Master Mix supplemented with random hexamer (Thermofisher, Cat #SO142) and oligo (dT) (Thermofisher, Cat #AM5730G) primers. V.sub.H/V.sub.HH genes were amplified using semi-nested PCR and cloned into the phagemid vector pMED1, followed by transformation of E. coli TG1 to construct two libraries with sizes of 1?10.sup.7 and 2?10.sup.7 independent transformants for Eva Green and Maple Red, respectively. Both libraries showed an insert rate of ?95%, as verified by DNA sequencing. Phage particles displaying the V.sub.Hs/V.sub.HHs were rescued from E. coli cell libraries using M13K07 helper phage (New England Biolabs, Cat #N0315S) as described in Hussack et al., 2011 and used for selection experiments described below.
Library Selection and Screening
[0364] Library selections (pannings) and screenings were performed essentially as described (Hussack et al., 2011; Rossotti et al., 2015). Library selections were performed on microtiter wells under 6 different phage binding/elution conditions designated P1-P6. Briefly, for the phage binding step, library phages were diluted at 1?10.sup.11 colony-forming units (cfu)/mL in PBSBT [PBS supplemented with 1% [w/v] BSA and 0.05% Tween? 20] and incubated in antigen-coated microtiter wells for 2 h at 4? C. For P1-P4, phages were added to wells with passively-adsorbed S (10 ?g/well; P1), passively-adsorbed S2 (10 ?g/well; P2), streptavidin-captured biotinylated S1 (0.5 ?g/well; P3) and streptavidin-captured biotinylated S1-RBD (0.5 ?g/well; P4). For P5, phages were pre-absorbed on passively-adsorbed S1-RBD wells (10 ?g/well) for 1 h at 4? C. and then the unbound phage in the solution was transferred to wells with streptavidin-captured biotinylated S1 (0.5 ?g/well) in the presence of non-biotinylated S1-RBD competitor in solution (10 ?g/well). Following the binding stage (P1-P5), wells were washed 10 times with PBST and bound phages were eluted by treatment with 100 mM glycine pH 2.2 for 10 min at room temperature, followed by immediate neutralization of phages with 2 M Tris. Similar to P4, in P6, phages were bound on streptavidin-captured biotinylated S1-RBD but elution of bound phages were carried out competitively with 50 nM ACE2-Fc following the washing step. For all pannings, a small aliquot of eluted phage was used to determine their titer on LB-agar/ampicillin plates and the remaining were used for their subsequent amplification in E. coli TG1 strain (Hussack et al., 2011). The amplified phages were used as input for the next round of selection as described above.
[0365] After two rounds of selection, 16 (Eva Green) or 12 (Maple Red) colonies from each of the P1-P6 selections were screened for antigen binding by monoclonal phage ELISA against S, S1, S2 and S1-RBD. Individual colonies from eluted-phage titer plates were grown in 96 deep well plates in 0.5 mL 2YT media/100 ?g/mL-carbenicillin/1% (w/v) glucose at 37? C. and 250 rpm to an OD.sub.600 of 0.5. Then, 10.sup.10 cfu M13K07 helper phage was added to each well and incubation continued for another 30 min under the same conditions. Cells were subsequently pelleted by centrifugation, the supernatant was discarded and the bacterial pellets were resuspended in 500 ?L 2YT/100 ?g/mL carbencillin/50 ?g/mL kanamycin and incubated overnight at 28? C. Next day, phage supernatants were recovered by centrifugation, diluted 3-fold in PBSTC and used in subsequent screening assays by ELISA. To this end, antigens were coated onto microtiter wells at 50 ng/well overnight at 4? C. Next day, plates were blocked with PBSC, washed five times with PBSTC, and 100 ?L of phage supernatants prepared above were added to wells, followed by incubation for 1 h at room temperature in an orbital shaking platform. After 10 washes, binding of phages was detected by adding 100 ?L/well of anti-M13:HRP (Santa Cruz, Cat #SC-53004HRP) at 40 ng/mL in PBSTC and incubating as above. After 10 washes, the peroxidase activity was determined as described previously. Following confirmation of success of library panning as determined by monoclonal phages ELISA, a total of ?1200 individual clones (2100 clones per panning strategy; ?600 clones per library) were colony-PCRed and subsequently sequenced, resulting in the identification of 35 (Eva Green) and 12 (Maple Red) potential spike-specific V.sub.HH antibodies.
Results and Discussion
[0366] Eva Green and Maple Red libraries were constructed with functional sizes (library sizes corrected for insert rate) of ?1?10.sup.7 and ?2?10.sup.7, respectively. Two rounds of selection under six different panning conditions (P1-P6) were subsequently performed for both libraries. To confirm the success of selection in enriching for binders, samples of 12-16 clones per panning condition were tested for binding against S, S1, S2 and S1-RBD by phage ELISA. The frequent occurrence of positive clones determined by monoclonal phage ELISA confirmed selections efficiently enriched for binders. Specificity patterns observed, i.e., binding against S vs S1 vs. S2 vs S1-RBD, in sample sets reflected the selection strategy as well as the immunization strategy (Eva Green was immunized with S once but predominantly [three times] with S1-RBD). In P3, P4 and P6, as expected based on the selection strategy, essentially all binders were S1-RBD specific. For Maple Red, the immunization with the whole spike S generated a strong bias against non-S1-RBD-specific antibodies, an observation recently seen with patients recovered from SARS-CoV-2 natural infection (Rogers et al., 2020) and rabbits immunized with SARS-CoV-2 S (Ravichandran et al. 2020). Panning against S (P1) essentially produced S2 binders as opposed to S1-RBD binders seen in the case of Eva Green library. Additionally, in contrast to what was observed in the case of the Eva Green, for the Maple Red P3 strategy, where panning was performed against S1, half of the binders tested were specific for non-S1-RBD region of S1. Nonetheless when selections were specifically directed towards S1-RBD binders, as in the P4 and P6 selection strategies, all tested binders were S1-RBD specific. Additionally, the P5 strategy almost exclusively selected for V.sub.HHs specific to non-RBD region of the S1 subunit. In summary, the immunization strategy was a key determinant of the outcome of in vivo generated V.sub.HHs with respect to spike subunit/domain specificity, and in vitro directed selection strategies effectively yielded intended binding specificities. Subsequently, a larger number of clones, >600 clones per library, were screened by DNA sequencing to obtain a large pool of potential binders. The unique sequences were subjected to binding validation, as described below.
Example 4: V.SUB.H.H Cloning/Expression in E. coli, Stability/Affinity Validation and Cross-Reactivity Studies
Introduction
[0367] Hits identified by monoclonal phage ELISA and DNA sequencing were cloned into the expression vector pMRo.BAP.H.sub.6 (Rossotti et al., 2019), produced as His6-tagged V.sub.HHs in the periplasmic space of E. coli BL21(DE3) and purified by immobilized metal-ion affinity chromatography (IMAC). V.sub.HHs were subsequently validated for binding and further explored for cross-reactivity soluble ELISA against SARS-CoV-2, SARS-CoV and MERS-CoV spike proteins. Additionally, V.sub.HHs were validated for aggregation resistance by size exclusion chromatography (SEC) and thermostability by circular dichroism T.sub.m measurement assays. Lead V.sub.HHs were produced in mammalian cells in fusion with human IgG1 Fc and were subsequently tested in a comprehensive cross-reactivity ELISA against a collection of various coronavirus spike proteins (S).
Materials and Methods
[0368] DNA Sequence Analysis and V.sub.HH Production in E. coli
[0369] Colonies were analyzed by DNA sequencing and identified V.sub.HH sequences were aligned using IMGT system. V.sub.HHs were subsequently cloned into pET expression vector (Novagen, Madison, WI) for their production in BL21(DE3) E. coli as monomeric soluble protein (Rosotti et al., 2019). Briefly, individual colonies were cultured overnight in 10 mL of LB supplemented with 50 ?g/mL of kanamycin (LB/Kan) at 37? C. and 250 rpm. After 16 h, cultures were added to 250 mL LB/Kan and grown to an OD.sub.600 of 0.6. Expression of V.sub.HHs was induced with 10 ?M of IPTG (isopropyl ?-D-1-thiogalactopyranoside) overnight at 28? C. and 250 rpm. The following day, bacterial pellets were harvested by centrifugation at 6,000 rpm for 15 min at 4? C. and V.sub.H/V.sub.HHs were extracted by sonication and purified by IMAC as described previously (Rosotti et al., 2019). In addition, for ELISA (see below), a small fraction was biotinylated by incubating 1 mg of purified V.sub.HHs with 10 ?M of ATP (Alfa Aesar, Cat #CAAAJ61125-09), 100 ?M of D-(+)-biotin (VWR, Cat #97061-446) and a bacterial cell extract overexpressing E. coli BirA as described previously (Rossotti et al., 2015b). The same procedure was followed to produce a biotinylated V.sub.HH-72 benchmark V.sub.HH (Wrapp et al., 2020), a SARS-CoV spike protein-specific V.sub.HH that cross-reacts with the SARS-CoV-2 spike protein receptor binding domain.
V.SUB.H.H Binding Validation and Preliminary Cross-Reactivity Studies by ELISA
[0370] Binding validation studies were performed with S1-RBD-specific clones. Briefly, microtiter well plates were coated with 50 ng/well SARS-CoV-2 S1-RBD in 100 ?L PBS overnight at 4? C. Plates were blocked with PBSC for 1 h at room temperature, then washed five times with PBST and incubated with decreasing concentrations of biotinylated V.sub.HHs. After 1 h incubation, plates were washed 10 times with PBST and binding of V.sub.HHs was probed using HRP-streptavidin (Jackson ImmunoResearch, Cat #016-030-084). Finally, plates were washed 10 times with PBST and peroxidase activity was determined as described above.
Stability Determinations by Size Exclusion Chromatography (SEC) and Circular Dichroism
[0371] Purified V.sub.HHs were subjected to SEC to validate their aggregation resistance. Briefly, 2 mg of each affinity purified V.sub.HH was injected into Superdex? 75 GL column (Cytiva) connected to an ?KTA FPLC protein purification system (Cytiva) as previously described (Henry et al., 2017). PBS was used as running buffer at 0.8 mL/min. Fractions corresponding to the monomeric peak were pooled and stored at 4? C. until use. To determine thermostability, V.sub.HH Ts were measured by circular dichroism as previously described (Henry et al., 2017). Ellipticity of V.sub.HHs were determined at 200 ?g/mL V.sub.HH concentrations and 205 nm wavelength in 100 mM sodium phosphate buffer, pH 7.4. Ellipticity measurements were normalized to percentage scale and T.sub.ms were determined from plot of % folded vs temperature and fitting the data to a Boltzmann distribution.
Production of V.sub.HHs in Mammalian Cells Infusion with Human IgG1 Fc
[0372] Codon-optimized genes for bivalent V.sub.HH-Fcs were synthesized (GenScript). For heterodimeric monovalent V.sub.HH-Fcs, V.sub.HH genes were PCR amplified as described previously and cloned into pTT5-hIgG1Fc between the genes for human V.sub.H leader sequence and the human IgG1 hinge/Fc sequences, using NarI/HindIII restriction sites. Bivalent V.sub.HH-Fcs were produced by transient transfection of HEK293-6E cells followed by protein A affinity chromatography as previously described (Rosotti et al., 2019). Heterodimeric monovalent V.sub.HH-Fcs were produced by co-transfection of HEK293-6E cells with two pTT5 vectors, one encoding for a 6?His-tagged heavy chain (V.sub.HH1-hinge-C.sub.H2-C.sub.H3-His.sub.6), the other for a non-tagged heavy chain of a different V.sub.HH (V.sub.HH2-hinge-C.sub.H2-C.sub.H3). The heterodimeric antibodies were purified by sequential protein A affinity chromatography and IMAC. For IMAC, antibodies were eluted using a linear 0-0.5 M imidazole gradient over 20 column volumes to separate species bearing one 6?His tag (heterodimeric, monovalent) from those bearing two 6?His tags (homodimeric, bivalent). Proteins were buffer exchanged using Amicon? Ultra-15 Centrifugal Filter Units (Millipore, Cat #UFC905024) with phosphate-buffered saline (PBS), pH 7.4. The same procedure was applied for the generation of the reference bio-V.sub.HH-72 and V.sub.HH-72-Fc using the sequence published by Wrapp et al., 2020. The sequence of the V.sub.HH was ordered as GeneBlock (IDT DNA) flanked by SfiI sites for cloning into pMRo.BAP.H6, and NarI/HindIII for cloning into pTT5-hIgG1Fc. Protein purity was evaluated by SDS-PAGE using 4-20% Mini-PROTEAN? TGX Stain-Free? Gels (Bio-Rad, Cat #17000435).
V.SUB.H.H-Fc Comprehensive Cross-Reactivity Studies by ELISA
[0373] Recombinant coronavirus spike proteins S (Table 1) were coated overnight onto NUNC? MaxiSorp? 4BX plates (Thermo Fisher) at 50 ng/well in 100 ?L of PBS, pH 7.4. The next day, plates were blocked with 200 ?L PBSC for 1 h at room temperature, then washed five times with PBST and incubated at room temperature for 1 h on rocking platform at 80 rpm with 1 ?g/mL V.sub.HH-Fc diluted in PBSTC. Plates were washed five times with PBSTC and binding of V.sub.HH-Fcs was detected using 1 ?g/mL HRP-conjugated goat anti-human IgG. Finally plates were washed five times and peroxidase (HRP) activity was measured as described above.
Results and Discussion
[0374] A total of ?1200 colonies were analyzed by DNA sequencing. Forty seven potential V.sub.HH binders were identified from the two libraries (35 from the Eva Green and 12 from the Maple Red library) by phage ELISA and DNA sequencing, with the vast majority (35 V.sub.HHs) coming from the Eva Green library (Tables 6 and 7). Some V.sub.HHs may be clonally related due to their high sequence identity in their CDRs. Examples include NRCoV2-1a, NRCoV2-1c and NRCoV2-1d from the Eva Green library (Table 6) and NRCoV2-MRed02 and NRCoV2-MRed04 from the Maple Red library (Table 7). V.sub.HH hits were cloned in E. coli, confirmed by DNA sequencing, and expressed and purified by IMAC. Following expression of V.sub.HHs, the binding of a sample set of V.sub.HHs was validated by ELISA. Affinities, expressed as EC.sub.50s, were high, ranging from 0.4 to 7.2 nM (data not shown). V.sub.HHs were also tested for aggregation resistance and stability, and cross-reactivity.
[0375] Aggregation resistance and stability are desirable attributes of biotherapeutics, as they affect both efficacy and manufacturability. By size exclusion chromatography, all V.sub.HHs tested were found to be aggregation resistant (
[0376] The results of cross-reactivity studies using SARS-CoV-2 variants and various coronaviruses are shown in
[0377] In a subsequent experiment (results shown in
[0378] When tested by SPR against SARS-CoV, 12 out of 14 ELISA-positive V.sub.HHs cross-reacted with SARS-CoV S, most with comparably high affinities (Table 11. Seven of these V.sub.HHs were S2-specific, four were RBD-specific and one was NTD-specific. Against the Alpha and Beta variants, the SPR cross-reactivity data, performed with 37 V.sub.HHs, were consistent with ELISA, except for NRCoV2-04 and NRCoV2-14, which were negative or very weak for binding to the Beta variant by SPR (Tables 11 and 12). All 37 V.sub.HHs tested bound the Alpha variant S protein, and 34 were also cross-reactive to the Beta variant S protein (
[0379] The cross-reactivity of the V.sub.HHs and V.sub.HH-Fcs is significant, as it is believed that the progenitor of SARS-CoV was generated by recombination among bat SARS-like coronaviruses that spread to humans via civet cat as an intermediate host (Zheng et al, 2020). Further, most new emerging viruses are derived from strains circulating in zoonotic reservoirs. Antibodies that can cross-react against a variety of animal and human coronaviruses have potential to be used for detection and/or treatment of emerging coronavirus outbreaks.
TABLE-US-00006 TABLE6 CDR-IMGTsequencesofanti-spikeproteinV.sub.HHs obtainedfromEvaGreenphagedisplaylibrary Sub- unit/ do- Selec- SEQ SEQ SEQ main tion CDR ID ID ID speci- stra- V.sub.HH 1 NO: CDR2 NO: CDR3 NO: ficity tegy NRCoV2- GSTL 1 VSSSDGST 45 AADYSMRP 90 S1-RBD P1, 1a.sup.2 DYYA LWVSRWHR P3, DYEY P4, P6 NRCoV2- GSIL 2 VSSSDGST 45 AADYSMRR 91 S1-RBD P1, 1c.sup.2 DYYA FAVGRWHR P3, DYEY P4, P6 NRCoV2- GSTL 1 VSSSDGNT 46 AADYSMRP 92 S1-RBD P1, 1d.sup.1 DYYA FAVGRWHR P3, DYEY P4, P6 NRCoV2- GFTF 3 ISGRGDDT 47 TKGPDLYY 93 S1-RBD P1, 02.sup.1 SNYA FGSGYSD P3, P4, P5, P6 NRCoV2- GITF 4 MSNMDST 48 NIYGPTYS 94 S1-RBD P4, 03.sup.1 SYYA TRRNEY P6 NRCoV2- GSPF 5 ISGGGIA 49 WSSYEST 95 S1-RBD P1, 04.sup.1 SNVV P3, P4, P6 NRCoV2- GFIF 6 INSGGGDT 50 SKGPVSSY 96 S1-RBD P1, 05.sup.1 SNYA YGSGYDY P3, P4, P6 NRCoV2- VSTF 7 IGFVGAT 51 NARHYGGS 97 S1-RBD P4, 06.sup.1 SSYA EY P6 NRCoV2- GVTL 8 ISSNGRRN 52 AAVQDVHG 98 S1-RBD P1, 07.sup.1 DYYA DNYYCTSP P3, NEYNV P4, P6 NRCoV2- GFTL 9 ISRSGTTT 53 AADYQYST 99 S1-RBD P4, 08.sup.2 DDYA YCLGYDAH P6 YEY NRCoV2- GNTF 10 ISSRGIS 54 YAADDLGD 100 S1-RBD P1, 10.sup.1 SRSN Y P3, P4 NRCoV2- GSSL 11 ISRYYSST 55 AARSRDFS 101 S1-RBD P1, 11.sup.1 DSYS SPFSATDT P5 YTS NRCoV2- GFTL 12 ISRYYEST 56 AARSRDFS 102 S1-RBD P5 11a.sup.2 DSYN SPISATDK YGS NRCoV2- GRTF 13 VAAISWGG 57 AADRGLSY 103 S1-RBD P1, 12.sup.2 RNYV TEI YYTRTTEY P4, NY P6 NRCoV2- GTTF 14 ISVFGST 58 HAVNADIG 104 S1-RBD P3 14.sup.1 SHYA GDY NRCoV2- GSTS 15 VSTSGAT 59 YAAYGGGG 105 S1-RBD P1, 15.sup.1 GRNT DY P3, P4, P6 NRCoV2- GSPF 16 ISPTGNR 60 QAANVNGG 106 S1-RBD P4 17.sup.1 SQLA DY NRCoV2- GITI 17 INSGGST 61 SLHTSHDY 107 S1-RBD P1, 18.sup.1 SGYN P3, P4, P5, P6 NRCoV2- GLTL 18 LTSGGTG 62 AADRARLR 108 S1-RBD P1, 19.sup.2 NSYA FGCSLNFR P4 REVAYDY NRCoV2- GRTF 19 VAVISGSD 63 AADRGMSY 109 S1-RBD P4 20.sup.1 SNYV TET YYTRATEY YY NRCoV2- GFTL 20 ISSGGST 64 AADHRGRS 110 S1-RBD P1, 21.sup.2 DYYA LRFGCSSS P4, TTDYLY P5 NRCoV2- GFTF 21 ISGNGGVT 65 AATGIRST 111 S1-NTD P3, SR01.sup.3 DNYA WSVYGCSR P5 LAGPYDY NRCoV2- EFTL 22 IRYSGGGI 66 AADRLYSR 112 S1-NTD P5 SR02.sup.3 NYYS ACPTAGGR NY NRCoV2- GSIF 23 ISSGGKT 67 NRGGWEYR 113 S1-NTD P3, SR03.sup.3 SNNH SSYYIMGP P5 H NRCoV2- GRTF 24 ISMGGNTN 68 NTAALVGN 114 S1-NTD P5 SR04.sup.3 SSHT YA RLLPMATI T NRCoV2- GSRF 25 ISSGGST 69 NMGGWDYR 115 S1-NTD P3, SR13.sup.3 GSKH SNTYIPGS P5 RSDY NRCoV2- GTTF 26 ISTSGAV 70 NTGGWDYR 116 S1-NTD P3, SR16.sup.3 SRYH SSTFIMGL P5 N NRCoV2- GRPY 27 KQRELVAA 71 NTGSLSYG 117 S2 P2 S2A3.sup.1 SNYA ISSGGTT GSVYYPSY DN NRCoV2- GSPF 28 ISTGGSR 72 HAAARDSH 118 S2 P2 S2A4.sup.1 RSNV GIYLLDT NRCoV2- ASTF 29 ISTGSNT 73 NYRSIYYG 119 S2 P2 S2B3.sup.2 GDSA QNF NRCoV2- GFTF 30 INSGDRDS 74 ALVFGYTS 120 S2 P2 S2H4.sup.2 NLYS TT RDYCLTPK RGNY NRCoV2- VRIL 31 ITSGGST 75 NLRDILSQ 121 S2 P2 S2F3.sup.1 SVPA PF NRCoV2- GSTF 32 ITSGGAT 76 YTTKRDDA 122 S2 P2 S2G3.sup.1 GIFL SVY NRCoV2- GSTF 33 ISSDGDK 77 NKHWWTGD 123 S2 P2 S2G4.sup.1 SGYA W NRCoV2- GITV 34 ISAGGST 78 NYGPGYRK 124 S2 P2 S202.sup.2 SRIG AA Subunit/domain specificities were determined by .sup.1SPR, .sup.2ELISA or .sup.3both. SPR assays and ELISAs were perfomed against various spike protein subunit/domains using V.sub.HHs and V.sub.HH-Fcs, respectively.
