Techniques regarding antigen-binding proteins that can bind to CoV (e.g., SARS-CoV-2) variants are provided. For example, one or more embodiments described herein can comprise an antigen-binding protein that can comprise a heavy polypeptide chain variable region with an amino acid sequence that is a variant of SEQ ID NO: 7. The amino acid sequence can comprise at least one amino acid substitution selected from the group consisting of: R50D, R50E, R50W, R50F, R50Y, R50L, R50V, R50I, R50Pho, I54D, I54E, I54W, I54F, I54Y, I54Pho, L55D, L55E, L55W, L55F, L55Y, and L55Pho.

This application incorporates by reference the Sequence Listing included in the ASCII text file titled “P202102482US01_SequenceListing”, created Feb. 24, 2022, and having a size of 70,155 bytes.

BACKGROUND

The subject disclosure relates to antigen-binding proteins, such as nanobodies, antibodies and/or antigen-binding fragments (“Fab”) thereof, that can bind to the spike protein of a CoV (e.g., SARS-CoV-2) variant, and more specifically, that can bind to the spike protein of a CoV (e.g., SARS-CoV-2) variant carrying the E484K,Q mutation and/or the L452R,Q mutation.

SUMMARY

According to an embodiment, an antigen-binding protein is provided. The antigen-binding protein can comprise a heavy polypeptide chain variable region with an amino acid sequence that is a variant of SEQ ID NO: 7. The amino acid sequence can comprise at least one amino acid substitution selected from the group consisting of: R50D, R50E, R50W, R50F, R50Y, R50L, R50V, R50I, R50Pho, I54D, I54E, I54W, I54F, I54Y, I54Pho, L55D, L55E, L55W, L55F, L55Y, and L55Pho.

According to another embodiment, an antigen-binding protein is provided. The antigen-binding protein can comprise a light polypeptide chain variable region with an amino acid sequence that is a variant of SEQ ID NO: 8. The amino acid sequence can comprise an amino acid substitution selected from the group consisting of: R96D, R96E, R96W, R96F, R96Y, R96L, R96V, R96I, and R96Pho.

According to another embodiment, an antigen-binding protein is provided. The antigen-binding protein can comprise a heavy polypeptide chain variable region with an amino acid sequence that is a variant of SEQ ID NO: 7. The amino acid sequence can comprise an amino acid substitution that substitutes at least one amino acid selected from the group consisting of R50, 154, and L55 with a negatively charged amino acid.

According to another embodiment, an antigen-binding protein is provided. The antigen-binding protein can comprise a heavy polypeptide chain variable region with an amino acid sequence that is a variant of SEQ ID NO: 7. The amino acid sequence can comprise an amino acid substitution that substitutes at least one amino acid selected from the group consisting of R50, 154, and L55 with a hydrophobic residue.

According to another embodiment, an antigen-binding protein is provided. The antigen-binding protein can comprise a heavy polypeptide chain variable region with an amino acid sequence that is a variant of SEQ ID NO: 7. The amino acid sequence can comprise an amino acid substitution that substitutes at least one amino acid selected from the group consisting of R50, 154, and L55 with an amino acid that forms a cation-π interaction with a receptor binding domain of a SARS-CoV-2 spike protein.

DETAILED DESCRIPTION

As used herein, the term “sequence identity” can refer to the relatedness between two amino acid sequences or between two nucleotide sequences. For example, the degree of sequence identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm. As used herein, the term “amino acid substitution” can refer to the replacement of one amino acid in an amino acid sequence with another amino acid. In various embodiments, the following nomenclature can be employed to define an amino acid substitution: [original amino acid][position of original amino acid in the amino acid sequence][substituted amino acid]. For example, the amino acid substitution E484K can delineate a replacement of glutamic acid (E) at the 484th position of the given amino acid sequence with lysine (K). Further, amino acids can be referenced herein by their standard single letter code. Additionally, the following nomenclature can be employed to delineate an amino acid: [amino acid][position of amino acid in the amino acid sequence]. For example, the amino acid E484 can be the glutamic acid (E) at the 484th position of the given amino acid sequence. Further, where a variant comprises a combination of amino acid substitutions, a “+” can be located between each respective amino acid substitution. Where different amino acids can be substituted at a given position, the possible substituents can be separated by a comma. For example, “R50D,E” can delineate two possible amino acid substitutions: R50D or R50E. Also, as used herein, the term “Pho” can represent a phosphorylated residue. For instance, an amino acid substitution comprising a phosphorylated residue can comprise an amino acid phosphorylated (e.g., via a post-translational modification (“PTM”)). Example phosphorylated residues can include, but are not limited to: serine, threonine, and/or tyrosine.