TABLE-US-00007 TABLE7 CDR-IMGTsequencesofanti-spikeproteinV.sub.HHs obtainedfromMapleRedV.sub.HHphagedisplay library Sub- unit/ do- Selec- SEQ SEQ SEQ main/ tion CDR ID ID ID speci- strat- V.sub.HH 1 NO: CDR2 NO: CDR3 NO: ficity egy NRCoV2- GNIF 35 IWSDS 79 AADRGFVV 125 S1-RBD P1, MRed02.sup.2 SINS RT RGQYDY P3, P4, P6 NRCoV2- GNSF 36 IWSDT 80 AADRGFVV 125 S1-RBD P4, MRed04.sup.1 SINT TT RGQYDY P6 NRCoV2- GFTL 20 ISSSD 81 ATDAFATC 126 S1-NTD P3, MRed03.sup.3 DYYA GST DSWYAQIA P5 QYDF NRCoV2- GFTL 20 ISSSD 81 ATGPQAYY 127 S1-RBD P4, MRed05.sup.2 DYYA GST SGSYYFQC P6 PQAGMDY NRCoV2- GFTL 37 ISSSD 82 ATDSFSSC 128 S1-NTD P3 MRed06.sup.3 AYYA GSA SDYESGMD F NRCoV2- GSIG 38 ITRGG 83 YANYGWAI 129 S1-NTD P3 MRed07.sup.3 PFNT VT PY NRCoV2- GFTF 39 INSGG 84 ATTISDGS 130 S2 P1, MRed11.sup.1 SSYA GST SWSTKSY P2 NRCoV2- TTVF 40 VSDGG 85 NYYNYYYG 131 S2 P2 MRed18.sup.1 GRNA TP RNF NRCoV2- TIIF 41 MTTSG 86 YMHSVYYG 132 S2 P2 MRed19.sup.1 KGQT SA IDY NRCoV2- GLSF 42 IRESG 87 AAKPPFYG 133 S2 P2 MRed20.sup.1 SSYD SGT SGTYSTPR AYLY NRCoV2- GSVF 43 ISSRG 88 NAREFTGF 134 S2 P2 MRed22.sup.1 ASNA ST DY NRCoV2- GHTF 44 ISWRG 89 AAEMWGTA 135 S2 P2 MRed25.sup.1 SRYG DST TIVASRYT Y Domain/subunit specificities were determined by .sup.1SPR, .sup.2ELISA or .sup.3both. SPR assays and ELISAs were performed against various spike protein domains/subunit using V.sub.HHs and V.sub.HH-Fcs, respectively.
Example 5: Binding Characteristics of V.SUB.H.Hs and V.SUB.H.H-Fcs: Surface Plasmon Resonance (SPR) and ELISA Binding Studies
Introduction
[0380] Binding of anti-SARS-CoV-2 V.sub.HHs against various SARS-CoV-2 spike protein fragments (Wuhan) was assayed using SPR and ELISA to determine their affinity and domain/sub-domain specificity. Binding of V.sub.HHs against SARS-CoV, SARS-CoV-2 UK (Alpha) variant and SARS-CoV-2 South African (Beta) variant spike protein S was also carried out to determine their virus cross-reactivity patterns.
Materials and Methods
Affinity/Specificity Detennination of V.SUB.H.Hs Against SARS-CoV Spike (S), SARS-CoV-2 Spike (S) and SARS-CoV-2 Spike Fragments by SPR
[0381] Standard SPR techniques were used for binding studies. All SPR assays were performed on a Biacore? T200 instrument (Cytiva) at 25? C. with HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% [v/v] Tween? 20, pH 7.4) and CM5 sensor chips (Cytiva). Prior to SPR analyses all analytes in flow (V.sub.HHs, ACE2 receptor) were SEC-purified on a Superdex? 75 Increase 10/300 GL column (Cytiva) in HBS-EP buffer at a flow rate of 0.8 mL/min to obtain monomeric proteins. SARS-CoV spike (S), SARS-CoV-2 spike trimer (S) and various SARS-CoV-2 spike fragments were immobilized on CM5 sensor chips through standard amine coupling (10 mM acetate buffer, pH 4.0; Cytiva). On the first sensor chip, 1983 response units (RUs) of SARS-CoV spike (Sino Biologicals, Cat #40634-V08B), 843 RUs of SARS-CoV-2 S1-RBD fused to human Fc (S1-RBD-Fc) and 972 RUs of EGFR (irrelevant control surface) were immobilized. On a second sensor chip, 2346 RUs of SARS-CoV-2 S, 1141 RUs of SARS-CoV-2 S1 subunit and 1028 RUs of SARS-CoV-2 S2 subunit were immobilized. The theoretical maximum binding response for V.sub.HHs binding to these surfaces ranged from 224-262 RUs. An ethanolamine blocked surface on each sensor chip served as a reference. Single cycle kinetics was used to determine V.sub.HH and ACE2 binding kinetics and affinities. V.sub.HHs at various concentration ranges (from 0.25-4 nM to 125-2000 nM) were flowed over all surfaces at a flow rate of 40 ?L/min with 180 s of contact time and 600 s of dissociation time. Surfaces were regenerated with a 12 s pulse of 10 mM glycine, pH 1.5, at a flow rate of 100 ?L/min. Injection of EGFR-specific V.sub.HH EK2 served as a negative control for the SARS-CoV and SARS-CoV-2 surfaces and as a positive control for the EGFR surface. The ACE2 affinity was determined using similar conditions by flowing a range of monomeric ACE2 concentrations (31.53-500 nM). All affinities were calculated by fitting reference flow cell-subtracted data to a 1:1 interaction model using BIA evaluation Software v3.0 (Cytiva).
[0382] For V.sub.HH 12 and MRed05, V.sub.HH-Fc formats were used in SPR experiments. Approximately 200 RUs of V.sub.HH-Fcs (2 ?g/mL) were captured on goat anti-human IgG surfaces (4000 RUs, Jackson ImmunoResearch, Cat #109-005-098) at a flow rate of 10 ?L/min for 30 s. A range of SEC-purified RBD fragments (Table 1; SARS-CoV, Wuhan, Alpha and Beta) at 0.62-10 nM were flowed over the captured V.sub.HH-Fc at a flow rate of 40 ?L/min with 180 s of contact time and 300 s of dissociation. Surfaces were regenerated with a 120 s pulse of 10 mM glycine, pH 1.5, at a flow rate of 50 ?L/min. Affinities were calculated from reference flow cell subtracted sensorgrams as described above.
Domain Specificity Determination of V.SUB.H.Hs by ELISA.
[0383] V.sub.HHs that bound to the S1 subunit but not its S1-RBD domain in SPR assays, were further examined by ELISA to determine if they were binding to the S1-NTD domain of S1. Briefly, S, S1, S1-NTD and S1-RBD were coated onto NUNC? MaxiSorp? 4BX plates (Thermo Fisher) at 100 ng/well in 100 ?L PBS, pH 7.4. The next day, plates were blocked with 200 ?L PBSC for 1 h at room temperature, then washed five times with PBST and incubated with fixed (13 nM) or decreasing concentrations of V.sub.HH-Fcs diluted in PBSTC. After 1 h, plates were washed 10 times with PBSTC and binding of V.sub.HH-Fc fusions was detected by incubating wells with 100 ?L of 1 ?g/mL HRP-conjugated goat anti-human IgG. Finally, plates were washed 10 times with PBST and peroxidase activity was determined as described above. EC.sub.50s for the binding of V.sub.HH-Fcs to S and S fragments were obtained from the plot of A450. (binding) vs V.sub.HH-Fc concentration. S1-NTD covering amino acids 16-305 of SARS-CoV-2 S (GenBank accession number: QHD43416.1) was expressed in CHO cells.
Affinity/Specificity Determination of V.sub.HHs Against Spike Protein S from SARS-CoV-2 Wuhan, UK (Alpha) and South African (Beta) Variants by SPR
[0384] Affinity and specificity of V.sub.HHs against spike protein S from SARS-CoV-2 Wuhan, UK and South African variants by SPR was determined essentially as described above.
Results and Discussion
[0385] V.sub.HHs were tested by SPR against SARS-CoV-2 S, S1, S1-RBD and S2 to determine their affinity and domain/sub-domain specificity. Binding data are presented in
[0386] As for the S1-RBD-specific V.sub.HHs, with the exception of NRCoV2-06, which had an affinity of 223 nM (Table 11), the remaining 16 cluster members displayed high affinities ranging from 0.02-10 nM, all vastly outperforming the benchmark V.sub.HH-72 V.sub.HH, which had a K.sub.D of 86.2 nM. Nine V.sub.HHs were S1-NTD-specific and, similar to S1-RBD-specific V.sub.HHs, displayed high affinities (K.sub.Ds) in the range of 0.1-5.2 nM. Lastly, 11 V.sub.HHs were S2 subunit-specific, with similarly high affinities (K.sub.Ds) ranging from 0.09-12.8 nM.
[0387] V.sub.HHs were tested against SARS-CoV (S) in SPR assays for quantitative determination of cross-reactivity. V.sub.HHs were first screened for cross-reactivity at fixed concentrations. Twelve out of 37 V.sub.HHs screened showed cross-reactivity to SARS-CoV S. These 12 V.sub.HHs were subsequently subjected to comprehensive binding analysis against both SARS-CoV S and SARS-CoV-2 S at multiple V.sub.HH concentrations. The SPR cross-reactivity results, which agreed with those from ELISAs, are presented in
[0388] Against the Alpha and Beta variants, SPR cross-reactivity data performed with 37 V.sub.HHs, were consistent with ELISA, except for NRCoV2-04 and NRCoV2-14 which were negative or very weak for binding to the Beta variant by SPR. All 37 V.sub.HHs tested bound the Alpha variant S protein, 34 of which were also cross-reactive to the Beta variant S protein (
TABLE-US-00008 TABLE 8 Kinetic and equilibrium constants for the binding of Eva Green V.sub.HHs to SARS-CoV-2 Wuhan spike protein fragments S1-RBD-Fc.sup.1 S.sup.1 V.sub.HH/ k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D ACE2 (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) NRCoV2-1d 5.62E+05 1.18E?03 2.10E?09 1.06E+06 1.17E?03 1.10E?09 NRCoV2-02 1.83E+06 1.41E?03 7.73E?10 2.14E+06 1.41E?03 6.61E?10 NRCoV2-03 1.66E+05 2.55E?04 1.53E?09 2.50E+05 3.34E?04 1.34E?09 NRCoV2-04 1.53E+06 1.92E?02 1.25E?08 1.97E+06 1.98E?02 1.00E?08 NRCoV2-05 2.51E+06 5.85E?03 2.33E?09 4.33E+06 7.43E?03 1.72E?09 NRCoV2-06 5.94E+04 4.15E?03 6.99E?08 3.02E+04 4.69E?03 1.55E?07 NRCoV2-07 3.18E+05 2.84E?04 8.94E?10 3.50E+05 4.03E?04 1.15E?09 NRCoV2-10 3.51E+05 9.32E?05 2.66E?10 4.83E+05 9.27E?05 1.92E?10 NRCoV2-11 8.92E+05 2.26E?04 2.53E?10 1.21E+06 4.73E?05 3.91E?11 NRCoV2-12 7.60E+05 3.69E?05 4.86E?11 nd NRCoV2-14 2.61E+05 9.44E?04 3.61E?09 5.28E+05 1.64E?03 3.10E?09 NRCoV2-15 6.82E+05 2.33E?04 3.42E?10 7.06E+05 2.21E?04 3.13E?10 NRCoV2-17 5.67E+05 2.24E?04 3.95E?10 6.59E+05 9.79E?05 1.49E?10 NRCoV2-18 2.68E+05 1.97E?04 7.36E?10 3.95E+05 1.52E?04 3.84E?10 NRCoV2-20 1.43E+06 1.23E?02 8.61E?09 2.37E+06 1.59E?02 6.73E?09 NRCoV2-SR01 ? ? ? 2.77E+06 1.23E?03 4.45E?10 NRCoV2-SR02 ? ? ? 9.67E+05 5.71E?04 5.90E?10 NRCoV2-SR03 ? ? ? 1.01E+06 1.02E?03 1.01E?09 NRCoV2-SR04 ? ? ? 2.39E+06 3.35E?04 1.40E?10 NRCoV2-SR13 ? ? ? 1.83E+06 4.81E?03 2.62E?09 NRCoV2-SR16 ? ? ? 6.57E+05 1.20E?03 1.82E?09 NRCoV2-S2A3 ? ? ? 8.40E+04 1.30E?04 1.55E?09 NRCoV2-S2A4 ? ? ? 3.49E+04 4.46E?04 1.28E?08 NRCoV2-S2G3 ? ? ? 1.62E+05 6.07E?04 3.74E?09 NRCoV2-S2G4 ? ? ? 8.93E+05 2.07E?04 2.32E?10 NRCoV2-S2F3 ? ? ? 1.56E+05 4.73E?04 3.03E?09 V.sub.HH-72.sup.2 6.67E+05 1.34E?01 2.00E?07 1.10E+06 1.56E?01 1.42E?07 ACE2-H6.sup.2 3.71E+04 1.18E?02 3.17E?07 6.02E+04 9.96E?03 1.65E?07 NRCsdAb022.sup.2 ? ? ? ? ? ? S1.sup.1 S2.sup.1 V.sub.HH/ k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D ACE2 (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) NRCoV2-1d 8.67E+05 1.14E?03 1.32E?09 ? ? ? NRCoV2-02 2.10E+06 1.38E?03 6.59E?10 ? ? ? NRCoV2-03 2.41E+05 2.63E?04 1.09E?09 ? ? ? NRCoV2-04 1.61E+06 1.80E?02 1.12E?08 ? ? ? NRCoV2-05 4.03E+06 8.06E?03 2.00E?09 ? ? ? NRCoV2-06 1.04E+05 1.33E?02 1.29E?07 ? ? ? NRCoV2-07 3.01E+05 3.08E?04 1.02E?09 ? ? ? NRCoV2-10 4.48E+05 9.54E?05 2.13E?10 ? ? ? NRCoV2-11 1.08E+06 1.80E?04 1.67E?10 ? ? ? NRCoV2-12 nd ? ? NRCoV2-14 3.19E+05 1.12E?03 3.49E?09 ? ? ? NRCoV2-15 6.75E+05 2.25E?04 3.33E?10 ? ? ? NRCoV2-17 6.29E+05 9.66E?05 1.53E?10 ? ? ? NRCoV2-18 3.08E+05 1.65E?04 5.36E?10 ? ? ? NRCoV2-20 1.96E+06 1.89E?02 9.63E?09 ? ? ? NRCoV2-SR01 3.63E+06 1.58E?03 4.37E?10 ? ? ? NRCoV2-SR02 1.05E+06 5.55E?04 5.30E?10 ? ? ? NRCoV2-SR03 9.05E+05 1.03E?03 1.13E?09 ? ? ? NRCoV2-SR04 2.18E+06 4.93E?04 2.26E?10 ? ? ? NRCoV2-SR13 2.25E+06 5.45E?03 2.43E?09 ? ? ? NRCoV2-SR16 7.39E+05 1.15E?03 1.55E?09 ? ? ? NRCoV2-S2A3 ? ? ? 4.60E+04 1.03E?04 2.23E?09 NRCoV2-S2A4 ? ? ? 2.81E+04 4.14E?04 1.47E?08 NRCoV2-S2G3 ? ? ? 1.47E+05 6.27E?04 4.28E?09 NRCoV2-S2G4 ? ? ? 9.25E+05 3.82E?04 4.13E?10 NRCoV2-S2F3 ? ? ? 1.01E+05 6.29E?04 6.22E?09 V.sub.HH-72.sup.2 9.40E+05 1.46E?01 1.56E?07 ? ? ? ACE2-H6.sup.2 6.21E+04 1.24E?02 2.00E?07 ? ? ? NRCsdAb022.sup.2 ? ? ? ? ? ? .sup.1For any given V.sub.HH, K.sub.D values across different spike fragments, S1-RBD-Fc, S1, S2 and S were in agreement. Lack of V.sub.HH binding for certain spike fragments is consistent with V.sub.HHs' subunit/domain specificities. Binding parameters were determined by flowing monomeric V.sub.HHs over sensorchip surfaces coated with various spike fragments, except for binding parameters for NRCoV2-12 which were obtained by flowing monomeric RBDs over V.sub.HH-Fc-captured surfaces. Dashes indicate lack of binding. nd, not done, .sup.2V.sub.HH-72 (Wrapp et al., 2020) and ACE2-H6 are positive binder controls, EGFR-specific V.sub.HH, NRCsdAb022 (Rossotti et al., 2019) is a negative control.
TABLE-US-00009 TABLE 9 Kinetic and equilibrium dissociation constants for the binding of Maple Red V.sub.HHs to various SARS-CoV-2 Wuhan spike protein fragments S1-RBD-Fc.sup.1 S.sup.1 V.sub.HH/ ka kd KD ka kd KD ACE2.sup.1 (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) NRCoV2-MRed03 ? ? ? 1.57E+05 8.14E?05 5.20E?10 NRCoV2-MRed04 8.55E+05 1.76E?03 2.06E?09 1.39E+06 1.50E?03 1.09E?09 NRCoV2-MRed05 6.50E+05 4.96E?04 7.63E?10 nd NRCoV2-MRed06 ? ? ? 1.85E+05 8.87E?04 4.80E?09 NRCoV2-MRed07 ? ? ? 1.60E+06 3.78E?04 2.36E?10 NRCoV2-MRed11 ? ? ? 2.34E+04 4.18E?04 1.78E?08 NRCoV2-MRed18 ? ? ? 2.02E+05 1.53E?03 7.56E?09 NRCoV2-MRed19 ? ? ? 1.59E+05 7.99E?04 5.01E?09 NRCoV2-MRed20 ? ? ? 1.60E+05 1.46E?05 9.18E?11 NRCoV2-MRed22 ? ? ? 3.47E+05 1.76E?04 5.06E?10 NRCoV2-MRed25 ? ? ? 1.12E+05 1.15E?04 1.02E?09 V.sub.HH-72.sup.2 6.82E+05 1.26E?01 1.85E?07 1.06E+06 1.50E?01 1.42E?07 ACE2-H6.sup.2 3.86E+04 1.06E?02 2.74E?07 8.63E+04 1.18E?02 1.37E?07 NRCsdAb022.sup.2 ? ? ? ? ? ? S1.sup.1 S2.sup.1 V.sub.HH/ ka kd KD ka kd KD ACE2.sup.1 (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) NRCoV2-MRed03 2.76E+05 6.15E?05 2.23E?10 ? ? ? NRCoV2-MRed04 1.09E+06 1.83E?03 1.68E?09 ? ? ? NRCoV2-MRed05 nd nd NRCoV2-MRed06 3.71E+05 1.57E?03 4.22E?09 ? ? ? NRCoV2-MRed07 1.13E+06 4.27E?04 3.78E?10 ? ? ? NRCoV2-MRed11 ? ? ? 4.09E+04 2.13E?04 5.21E?09 NRCoV2-MRed18 ? ? ? 1.52E+05 1.03E?03 6.80E?09 NRCoV2-MRed19 ? ? ? 1.02E+05 8.40E?04 8.20E?09 NRCoV2-MRed20 ? ? ? 1.13E+05 3.38E?05 2.99E?10 NRCoV2-MRed22 ? ? ? 2.70E+05 2.84E?04 1.05E?09 NRCoV2-MRed25 ? ? ? 1.22E+05 1.90E?04 1.56E?09 V.sub.HH-72.sup.2 8.30E+05 1.61E?01 1.94E?07 ? ? ? ACE2-H6.sup.2 7.29E+04 1.32E?02 1.82E?07 ? ? ? NRCsdAb022.sup.2 ? ? ? ? ? ? .sup.1For any given V.sub.HH, K.sub.D values across different spike fragments, S1-RBD-Fc, S1, S2 and S were in agreement. Lack of V.sub.HH binding for certain spike fragments was consistent with V.sub.HHs' subunit/domain specificities. Binding parameters were determined by flowing monomeric V.sub.HHs over sensorchip surfaces coated with various spike fragments, except for NRCoV2-MRed05, for which binding parameters were obtained by flowing monomeric RBDs over V.sub.HH-Fc-captured surfaces. Dashes indicate lack of binding, nd, not done. .sup.2V.sub.HH-72 (Wrapp et al., 2020) and ACE2-H6 are positive binder controls, EGFR-specific V.sub.HH, NRCsdAb022 (Rossotti et al., 2019) is a negative control.
TABLE-US-00010 TABLE 10 ELISA data for the binding of V.sub.HH-Fcs to various spike protein fragments EC.sub.50app (nM).sup.2 Subdomain V.sub.HH-Fc.sup.1 S S1 S1-NTD S1-RBD specificity NRCoV2-SR01 0.13 0.23 0.19 S1-NTD NRCoV2-SR02 0.11 0.13 0.34 S1-NTD NRCoV2-SR03 0.16 0.20 0.17 S1-NTD NRCoV2-SR04 0.20 0.25 0.17 S1-NTD NRCoV2-SR13 0.43 0.43 0.23 S1-NTD NRCoV2-SR16 0.59 2.70 0.36 S1-NTD NRCoV2-MRed03 0.20 0.48 0.70 S1-NTD NRCoV2-MRed06 0.40 0.59 0.40 S1-NTD NRCoV2-MRed07 1.20 1.50 0.80 S1-NTD NRCoV2-02 0.10 0.11 0.21 S1-RBD .sup.1S1-specfic V.sub.HHs that did not bind to S1-RBD by SPR (Table 8 and Table 9), were tested for specificity against S1-NTD. The S1-RBD-specific NRCoV2-02 internal control gave the expected specificity binding profile. .sup.2EC.sub.50app, apparent EC.sub.50.
TABLE-US-00011 TABLE 11 Kinetic and equilibrium dissociation constants for the binding of V.sub.HHs to SARS-CoV-2 Wuhan, SARS-CoV-2 Alpha, SARSCoV-2 Beta and SARS-CoV spike glycoproteins SARS-CoV-2 SARS-CoV-2 Wuhan.sup.a Alpha.sup.a V.sub.HH/ k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D ACE2 (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) RBD-specific V.sub.HH NRCoV2-1d 1.39E+06 1.05E?03 7.50E?10 1.13E+06 1.02E?03 9.07E?10 NRCoV2-02 2.04E+06 1.27E?03 6.24E?10 1.64E+06 2.22E?02 1.36E?08 NRCoV2-03 1.16E+05 1.81E?04 1.56E?09 1.03E+05 1.54E?04 1.49E?09 NRCoV2-04 1.84E+06 1.88E?02 1.02E?08 1.67E+06 1.96E?02 1.17E?08 NRCoV2-05 2.76E+06 7.09E?03 2.57E?09 2.28E+06 2.61E?02 1.14E?08 NRCoV2-06 2.05E+04 4.56E?03 2.23E?07 2.05E+04 4.68E?03 2.29E?07 NRCoV2-07 3.78E+05 3.55E?04 9.39E?10 3.39E+05 3.78E?04 1.12E?09 NRCoV2-10 5.81E+05 1.16E?04 1.99E?10 4.84E+05 1.02E?04 2.10E?10 NRCoV2-11 9.64E+05 1.71E?05 1.77E?11 9.28E+05 1.59E?05 1.71E?11 NRCoV2-12.sup.a 7.47E+05 3.50E?05 4.69E?11 9.47E+05 4.38E?05 4.63E?11 NRCoV2-14 3.89E+05 1.01E?03 2.60E?09 3.75E+05 9.17E?04 2.44E?09 NRCoV2-15 6.91E+05 2.22E?04 3.21E?10 6.37E+05 1.94E?04 3.05E?10 NRCoV2-17 6.14E+05 9.45E?05 1.54E?10 6.14E+05 7.64E?05 1.25E?10 NRCoV2-18 2.95E+05 9.39E?05 3.18E?10 2.78E+05 9.79E?05 3.53E?10 NRCoV2-20 2.64E+06 1.16E?02 4.39E?09 2.48E+06 1.23E?02 4.97E?09 NRCoV2- 1.51E+06 1.31E?03 8.63E?10 1.34E+06 1.22E?03 9.09E?10 MRed04 NRCoV2- 5.62E+05 5.09E?04 9.05E?10 5.73E+05 1.76E?04 3.07E?10 MRed05.sup.a NTD-specific V.sub.HH NRCoV2-SR01 1.43E+06 8.05E?04 5.64E?10 1.01E+06 5.94E?04 5.91E?10 NRCoV2-SR02 4.98E+06 6.69E?04 1.35E?10 4.54E+06 2.54E?04 5.59E?11 NRCoV2-SR03 6.70E+05 1.13E?03 1.69E?09 5.55E+05 9.55E?04 1.72E?09 NRCoV2-SR04 3.68E+06 5.15E?04 1.40E?10 2.80E+06 7.50E?04 2.67E?10 NRCoV2-SR13 1.06E+06 3.76E?03 3.56E?09 5.92E+05 3.44E?03 5.82E?09 NRCoV2-SR16 4.88E+05 9.57E?04 1.96E?09 4.04E+05 6.33E?04 1.57E?09 NRCoV2- 2.37E+05 1.21E?04 5.08E?10 2.71E+05 9.89E?05 3.64E?10 MRed03 NRCoV2- 1.92E+05 9.96E?04 5.19E?09 2.27E+05 1.30E?03 5.72E?09 MRed06 NRCoV2- 4.58E+06 4.81E?04 1.05E?10 3.90E+06 1.03E?03 2.64E?10 MRed07 S2-specific V.sub.HH NRCoV2-S2A3 9.83E+04 5.51E?05 5.61E-10 8.70E+04 1.89E?04 2.18E?09 NRCoV2-S2A4 3.49E+04 4.46E?04 1.28E?08 2.48E+05 2.36E?03 9.52E?09 NRCoV2-S2F3 1.56E+05 4.73E?04 3.03E?09 + NRCoV2-S2G3 3.24E+05 6.06E?04 1.87E?09 2.98E+05 5.30E?04 1.78E?09 NRCoV2-S2G4 8.93E+05 2.07E?04 2.32E?10 1.56E+06 2.92E?04 1.87E?10 NRCoV2- 4.57E+04 2.83E?04 6.20E?09 3.11E+04 4.26E?04 1.37E?08 MRed11 NRCoV2- 1.97E+05 1.19E?03 6.03E?09 3.82E+05 4.93E?03 1.29E?08 MRed18 NRCoV2- 1.31E+05 1.18E?03 9.07E?09 1.83E+05 3.70E?03 2.02E?08 MRed19 NRCoV2- 1.60E+05 1.46E?05 9.18E?11 2.78E+05 1.53E?04 5.50E?10 MRed20 NRCoV2- 3.47E+05 1.76E?04 5.06E?10 8.96E+05 2.20E?04 2.46E?10 MRed22 NRCoV2- 1.12E+05 1.15E?04 1.02E?09 1.02E+06 2.89E?04 2.83E?10 MRed25 Control ACE2-H.sub.6.sup.b 6.38E+04 9.79E?03 1.53E?07 8.52E+04 1.56E?03 1.83E?08 VHH-72.sup.b 1.23E+06 1.06E?01 8.62E?08 1.05E+06 1.01E?01 9.60E?08 NRCsdAb022.sup.b ? ? ? ? ? ? SARS-CoV-2 Beta.sup.a SARS-CoV.sup.a V.sub.HH/ k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D ACE2 (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) RBD-specific V.sub.HH NRCoV2-1d 8.12E+05 9.56E?04 1.18E?09 ? ? ? NRCoV2-02 ? ? ? ? ? ? NRCoV2-03 5.54E+04 2.26E?04 4.08E?09 ? ? ? NRCoV2-04 ? ? ? ? ? ? NRCoV2-05 ? ? ? ? ? ? NRCoV2-06 2.48E+04 6.14E?03 2.48E?07 ? ? ? NRCoV2-07 3.05E+05 3.21E?04 1.05E?09 1.20E+05 1.46E?03 1.22E?08 NRCoV2-10 2.70E+05 2.62E?03 9.73E?09 ? ? ? NRCoV2-11 7.57E+05 1.72E?05 2.27E?11 1.93E+06 2.70E?05 1.40E?11 NRCoV2-12.sup.a 9.70E+05 3.89E?05 4.01E?11 3.76E+05 1.01E?03 2.69E?09 NRCoV2-14 ? ? ? ? ? ? NRCoV2-15 1.48E+05 3.28E?03 2.22E?08 ? ? ? NRCoV2-17 1.14E+06 5.88E?03 5.14E?09 ? ? ? NRCoV2-18 2.76E+05 1.01E?04 3.65E?10 ? ? ? NRCoV2-20 2.08E+06 1.14E?02 5.47E?09 ? ? ? NRCoV2- 1.14E+06 1.23E?03 1.07E?09 3.23E+05 9.70E?02 3.00E?07 MRed04 NRCoV2- 6.12E+05 5.44E?04 8.88E?10 ? MRed05.sup.a NTD-specific V.sub.HH NRCoV2-SR01 1.09E+06 2.21E?04 2.02E?10 6.81E+05 1.05E?04 1.54E?10 NRCoV2-SR02 4.19E+06 6.28E?04 1.50E?10 ? ? ? NRCoV2-SR03 5.64E+05 1.40E?03 2.49E?09 ? ? ? NRCoV2-SR04 2.24E+06 7.23E?04 3.24E?10 ? ? ? NRCoV2-SR13 5.71E+05 4.01E?03 7.02E?09 ? ? ? NRCoV2-SR16 3.95E+05 1.01E?03 2.57E?09 ? ? ? NRCoV2- 2.13E+05 1.43E?04 6.72E?10 ? ? ? MRed03 NRCoV2- 1.39E+05 1.01E?03 7.24E?09 ? ? ? MRed06 NRCoV2- 2.38E+06 5.52E?04 2.31E?10 ? ? ? MRed07 S2-specific V.sub.HH NRCoV2-S2A3 6.69E+04 5.71E?05 8.53E?10 ? ? ? NRCoV2-S2A4 5.88E+4 8.98E?4 1.53E?8 ? ? ? NRCoV2-S2F3 + 2.82E+05 1.39E?03 4.91E?09 NRCoV2-S2G3 3.04E+05 5.63E?04 1.85E?09 1.87E+05 8.00E?04 4.27E?09 NRCoV2-S2G4 + 9.20E+05 7.35E?04 7.99E?10 NRCoV2- 4.54E+04 2.82E?04 6.21E?09 ? ? ? MRed11 NRCoV2- 3.69E+05 2.39E?03 6.48E?09 3.00E+05 6.77E?03 2.25E?08 MRed18 NRCoV2- 1.29E+05 1.04E?03 8.07E?09 2.49E+05 6.14E?03 2.46E?08 MRed19 NRCoV2- 1.39E+05 6.33E?05 4.55E?10 3.80E+05 4.06E?03 1.07E?08 MRed20 NRCoV2- + ? ? ? MRed22 NRCoV2- 2.05E+06 3.29E?03 1.60E?09 2.18E+04 5.01E?05 2.29E?09 MRed25 Control ACE2-H.sub.6.sup.b 3.66E+04 4.78E?03 1.31E?07 1.11E+05 3.89E?02 3.51E?07 VHH-72.sup.b 8.01E+05 9.92E?02 1.24E?07 1.01E+06 6.56E?03 6.52E?09 NRCsdAb022.sup.b ? ? ? ? ? ? .sup.aBinding parameters were determined by flowing monomeric V.sub.HHs over sensorchip surfaces coated with S, except for V.sub.HH NRCoV2-12 and MRed05, which were obtained by flowing monomeric RBDs (aa319-541 [SARS-CoV-2]; aa306-527 [SARS-CoV]) over V.sub.HH-Fc-captured surfaces. Dashes indicate lack of binding. nd, not determined. .sup.bACE2-H.sub.6 and V.sub.HH-72 (Wrapp et al., 2020), positive controls, EGFR-specific V.sub.HH NRCsdAb022 (Rossotti et al., 2019) negative control.
TABLE-US-00012 TABLE 12 SPR affinity (K.sub.D) of V.sub.HHs against trimeric spikes protein S from the Wuhan-Hu-1 (Wuhan), UK B.1.1.7 (Alpha) and South Africa B.1.351 (Beta) SARS-CoV-2 variants Subunit/domain K.sub.D (nM) V.sub.HH/ACE2 specificity/epitope bin Wuhan Alpha Beta ACE2 S1-RBD 153 18.3 131 NRCoV2-1d S1-RBD/Bin 1 0.75 0.91 1.2 NRCoV2-07 0.94 1.1 1.1 NRCoV2-12 0.047 0.046 0.04 NRCoV2-18 0.32 0.35 0.37 NRCoV2-20 4.39 4.97 5.47 NRCoV2-MRed04 0.86 0.91 1.07 V.sub.HH-72.sup.1 86.2 96 124 NRCoV2-02 S1-RBD/Bin 2, 3 0.62 13.6 .sup.3 NRCoV2-05 2.6 11.4 NRCoV2-10 S1-RBD/Bin 2, 3, 4 0.2 0.21 9.73 NRCoV2-15 0.32 0.31 22.2 NRCoV2-MRed05 0.91 0.31 0.89 NRCoV2-14 S1-RBD/Bin 2, 4 2.6 2.44 weak binding NRCoV2-17 S1-RBD/Bin 3, 4 0.15 0.13 5.1 NRCoV2-04 S1-RBD/Bin 4 10.2 11.7 NRCoV2-06 S1-RBD/Bin 5 223 229 248 NRCoV2-11 0.018 0.017 0.023 NRCoV2-03 S1-RBD/Bin 6 1.56 1.49 4.08 NRCoV2-SR01 S1-NTD/Bin 7, 9, 10 0.56 0.59 0.2 NRCov2-SR03 1.69 1.72 2.49 NRCoV2-SR13 3.6 5.8 7 NRCoV2-SR16 2 1.6 2.6 NRCoV2-MRed03 S1-NTD/Bin 8 0.51 0.36 0.67 NRCov2-MRed06 5.2 5.72 7.24 NRCoV2-MRed07 S1-NTD/Bin 9 0.11 0.26 0.23 NRCoV2-SR04 S1-NTD/Bin 7, 9 0.14 0.27 0.32 NRCoV2-SR02 S1-NTD/Bin 10 0.47 0.11 0.53 NRCoV2-S2A3 S2/Bin 11 0.56 2.18 0.85 NRCoV2-S2A4 S2/Bin 12 12.8 9.5 15.3 NRCoV2-S2F3 S2/Bin 13 3.03 +.sup.4 +.sup.4 NRCoV2-MRed18 6.03 12.9 6.48 NRCoV2-MRed19 9.07 20.2 8.07 NRCoV2-MRed20 0.092 0.55 0.45 NRCoV2-MRed22 0.51 0.25 +.sup.4 NRCoV2-S2G3 S2/Bin 14 1.87 1.78 1.85 NRCoV2-S2G4 S2/Bin 15 0.23 0.19 +.sup.4 NRCoV2-MRed11 S2/Bin 16 6.2 13.7 6.2 NRCoV2-MRed25 S2/Bin 17 1.02 0.28 1.6 .sup.1V.sub.HH-72 is the benchmark (Wrapp et al., 2020); .sup.2nd, not determined; .sup.3, no binding; .sup.4+, V.sub.HH bound, but poor fitting precluded K.sub.D determination. Epitope bin numbers correspond to the bins shown in FIG. 9G.
Example 6: Cell Binding Assays by Flow Cytometry
Introduction
[0389] In the previous Examples, lead V.sub.HHs were shown to be binding to SARS-CoV-2 S in its purified form. In this Example, it was confirmed whether the V.sub.HHs also bind to SARS-CoV-2 S in its more natural context, i.e., displayed on the cell membrane of CHO cells.
Materials and Methods
[0390] A stable Chinese hamster ovary (CHO) cell line CHO.sup.BRI TM/55E1 (Stuible et al., 2021) overexpressing SARS-CoV-2 S (CHO-S) was grown in BalanCD? CHO Growth A medium (Irvine Scientific) supplemented with 50 ?M of methionine sulfoximine (MSX) at 120 rpm and 37? C. in a humidified 5% CO.sub.2 atmosphere. When the cell count reached 2?10.sup.6/mL, the expression of the membrane anchored SARS-CoV-2 trimeric spike protein (SmT1, described in Stuible et al, 2021) was induced by adding cumate at 2 ?g/mL. Expression was carried out for 48 h at 32? C. For flow cytometry experiments, cells were harvested by centrifugation and resuspended at 1?10.sup.6 cells/mL in PBSB (1% PBS containing 1% BSA and 0.05 [v/v] sodium azide). Cells were kept on ice until use. Serially, three-fold dilutions of V.sub.HH-Fcs were prepared in V-Bottom 96-well microtest plates (Globe Scientific, Cat #120130) and mixed with 50 ?L of CHO-S cells. Plates were incubated for 1 h on ice, washed twice with PBSB by centrifugation 5 min at 1200 rpm and then incubated for an additional hour with 50 ?L of R-Phycoerythrin AffiniPure F(ab).sub.2 Fragment Goat Anti-Human IgG (Jackson Immunoresearch, Cat #109-116-170) at 250 ng/mL diluted in PBSB. After a final wash, cells were resuspended in 100 ?L PBSB and data were acquired on a Beckman Culter CytoFlex S and analyzed by FlowJo? (FlowJo LLC, vi 0.6.2, Ashland).
Results and Discussion
[0391] Interestingly, four V.sub.HH-Fcs (NRCoV2-08, NRCoV2-19, NRCoV2-21, NRCoV2-S202) which bound to SARS-CoV-2 S in purified form did not bind to SARS-CoV-2 S-displaying target cells. The remaining 41 V.sub.HH-Fcs, however, bound to cells in a dose dependent manner (
TABLE-US-00013 TABLE 13 Summary of V.sub.HH-Fc bindings to SARS-CoV-2 S expressing CHOS cells S1-RBD-specific S1-NTD-specific S2-specific EC.sub.50 EC.sub.50 EC.sub.50 V.sub.HH-Fc (nM) B.sub.max V.sub.HH-Fc (nM) B.sub.max V.sub.HH-Fc (nM) B.sub.max NRCoV2- 1.1 17186 NRCoV2- 6.6 15389 NRCoV2- 0.44 9819 1a SR01 S2A3 NRCoV2- 1.1 13817 NRCoV2- 1.2 14416 NRCoV2- 0.1 6858 1d SR02 S2A4 NRCoV2- 0.3 18553 NRCoV2- 7.1 19857 NRCoV2- 6.5 6665 02 SR03 S2B3 NRCoV2- 78.9 16242 NRCoV2- 8.2 10119 NRCoV2- 2.7 6953 03 SR04 S2F3 NRCoV2- 1.3 15489 NRCoV2- 7.0 17808 NRCoV2- 0.3 7529 04 SR13 S2G3 NRCoV2- 0.5 17419 NRCoV2- 1.4 11553 NRCoV2- 0.3 6897 05 SR16 S2G4 NRCoV2- 8.1 13615 NRCoV2- 15.1 11990 NRCoV2- 1.0 7906 06 MRed03 S2H4 NRCoV2- 1.1 10620 NRCoV2- 8.5 9023 NRCoV2- ? ? 07 MRed06 S202 NRCoV2- ? ? NRCoV2- 132.4 8673 NRCoV2- 2.9 3249 08 MRed07 MRed11 NRCoV2- 1.3 28044 NRCoV2- 0.9 10205 10 MRed18 NRCoV2- 4.3 18230 NRCoV2- 1.3 5482 11 MRed19 NRCoV2- 2.0 11963 NRCoV2- 6.5 6665 11a MRed20 NRCoV2- 0.8 21821 NRCoV2- 0.2 5324 12 MRed22 NRCoV2- 0.9 18801 NRCoV2- 0.8 4421 14 MRed25 NRCoV2- 0.8 21053 15 NRCoV2- 0.4 19019 17 NRCoV2- 0.9 18266 18 NRCoV2- ? ? 19 NRCoV2- 0.7 12056 20 NRCoV2- ? ? 21 NRCoV2- 4.2 14916 MRed04 NRCoV2- 0.5 17349 MRed05 VHH-72 0.2 8026 ?, No detectable binding observed.
Example 7: Epitope Studies
Introduction
[0392] Western blotting experiments were performed to determine if V.sub.HHs bind to conformational or linear epitopes. Additionally, competitive sandwich ELISA as well as SPR were performed to differentiate V.sub.HHs with respect to recognizing non-overlapping epitopes.
Materials and Methods
Epitope Typing by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis/Western Blotting (SDS-PAGE/WB)
[0393] A standard SDS-PAGE/WB was performed to detect the binding of V.sub.HHs to nitrocellulose-immobilized, denatured SARS-CoV-2 S. Briefly, 10 ?g/lane of S was run on 4-20% Mini-PROTEAN? TGX Stain-Free? Protein Gels (Bio-Rad, Cat #4568081), transferred to nitrocellulose (Sigma, Cat #GE10600002) and blocked with 1% PBSC overnight at 4? C. Then, 0.5-cm nitrocellulose strips containing the denatured S were placed on Mini Incubation Trays (Bio-Rad, Cat #1703902) and incubated with 1 mL of 1 ?g/mL V.sub.HH-Fcs or biotinylated V.sub.HHs (V.sub.HH-BAP-His6). After 1 h incubation at room temperature, strips were washed 10 times with PBST and the binding of V.sub.HH-Fcs or biotinylated V.sub.HHs to denatured S was probed, respectively, by incubating strips with 1 mL of 100 ng/mL anti-human Ig Fc antibody-peroxidase conjugate or streptavidin-peroxidase conjugate (Jackson ImmunoResearch, Cat #016-030-084) at room temperature for 1 h. Finally, strips were washed 10 times with PBST and peroxidase activity was detected using chemiluminescent reagent (SuperSignal? West Pico PLUS Chemiluminescent Substrate, ThermoFisher, Cat #34580). Images of developed strips were acquired on Molecular Imager? Gel Doc? XR System (Bio-Rad, Cat #1708195EDU).