As used herein, the term “coronavirus” and/or “CoV” refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, SARS-CoV-1, and/or Mers-Cov. As used herein, the term “SARS-CoV-2” refers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a positive-sense single-stranded ribonucleic acid (“RNA”) virus that causes the ongoing coronavirus disease 2019 (“COVID-19”). SARS-CoV-2 binds via a viral spike protein to human host cell receptor angiotensin-converting enzyme 2 (“ACE2”). The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus. The SARS-CoV-2 spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle. Further, the spike protein binds to its cognate receptor via a receptor binding domain (“RBD”). The amino acid sequence of the RBD of the SARS-CoV-2 spike protein can be defined by the amino acid sequence provided in SEQ ID NO: 1.

As used herein, the term “coronavirus infection” and/or “CoV infection,” can refer to infection with a coronavirus such as SARS-CoV-2, SARS-CoV-1, and/or Mers-Cov. A coronavirus infection can include coronavirus respiratory tract infections, often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (e.g., kidney failure and renal dysfunction), and/or the like.

As used herein, the term “antibody” can refer to immunoglobulin molecules comprising four polypeptide chains: two heavy polypeptide chains and two light polypeptide chains. Further, the heavy polypeptide chains can each comprise a heavy chain variable region (“VH”) and multiple heavy chain constant regions (“CH”). Likewise, the light polypeptide chains can each comprise a light chain variable region (“VL”) and a light chain constant regions (“CL”). As used herein, the term “antigen-binding fragment” (“Fab”) can refer to the variable region and constant region of the heavy polypeptide chains and light polypeptide chains that bind to a target antigen (e.g., bind to the RBD of the SARS-Cov-2 spike protein. The Fab of an antibody can comprise at least one variable region, which can be of any size or amino acid composition. In Fabs having a VHassociated with a VL, the VHand VLcan be situated relative to one another in any suitable arrangement. Alternatively, the Fab of an antibody can contain a monomeric VHor VL. In various embodiments, the Fab can comprise at least one variable region covalently linked to at least one constant region. Moreover, Fabs described herein can comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant region configurations. Further, in one or more embodiments, an antibody and/or Fab described herein can be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”) (e.g., such as an anti-viral compound, a second anti-influenza antibody, or any other therapeutic moiety useful for treating a viral infection). Moreover, in one or more embodiments, an antibody and/or Fab thereof can be, for example, a chimeric antibody, hybrid antibody and/or recombinant antibody.

Various embodiments described herein can include monoclonal anti-CoV antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term “monoclonal antibody” can refer to a population of substantially homogeneous antibodies. For example, the antibody molecules comprising a population of monoclonal antibodies can be substantially identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts.

As used herein, the terms “isolated antigen-binding proteins,” “isolated antibodies,” and/or “isolated Fabs” can refer to polypeptides, polynucleotides and/or vectors that are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, nanobody, other antibodies, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody and/or Fab can further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof.

As used herein, the term “conservatively modified variant” and/or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). Such changes can be made without significantly disrupting the biological activity of the antibody or fragment. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. In addition, substitutions of structurally or functionally similar amino acids can be less likely to significantly disrupt biological activity. Particularly, the variable regions (e.g., “VH” and “VL” in the heavy and light chains respectively) can be less conserved than the constant regions in a Fab. For example, mutations, such as amino acid substitutions, can be observed in the variable regions of the antigen-binding protein (e.g., nanobody, antibody and/or Fab thereof), which can allow the Fab to bind new targets. Also, as used herein, the term “neutralizing anti-CoV antigen-binding protein,” can refer to a molecule (e.g., a nanobody, antibody and/or Fab thereof) that can inhibit an activity of CoV to a detectable degree (e.g., can inhibit the ability of SARS-CoV-2 spike protein to bind to a receptor, such as ACE2).