Epitope Binning by SPR
[0394] Standard SPR techniques were used for binding studies. All SPR assays were performed on a Biacore? T200 instrument (Cytiva) at 25? C. with HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% [v/v] Tween? 20, pH 7.4) and CM5 sensor chips (Cytiva). Prior to SPR analyses all analytes in flow (V.sub.HHs, ACE2 receptor) were SEC-purified on a Superdex? 75 Increase 10/300 GL column (Cytiva) in HBS-EP buffer at a flow rate of 0.8 mL/min to obtain monomeric proteins. V.sub.HH epitope binning was performed by SPR dual injection experiments on the SARS-CoV-2 S at a flow rate of 40 ?L/min in HBS-EP buffer. Dual injections consisted of injection of V.sub.HH1 (at 50-100?K.sub.D concentration) for 150 s, followed by immediate injection of a mixture of V.sub.HH1+V.sub.HH2 (both at 50-100?K.sub.D concentration) for 150 s. The opposite orientation was also performed (V.sub.HH2 followed by V.sub.HH2+V.sub.HH1) (
Epitope Binning by ELISA
[0395] The pairwise ability of V.sub.HHs to bind to their antigen in a sandwich ELISA format was evaluated as described previously (Rosotti et al., 2015a; Delfin-Riela et al., 2020), (
Results and Discussion
[0396] To determine whether V.sub.HHs recognize conformational or linear epitopes, they were subjected to binding analysis against SARS-CoV-2 S by denaturing, SDS-PAGE/Western blot. As shown in
[0397] To identify the number of distinct (non-overlapping) epitopes, V.sub.HHs were subjected to epitope binning experiments by SPR and sandwich ELISA. In SPR epitope binning assays, the first V.sub.HH (V.sub.HH1) was flowed over a spike protein-immobilized sensorchip and allowed to saturate its epitope, followed by the addition of the second, V.sub.HH2 applied as a mixture of V.sub.HH1+V.sub.HH2 to keep the V.sub.HH1 epitope saturated during the binding of V.sub.HH2. Assays were performed in a second orientation as well to cross-confirm results: V.sub.HH2+(V.sub.HH2+V.sub.HH1).
TABLE-US-00014 TABLE14 Summaryofepitopebinningresults Do- main/ sub- SEQ SEQ SEQ unit Epi- CDR ID ID ID speci- tope V.sub.HH 1 NO: CDR2 NO: CDR3 NO: ficity Bin NRCoV2- GFTF 21 ISGNG 65 AATGIRST 111 S1-NTD 7/9/ SR01 DNYA GVT WSVYGCSR 10 LAGPYDY NRCoV2- GSIF 23 ISSGG 67 NRGGWEYR 113 S1-NTD 7/9/ SR03 SNNH KT SSYYIMGP 10 H NRCoV2- GSRF 25 ISSGG 69 NMGGWDYR 115 S1-NTD 7/9/ SR13 GSKH ST SNTYIPGS 10 RSDY NRCoV2- GTTF 26 ISTSG 70 NTGGWDYR 116 S1-NTD 7/9/ SR16 SRYH AV SSTFIMGL 10 N NRCoV2- GRTF 24 ISMGG 68 NTAALVGN 114 S1-NTD 7/9 SR04 SSHT NTNYA RLLPMATI T NRCoV2- GFTL 20 ISSSD 81 ATDAFATC 126 S1-NTD 8 MRed03 DYYA GST DSWYAQIA QYDF NRCoV2- GFTL 37 ISSSD 82 ATDSFSSC 128 S1-NTD 8 MRed06 AYYA GSA SDYESGMD F NRCoV2- GSIG 38 ITRGG 83 YANYGWAI 129 S1-NTD 9 MRed07 PFNT VT PY NRCoV2- EFTL 22 IRYSG 66 AADRLYSR 112 S1-NTD 10 SR02 NYYS GGI ACPTAGGR NY NRCoV2- GSTL 1 VSSSD 45 AADYSMRP 90 S1-RBD 1 1a DYYA GST LWVSRWHR DYEY NRCoV2- GSIL 2 VSSSD 45 AADYSMRR 91 S1-RBD 1 1c DYYA GST FAVGRWHR DYEY NRCoV2- GSTL 1 VSSSD 46 AADYSMRP 92 S1-RBD 1 1d DYYA GNT FAVGRWHR DYEY NRCoV2- GVTL 8 ISSNG 52 AAVQDVHG 98 S1-RBD 1 07 DYYA RRN DNYYCTSP NEYNV NRCoV2- GRTF 13 VAAIS 57 AADRGLSY 103 S1-RBD 1 12 RNYV WGGTE YYTRTTEY I NY NRCoV2- GITI 17 INSGG 61 SLHTSHDY 107 S1-RBD 1 18 SGYN ST NRCoV2- GRTF 19 VAVIS 63 AADRGMSY 109 S1-RBD 1 20 SNYV GSDTE YYTRATEY T YY NRCoV2- GNIF 35 IWSDS 79 AADRGFVV 125 S1-RBD 1 MRed02 SINS RT RGQYDY NRCoV2- GNSF 36 IWSDT 80 AADRGFVV 125 S1-RBD 1 MRed04 SINT TT RGQYDY NRCoV2- GSPF 5 ISGGG 49 WSSYEST 95 S1-RBD 4 04 SNVV IA NRCoV2- VSTF 7 IGFVG 51 NARHYGGS 97 S1-RBD 5 06 SSYA AT EY NRCoV2- GITF 4 MSNMD 48 NIYGPTYS 94 S1-RBD 6 03 SYYA ST TRRNEY NRCoV2- GFTL 9 ISRSG 53 AADYQYST 99 S1-RBD 6 08 DDYA TTT YCLGYDAH YEY NRCoV2- GSSL 11 ISRYY 55 AARSRDFS 101 S1-RBD 6 11 DSYS SST SPFSATDT YTS NRCoV2- GFTL 12 ISRYY 56 AARSRDFS 102 S1-RBD 6 11a DSYN EST SPISATDK YGS NRCoV2- GFTF 3 ISGRG 47 TKGPDLYY 93 S1-RBD 2,3 02 SNYA DDT FGSGYSD NRCoV2- GFIF 6 INSGG 50 SKGPVSSY 96 S1-RBD 2,3 05 SNYA GDT YGSGYDY NRCoV2- GNTF 10 ISSRG 54 YAADDLGD 100 S1-RBD 2,3, 10 SRSN IS Y 4 NRCoV2- GSTS 15 VSTSG 59 YAAYGGGG 105 S1-RBD 2,3, 15 GRNT AT DY 4 NRCoV2- GFTL 20 ISSSD 81 ATGPQAYY 127 S1-RBD 2,3, MRed05 DYYA GST SGSYYFQC 4 PQAGMDY NRCoV2- GTTF 14 ISVFG 58 HAVNADIG 104 S1-RBD 2,4 14 SHYA ST GDY NRCoV2- GSPF 16 ISPTG 60 QAANVNGG 106 S1-RBD 3,4 17 SQLA NR DY NRCoV2- GLTL 18 LTSGG 62 AADRARLR 108 S1-RBD ud 19 NSYA TG FGCSLNFR REVAYDY NRCoV2- GFTL 20 ISSGG 64 AADHRGRS 110 S1-RBD ud 21 DYYA ST LRFGCSSS TTDYLY NRCoV2- GRPY 27 KQREL 71 NTGSLSYG 117 S2 11 S2A3 SNYA VAAIS GSVYYPSY SGGTT DN NRCoV2- GSPF 28 ISTGG 72 HAAARDSH 118 S2 12 S2A4 RSNV SR GIYLLDT NRCoV2- VRIL 31 ITSGG 75 NLRDILSQ 121 S2 13 S2F3 SVPA ST PF NRCoV2- TTVF 40 VSDGG 85 NYYNYYYG 131 S2 13 MRed18 GRNA TP RNF NRCoV2- TIIF 41 MTTSG 86 YMHSVYYG 132 S2 13 MRed19 KGQT SA IDY NRCoV2- GLSF 42 IRESG 87 AAKPPFYG 133 S2 13 MRed20 SSYD SGT SGTYSTPR AYLY NRCoV2- GSVF 43 ISSRG 88 NAREFTGF 134 S2 13 MRed22 ASNA ST DY NRCoV2- GSTF 33 ISSDG 77 NKHWWTGD 123 S2 14 S2G4 SGYA DK W NRCoV2- GSTF 32 ITSGG 76 YTTKRDDA 122 S2 15 S2G3 GIFL AT SVY NRCoV2- GFTF 39 INSGG 84 ATTISDGS 130 S2 16 MRed11 SSYA GST SWSTKSY NRCoV2- GHTF 44 ISWRG 89 AAEMWGTA 135 S2 17 MRed25 SRYG DST TIVASRYT Y NRCoV2- ASTF 29 ISTGS 73 NYRSIYYG 119 S2 nd S2B3 GDSA NT QNF NRCoV2- GFTF 30 INSGD 74 ALVFGYTS 120 S2 nd S2H4 NLYS RDSTT RDYCLTPK RGNY NRCoV2- GITV 34 ISAGG 78 NYGPGYRK 124 S2 nd S202 SRIG ST AA nd =no data, ud =undetermined. Epitope bin numbers correspond to the bins shown in FIG. 9G.
Example 8: Surrogate Virus Neutralization Assays
Introduction
[0398] Surrogate neutralization assays were performed to identify potential neutralizing V.sub.HHs/V.sub.HH-Fcs, i.e., V.sub.HHs/V.sub.HH-Fcs inhibiting SARS-CoV-2 viruses from entering host cells. Three different surrogate assays were performed: ELISA, SPR and flow cytometry. In ELISA and SPR, ACE2 and SARS-CoV-2 S acted as surrogates for an ACE2-containing host cell and an S-containing invading virus, respectively. In flow cytometry assays, which were performed directly against the host cell (Vero E6), S1-RBD or S served as surrogate virus. Antibodies that interfered with the binding of spike fragment proteins to ACE2 in the surrogate assays were considered to be neutralizing antibodies.
Materials and Methods
ACE2 Competition Assay by ELISA
[0399] Wells of NUNC? MaxiSorp? microtiter plates (Thermo Fisher) were coated overnight at 4? C. with 50 ng/well of S in 100 ?L PBS, pH 7.4. Next day, plates were blocked with 250 ?L PBSC for 1 h at room temperature. For ACE2/V.sub.HH competition binding to SARS-CoV-2 S, 50 ?L of ACE2-Fc (ACROBiosystems, Cat #AC2-H5257) at 400 ng/mL was mixed with 50 ?L of V.sub.HH at 1 ?M, and then transferred to SARS-CoV-2 S coated microtiter plate wells. After 1 h incubation at room temperature, plates were washed 10 times with PBST and the ACE2-Fc binding was detected using 1 ?g/mL goat anti-human IgG (Fc specific)-peroxidase antibody (SIGMA, Cat #A0170) in 100 ?L PBSCT. After 10 washes with PBST, the peroxidase activity was determined as described above.
ACE2 competition assay by SPR
[0400] Standard SPR techniques were used for binding studies. All SPR assays were performed on a Biacore? T200 instrument (Cytiva) at 25? C. with HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% [v/v] Tween? 20, pH 7.4) and CM5 sensor chips (Cytiva). Prior to SPR analyses, all analytes in flow (V.sub.HHs, ACE2 receptor) were SEC-purified on a Superdex? 75 Increase 10/300 GL column (Cytiva) in HBS-EP buffer at a flow rate of 0.8 mL/min to obtain monomeric proteins. V.sub.HHs were analyzed for their ability to block the SARS-CoV-2 spike trimer (S) interaction with ACE2 using SPR dual injection experiments. V.sub.HHs and ACE2 were flowed over the SARS-CoV-2 S surface at 40 ?L/min in HBS-EP buffer. Dual injections consisted of injection of ACE2 (1 ?M) for 150 s, followed by immediate injection of a mixture of ACE2 (1 ?M)+V.sub.HH (at 20-40?K.sub.D concentration) for 150 s. The opposite orientation was also performed (V.sub.HH followed by V.sub.HH+ACE2). Surfaces were regenerated using a 12 s pulse of 10 mM glycine, pH 1.5, at a flow rate of 100 ?L/min. All pairwise combinations of V.sub.HHs and ACE2 were analyzed. V.sub.HHs that competed with ACE2 for SARS-CoV-2 spike trimer binding showed no increase in binding response during the second injection. Conversely, a binding response was seen during the second injection for V.sub.HHs that did not compete with ACE2.
ACE2 Competition Assay by Flow Cytometry
[0401] Experiments were performed essentially as described in Example 2. Briefly, 400 ng of chemically biotinylated trimeric SARS-CoV-2 S was mixed with 5?10.sup.4 Vero E6 cells in the presence of decreasing concentrations of V.sub.HHs or V.sub.HH-Fcs in a final volume of 150 ?L. Following 1 h incubation on ice, cells were washed twice with PBSB by centrifugation at 1200 rpm for 5 min and then incubated for an additional hour with 50 ?L Streptavidin, R-Phycoerythrin Conjugate (SAPE, ThermoFisher, Cat #S866) at 250 ng/mL diluted in PBSB. After a final wash, cells were resuspended in 100 ?L PBSB and data were acquired on a CytoFlex? S flow cytometer (Beckman Culter) and analyzed by FlowJo? (FlowJo LLC, v10.6.2, Ashland, OR). As an internal reference for competition experiments, a competition assay with recombinant human ACE2-His6 in lieu of V.sub.HH was also included. A20.1, a C. difficile toxin A-specific V.sub.HH (Hussack et al., 2011) was used as negative control V.sub.HH. Percent inhibition (neutralization) was calculated according to the following formula: % inhibition=100?[1?(F.sub.n?F.sub.min)/(F.sub.max?F.sub.min)], where, F.sub.n is the measured fluorescence at any given competitor V.sub.HH concentration, F.sub.min is the background fluorescence measured in the presence of cells and SAPE only, and F.sub.max is the maximum fluorescence, measured in the absence of V.sub.HH competitor.
Results and Discussion
[0402] Initially, a total of 26 V.sub.HHs (14 S1-RBD-specific, 6 S1-NTD-specific and 6 S2-specific) were subjected to competitive ELISA, to identify those that are neutralizing, i.e., reduce the binding of ACE2-Fc to S. As shown in
[0403] Finally, a quantitative surrogate neutralization assay was performed by flow cytometry, where antibodies were assessed based on their ability to block the interaction of trimeric SARS-CoV-2 S with ACE2 on the surface of Vero E6 cells (African green monkey kidney cells). (Vero E6 cells are known to be highly susceptible to infection by SARS-CoV-2 and SARS-CoV.) Both monomeric V.sub.HHs and bivalent V.sub.HH-Fcs were assessed. IC.sub.50s, IC.sub.99s and I.sub.max% values, measures of potency and efficacy were used to rank neutralizing antibodies. A preliminary screen performed at a single concentration with S1-RBD-, S1-NTD- and S2-specific V.sub.HHs showed that many of the S1-RBD-specific V.sub.HHs were potent neutralizers (
[0404] To increase the neutralization potency and efficacy of the V.sub.HHs, they were reformatted as bivalent V.sub.HH-Fcs. The increase in size (from 16 kDa V.sub.HH to 80 kDa V.sub.HH-Fc) as well as avidity (from monovalent V.sub.HH to bivalent V.sub.HH-Fc) could sterically hinder the binding of S to ACE2 and increase V.sub.HHs' apparent affinity leading to their improved neutralization potency and efficacy. Thus, V.sub.HH-Fcs were generated and tested in flow cytometry surrogate neutralization assays as described above. The majority of V.sub.HH-Fcs demonstrated high potencies and efficacies (
[0405] The surrogate neutralization assays were then extended to variants Alpha, Beta, Gamma, Delta, Kappa and Omicron using all of the RBD-specific and a subset of NTD-specific V.sub.HH-Fcs (Table 15). In this assay Wuhan was included and performed again as an internal reference. Several observations were made. First, for cross-neutralizing V.sub.HHs, the IC.sub.50s across variants did not change significantly. Second, while all Wuhan neutralizers also remained Alpha neutralizers, some lost their capability to inhibit Beta, Gamma, Delta and Kappa with variable cross-neutralizing patterns. In particular, with respect to the RBD-specific V.sub.HHs, the cross-neutralization profiles for Beta vs Gamma and Delta vs Kappa were identical, which is likely reflective of the key escape mutations in these variants (K417N, E484K and N501Y for Beta vs K417T, E484K and N501Y for Gamma; L452R and T478K for Delta vs L452R and E484Q for Kappa). Third, and importantly, 12 out of 20 V.sub.HH-Fcs (10 RBD-specific, two NTD-specific) were Delta neutralizers, nine of which (eight RBD-specific, one NTD-specific) neutralized across all variants. Fourth, the majority of these nine pan-neutralizers (six RBD-specific, one NTD-specific) also neutralized SARS-CoV. Fifth, Omicron mutations had a major impact on antibodies targeting bin 1, from which only NRCoV2-12 and 20 were able to neutralize with comparable potency to Wuhan or the other variants tested. The neutralization ability of the benchmark V.sub.HH-72 was abolished by Omicron mutations. Antibodies from bin 2/3/4 were able to neutralize Omicron with comparable IC.sub.50 to Wuhan, except for NRCoV-2-02/05 and MRed05, which were negative. NRCoV2-11 (anti-RBD) and SR01 (anti-NTD) were also efficient, achieving neutralization as potent as was observed against Wuhan spike protein. From the list of antibodies tested NRCoV2-12, -20, -11 and -SR01 are the leads, showing efficient pan-neutralization against the SARS-CoV-2 variants generated so far, and outperforming the benchmark V.sub.HH-72.