Various embodiments described herein can regard neutralizing antigen-binding proteins that can bind to the RBD of one or more variants of the CoV spike protein. For example, the RBD of the SAR-COV-2 spike protein can span from the 333rdto the 527thposition of the amino acid sequence of the SAR-COV-2 spike protein and can be delineated by the amino acid sequence shown inFIG.1as SEQ ID NO: 1. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. A first variant of the RBD of the SAR-COV-2 spike protein can comprise at least the amino acid substitution E484K, as shown inFIG.1as SEQ ID NO: 2. A second variant of the RBD of the SAR-COV-2 spike protein can comprise at least the amino acid substitution E484Q, as shown inFIG.1as SEQ ID NO: 3. A third variant of the RBD of the SAR-COV-2 spike protein can comprise at least the amino acid substitution L452R, as shown inFIG.2as SEQ ID NO: 4. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. A fourth variant of the RBD of the SAR-COV-2 spike protein can comprise at least the amino acid substitution L452Q, as shown inFIG.2as SEQ ID NO: 5. A fifth possible variant of the RBD of the SAR-COV-2 spike protein can comprise a combination of amino acid substitutions at E484 and L452, such as at least the amino acid sequence substitutions E484K and the L452R amino acid, as shown inFIG.3as SEQ ID NO: 6. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. One or more embodiments described herein can comprise neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) that can exhibit an affinity to bind to the first, second, third, fourth, and/or fifth variants of the RBD of the CoV (e.g., SAR-CoV-2) spike protein. For example, one or more embodiments described herein can comprise one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) that can exhibit an affinity to bind to an RBD of CoV (e.g., SARS-CoV-2) spike protein having at least 50% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6.

The RBD of the CoV (e.g., SARS-CoV-2) spike protein (e.g., having the amino acid sequence of SEQ ID NO: 1) can bind strongly with a Fab of the Bamlanivimab antibody through two salt bridges. For example, the Fab of Bamlanivimab can have: a VHwith the amino acid sequence of SEQ ID NO: 7 (e.g., shown inFIG.4); and/or a VLwith the amino acid sequence of SEQ ID NO: 8 (e.g., shown inFIG.5). Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. The E484 amino acid in SEQ ID NO: 1 (e.g., RBD of SARS-COV-2 spike protein) can form a salt bridge with the R50 amino acid in SEQ ID NO: 7 (e.g., VHof Bamlanivimab) and the R96 amino acid in SEQ ID NO: 8 (e.g., VLof Bamlanivimab). For instance, both the R50 and R96 amino acids of Bamlanivimab can be positively charged, whereas the E484 amino acid of the RBD of the SARS-CoV-2 spike protein can be negatively charged; thereby a local electrostatic interaction can be achieved via salt bridges formed simultaneous between: the R50 amino acid of Bamlanivimab and the E484 amino acid of the RBD of the SARS-CoV-2 spike protein; and the R96 amino acid of Bamlanivimab and the E484 amino acid of the RBD of the SARS-CoV-2 spike protein.

However, after the E484K,Q amino acid substitution in the RBD of a SARS-CoV-2 spike protein variant, the resulting positively charged lysine amino acid K484 or glutamine Q484 cannot coordinate with the R50 (e.g., of SEQ ID NO: 7) and/or R96 (e.g., of SEQ ID NO: 8) amino acids in the Fab of Bamlanivimab. The three positively charged residues (i.e., R50 amino acid of SEQ ID NO: 7, R96 amino acid of SEQ ID NO: 8, and K484 or Q484 amino acid of SEQ ID NO: 2, SEQ ID: 3 SEQ ID NO: 6) cannot achieve sufficient coordination due to at least electrostatic repulsion. For instance, the E484K amino acid substitution of SEQ ID NO: 2 and/or SEQ ID NO: 4 can increase the local binding free energy by, for example, 40.65 kilocalories per mol (kcal/mol); thereby destabilizing the interfacial interaction. Various embodiments described herein can regard amino acid substitutions to the Fab of Bamlanivimab (e.g., to SEQ ID NO: 7 and/or SEQ ID NO: 8) to enhance the binding affinity of the Fab of Bamlanivimab towards the K484 or Q484 amino acid of the RBD of the one or more SARS-CoV-2 spike protein variants (e.g., of SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 6).

For example, one or more embodiments described herein can comprise amino acid substations to the Fab of Bamlanivimab (e.g., to SEQ ID NO: 7 and/or SEQ ID NO: 8) in order to: form a salt bridge with the RBD of the one or more CoV (e.g., SARS-CoV-2) spike protein variants; incorporate a hydrophobic residue into the Fab to interact with the carbon chains of the RBD of the one or more CoV (e.g., SARS-CoV-2) spike protein variants; and/or establish cation-π interactions between the Fab and the RBD of the one or more CoV (e.g., SARS-CoV-2) spike protein variants.

For instance, one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fab thereof) can comprise one or more amino acid substitutions to the VHand/or VLof Bamlanivimab, substituting R50 of SEQ ID NO: 7 and/or R96 of SEQ ID NO: 8 with negatively charged aspartic acid (D) and/or glutamic acid (E). In another instance, one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fab thereof) can comprise one or more amino acid substitutions to the VHand/or VLof Bamlanivimab, substituting R50 of SEQ ID NO: 7 and/or R96 of SEQ ID NO: 8 with a hydrophobic amino acid, including, but not limited to: leucine (L), valine (V), and/or isoleucine (I). In a further instance, one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fab thereof) can comprise one or more amino acid substitutions to the VHand/or VLof Bamlanivimab, substituting R50 of SEQ ID NO: 7 and/or R96 of SEQ ID NO: 8 with tryptophan (W), phenylalanine (F), and/or tyrosine (Y) to establish cation-n interactions with the K484 or Q484 amino acid of the RBD of the one or more SARS-COV-2 spike protein variants (e.g., of SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4).

For example, as shown inFIG.4, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VHwith at least 50% sequence identity with SEQ ID NO: 7 and one of the following amino acid substitutions of SEQ ID NO: 7: R50D, R50E, R50W, R50F, R50Y, R50L, R50V, R50I, or R50Pho. For instance, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VHhaving at least at least 50% (e.g., at least 80%) sequence identity with one of the following amino acid sequences: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16. The VLof the neutralizing antigen-binding protein can have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 8.

In another example, as shown inFIG.5, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VLwith at least 50% sequence identity with SEQ ID NO: 8 and one of the following amino acid substitutions of SEQ ID NO: 8: R96D, R96E, R96W, R96F, R96Y, R96L, R96V, R96I, or R96Pho. For instance, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., an antibody and/or Fab thereof) comprising a VLhaving at least 50% (e.g., at least 80%) sequence identity with one of the following amino acid sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The VHof the neutralizing antigen-binding protein can have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 7.

Additionally, various embodiments described herein can include one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fab thereof) having at least 50% sequence identity with Bamlanivimab's variable domain VH(e.g., defined by SEQ ID NO: 7), and comprising one or more amino acid substitutions with regards to the 154 and/or L55 amino acids of SEQ ID NO: 7. In one or more embodiments, one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fab thereof) having at least 50% sequence identity with Bamlanivimab's variable domain VH(e.g., defined by SEQ ID NO: 7), and comprising one or more amino acid substitutions with regards to the 154 and/or L55 amino acids of SEQ ID NO: 7 can exhibit enhanced binding affinity for RBD variants of SARS-CoV-2 spike proteins carrying the amino acid substitution L452R,Q (e.g., for RBD of SARS-CoV-2 spike protein having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6). _For instance, after the L452R,Q mutation, the charged R452 or Q452 amino acid cannot coordinate well with the hydrophobic 154 and/or L55 amino acids.