TABLE-US-00015 TABLE 15 Flow cytometry SVNAs against SARS-CoV-2 variants and SARS-CoV SVNA IC.sub.50 (nM) SARS-CoV-2 S V.sub.HH-Fc Wuhan Alpha Beta Gamma Delta Kappa Omicron SARS-CoV S bin RBD-specific V.sub.HH 1 VHH-72 5.6 10.6 5.1 3.3 10.5 8.5 ? 7.8 1d 4.7 6.1 13.1 4.8 6.4 5.5 ? ? 07 4.7 5.7 3.6 3.2 2.3 3.6 ? 4.2 12 3.5 5.2 8 3.4 6.4 6.2 17.2 3.1 18 9.1 12.2 16.6 12.1 10.2 17.4 ? 12.7 20 6.5 5.2 11.9 7.5 12.6 4.1 13.1 10.3 MRed04 5 6.4 24.4 8.7 11.8 10.4 ? 24.6 2, 3 02 4.7 4.2 ? ? 8.4 7.2 ? ? 05 4.9 4.8 ? ? 7.6 6.8 ? ? 2, 4 14 7.5 18 58 177 ? ? 5.2 ? 3, 4 17 8.6 10.6 26.4 214 ? ? 3.4 ? 2, 3, 4 10 8.8 11.3 10.8 21.8 ? ? 4.3 ? 15 5.8 9.8 12.2 10.8 ? ? 3 ? MRed05 4.3 4.2 4.4 4.7 4.8 4.8 ? ? 4 04 10.8 21.9 ? ? ? ? 18.4 ? 5 06 ? ? ? ? ? ? ? ? 6 03 ? ? ? ? ? ? ? ? 11 3.2 6.6 10.7 4.7 7.7 3.3 8.8 7.7 bin NTD-specific V.sub.HH 7, 9, 10 SR01 4.2 3.1 8.8 2.1 3.1 2.3 4.4 5.1 SR13 7.7 22.4 ? 16.5 ? 12.2 ? 15 10 SR02 1.7 7.3 ? 4.7 6.1 3.1 ? ? ? dash indicates lack of surrogate neutralization
TABLE-US-00016 TABLE 16 Eva Green V.sub.HH binding data obtained by tandem SPR surrogate virus neutralization assays against surface-immobilized SARS-CoV-2 S. Summary orientation #1: V.sub.HH followed by V.sub.HH + ACE2 Summary orientation #2: ACE2 followed by ACE2 + V.sub.HH End End End End 1.sup.st 2.sup.nd inj 1.sup.st 2.sup.nd inj Solution Solution inj.sup.1 inj.sup.1 2 ? 1 Solution Solution inj.sup.1 inj.sup.1 2 ? 1 Cycle 1 2 (RU) (RU) (?RU) Blocker Cycle 1 2 (RU) (RU) (?RU) Blocker 1 Buffer ACE2 ?3.1 56 59.1 No 1 ? ? ? ? ? ? 2 NRCoV2- NRCoV2-1d + 26.3 40 13.7 Yes 2 ACE2 NRCoV2-1d + 57.1 56.4 ?0.7 Yes 1d ACE2 ACE2 3 NRCoV2- NRCoV2-02 + 33 39.3 6.3 Yes 3 ACE2 NRCoV2-02 + 55.8 59.6 3.8 Yes 02 ACE2 ACE2 4 NRCoV2- NRCoV2-03 + 18.1 82.3 64.2 No 4 ACE2 NRCoV2-03 + 52.8 75.3 22.5 No 03 ACE2 ACE2 5 NRCoV2- NRCoV2-04 + 41.4 95.8 54.4 No 5 ACE2 NRCoV2-04 + 52.4 98.8 46.4 No 04 ACE2 ACE2 6 NRCoV2- NRCoV2-05 + 36.9 46.8 9.9 Yes 6 ACE2 NRCoV2-05 + 52.2 60.5 8.3 Yes 05 ACE2 ACE2 7 NRCoV2- NRCoV2-06 + 38.5 92.2 53.7 No 7 ACE2 NRCoV2-06 + 52.3 94.4 42.1 No 06 ACE2 ACE2 8 NRCoV2- NRCoV2-07 + 19.1 37.5 18.4 Yes 8 ACE2 NRCoV2-07 + 52 59.1 7.1 Yes 07 ACE2 ACE2 10 NRCoV2- NRCoV2-10 + 9.6 58.8 49.2 No 10 ACE2 NRCoV2-10 + 52.1 64.1 12 No 10 ACE2 ACE2 11 NRCoV2- NRCoV2-11 + 21.8 80.7 58.9 No 11 ACE2 NRCoV2-11 + 52.3 83.6 31.3 No 11 ACE2 ACE2 12 NRCoV2- NRCoV2-SR13 + 36.9 86.8 49.9 No 12 ACE2 NRCoV2-SR13 + 52.3 92.8 40.5 No SR13 ACE2 ACE2 13 NRCoV2- NRCoV2-14 + 26.9 70.4 43.5 +/? 13 ACE2 NRCoV2-14 + 52.3 75.9 23.6 No 14 ACE2 ACE2 14 NRCoV2- NRCoV2-15 + 10.1 50.7 40.6 +/? 14 ACE2 NRCoV2-15 + 50 59.7 9.7 No 15 ACE2 ACE2 15 NRCoV2- NRCoV2-SR16 + 37.4 88.8 51.4 No 15 ACE2 NRCoV2-SR16 + 52.1 95.7 43.6 No SR16 ACE2 ACE2 16 NRCoV2- NRCoV2-17 + 16.7 69.5 52.8 No 16 ACE2 NRCoV2-17 + 52 71.7 19.7 No 17 ACE2 ACE2 17 NRCoV2- NRCoV2-18 + 12.6 52.5 39.9 +/? 17 ACE2 NRCoV2-18 + 50.9 61.7 10.8 +/? 18 ACE2 ACE2 18 NRCoV2- NRCoV2-20 + 27.1 60.5 33.4 +/? 18 ACE2 NRCoV2-20 + 51.3 66.2 14.9 +/? 20 ACE2 ACE2 19 NRCoV2- NRCoV2-SR01 + 39.4 92 52.6 No 19 ACE2 NRCoV2-SR01 + 51.4 97.2 45.8 No SR01 ACE2 ACE2 20 NRCoV2- NRCoV2-SR02 + 10.3 60.2 49.9 No 20 ACE2 NRCoV2-SR02 + 49.1 67.8 18.7 No SR02 ACE2 ACE2 21 NRCoV2- NRCoV2-SR03 + 18.5 70.7 52.2 No 21 ACE2 NRCoV2-SR03 + 50 76.4 26.4 No SR03 ACE2 ACE2 22 NRCoV2- NRCoV2-SR04 + 10.9 63.6 52.7 No 22 ACE2 NRCoV2-SR04 + 50.5 68.5 18 No SR04 ACE2 ACE2 23 NRCoV2- NRCoV2-S2A3 + 15 82 67 No 23 ACE2 NRCoV2-S2A3 + 50.8 72.2 21.4 No S2A3 ACE2 ACE2 24 NRCoV2- NRCoV2-S2A4 + 11.6 73.3 61.7 No 24 ACE2 NRCoV2-S2A4 + 50.9 71.1 20.2 No S2A4 ACE2 ACE2 25 NRCoV2- NRCoV2-S2G3 + 59 113.2 54.2 No 25 ACE2 NRCoV2-S2G3 + 51 114.1 63.1 No S2G3 ACE2 ACE2 26 NRCoV2- NRCoV2-S2G4 + 21.3 81.7 60.4 No 26 ACE2 NRCoV2-S2G4 + 50.9 79.3 28.4 No S2G4 ACE2 ACE2 27 NRCoV2- NRCoV2-S2F3 + 50 111.1 61.1 No 27 ACE2 NRCoV2-S2F3 + 50.7 106.9 56.2 No S2F3 ACE2 ACE2 28 V.sub.HH-72 V.sub.HH-72 + 25.8 37.8 12 Yes 28 ACE2 V.sub.HH-72 + 51 51.2 0.2 Yes ACE2 ACE2 V.sub.HHs were used at 20-40x K.sub.D concentrations, ACE2 at 1 ?M. .sup.1inj, injection.
TABLE-US-00017 TABLE 17 Neutralization capabilities of SARS-CoV-2-specific V.sub.HHs/V.sub.HH-Fcs obtained by surrogate virus neutralization flow cytometry assays against SARS-CoV-2 S (Wuhan) Domain/ V.sub.HH/ACE2-H.sub.6.sup.2 V.sub.HH-Fc/ACE2-Fc.sup.2 V.sub.HH/ subdomain IC.sub.50.sup.3 IC.sub.99.sup.3 IC.sub.50.sup.3 IC.sub.99.sup.3 ACE2 specificity (nM) (nM) I.sub.max %.sup.3 (nM) (nM) I.sub.max %.sup.3 ACE2 S1-RBD 24.5 1506 94% 5.1 328.1 97% V.sub.HH-72.sup.1 S1-RBD 59 9409 43% 7.2 238.5 57% NRCoV2-1a S1-RBD nd nd nd 9.2 89.4 94% NRCoV2-1d S1-RBD 8.6 40.8 72% 5.4 42 89% NRCoV2-02 S1-RBD 5.1 15.5 100% 5 30 94% NRCoV2-03 S1-RBD ? ? ? ? ? ? NRCoV2-04 S1-RBD ? ? ? 11.7 420.4 51% NRCoV2-05 S1-RBD 9.5 14.2 97% 6.7 37 95% NRCoV2-06 S1-RBD ? ? ? ? ? ? NRCoV2-07 S1-RBD 7.5 65.3 86% 6.8 98.9 89% NRCoV2-08 S1-RBD nd nd nd ? ? ? NRCoV2-10 S1-RBD 16.1 121 50% 7.7 77.3 91% NRCoV2-11 S1-RBD ? ? ? 9.7 99.8 63% NRCoV2-11a S1-RBD nd nd nd 9.7 67.6 46% NRCoV2-12 S1-RBD nd nd nd 7.3 839.9 90% NRCoV2-14 S1-RBD 21.3 17352 54% 9.9 160.5 83% NRCoV2-15 S1-RBD 12.1 233 44% 8.1 71.5 95% NRCoV2-17 S1-RBD ? ? ? 8.6 100.6 88% NRCoV2-18 S1-RBD 8.9 133 51% 12 680.5 90% NRCoV2-19 S1-RBD nd nd nd ? ? ? NRCoV2-20 S1-RBD 5.1 1371 43% 8.7 70.3 66% NRCoV2-21 S1-RBD nd nd nd ? ? ? NRCoV2-MRed04 S1-RBD 6.1 124 53% 4.6 262 73% NRCoV2-MRed05 S1-RBD 15 184 88% 6.1 36.3 95% NRCoV2-SR01 S1-NTD ? ? ? 6.6 56.9 59% NRCoV2-SR02 S1-NTD ? ? ? 5.8 49.8 53% NRCoV2-SR03 S1-NTD ? ? ? ? ? ? NRCoV2-SR04 S1-NTD ? ? ? ? ? ? NRCoV2-SR13 S1-NTD ? ? ? 23.8 1974.5 82% NRCoV2-SR16 S1-NTD ? ? ? ? ? ? NRCoV2-MRed03 S1-NTD ? ? ? ? ? ? NRCoV2-MRed06 S1-NTD ? ? ? ? ? ? NRCoV2-MRed07 S1-NTD ? ? ? ? ? ? NRCoV2-S2A3 S2 ? ? ? ? ? ? NRCoV2-S2A4 S2 ? ? ? ? ? ? NRCoV2-S2F3 S2 ? ? ? ? ? ? NRCoV2-S2G3 S2 ? ? ? ? ? ? NRCoV2-S2G4 S2 ? ? ? ? ? ? NRCoV2-S2H4 S2 nd nd nd ? ? ? NRCoV2-S202 S2 nd nd nd ? ? ? NRCoV2-MRed11 S2 ? ? ? ? ? ? NRCoV2-MRed18 S2 ? ? ? ? ? ? NRCoV2-MRed19 S2 ? ? ? ? ? ? NRCoV2-MRed20 S2 ? ? ? ? ? ? NRCoV2-MRed22 S2 ? ? ? ? ? ? NRCoV2-MRed25 S2 ? ? ? ? ? ? .sup.1V.sub.HH-72 benchmark is SARS-CoV S-specific V.sub.HH that cross-reacts with SARS-CoV-2 S (Wrapp et al., 2020). .sup.2ACE2-H.sub.6 is His.sub.6-tagged monomeric ACE2, ACE2-Fc, human Ig Fc-fused dimeric ACE2. .sup.3IC.sub.50, concentration of V.sub.HH/V.sub.HH/Fc giving 50% neutralization; IC.sub.99, concentration of V.sub.HH/V.sub.HH/Fc giving 99% neutralization; I.sub.max %, maximal inhibitory effect; IC.sub.50, IC.sub.99 and I.sub.max % values were extracted from graphs exemplified in FIGS. 12B and FIGS. 13B. Dash indicate V.sub.HH/V.sub.HH-Fc does not neutralize the interaction between Vero E6 cell-displayed ACE2 and soluble S. .sup.4ICs cannot be determined with certainty due to low I.sub.max % values. nd, not determined, due to lack of sufficient quantities and/or neutralization as V.sub.HH-Fc.
Example 9: Live-Virus Neutralization Assays
Introduction
[0406] V.sub.HH-Fcs were subjected to authentic-virus neutralizations assays, i.e., microneutralization assays, to identify those that neutralized infection of host cells by the invading SARS-CoV-2 virus.
Materials and Methods
Authentic-Virus Neutralizations Assays
[0407] Neutralization activity of antibodies to SARS-CoV-2 was determined with the microneutralization assay. In brief, antibody (V.sub.HH-Fc and V.sub.HH) stocks were prepared at 1 mg/mL in PBS and sterilized by passing through 0.22 ?M filters. 1:5 serial dilutions of 50 ?g/mL of each antibody was carried out in DMEM, high glucose media supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 U/ml penicillin-streptomycin, and 1% heat-inactivated fetal bovine serum. SARS-CoV-2 (strain SARS-CoV-2/Canada/VIDO-01/2020) was incubated at 250 pfu with antibody dilution in 1:1 ratio at 37? C. for 1 h. Vero E6 cells seeded in 96-well plates were infected with virus/antibody mix and incubated at 37? C. in humidified/5% CO.sub.2 incubator for 72 hours post-infection (hpi). Cells were then fixed in 10% formaldehyde overnight and virus infection was detected with mouse anti-SARS-CoV-2 nucleocapsid antibody (R&D Systems, clone #1035111) and counterstained with rabbit anti-mouse IgG-HRP (Rockland Inc.). Colorimetric development was obtained with o-phenylenediamine dihydrochloride peroxidate substrate (Sigma-Aldrich) and detected on Biotek Synergy H1 plate reader at 490 nm. IC.sub.50 was determined from non-linear regression on GraphPad Prism 9. For determining neutralization potencies by measuring cytopathic effect (CPE), infected Vero E6 cells were incubated at 37? C. for 96 h until the virus-only control wells had nearly 100% CPE (cell-only controls were also included). Neutralization was scored by MN.sub.100, lowest antibody concentration that gave no CPE, i.e., 100% neutralization. Assays were performed in technical duplicates.
Results and Discussion
[0408] A select set of lead V.sub.HH-Fcs were subjected to preliminary authentic-virus microneutralization assays to assess their SARS-CoV-2 virus-neutralizing activity. These included five S1-RBD-specific V.sub.HHs and two S1-NTD-specfic V.sub.HHs. Neutralization was scored by MN.sub.100, the lowest antibody concentration that gave no cytopathic effect (100% neutralization). Results are shown in
[0409] A more comprehensive authentic neutralization assay was performed to determine the IC.sub.50 of V.sub.HH-Fcs (
[0410] The live virus neutralization assays were then extended to include Alpha and Beta variants. With the exception of V.sub.HH-Fc NRCoV2-06, all remaining 16 RBD-specific Wuhan neutralizers maintained their ability to neutralize Alpha (Table 19,
TABLE-US-00018 TABLE 18 Neutralization capabilities (MN.sub.100) of SARS-CoV-2-specific V.sub.HH-Fcs obtained by authentic-virus (aka live virus) neutralization assays Domain/subunit V.sub.HH specificity MN.sub.100 (nM).sup.2 NRCoV2-1d S1-RBD 0.25 NRCoV2-02 S1-RBD ?0.01.sup.3 NRCoV2-03 S1-RBD 6.25 NRCoV2-04 S1-RBD 1.25 NRCoV2-07 S1-RBD 1.25 NRCoV2-SR01 S1-NTD 6.25 NRCoV2-SR02 S1-NTD 6.25 NRCoV2-15 ? S1-RBD ? null 1.25 A26.8 V.sub.HH72 ? S1-RBD ? null A26.8 NRCoV2-S2A4 ? S2 ? null A26.8 NRCoV2-07 ? S1-RBD ? null 31.25 A26.8 NRCoV2-1d ? S1-RBD ? null 6.25 A26.8 NRCoV2-SR01 ? S1-NTD ? null 31.25 A26.8 NRCoV2-02 ? S1-RBD ? null 1.25 A26.8 V.sub.HH-72.sup.1 S1-RBD 1.25.sup.3 A20.1.sup.1 null .sup.1V.sub.HH-72 benchmark is a SARS-CoV S-specific V.sub.HH that cross-reacts with SARS-CoV-2 S (Wrapp et al., 2020); A20.1 and A26.8 are C. difficile toxin A-specific negative control V.sub.HH (Hussack et al., 2011). .sup.2MN.sub.100 is the lowest antibody concentration that gave no cytopathic effect (100% neutralization). Dash indicate V.sub.HH-Fc does not neutralize SARS-CoV-2 virus at the highest V.sub.HH-Fc concentration used. MN.sub.100 values were used to construct FIG. 14A-B graphs. .sup.3The MN.sub.100 of monovalent V.sub.HH-72 and NRCoV2-02 V.sub.HHs were 156.25 and 1.25 nM, respectively.
TABLE-US-00019 TABLE 19 Neutralization capabilities (IC.sub.50) of SARS-CoV-2-specific V.sub.HH-Fcs obtained by authentic-virus (aka live virus) neutralization assays Epitope LVNA IC.sub.50 (nM) V.sub.HH-Fc bin Wuhan Alpha Beta RBD-specific V.sub.HH 1d 1 1.94 0.37 2.14 07 6.15 0.42 3.18 12 2.82 1.35 2.62 18 6.4 2.82 9.48 20 11.2 1.94 2.88 MRed04 9.61 4.5 5.73 02 2, 3 0.12 0.09 05 0.0008 0.03 14 2, 4 3.1 0.88 32.8 17 3, 4 2.82 0.61 34.7 10 2, 3, 4 1.28 0.47 2.25 15 0.73 0.16 0.43 MRed05 0.17 0.13 0.11 04 4 1.65 2.3 06 5 76 03 6 58 16 62 11 9.9 2.3 18.5 NTD-specific V.sub.HH SR01 7, 9, 10 9.42 3.77 70.3 SR03 ~500 22.2 SR13 ~100 ~100 SR16 54.2 17.8 100 SR04 7/9 ~500 MRed03 8 MRed06 8 MRed07 9 SR02 10 14.13 9.05 ~300 S2-specific V.sub.HH S2A3 11 12.2 31 54 S2A4 12 S2F3 MRed18 ~400 MRed19 13 MRed20 MRed22 S2G3 14 ~200 S2G4 15 ~200 MRed11 16 MRed25 17 Reference VHH-72.sup.1 1 8.46 1.86 9.34 A20.1.sup.1 null .sup.1V.sub.HH-72 benchmark is a SARS-CoV S-specific V.sub.HH that cross-reacts with SARS-CoV-2 S (Wrapp et al., 2020); A20.1 is C. difficile toxin A-specific negative control V.sub.HH (Hussack et al., 2011. Epitope bin numbers correspond to the bins shown in FIG. 9G.
Example 10: Stability of V.SUB.H.Hs Against Aerosolization
Introduction
[0411] One effective therapeutic approach against COVID-19 might be the direct delivery of aerosolized antibodies to the nasal and lung epithelia by inhalation. V.sub.HHs in particular, are advantageously fit for such administration approach due to their high stability and robustness. Since aerosolization could compromise the structural integrity and function of antibodies that lack sufficient stability, such as mAbs (Detalle et al., 2016; Respaud et al., 2015), the effect of aerosolization on the stability of V.sub.HHs was tested.
Materials and Methods
Aerosolization Studies
[0412] Prior to aerosolization, 4 mg of each V.sub.HH was purified by size-exclusion chromatography using a Superdex? 75 GL column (Cytiva) and PBS as running buffer, as described above. Protein fractions corresponding to the chromatogram's monomeric peak were pooled, quantified and the concentration adjusted to 0.5 mg/mL. One mL of each V.sub.HH was subsequently aerosolized at room temperature with a portable mesh nebulizer (AeroNeb? Solo, Aerogen, Galway, Ireland), which produces 3.4-?m particles. Aerosolized V.sub.HHs were collected into 15 mL Round-Bottom Polypropylene test tubes (Falcon, Cat #C352059) for 5 min to allow condensation and were subsequently quantified and kept at 4? C. until use. Then 200 ?L aliquots of pre- and post-aerosolized V.sub.HHs were subjected to SEC to obtain chromatogram profiles. Additionally, condensed V.sub.HHs were closely monitored for the formation of any visible aggregates, and in cases where aggregate formation was observed, aggregates were removed by centrifugation prior to concentration determination, SEC analysis and ELISA. % soluble aggregate was determined as the proportion of a V.sub.HH that gave elution volumes (V.sub.es) smaller than that of the monomeric V.sub.HH fraction. % recovery was determined as the proportion of a V.sub.HH that remained monomerically soluble following aerosolization.
[0413] To assess the effect of aerosolization on the functionality of V.sub.HHs, the activities of post-aerosolized V.sub.HHs were determined by ELISA and compared to those for pre-aerosolized V.sub.HHs. To perform ELISA, S1-Fc (ACRO Biosystems, Cat #S1N-05255) was diluted in PBS to 500 ng/mL, and 100 ?L/well were coated overnight at 4? C. The next day, plates were washed with PBST and blocked with 200 ?L PBSC for 1 h at room temperature. After five washes with PBST, serial dilutions of the pre- and post-aerosolized V.sub.HHs were added to wells and incubated for 1 h at room temperature. Then plates were washed 10 times with PBST and binding of V.sub.HHs to S1-Fc was detected with rabbit anti-6?His Tag antibody HRP Conjugate (Bethyl, Cat #A190-114P), diluted at 10 ng/mL in PBST and added at 100 ?L/well. Finally, after 1 h incubation at room temperature, peroxidase activity was detected as described previously.
Results and Discussion
[0414] V.sub.HHs including the benchmark V.sub.HH-72 were examined for their aggregation resistance/stability against aerosolization. For a few V.sub.HHs, e.g., NRCoV2-MRed20, NRCoV2-S2A4, as well as the V.sub.HH-72 benchmark, aerosolization induced some soluble aggregation formation as determined by SEC (
TABLE-US-00020 TABLE 20 Stability of V.sub.HHs against aerosolization Soluble aggregates (%).sup.2 Recovery Pre- Post- ?Soluble Visible V.sub.HH (%).sup.1 aerosolization aerosolization agg..sup.3 aggregates NRCoV2-1c 91 3 5 2 No NRCoV2-1d 83 2 4 2 No NRCoV2-02 89 2 2 0 No NRCoV2-03 81 2 1 ?1 No NRCoV2-04 62 6 5 ?1 Yes NRCoV2-05 89 2 5 3 No NRCoV2-06 51 7 5 ?2 Yes NRCoV2-07 75 2 3 1 No NRCoV2-10 83 5 5 0 No NRCoV2-11 24 6 5 ?1 Yes NRCoV2-14 55 6 6 0 Yes NRCoV2-15 69 4 5 1 Yes NRCoV2-17 85 5 6 1 No NRCoV2-18 99 9 5 ?4 No NRCoV2-20 97 2 1 ?1 No NRCoV2-SR03 43 3 11 8 Yes NRCoV2-SR04 52 5 3 ?2 Yes NRCoV2-SR13 83 4 6 2 No NRCoV2-S2A4 84 7 11 4 No NRCoV2-S2G4 91 3 6 3 No NRCoV2-MRed02 73 1 1 0 No NRCoV2-MRed03 96 3 2 ?1 No NRCoV2-MRed04 59 4 4 0 Yes NRCoV2-MRed07 90 10 3 ?7 No NRCoV2-MRed11 89 4 5 1 No NRCoV2-MRed18 96 3 10 7 No NRCoV2-MRed19 87 5 9 4 No NRCoV2-MRed20 76 2 18 16 No NRCoV2-MRed22 86 3 9 6 No NRCoV2-MRed25 44 3 3 0 Yes V.sub.HH-72 78 1 14 13 No .sup.1% recovery were determined as described in Examples 10. .sup.2% soluble aggregate was determined as the proportion of a V.sub.HH that gave elution volumes (V.sub.es) smaller than that of the monomeric V.sub.HH fraction. .sup.3?Soluble agg. = Post-aerosolization ? Pre-aerosolization.