For instance, one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fab thereof) can comprise one or more amino acid substitutions to the VHof Bamlanivimab, substituting the 154 and/or L55 amino acids of SEQ ID NO: 7 with negatively charged aspartic acid (D) and/or glutamic acid (E). In another instance, one or more neutralizing antigen-binding proteins (e.g., antibodies and/or Fab thereof) can comprise one or more amino acid substitutions to the VHof Bamlanivimab, substituting the 154 and/or L55 amino acids of SEQ ID NO: 7 with a negatively charged amino acid, including, but not limited to: D54, E54, D55, and/or E55. In a further instance, one or more neutralizing antigen-binding proteins (e.g., antibodies and/or Fab thereof) can comprise one or more amino acid substitutions to the VHof Bamlanivimab, substituting the 154 and/or L55 amino acids of SEQ ID NO: 7 with tryptophan (W), phenylalanine (F), and/or tyrosine (Y) to establish cation-n interactions with the R452 or Q452 amino acid of the RBD of the one or more SARS-CoV-2 spike protein variants (e.g., of SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6).

For example, as shown inFIG.6, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VHwith at least 50% sequence identity with SEQ ID NO: 7 and one of the following amino acid substitutions of SEQ ID NO: 7: I54D, I54E, I54W, I54F, I54Y, I54Pho. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. For instance, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VHhaving at least 50% sequence identity with one of the following amino acid sequences: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29. The VLof the neutralizing antigen-binding protein can have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

In another example, as shown inFIG.7, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VHwith at least 50% sequence identity with SEQ ID NO: 7 and one of the following amino acid substitutions of SEQ ID NO: 7: L55D, L55E, L55W, L55F, L55Y, L55Pho. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. For instance, various embodiments described herein can include a neutralizing antigen-binding protein (e.g., a nanobody, antibody and/or Fab thereof) comprising a VHhaving at least 50% sequence identity with one of the following amino acid sequences: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. The VLof the neutralizing antigen-binding protein can have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24.

One or more embodiments described herein can include one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) comprising: a VHwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 10 (e.g., comprising the amino acid substitution R50E of SEQ ID NO: 7), and a VLwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 18 (e.g., comprising the amino acid substitution R96E of SEQ ID NO: 8). For example, the K484 amino acid of the RBD variant of SARS-CoV-2 spike protein having SEQ ID NO: 2 or SEQ ID NO: 6 (e.g., carrying the E484K amino acid substitution) can form a first salt bridge with the E50 amino acid (e.g., comprised within the VH) and/or a second salt bridge with the E96 amino acid (e.g., comprised within the VL) of the one or more neutralizing antigen-binding proteins (e.g., antibodies and/or Fabs thereof). At least because the E50 and E96 amino acids of the neutralizing antigen-binding protein can be negatively charged, the K484 amino acid of the RBD of the SARS-CoV-2 spike protein can electrostatically favor the interaction with the E50 and E96 amino acids.

Table 1, shown inFIG.8, depicts protein-protein interaction energies calculated via molecular mechanics-generalized Born surface area (“MMGBSA”). Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. In Table 1: “RBD-E484 (WT)” represents a SARS-CoV-2 spike protein comprising an RBD with the amino acid sequence of SEQ ID NO: 1; “RBD-K484” represents a SARS-CoV-2 spike protein comprising an RBD with the amino acid sequence of SEQ ID NO: 2; and “Bamlanivimab-EE” represents an antigen-binding protein (e.g., a variant of Bamlanivimab) comprising a VHwith an amino acid sequence having at least 50% sequence identity with of SEQ ID NO: 10 (e.g., comprising the amino acid substitution R50E of SEQ ID NO: 7), and a VLwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 18 (e.g., comprising the amino acid substitution R96E of SEQ ID NO: 8) in accordance with various embodiments described herein. As shown in Table 1, the binding free energy for a complex of Bamlanivimab and the SARS-CoV-2 spike protein comprising a RBD having the amino acid sequence of SEQ ID NO: 1 can be −157.87 kcal/mol. However, after the E484K mutation, the binding free energy can increase to −108.24 kcal/mol (e.g., suggesting that the interfacial interaction is substantially weakened. In contrast, the binding free energy for a complex of an antigen-binding protein (e.g., a variant of Bamlanivimab) comprising a VHwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 10 (e.g., comprising the amino acid substitution R50E of SEQ ID NO: 7), and a VLwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 18 (e.g., comprising the amino acid substitution R96E of SEQ ID NO: 8) and the SARS-CoV-2 spike protein carrying the E484K mutation can be −156.79 kcal/mol; thereby demonstrating that the various neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) can achieve a binding affinity towards the SARS-CoV-2 variant that is comparable to that between Bamlanivimab and the RBD of the SARS-CoV-2 nonvariant.