TABLE-US-00021 TABLE 21 Affinities (EC.sub.50s) of pre-aerosolized (Pre) vs post-aerosolized (Post) V.sub.HHs EC.sub.50 (nM) V.sub.HH Pre Post NRCoV2-1d 1.1 1.3 NRCoV2-02 0.2 0.2 NRCoV2-07 1.1 1 NRCoV2-11 0.2 0.2
Example 11: V.SUB.H.Hs for Diagnosis and Capture of SARS-CoV-2
[0415] introduction
[0416] V.sub.HHs described herein are promising diagnostic/capture agents against SARS-CoV-2, SARS-CoV and related viruses as well as their spike proteins. To explore the use of these V.sub.HHs as capture agents, four of the V.sub.HHs were tested in sandwich ELISA for their diagnostic/capturing capability against SARS-CoV-2.
Materials and Methods
Sandwich ELISA
[0417] NUNC? MaxiSorp? 4 HBX plates (Thermo Fisher) were coated overnight at 4? C. with 4 ?g/mL streptavidin (Jackson ImmunoResearch, Cat #016-000-113) in 100 ?L PBS, pH 7.4. Wells were blocked with 200 ?L PBSC for 1 h at room temperature followed by capturing biotinylated NRCoV2-02 V.sub.HH (10 ?g/mL in 100 ?L PBSCT) for 1 h at room temperature. Wells were washed five times with PBST and incubated with variable concentrations of SARS-CoV-2 S, S1 or S1-RBD diluted in PBSCT for 1 h. Well were washed and incubated with detecting V.sub.HH-Fcs at 1 ?g/mL. The binding of V.sub.HH-Fcs to spike protein fragments was probed using 100 ?L 1 ?g/mL HRP-conjugated goat anti-human IgG (SIGMA, Cat #A0170). Finally, plates were washed 10 times with PBST and peroxidase activity was determined as described above.
Results and Discussion
[0418] To provide proof of concept for the utility of the V.sub.HHs as detecting/capturing agents against SARS-CoV-2, SARS-CoV and related viruses, sandwich ELISAs were performed with four V.sub.HHs using SARS-CoV-2 spike protein fragments as surrogates for the virus. Wells were coated with NRCoV2-02 V.sub.HH as the capturing antibody, followed by the capture of antigens S, S1, or S1-RBD added at variable concentrations. Then a second, V.sub.HH-Fc that binds to a non-overlapping epitope in relation to NRCoV2-02 was added as the detecting antibody followed by the addition of a HRP-conjugated probing antibody binding to the detecting antibody. The different V.sub.HH-Fcs tested as detecting antibodies were: NRCoV2-1d, NRCoV2-04, NRCoV2-07, and NRCoV2-11. Very low SC.sub.50 values were obtained in ELISA assays (
TABLE-US-00022 TABLE 22 SC.sub.50 values obtained in ELISA assays SC.sub.50 (ng/mL) S S1 S1-RBD NRCoV2-1d 25 16 1.7 NRCoV2-04 39 25 3.3 NRCoV2-07 18 5 0.7 NRCoV2-11 20 6 0.8
TABLE-US-00023 TABLE 23 Limit of detection (ng/mL) NRCoV2-1d NRCoV2-04 NRCoV2-07 NRCoV2-11 S 1.4 4.1 1.4 1.4 S1 1.37 4.12 0.46 0.46 S1-RBD 0.15 0.46 0.08 0.08
Example 12: In Vivo Therapeutic Efficacy of V.SUB.H.H-Fcs
[0419] Before testing V.sub.HH-Fcs in hamsters for in vivo efficacy, they were assessed for in vivo stability and persistence. NRCov2-1d V.sub.HH-Fc was chosen as a representative V.sub.HH and V.sub.HH-72 V.sub.HH-Fc, whose modified/enhanced version is currently in a phase 1 clinical trial, was included as a reference. Hamsters were injected intraperitoneally (IP) with 1 mg of each antibody and serum antibody concentration was monitored for up to four days by ELISA. Significant and comparable V.sub.HH-Fc concentrations were present in the hamster sera for both 1 d and V.sub.HH-72 V.sub.HH-Fcs on days 1 and 4 post injection (
[0420] The in vivo therapeutic efficacy of V.sub.HH-Fcs which were neutralizing by live virus neutralization assay was then assessed in a hamster model of SARS-CoV-2 infection. Five V.sub.HH-Fcs were selected to cover a wide range of important attributes including in vitro neutralization potencies and breadth, epitope bin, subunit/domain specificity and cross-reactivity pattern. These included three RBD-specific (1d, 05, MRed05), one NTD-specific (SR01) and one S2-specific (S2A3) V.sub.HH-Fcs. Cocktails of two V.sub.HH-Fcs were also included to explore synergy between the antibody pairs recognizing distinct epitopes within the RBD (1 d/1d/MRed05) or RBD and NTD (1d/SR01).
[0421] Hamsters were administered IP with 1 mg of V.sub.HH-Fcs 24 h prior to intranasal challenge with SARS-CoV-2 Wuhan isolate. Daily weight change and clinical symptoms were monitored. At 5 dpi, lungs were collected to determine viral titers. Viral titer decrease and reversal of weight loss in antibody treated versus control animals were taken as measures of antibody efficacy. Animals treated with RBD binders 1 d, 05, and MRed05 showed reduced lung viral burden by three, five and six orders of magnitude, respectively, relative to PBS or V.sub.HH-Fc isotype controls, with 05 and MRed05 reducing viral burden to below detectable levels (
[0422] Subsequent immunohistochemistry studies corroborated the viral titer and weight change results. First, in agreement with the viral titer observations, substantial viral antigen (nucleocapsid) reductions in hamster lungs were observed with antibody treatments (
[0423] The preceding examples have been provided to illustrate various aspects of the disclosure and are non-limiting. The scope of the claims is not limited to specific details provided in the examples; rather the claims are to be given the broadest interpretation consistent with the teachings of the disclosure as a whole.
TABLE-US-00024 TABLE24 Listofsequencesdescribedinthespecification SEQ Antibody(ies) ID Seq. including NO: Sequence Type sequence 1 GSTLDYYA CDR1 NRCoV2-1a NRCoV2-1d 2 GSILDYYA CDR1 NRCoV2-1c 3 GFTFSNYA CDR1 NRCoV2-02 4 GITFSYYA CDR1 NRCoV2-03 5 GSPFSNVV CDR1 NRCoV2-04 6 GFIFSNYA CDR1 NRCoV2-05 7 VSTFSSYA CDR1 NRCoV2-06 8 GVTLDYYA CDR1 NRCoV2-07 9 GFTLDDYA CDR1 NRCoV2-08 10 GNTFSRSN CDR1 NRCoV2-10 11 GSSLDSYS CDR1 NRCoV2-11 12 GFTLDSYN CDR1 NRCoV2-11a 13 GRTFRNYV CDR1 NRCoV2-12 14 GTTFSHYA CDR1 NRCoV2-14 15 GSTSGRNT CDR1 NRCoV2-15 16 GSPFSQLA CDR1 NRCoV2-17 17 GITISGYN CDR1 NRCoV2-18 18 GLTLNSYA CDR1 NRCoV2-19 19 GRTFSNYV CDR1 NRCoV2-20 20 GFTLDYYA CDR1 NRCoV2-21 NRCoV2-MRed03 NRCoV2-MRed05 21 GFTFDNYA CDR1 NRCoV2-SR01 22 EFTLNYYS CDR1 NRCoV2-SR02 23 GSIFSNNH CDR1 NRCoV2-SR03 24 GRTFSSHT CDR1 NRCoV2-SR04 25 GSRFGSKH CDR1 NRCoV2-SR13 26 GTTFSRYH CDR1 NRCoV2-SR16 27 GRPYSNYA CDR1 NRCoV2-S2A3 28 GSPFRSNV CDR1 NRCoV2-S2A4 29 ASTFGDSA CDR1 NRCoV2-S2B3 30 GFTFNLYS CDR1 NRCoV2-S2H4 31 VRILSVPA CDR1 NRCoV2-S2F3 32 GSTFGIFL CDR1 NRCoV2-S2G3 33 GSTFSGYA CDR1 NRCoV2-S2G4 34 GITVSRIG CDR1 NRCoV2-S202 35 GNIFSINS CDR1 NRCoV2-MRed02 36 GNSFSINT CDR1 NRCoV2-MRed04 37 GFTLAYYA CDR1 NRCoV2-MRed06 38 GSIGPFNT CDR1 NRCoV2-MRed07 39 GFTFSSYA CDR1 NRCoV2-MRed11 40 TTVFGRNA CDR1 NRCoV2-MRed18 41 TIIFKGQT CDR1 NRCoV2-MRed19 42 GLSFSSYD CDR1 NRCoV2-MRed20 43 GSVFASNA CDR1 NRCoV2-MRed22 44 GHTFSRYG CDR1 NRCoV2-MRed25 45 VSSSDGST CDR2 NRCoV2-1a NRCoV2-1c 46 VSSSDGNT CDR2 NRCoV2-1d 47 ISGRGDDT CDR2 NRCoV2-02 48 MSNMDST CDR2 NRCoV2-03 49 ISGGGIA CDR2 NRCoV2-04 50 INSGGGDT CDR2 NRCoV2-05 51 IGFVGAT CDR2 NRCoV2-06 52 ISSNGRRN CDR2 NRCoV2-07 53 ISRSGTTT CDR2 NRCoV2-08 54 ISSRGIS CDR2 NRCoV2-10 55 ISRYYSST CDR2 NRCoV2-11 56 ISRYYEST CDR2 NRCoV2-11a 57 VAAISWGGTEI CDR2 NRCoV2-12 58 ISVFGST CDR2 NRCoV2-14 59 VSTSGAT CDR2 NRCoV2-15 60 ISPTGNR CDR2 NRCoV2-17 61 INSGGST CDR2 NRCoV2-18 62 LTSGGTG CDR2 NRCoV2-19 63 VAVISGSDTET CDR2 NRCoV2-20 64 ISSGGST CDR2 NRCoV2-21 65 ISGNGGVT CDR2 NRCoV2-SR01 66 IRYSGGGI CDR2 NRCoV2-SR02 67 ISSGGKT CDR2 NRCoV2-SR03 68 ISMGGNTNYA CDR2 NRCoV2-SR04 69 ISSGGST CDR2 NRCoV2-SR13 70 ISTSGAV CDR2 NRCoV2-SR16 71 KQRELVAAISSGGTT CDR2 NRCoV2-S2A3 72 ISTGGSR CDR2 NRCoV2-S2A4 73 ISTGSNT CDR2 NRCoV2-S2B3 74 INSGDRDSTT CDR2 NRCoV2-S2H4 75 ITSGGST CDR2 NRCoV2-S2F3 76 ITSGGAT CDR2 NRCoV2-S2G3 77 ISSDGDK CDR2 NRCoV2-S2G4 78 ISAGGST CDR2 NRCoV2-S202 79 IWSDSRT CDR2 NRCoV2-MRed02 80 IWSDTTT CDR2 NRCoV2-MRed04 81 ISSSDGST CDR2 NRCoV2-MRed03 NRCoV2-MRed05 82 ISSSDGSA CDR2 NRCoV2-MRed06 83 ITRGGVT CDR2 NRCoV2-MRed07 84 INSGGGST CDR2 NRCoV2-MRed11 85 VSDGGTP CDR2 NRCoV2-MRed18 86 MTTSGSA CDR2 NRCoV2-MRed19 87 IRESGSGT CDR2 NRCoV2-MRed20 88 ISSRGST CDR2 NRCoV2-MRed22 89 ISWRGDST CDR2 NRCoV2-MRed25 90 AADYSMRPLWVSRWHRDYEY CDR3 NRCoV2-1a 91 AADYSMRRFAVGRWHRDYEY CDR3 NRCoV2-1c 92 AADYSMRPFAVGRWHRDYEY CDR3 NRCoV2-1d 93 TKGPDLYYFGSGYSD CDR3 NRCoV2-02 94 NIYGPTYSTRRNEY CDR3 NRCoV2-03 95 WSSYEST CDR3 NRCoV2-04 96 SKGPVSSYYGSGYDY CDR3 NRCoV2-05 97 NARHYGGSEY CDR3 NRCoV2-06 98 AAVQDVHGDNYYCTSPNEYNV CDR3 NRCoV2-07 99 AADYQYSTYCLGYDAHYEY CDR3 NRCoV2-08 100 YAADDLGDY CDR3 NRCoV2-10 101 AARSRDFSSPFSATDTYTS CDR3 NRCoV2-11 102 AARSRDFSSPISATDKYGS CDR3 NRCoV2-11a 103 AADRGLSYYYTRTTEYNY CDR3 NRCoV2-12 104 HAVNADIGGDY CDR3 NRCoV2-14 105 YAAYGGGGDY CDR3 NRCoV2-15 106 QAANVNGGDY CDR3 NRCoV2-17 107 SLHTSHDY CDR3 NRCoV2-18 108 AADRARLRFGCSLNFRREVAYDY CDR3 NRCoV2-19 109 AADRGMSYYYTRATEYYY CDR3 NRCoV2-20 110 AADHRGRSLRFGCSSSTTDYLY CDR3 NRCoV2-21 111 AATGIRSTWSVYGCSRLAGPYDY CDR3 NRCoV2-SR01 112 AADRLYSRACPTAGGRNY CDR3 NRCoV2-SR02 113 NRGGWEYRSSYYIMGPH CDR3 NRCoV2-SR03 114 NTAALVGNRLLPMATIT CDR3 NRCoV2-SR04 115 NMGGWDYRSNTYIPGSRSDY CDR3 NRCoV2-SR13 116 NTGGWDYRSSTFIMGLN CDR3 NRCoV2-SR16 117 NTGSLSYGGSVYYPSYDN CDR3 NRCoV2-S2A3 118 HAAARDSHGIYLLDT CDR3 NRCoV2-S2A4 119 NYRSIYYGQNF CDR3 NRCoV2-S2B3 120 ALVFGYTSRDYCLTPKRGNY CDR3 NRCoV2-S2H4 121 NLRDILSQPF CDR3 NRCoV2-S2F3 122 YTTKRDDASVY CDR3 NRCoV2-S2G3 123 NKHWWTGDW CDR3 NRCoV2-S2G4 124 NYGPGYRKAA CDR3 NRCoV2-S202 125 AADRGFVVRGQYDY CDR3 NRCoV2-MRed02 NRCoV2-MRed04 126 ATDAFATCDSWYAQIAQYDF CDR3 NRCoV2-MRed03 127 ATGPQAYYSGSYYFQCPQAGMDY CDR3 NRCoV2-MRed05 128 ATDSFSSCSDYESGMDF CDR3 NRCoV2-MRed06 129 YANYGWAIPY CDR3 NRCoV2-MRed07 130 ATTISDGSSWSTKSY CDR3 NRCoV2-MRed11 131 NYYNYYYGRNF CDR3 NRCoV2-MRed18 132 YMHSVYYGIDY CDR3 NRCoV2-MRed19 133 AAKPPFYGSGTYSTPRAYLY CDR3 NRCoV2-MRed20 134 NAREFTGFDY CDR3 NRCoV2-MRed22 135 AAEMWGTATIVASRYTY CDR3 NRCoV2-MRed25 136 EVKLVQSGGGSVQPGGSLRLSCAASGSTLDYYAIGWF V.sub.HH NRCoV2-1a RQAPGKEREWVSCVSSSDGSTLYADSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYVCAADYSMRPLWVSRWH RDYEYWGQGTQVTVSS 137 EVQLVESGGGSVQPGGSLRLSCAASGSILDYYAVGWF V.sub.HH NRCoV2-1c RQAPGKEREWVSSVSSSDGSTLYADSVKGRFTISRDDA KNTIYLQMDNLEPEDTAVYVCAADYSMRRFAVGRWHR DYEYWGQGTQVTVSS 138 EVQLVESGGGSVQPGGSLRLSCAASGSTLDYYAIGWF V.sub.HH NRCoV2-1d RQAPGKEREWVSSVSSSDGNTLYADSVKGRFTISRDNA KNTVYLQMNSLKAEDTAVYVCAADYSMRPFAVGRWH RDYEYWGQGTQVTVSS 139 EVQLVESGGGLVQAGGSLRLSCAASGFTFSNYAMNWV V.sub.HH NRCoV2-02 RQAPGKGLEWVSGISGRGDDTRYADSVKGRFTISRDN AKNTLFLQMRSLRPEDTGVYRCTKGPDLYYFGSGYSD RGQGTQVTVSS 140 EVQLVSSGGGLVQAGGSLRLSCTASGITFSYYAMGWY V.sub.HH NRCoV2-03 RQAPGQPRELVASMSNMDSTIYADSVKGRFTISRDNAK TTIYLQMNNLKPEDTAVYFCNIYGPTYSTRRNEYWGQG TQVTVSS 141 AVQLVDSGGGLVQPGGSLRLSCAASGSPFSNVVMAWY V.sub.HH NRCoV2-04 RQAPGKQRERVAFISGGGIADYIMSVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCWSSYESTWGQGTQVTVSS 142 EVKLVQSGGGLVQPGGSLRLSCAASGFIFSNYAMNWV V.sub.HH NRCoV2-05 RQAPGKGLEWVSGINSGGGDTRYADSVKGRFTVSRDN AKNTLYLQMNSLKPEDTGVYYCSKGPVSSYYGSGYDYR GQGTQVTVSS 143 EVQLVQSGGGLVQAGESLRLSCAASVSTFSSYAMGWY V.sub.HH NRCoV2-06 RQAPGKQRELVASIGFVGATYYIDSVKGRFTISRDNAKK TAYLQMNDLKPDDTAVYYCNARHYGGSEYWGQGTQV TVSS 144 QVQLVQSGGGLVQPGGSLRLSCAASGVTLDYYAIGWF V.sub.HH NRCoV2-07 RQAPGKEREAVSCISSNGRRNHYVASVRGRFTISRDNA KSTVYLQMNSLKPEDTAVYYCAAVQDVHGDNYYCTSP NEYNVWGQGTQVTVSS 145 EVQLQQSGGGLVQPGGSLRLSCAASGFTLDDYAIGWF V.sub.HH NRCoV2-08 RQSPGKEREWVTCISRSGTTTYYTASVKGRFTFSRDNA KNTAYLQMNSLRPEDTAVYYCAADYQYSTYCLGYDAH YEYWGQGTQVTVSS 146 QLQLQESGGGLVQPGGSLTLSCAASGNTFSRSNMHWY V.sub.HH NRCoV2-10 RQAPGAQREWVAAISSRGISTYAYSAKGRFTISRDNAKN TVSLQMNSLKPEDTAVYYCYAADDLGDYWGQGTQVTV SS 147 EVQLVSSGGGLVQPGGSLRLSCAASGSSLDSYSVSWFR V.sub.HH NRCoV2-11 QAPGKEREWISFISRYYSSTYYTDSVKGRFTTSRDGDQ KTVHLQMNSLKPEDTAVYYCAARSRDFSSPFSATDTYT SWGQGTQVTVSS 148 EVQLQQSGGGLVQPGGSLRLSCAASGFTLDSYNIAWF V.sub.HH NRCoV2-11a RQAPGKEREWISYISRYYESTYYSDSVKGRFTTSRDGD KKTVSLQMNSLKSEDTAVYYCAARSRDFSSPISATDKY GSWGQGTQVTVSS 149 EVQLQQSGGGLVQAGGSLRLSCAASGRTFRNYVMGW V.sub.HH NRCoV2-12 FRQAPQAPGKDHEFVAAISWGGTEIYYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAADRGLSYYYTR TTEYNYWGQGTQVTVSS 150 EVQLVSSGGGLVQAGGSLRLSCEASGTTFSHYAVGWY V.sub.HH NRCoV2-14 RQAPGKQREWVASISVFGSTTYGGSVAGRFTISRDNDK NTVDLQMNSLKPEDTAVYYCHAVNADIGGDYWGQGTQ VTVSS 151 EVQLVSSGGGLVEAGGSLRLSCIASGSTSGRNTMGWF V.sub.HH NRCoV2-15 RQAPGKQREWVAIVSTSGATNYAGSVKGRFTLSRDNA KNAVYLQMNNLKPEDTAVYYCYAAYGGGGDYWGQGT QVTVSS 152 EVQLQQSGGGLVQTGGSLRLSCAAAGSPFSQLAMSWY V.sub.HH NRCoV2-17 RQISGKERAWVASISPTGNRSYSKIAKGRFTISRDNAKN TVTLQMTSLKPEDTAAYICQAANVNGGDYWGQGTQVT VSS 153 EVQLVESGGGLVQAGGSLRLSCVASGITISGYNMAWW V.sub.HH NRCoV2-18 RQTRGKQTERVAFINSGGSTTYSDSVKGRFTISRDNGK NTAYLQMNSLNAEDTADYFCSLHTSHDYWGQGTQVTV SS 154 EVQLLESGGGLVLPGGSLRLSCAVSGLTLNSYAIGWFR V.sub.HH NRCoV2-19 QAPGKEREGLSCLTSGGTGVYAESVKGRFTISRDNAEN TVYLQMNSLKPEDTAVYYCAADRARLRFGCSLNFRRE VAYDYWGQGTQVTVSS 155 EVQLQQSGGGLVQPGGSLRLSCAASGRTFSNYVVGWF V.sub.HH NRCoV2-20 RQAPQAPGKDHEFVAVISGSDTETYYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCAADRGMSYYYTRA TEYYYWGQGTQVTVSS 156 EVQLVESGGGLVQPGGSLRLSCATSGFTLDYYAIGWFR V.sub.HH NRCoV2-21 QAPGKEREWVSCISSGGSTFYVDSVKGRFTISRDNAKD TVYLQMSSLKPDDTAVYYCAADHRGRSLRFGCSSSTT DYLYWGQGTQVTVSS 157 EVQLVQSGGGSVQAGGSLRLSCVASGFTFDNYAIGWF V.sub.HH NRCoV2-SR01 RQAPGKEREGVSCISGNGGVTIYADSVKGRFTISRDNA KNLVYLQMNSLKPEDTAVYYCAATGIRSTWSVYGCSRL AGPYDYWGQGTQVTVSS 158 EVQLVDSGGGLVQAGGSLRLSCTASEFTLNYYSIGWFR V.sub.HH NRCoV2-SR02 QSPGKEREGVSCIRYSGGGIDYADSVKGRFTISRDNAK NTVYLTMNSLKPEDTAVYYCAADRLYSRACPTAGGRN YWGQGTQVTVSS 159 AVQLVDSGGGLVQAGGSLRLSCAASGSIFSNNHMGWY V.sub.HH NRCoV2-SR03 RQAPGKQRELVAAISSGGKTNYADFAKGRFTISRDNAK NMVYLQMNSLKPEDTAVYYCNRGGWEYRSSYYIMGPH WGQGTQVTVSS 160 QVQLVESGGGLVQAGGSLRLSCAASGRTFSSHTVGWY V.sub.HH NRCoV2-SR04 RQAPGKQRDLVAAISMGGNTNYADYADSVKGRFTISRD NAKNTLYLQMNSLKPEDTAVYYCNTAALVGNRLLPMAT ITWGQGTQVTVSS 161 EVQLVESGGGLVTTGGSLRLSCATSGSRFGSKHMAWY V.sub.HH NRCoV2-SR13 RQAPGKQRDLVAAISSGGSTHYGSSVKGRFTISRDNAK STVYLQMNSLNPEDTAVFYCNMGGWDYRSNTYIPGSR SDYWGQGTQVTVSS 162 QVQLVQSAGGLVQAGGSLRLSCVVSGTTFSRYHMGW V.sub.HH NRCoV2-SR16 HRQAPGKQRDFVAGISTSGAVTYADSAKGRFTISRDNA KNTVYLEMNSLKLEDTALYYCNTGGWDYRSSTFIMGL NWGQGTQVTVSS 163 EVQLQQSGGGLVQAGGSLRLSCAASGRPYSNYAMAWY V.sub.HH NRCoV2-S2A3 RQAPGKQHELVAGKQRELVAAISSGGTTKYADSVKAR FTISRDNAKNTVYLQMNILRPEDTAVYYCNTGSLSYGGS VYYPSYDNWGQGTQVTVSS 164 QVQLVQSGGGLVQAGGSLRLSCAVSGSPFRSNVMEWY V.sub.HH NRCoV2-S2A4 RQAPGKQRELVASISTGGSRTYTDSVKGRFTISRDNAK NEAFLQMNSLKPEDTAVYYCHAAARDSHGIYLLDTWG QGTQVTVSS 165 QVQLVDSGGGLVQAGGSLRLSCAASASTFGDSAMGYY V.sub.HH NRCoV2-S2B3 RQAPGKQRELVATISTGSNTNYADSVKGRFTISRDDAK NTVYLQMNSLKPEDTAVYYCNYRSIYYGQNFWGQGTQ VTVSS 166 QVQLVQSGGGLVQAGGSLRLSCAASGFTFNLYSIAWFR V.sub.HH NRCoV2-S2H4 QAPGKEREGVSCINSGDRDSTTYYADSVKGRFTISRDN AKHTAYLQMDSLKPEDTAVYYCALVFGYTSRDYCLTPK RGNYWGQGTQVTVSS 167 EVQLVQSGGGLVQAGGSLRLSCATSVRILSVPAMGWY V.sub.HH NRCoV2-S2F3 RQAPGKEREMVAVITSGGSTNYADSVKGRFTISRDNAK NTVYLQMNSLKLEDTAVYQCNLRDILSQPFWGQGTQV TVSS 168 QVQLVQSGGGSVQAGGSLRLSCAASGSTFGIFLMGWR V.sub.HH NRCoV2-S2G3 RQAPGKQRELVAHITSGGATNYADSVKGRFTISRDNAK NTVYLQMNSLEPEDTAVYYCYTTKRDDASVYWGQGTQ VTVSS 169 QVQLVQSGGGLVQAGGSLTLSCAPSGSTFSGYATNWY V.sub.HH NRCoV2-S2G4 RQAPGKQRELVATISSDGDKNYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCNKHWWTGDWWGQGTQ VTVSS 170 QVQLVQSGGGLVQAGGSLRLSCAASGITVSRIGMGWY V.sub.HH NRCoV2-S202 RQAPGKQRDMVAVISAGGSTNYADSVKGRFTISRDNAK NTVYLQMDSLKPEDTAVYYCNYGPGYRKAAWGQGTQ VTVSS 171 EVQLVESGGGLVQPGGSLRLSCAASGNIFSINSMGWFR V.sub.HH NRCoV2-MRed02 QAPGKERDVVATIWSDSRTSYADSVKGRFTISTDNTRT KVYLQMSSLNPEDTAVYYCAADRGFVVRGQYDYWGQ GTQVTVSS 172 EVQLVESGGGLVQPGGSLRLSCAAIGFTLDYYAIGWFR V.sub.HH NRCoV2-MRed03 QAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCATDAFATCDSWYAQIAQ YDFRGQGTQVTVSS 173 EVQLVESGGGLVQPGRSLRLSCAASGNSFSISTMGWF V.sub.HH NRCoV2-MRed04 RQAPGKERELVASIWSDTTTSYADSVKGRFTISTDNTRT KVYLQMSSLNPEDTAVYYCAADRGFVVRGQYDYWGQ GTQVTVSS 174 EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFR V.sub.HH NRCoV2-MRed05 QAPGKEREGVSCISSSDGSTLYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCATGPQAYYSGSYYFQCP QAGMDYWGKGTQVTVSS 175 EVQLVESGGGLVQAGGSLRLSCAASGFTLAYYAIGWFR V.sub.HH NRCoV2-MRed06 QAPGKEREGVSCISSSDGSAHYADSVKGRFTISRDNAK NTVSLQMNSLKPEDTAVYYCATDSFSSCSDYESGMDF WGKGTQVTVSS 176 EVQLVESGGGLVQPGGSLTLSCAASGSIGPFNTMGWY V.sub.HH NRCoV2-MRed07 RQAPGNQREPAAIITRGGVTNYADSVKGRFTISRDNAK NAVYLQMDSLKPDDTAVYYCYANYGWAIPYWGNGTQV TVSS 177 EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYAMSWH V.sub.HH NRCoV2-MRed11 RQAPGKGLEWVSAINSGGGSTSYADSVKGRFAISRDNA KNTLYLQMNSLKPEDTAVYYCATTISDGSSWSTKSYRG QGTQVTVSS 178 EVQLVESGGGLVQPGGSLRLSCAASTTVFGRNAMGWY V.sub.HH NRCoV2-MRed18 RQAPGKERELVATVSDGGTPNYADSVKGRFTISRDNAK NTIYLQMNSLEPEDTAVYYCNYYNYYYGRNFWGQGTQ VTVSS 179 EVQLVESGGGLVQPGGSLRLSCAASTIIFKGQTMGWF V.sub.HH NRCoV2-MRed19 RQAPGNERELVATMTTSGSANYADSVKGRFTISRDNEK TVTLQMNSLKPEDTALYYCYMHSVYYGIDYWGKGTQV TVSS 180 EVQLVESGGGSVQAGGSLRLSCAASGLSFSSYDMGWF V.sub.HH NRCoV2-MRed20 RQAPGKEREFVAAIRESGSGTYYADSVKGRFTISRDNA KNTVYLQVSSLKPEDTAVYTCAAKPPFYGSGTYSTPRA YLYWGQGTQVTVSS 181 EVQLVESGGGLVQPGGSLRLSCAASGSVFASNAMGWY V.sub.HH NRCoV2-MRed22 RQAPGKQRELVATISSRGSTNYADSVKGRFTISRDNAK NTVYLQMNSLGPEDTAVYYCNAREFTGFDYWGQGTQV TVSS 182 EVQLVESGGGLVQAGGSLRLSCAASGHTFSRYGMGW V.sub.HH NRCoV2-MRed25 FRQAPGKEREFVAAISWRGDSTYYRDSVNGRFTISRDN AKNTVYLGMNSLKPEDTAVYYCAAEMWGTATIVASRY TYWGQGTQVTVSS 183 XXXLXXSXGGXVXXGXSLXLSCXXXXXXXXXXXXXXX V.sub.HH Consensussequence RQXXXXXGXXXXXXXXXXXXXXXXXXXXXXXXXXXXX cons. forV.sub.HHsequences, XXXXXRFXXSXDXXXXXXXLXXXXLXXXDTXXXXCXX seq. generatedbasedon XXXXXXXXXXXXXXXXXXXXXXGXGTQVTVSS thesequence Xatposition1isAla,Glu,orGln alignmentshown Xatposition2isLeuorVal inFIG.18 Xatposition3isLysorGln Xatposition5isLeu,Gln,orVal Xatposition6isAsp,Glu,Gln,orSer Xatposition8isAlaorGly Xatposition11isLeuorSer Xatposition13isGlu,Leu,Gln,orThr Xatposition14isAla,Pro,orThr Xatposition16isGlu,Gly,orArg Xatposition19isArgorThr Xatposition23isAla,Glu,Ile,Thr,orVal Xatposition24isAla,Pro,Thr,orVal Xatposition25isAla,Ile,orSer Xatposition26isAla,Glu,Gly,Thr,orVal Xatposition27isPhe,His,Ile,Leu,Asn, Arg,Ser,Thr,orVal Xatposition28isIle,Pro,Arg,Ser,Thr, orVal Xatposition29isPhe,Gly,Ile,Leu,Ser, Val,orTyr Xatposition30isAla,Asp.Gly,Lys,Asn, Pro,Arg,orSer Xatposition31isAsp,Phe,Gly,His,Ile, Leu,Asn,Gln,Arg,Ser,Val,Tyr Xatposition32isPhe,His,Ile,Lys,Leu, Asn,Pro,Gln,Ser,Val,orTyr Xatposition33isAla,Asp,Gly,His,Leu, Asn,Ser,Thr,orVal Xatposition34isIle,Met,Thr,orVal Xatposition35isAla,Glu,Gly,His,Asn, orSer Xatposition36isTrporTyr Xatposition37isPhe,His,Arg,Val,Trp, orTyr Xatposition40isAla,Ile,Ser,orThr Xatposition41isProorabsent Xatposition42isGlnorabsent Xatposition43isAlaorabsent Xatposition44isPro,Arg,orSer Xatposition46isAla,Lys,Asn,orGln Xatposition47isAsp,Glu,Gly,Pro,or Gln Xatposition48isHisorabsent Xatposition49isGluorabsent Xatposition50isLeuorabsent Xatposition51isValorabsent Xatposition52isAlaorabsent Xatposition53isGlyorabsent Xatposition54isLysorabsent Xatposition55isGlnorabsent Xatposition56isHis,Leu,Arg,orThr Xatposition57isAla,Asp,orGlu Xatposition58isAla,Phe,Gly,Leu,Met, Pro,Arg,Val,orTrp Xatposition59isAla,Ile,Leu,orVal Xatposition60isAla,Ser,orThr Xatposition61isAla,Cys,Phe,Gly,His, Ile,Ser,Thr,Val,orTyr Xatposition62isIle,Leu,Met,orVal Xatposition63isGly,Asn,Arg,Ser,Thr, orTrp Xatposition64isAla,Asp,Glu,Phe,Gly, Met,Asn,Pro,Arg,Ser,Thr,Val,Trp,orTyr Xatposition65isAsp,Phe,Gly,Met,Asn, Arg,Ser,Thr,Val,Tyr,orabsent Xatposition66isAsp,Gly,Ser,Thr,orTyr Xatposition67isAla,Asp,Glu,Gly,Ile, Lys,Asn,Arg,Ser,Thr,orVal Xatposition68isAla,Asp,Glu,Gly,Lys, Asn,Pro,Arg,Ser,Thr,orVal Xatposition69isAsn,Ser,orabsent Xatposition70isThr,Tyr,orabsent Xatposition71isAla,Gly,Ile,Asn,Thr, orabsent Xatposition72isAsp,Phe,His,Ile,Lys, Leu,Asn,Arg,Ser,Thr,Val,Tyr,orabsent Xatposition73isTyrorabsent Xatposition74isAla,Gly,Ile,Arg,Ser, Thr,orVal Xatposition75isAla,Asp,Glu,Gly,Lys, Met,Ser,orTyr Xatposition76isPhe,Ile,orSer Xatposition77isAlaorVal Xatposition78isAla,Lys,Asn,orVal Xatposition79isAlaorGly Xatposition82isAlaorThr Xatposition83isPhe,Ile,Leu,Thr,orVal Xatposition85isArgorThr Xatposition87isAsp,Gly,orAsn Xatposition88isAla,Asp,Glu,Gly,orThr Xatposition89isGlu,Lys,Gln,orArg Xatposition90isAsp,His,Lys,Asn,Ser, Thr,orabsent Xatposition91isAla,Glu,Lys,Leu,Met, orThr Xatposition92isAla,Ile,Leu,orVal Xatposition93isAsp,Phe,His,Ser,Thr, orTyr Xatposition95isGlu,Gly,Gln,orThr Xatposition96isMetorVal Xatposition97isAsp,Asn,Arg,Ser,orThr Xatposition98isAsp,Ile,Asn,orSer Xatposition100isGlu,Gly,Lys,Asn,orArg Xatposition101isAla,Leu,Pro,orSer Xatposition102isAsporGlu Xatposition105isAlaorGly Xatposition106isAla,Asp,Leu,orVal Xatposition107isPheorTyr Xatposition108isPhe,Ile,Gln,Arg,Thr, Val,orTyr Xatposition110isAla,His,Asn,Gln,Ser, Thr,Trp,orTyr Xatposition111isAla,Ile,Lys,Leu,Met, Arg,Ser,Thr,orTyr Xatposition112isAla,Asp,Glu,Gly,His, Lys,Asn,Arg,Ser,Thr,Val,orTyr Xatposition113isAla,Asp,Glu,Phe,Gly, His,Ile,Lys,Asn,Pro,Gln,Arg,Ser,Thr, Tyr,orabsent Xatposition114isAla,Asp,Phe,Gly,Ile, Leu,Pro,Gln,Arg,Ser,Val,Trp,orabsent Xatposition115isAla,Asp,Glu,Phe,Gly, Leu,Met,Arg,Ser,Thr,Val,Tyr,orabsent Xatposition116isPhe,Gly,His,Leu,Ser, Thr,Trp,Tyr,orabsent Xatposition117isCys,Gly,His,Asn,Arg, Ser,Thr,Tyr,orabsent Xatposition118isAla,Asp,Phe,Gly,Arg, Ser,Thr,Trp,orabsent Xatposition119isAla,Cys,Asp,Gly,Ile, Leu,Asn,Pro,Arg,Ser,Tyr,orabsent Xatposition120isCys,Gly,Ile,Asn,Pro, Arg,Ser,Thr,Val,Trp,Tyr,orabsent Xatposition121isAsp,Phe,Gly,Leu,Ser, Trp,Tyr,orabsent Xatposition122isAla,Asp,Gly,Ile,Leu, Thr,Val,Trp,Tyr,orabsent Xatposition123isCys,Phe,Asn,Val,or absent Xatposition124isPhe,Gly,Ile,Leu,Gln, Ser,Tyr,orabsent Xatposition125isAla,Cys,Asp,Gly,Pro, Arg,Ser,Thr,orabsent Xatposition126isAla,Phe,Gly,Leu,Met, Pro,Gln,Arg,Ser,Thr,Val,Trp,Tyr,or absent Xatposition127isAla,Asp,Glu,Gly,His, Ile,Pro,Gln,Arg,Ser,Thr,Trp,orabsent Xatposition128isAla,Asp,Gly,Ile,Lys, Met,Asn,Arg,Ser,Thr,Val,Trp,Tyr,or absent Xatposition129isAla,Asp,Glu,Gly,His, Lys,Leu,Pro,Gln,Arg,Ser,Thr,orabsent Xatposition130isGlu,Phe,Gly,His,Ile, Lys,Leu,Met,Asn,Gln,Arg,Ser,Thr,orTyr Xatposition131isAla,Asp,Glu,Gly,Ile, Leu,Asn,Pro,Ser,Thr,Val,orTyr Xatposition132isAla,Asp,Phe,His,Asn, Ser,Thr,Val,Trp,orTyr Xatposition133isArgorTrp Xatposition135isLys,Asn,orGln 184 XVQLVXSXGGXVXXGGSLXLSCXXXXXXXXXXXXXWX V.sub.HH Consensussequence RQXPGXXRXXXXXIXXXXGXXXYXXXXXXXKGRFTISR cons. forS1-NTDspecific DNAKXXXXLXMXSLXXXDTAXXYCXXXXXXXXXXXXX seq. V.sub.HHs,generated XXXXXXXXXXXGXGTQVTVSS basedonthe Xatposition1isAla,Glu,orGln sequencealignment Xatposition6isAsp.Glu,orGln showninFIG.19 Xatposition8isAlaorGly Xatposition11isLeuorSer Xatposition13isGlnorThr Xatposition14isAla,Pro,orThr Xatposition19isArgorThr Xatposition23isAla,Thr,orVal Xatposition24isAla,Thr,orVal Xatposition25isIleorSer Xatposition26isGluorGly Xatposition27isPhe,Arg,Ser,orThr Xatposition28isIle,Arg,orThr Xatposition29isPhe,Gly,orLeu Xatposition30isAsp,Ala,Gly,Asn,Pro, orSer Xatposition31isPhe,Asn,Arg,Ser,or Tyr Xatposition32isHis,Lys,Asn,orTyr Xatposition33isAla,His,Ser,orThr Xatposition34isIle,Met,orVal Xatposition35isAlaorGly Xatposition37isPhe,His,orTyr Xatposition40isAlaorSer Xatposition43isLysorAsn Xatposition44isGluorGln Xatposition46isAsporGlu Xatposition47isPhe,Gly,Leu,orPro Xatposition48isAlaorVal Xatposition49isAlaorSer Xatposition50isAla,Cys,Gly,orIle Xatposition52isArg,Ser,orThr Xatposition53isGly,Met,Arg,Ser,Thr, orTyr Xatposition54isGly,Asn,orSer Xatposition55isAsp,Gly,orabsent Xatposition57isAla,Gly,Lys,Asn,Ser, orVal Xatposition58isAla,Ile,Thr,orVal Xatposition59isAsp,His,Ile,Asn,Thr, orTyr Xatposition61isAla,Gly,orabsent Xatposition62isAsporabsent Xatposition63isTyrorabsent Xatposition64isAlaorabsent Xatposition65isAsporSer Xatposition66isPheorSer Xatposition67isAlaorVal Xatposition80isAsnorSer Xatposition81isAla,Leu,Met,orThr Xatposition82isLeuorVal Xatposition83isSerorTyr Xatposition85isGlu,Gln,orThr Xatposition87isAsporAsn Xatposition90isLysorAsn Xatposition91isLeuorPro Xatposition92isAsporGlu Xatposition96isLeuorVal Xatposition97isPheorTyr Xatposition100isAla,Asn,orTyr Xatposition101isAla,Met,Arg,orThr Xatposition102isAsp,Ala,Gly,Asn,or Thr Xatposition103isAla,Gly,Arg,Ser,or absent Xatposition104isPhe,Ile,Leu,Trp,or absent Xatposition105isAsp,Glu,Ala,Arg,Ser, Val,Tyr,orabsent Xatposition106isGly,Ser,Thr,Tyr,or absent Xatposition107isCys,Asn,Arg,Thr,or absent Xatposition108isAsp,Ser,Trp,orabsent Xatposition109isAsp,Asn,Ser,orabsent Xatposition110isThr,Val,Trp,Tyr,or absent Xatposition111isGlu,Phe,Arg,Tyr,or absent Xatposition112isGlyorabsent Xatposition113isAla,Cys,orabsent Xatposition114isCys,Ser,orabsent Xatposition115isAla,Ile,Leu,Pro,Arg, Ser,orabsent Xatposition116isGln,Leu,Met,Pro,Thr, Tyr,orabsent Xatposition117isAla,Gly,Ile,Pro,or absent Xatposition118isAla,Gly,Leu,Met,Pro, Ser,Trp,orabsent Xatposition119isGln,Ala,Gly,Pro,Arg, orabsent Xatposition120isIle,Met,Arg,Ser,Thr, Tyr,orabsent Xatposition121isAsp,Ile,Asn,Pro,or absent Xatposition122isPhe,His,Asn,Thr,or Tyr Xatposition123isArgorTrp Xatposition125isGln,Lys,orAsn 185 XVQLXXSGGGXVQXGGSLXLSCAXSXXXXXXXXXXXX V.sub.HH Consensussequence RQAPGXXXXXXXXXXXXXVXXXXXXXXXXXXXYXDSV cons. forS2specificV.sub.HHs, XXRFXISRDXXKXXXXLXXXXLXXEDTAXYXCXXXXXX seq. generatedbasedon XXXXXXXXXXXXXXXGXGTQVTVSS thesequence Xatposition1isAlaorPro alignmentshownin Xatposition5isProorVal FIG.20 Xatposition6isGln,Ala,orPro Xatposition11isLysorSer Xatposition14isAsporAsn Xatposition19isArgorThr Xatposition24isAsp,Asn,Thr,orVal Xatposition26isAsp,Phe,Thr,orVal Xatposition27isCys,Gly,His,Lys,Arg, Ser,orThr Xatposition28isHis,Asn,Ser,Thr,or Val Xatposition29isCys,Lys,Val,orTyr Xatposition30isAsp,Phe,Ile,Met,Arg, orSer Xatposition31isGln,Phe,His,Lys,Met, Arg,Ser,orVal Xatposition32isCys,His,Met,Asn,Pro, Ser,orTyr Xatposition33isAsp,Gln,Phe,Lys,Ser, Thr,orVal Xatposition34isHis,Leu,orThr Xatposition35isAsp,Ala,Phe,Met,or Ser Xatposition36isTrporTyr Xatposition37isCys,Gly,Arg,orTyr Xatposition43isIleorMet Xatposition44isAla,Phe,orPro Xatposition45isGly,Lys,orabsent Xatposition46isAlaorabsent Xatposition47isLysorabsent Xatposition48isValorabsent Xatposition49isAsporabsent Xatposition50isPheorabsent Xatposition51isIleorabsent Xatposition52isProorabsent Xatposition53isArgorabsent Xatposition54isGlnorAla Xatposition55isCys,Phe,Lys,Leu,or Trp Xatposition57isAsporSer Xatposition58isAsp,Glu,Gly,Ser,Thr, orVal Xatposition59isHis,Leu,orVal Xatposition60isMet,Arg,Ser,orabsent Xatposition61isAla,Ser,Thr,orTrp Xatposition62isAsp,Gln,Phe,Arg,Ser, orThr Xatposition63isGlnorabsent Xatposition64isArgorabsent Xatposition65isGln,Phe,Arg,orSer Xatposition66isGln,Phe,orSer Xatposition67isAsp,Gln,Phe,Met,Ser, orThr Xatposition68isAsp,Ile,Asn,Arg,orThr Xatposition69isIle,Met,Ser,Thr,orTyr Xatposition71isAsp,Arg,orThr Xatposition75isIleorMet Xatposition76isAsporPhe Xatposition79isAsporThr Xatposition84isGlnorMet Xatposition85isAsporAla Xatposition87isGly,Met,orabsent Xatposition88isAlaorThr Xatposition89isAsp,His,Lys,orVal Xatposition90isCys,Thr,orTyr Xatposition92isPheorPro Xatposition93isLeuorVal Xatposition94isGln,Met,orSer Xatposition95isHisorSer Xatposition97isAla,Phe,Ile,orArg Xatposition98isLysorAsn Xatposition103isLysorVal Xatposition105isPro,Thr,orTyr Xatposition107isAsp,Gly,Met,orTyr Xatposition108isAsp,ile,Lys,Leu,Thr, orTyr Xatposition109isAsp,Ala,Phe,Gly,Ile, Arg,Thr,Val,orTyr Xatposition110isAsp,Gln,Ala,Cys,His, Ile,Met,Asn,Ser,orabsent Xatposition111isPhe,His,Lys,Asn,Arg, Ser,Val,Tyr,orabsent Xatposition112isGln,Cys,Leu,Ser,Tyr, orabsent Xatposition113isPhe,Ser,Thr,Trp,Tyr, orabsent Xatposition114isPhe,Gly,Ser,orabsent Xatposition115isPhe,Arg,Ser,Thr,or absent Xatposition116isAsp,Gln,Phe,orabsent Xatposition117isThr,Tyr,orabsent Xatposition118isGlu,His,Ser,Tyr,or absent Xatposition119isLys,Ser,Val,orabsent Xatposition120isSer,Thr,Val,Tyr,or absent Xatposition121isAsp,Gln,Cys,His,Asn, Trp,Tyr,orabsent Xatposition122isGln,Ile,Lys,Asn,Arg, Ser,Thr,Trp,orTyr Xatposition123isAsp,Phe,Lys,Arg,Ser, orThr Xatposition124isCys,Phe,His,Ile,Lys, Pro,Arg,Ser,orTyr Xatposition125isAsp,Gln,Lys,Met,Asn, Ser,Thr,orVal Xatposition126isAsp,Cys,Met,Thr,Trp, orTyr Xatposition127isArgorTrp Xatposition129isIleorPro 186 XXXLXXSGGGXVXXGXSLXLSCXXXXXXXXXXXXXWX V.sub.HH Consensussequence RQXXXXXGXXXXXXXXXXXXXXXXXYXXXXXGRFTXS cons. forS1-RBDspecific XDXXXXXXXLQMXXLXXXDTXXYXCXXXXXXXXXXXX seq. V.sub.HHs,generated XXXXXXXXXXXXGXGTQVTVSS basedonthe Xatposition1isAsp,Ala,orPro sequencealignment Xatposition2isLysorVal showninFIG.21 Xatposition3isIleorPro Xatposition5isLys,Pro,orVal Xatposition6isGln,Ala,Pro,orSer Xatposition11isLysorSer Xatposition13isAla,Lys,orPro Xatposition14isAsp,Asn,orThr Xatposition16isAla,Phe,orArg Xatposition19isArgorThr Xatposition23isAsp,Ala,His,Thr,or Val Xatposition24isAsp,Thr,orVal Xatposition25isAsporSer Xatposition26isPheorVal Xatposition27isCys,His,Lys,Met,Arg, Ser,Thr,orVal Xatposition28isHis,Asn,Ser,orThr Xatposition29isCys,His,Lys,orSer Xatposition30isGln,Phe,Met,Arg,or Ser Xatposition31isGln,Phe,Gly,His,Met, Pro,Arg,Ser,orTyr Xatposition32isLys,Met,Ser,Val,or Tyr Xatposition33isAsp,Met,Ser,Thr,or Val Xatposition34isHis,Leu,orVal Xatposition35isAsp,Phe,Gly,Met,or Ser Xatposition37isCys,Val,Trp,orTyr Xatposition40isAsp,His,Ser,orThr Xatposition41isAsnorabsent Xatposition42isProorabsent Xatposition43isAsporabsent Xatposition44isAsn,Arg,orSer Xatposition46isAsp,Ile,orPro Xatposition47isGln,Ala,Phe,Asn,or Pro Xatposition48isGly,Lys,Arg,orThr Xatposition49isAsp,Gln,orAla Xatposition50isAsp,Cys,Phe,Lys, Arg,Val,orTrp Xatposition51isHis,Lys,orVal Xatposition52isAsp,Ser,orThr Xatposition53isAsp,Glu,Cys,Phe, His,Ser,Thr,Val,orTyr Xatposition54isHis,Lys,Leu,orVal Xatposition55isPhe,Met,Ser,Thr,or Trp Xatposition56isCys,Phe,Met,Asn, Arg,Ser,Thr,Val,Trp,orabsent Xatposition57isPhe,Met,Arg,Ser, Tyr,orabsent Xatposition58isGln,Cys,Phe,Leu, Arg,Ser,Thr,Val,orTyr Xatposition59isGln,Ala,Phe,Arg, Ser,orThr Xatposition60isAsp,Gln,Ala,His, Met,Arg,Ser,orThr Xatposition61isAsp,Phe,His,Met, Arg,Ser,orThr Xatposition62isGln,Cys,Gly,His, Lys,Met,Arg,Ser,Thr,Val,orTyr Xatposition64isAsp,Phe,His,Ser, Thr,orVal Xatposition65isAsp,Gln,Ala,Phe, Ile,Leu,orTyr Xatposition66isHisorSer Xatposition67isAsporVal Xatposition68isAsp,Ile,orArg Xatposition73isCys,His,Lys,Thr, orVal Xatposition75isArgorThr Xatposition77isGln,Phe,orMet Xatposition78isAsp,Gln,Phe,orThr Xatposition79isAla,Ile,Pro,orArg Xatposition80isGln,Ile,Met,Ser,or Thr Xatposition81isAsp,Ile,orThr Xatposition82isAsp,His,Lys,orVal Xatposition83isGln,Cys,Gly,Ser,Thr, orTyr Xatposition87isGln,Met,Arg,Ser,or Thr Xatposition88isGln,Met,orSer Xatposition90isAla,Ile,Met,orArg Xatposition91isAsp,Asn,orSer Xatposition92isGlnorAla Xatposition95isAsporPhe Xatposition96isAsp,Gln,orVal Xatposition98isCys,His,Arg,Val, orTyr Xatposition100isAsp,Gly,Met,Pro, Ser,Thr,Trp,orTyr Xatposition101isAsp,His,Ile,Lys, Ser,orThr Xatposition102isAsp,Gln,Phe,Gly, Arg,Ser,Val,orTyr Xatposition103isPhe,Gly,Met,Asn, Pro,Arg,Ser,Tyr,orabsent Xatposition104isAsp,Gln,Phe,Asn, Pro,Arg,Val,orabsent Xatposition105isAsp,Gln,Cys,Phe, Lys,Leu,Arg,Ser,Thr,Val,Tyr,or absent Xatposition106isCys,Gly,Lys,Met, Arg,Ser,Val,Tyr,orabsent Xatposition107isAsp,Ala,Arg,Ser, Val,Tyr,orabsent Xatposition108isCys,Phe,Lys,Leu, Ser,Tyr,orabsent Xatposition109isGln,Phe,Arg,or absent Xatposition110isGlu,Met,Asn,Arg, Ser,orabsent Xatposition111isCys,Lys,Ser,Thr, Tyr,orabsent Xatposition112isAsp,Phe,Lys,Trp, Tyr,orabsent Xatposition113isGlu,Cys,Met,Asn, Val,Tyr,orabsent Xatposition114isCys,Phe,His,Lys, Pro,Ser,Tyr,orabsent Xatposition115isGlu,Phe,Arg,Ser, Thr,orabsent Xatposition116isAsp,Asn,Arg,Ser, Trp,Tyr,orabsent Xatposition117isAsp,Gln,Ala,Phe, Gly,Asn,Pro,Arg,Thr,Val,Tyr,or absent Xatposition118isAsp,Gln,Phe,His, Met,Arg,Ser,Thr,Val,orabsent Xatposition119isAsp,Gln,Ala,Phe, Gly,Ile,Lys,Pro,Arg,Ser,Thr,or absent Xatposition120isPhe,Gly,Leu,Met, Ser,Tyr,orabsent Xatposition121isGln,Ala,Phe,Lys, Met,Ser,Thr,orTyr Xatposition122isGln,Ser,Thr,Val,orTyr Xatposition123isArgorTrp Xatposition125isIleorPro Bold highlighting in V.sub.HH sequences indicates the locations of CDR sequences, as defined using the IMGT numbering system described in Lefranc et al (2003).
REFERENCES
[0424] All publications identified herein, including each of the references listed below and any published sequences that are identified by name and/or accession number, are hereby incorporated by reference in their entirety. [0425] Adejare, A. (ed.) (2021) Remington (23.sup.rd Edition), The Science and Practice of Pharmacy Academic Press, ISBN 978-O-12-820007-0. [0426] Akache B, et al. Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant. Sci Rep 11, 21849 (2021). [0427] Algaissi, A., and Hashem, A. M. (2020) Evaluation of MERS-CoV Neutralizing Antibodies in Sera Using Live Virus Microneutralization Assay. Methods Mol Biol 2099, 107-116. [0428] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J., Basic local alignment search tool, Journal of Molecular Biology, 1990, 215(3): 403-410. [0429] Altschul, S.F., Madden, T. L., Sch?ffer, A. A., Zhang, J., Zhang, Z., Miller, W., Lipman, D. J., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Research, 1997, 25(17):3389-402. [0430] Arbabi-Ghahroudi, M. Desmyter A, Wyns L, Hamers R., and Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies, FEBS Lett 414, 521-526. [0431] Arora, P, Jafferany, M, Lotti, T, Sadoughifar, R, Goldust, M. Learning from history: Coronavirus outbreaks in the past. Dermatologic Therapy. 2020; 33:e13343. [0432] Chi, X., Yan, R., Zhang, J., Zhang, G., Zhang, Y., Hao, M., Zhang, Z., Fan, P., Dong, Y., Yang, Y., Chen, Z., Guo, Y., Zhang, J., Li, Y., Song, X., Chen, Y., Xia, L., Fu, L., Hou, L., Xu, J., Yu, C., Li, J., Zhou, Q., and Chen, W. (2020) A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 369, 650-655. [0433] Colwill K, et al. A Made-in-Canada serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination. medRxiv, 2021.2010.2025.21265476 (2021). [0434] Coughlin, M. M., Babcook, J., and Prabhakar, B. S. (2009) Human monoclonal antibodies to SARS-coronavirus inhibit infection by different mechanisms. Virology 394, 39-46. [0435] Davies J., and L. Riechmann, Affinity improvement of single antibody V.sub.H domains: residues in all three hypervariable regions affect antigen binding. Immunotechnology 2 (1996) 169-179. [0436] Delfin-Riela, T., Rossotti, M. A., Alvez-Rosado R, Leizagoyen C, and Gonzalez-Sapienza, G. (2020) Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISAA nanobody-based test for highly sensitive detection of hemoglobin in fecal samples. Biomolecules, 10 (12):1652. [0437] Detalle, L., Stohr, T., Palomo, C., Piedra, P. A., Gilbert, B. E., Mas, V., Millar, A., Power, U. F., Stortelers, C., Allosery, K., Melero, J. A., and Depla, E. (2016) Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection. Antimicrob Agents Chemother 60, 6-13. [0438] Dondelinger, M., Filee, P., Sauvage, E., Quinting, B., Muyldermans, S., Galleni, M., & Vandevenne, M. S. (2018). Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Frontiers in immunology, 9, 2278. [0439] Dumoulin, M., Conrath, K., Van Meirhaighe, A., Meersman, F., Heremans, K., Frenken, L. G., Muyldermans, S., Wyns, L, and Matagne, A. (2002) Protein Sci 11, 500-S15. [0440] Galipeau Y, et al. Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2. EBioMedicine 74, 103700 (2021). [0441] Hamers-Casterman, C, Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C, Songa, E. B., Bendahman, N., and Hamers, R. (1993) Nature 363, 446-448 Henry, K. A., Hussack, G., Collins, C., Zwaagstra, J. C., Tanha, J., and MacKenzie, C. R. (2016) Isolation of TGF-beta-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing. Protein Eng Des Sel 29, 439-443. [0442] Henry, K. A., Kim, D. Y., Kandalaft, H., Lowden, M. J., Yang, Q., Schrag, J. D., Hussack, G., MacKenzie, C. R., and Tanha, J. (2017) Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human V.sub.H/V.sub.L Single-Domain Antibodies from In Vitro Display Libraries. Front Immunol 8, 1759. [0443] Henry, K. A., Tanha, J., and Hussack, G. (2015) Identification of cross-reactive single-domain antibodies against serum albumin using next-generation DNA sequencing. Protein Eng Des Sel 28, 379-383. [0444] Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K., MacKenzie, R., and Tanha, J. (2011) Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. J Biol Chem 286, 8961-8976. [0445] Jespers, L, Schon, O., Famm, K., and Winter, G. (2004) Nat. Biotechnol. 22, 1161-1165. [0446] Karlin, S., Altschul, S.F., Applications and statistics for multiple high-scoring segments in molecular sequences. PNAS, 1993, 90(12): 5873-5877. [0447] Karlin, S., Altschul, S.F., Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. PNAS, 1990, 87(6): 2264-2268. [0448] Kim, D. Y., Kandalaft, H., Ding, W., Ryan, S., van Fassen, H., Hirama, T., Foote, S. J., MacKenzie, R., and Tanha, J. (2012) PEDS advance access Aug. 30, 2012, 1-9. [0449] Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q., Zhang, L., Wang, X. (2020), Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220. [0450] Lefranc, M.-P. et al., (2003) Dev. Comp. Immunol., 27, 55-77. [0451] Li, R. (2016) Structure, Function, and Evolution of Coronavirus Spike Proteins, Annual Review of Virology 3, 237-261. [0452] Li S, Zheng W, Kuolee R, Hirama T, Henry M, Makvandi-Nejad S, Fjallman T, Chen W, Zhang J. Pentabody-mediated antigen delivery induces antigen-specific mucosal immune response. Mol Immunol 2009; 46:1718-26. [0453] Myers, E.W., Miller, W., Optimal alignments in linear space, Bioinformatics, 4(1): 11-17. Sliepen K, van Montfort T, Melchers M, Isik G, Sanders R W, 2015. Immunosilencing a highly immunogenic protein trimerization domain. J Biol Chem 290, 7436-7442. [0454] Nuttall, S.D., Krishnan, U. V., Doughty, L., Pearson, K., Ryan, M. T., Hoogenraad, N.J., Hattarki, M., Carmichael, J.A., Irving, R. A., and Hudson, P. J. (2003) Eur. J. Biochem. 270, 3543-3554. [0455] Ravichandran, S., Coyle, E. M., Klenow, L., Tang, J., Grubbs, G., Liu, S., Wang, T., Golding, H., and Khurana, S. (2020) Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits. Sci Transl Med 12. [0456] Respaud, R., Vecellio, L., Diot, P., and Heuze-Vourc'h, N. (2015) Nebulization as a delivery method for mAbs in respiratory diseases. Expert Opin Drug Deliv 12, 1027-1039. [0457] Rogers, T. F., Zhao, F., Huang, D., Beutler, N., Burns, A., He, W. T., Limbo, O., Smith, C., Song, G., Woehl, J., Yang, L., Abbott, R. K., Callaghan, S., Garcia, E., Hurtado, J., Parren, M., Peng, L., Ramirez, S., Ricketts, J., Ricciardi, M. J., Rawlings, S. A., Wu, N. C., Yuan, M., Smith, D. M., Nemazee, D., Teijaro, J. R., Voss, J. E., Wilson, I. A., Andrabi, R., Briney, B., Landais, E., Sok, D., Jardine, J. G., and Burton, D. R. (2020) Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science 369, 956-963. [0458] Rossotti, M. A., Henry, K. A., van Faassen, H., Tanha, J., Callaghan, D., Hussack, G., Arbabi-Ghahroudi, M., and MacKenzie, C. R. (2019) Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling. Biochem J 476, 39-50. [0459] Rossotti, M. A., Pirez, M., Gonzalez-Techera, A., Cui, Y., Bever, C. S., Lee, K. S., Morisseau, C., Leizagoyen, C., Gee, S., Hammock, B. D., and Gonzalez-Sapienza, G. (2015a) Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays. Anal Chem 87, 11907-11914. [0460] Rossotti, M., Tabares, S., Alfaya, L., Leizagoyen, C., Moron, G., and Gonzalez-Sapienza, G. (2015b) Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells. Biochim Biophys Acta 1850, 1397-1404. [0461] Stuible, M. Gervais, C., Lord-Dufour, S., Perret, S., L'Abbe, D., Schrag, J., St-Laurent, G., Durocher, Y. (2021), Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells. Journal of Biotechnology 326, 21-27. [0462] Sulea T, Baardsnes J, Stuible M, Rohani N, Tran A, Parat M, Cepero Donates Y, Duchesne M, Plante P, Kour G, Durocher Y. (2022) Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody. PLoS One. 17(3):e0266250. [0463] Wrapp, D., De Vlieger, D., Corbett, K. S., Torres, G. M., Wang, N., Van Breedam, W., Roose, K., van Schie, L., Team, V.-C. C.-R, Hoffmann, M., Pohlmann, S., Graham, B. S., Callewaert, N., Schepens, B., Saelens, X., and McLellan, J. S. (2020) Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 181, 1004-1015 e1015. [0464] Wrapp D, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263 (2020). [0465] Zheng M, Zhao X, Zheng S, et al. Bat SARS-Like WIV1 coronavirus uses the ACE2 of multiple animal species as receptor and evades IFITM3 restriction via TMPRSS2 activation of membrane fusion. Emerg Microbes Infect. 2020; 9(1):1567-1579. [0466] Zhou, P., Yang, XL., Wang, X G. et al. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273.