One or more embodiments described herein can include one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) comprising: a VHwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 10 (e.g., comprising the amino acid substitution R50E of SEQ ID NO: 7), and a VLwith an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 19 (e.g., comprising the amino acid substitution R96W of SEQ ID NO: 8). For example, the K484 amino acid of the RBD variant of SARS-CoV-2 spike protein having SEQ ID NO: 2 or SEQ ID NO: 6 (e.g., carrying the E484K amino acid substitution) can form a salt bridge with the E50 amino acid (e.g., comprised within the VH) and/or favorable cation-π interactions with the W96 amino acid (e.g., comprised within the VL) of the one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof).

In one or more embodiments, the various neutralizing antigen-binding proteins described herein can be isolated antibodies. In various embodiments, the VHand/or VLregions described herein can be conservatively modified variants of the example amino acid sequences. For example, conservatively modified variants of SEQ ID NO: 9- SEQ ID NO: 34. Additionally, in accordance with the various embodiments described herein, the various neutralizing antigen-binding proteins described herein can be included in one or more therapeutic compounds. For example, the one or more neutralizing antigen-binding proteins can be included in a composition with a pharmaceutically acceptable carrier and/or other therapeutic agent (e.g., an anti-inflammatory agent, an antibody that binds to TMPRSS2, and/or an antibody that binds to the SARS-CoV-2 spike protein). In another example, the one or more neutralizing antigen-binding proteins described herein can be included in one or more combination therapies (e.g., therapies that further include Bamlanivimab, remdesivir, and/or dexamethasone).

FIG.9illustrates a flow diagram of an example, non-limiting method 900 that can facilitate treating a CoV infection with one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) in accordance with one or more embodiments described herein. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. In various embodiments, method 900 can be employed to facilitate treatment of a CoV infection comprising SARS-CoV-2 carrying the E484K,Q mutation.

At 902, the method 900 can comprise contacting one or more antigen-binding proteins with the RBD of a SARS-CoV-2 spike protein variant carrying the E484K,Q mutation. For example, the RBD of the SARS-CoV-2 spike protein variant can have the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In various embodiments, the one or more antigen-binding proteins can comprise a variant of Bamlanivimab's VHand/or VLregions, and can comprise one or more amino acid substitutions of the R50, 154, L55, and/or R96 amino acids in accordance with various embodiments described herein. For instance, the one or more antigen-binding proteins can be a variant of Bamlanivimab's VHand/or VLregions comprising one or more amino acid substitutions of the R50 and/or R96 amino acids with: one or more negatively charged amino acids (e.g., D and/or E), one or more hydrophobic residues (e.g., L, V, and/or I), one or more phosphorylated residues (e.g., serine, threonine, or tyrosine), and/or one or more molecules (e.g., amino acids W, F, and/or Y) that can establish cation-π interactions with the RBD.

In various embodiments, the contacting at 902 can be facilitated by administering an effective concentration (e.g., 35 milligrams per milliliter (mg/mL)) of the one or more antigen-binding proteins to a patient inflicted with a CoV infection. For example, the one or more antigen-binding proteins can be included in one or more therapeutic compositions (e.g., comprising a pharmaceutically acceptable carrier) that can be injected into the patient via a needle.

At 904, the method 900 can comprise inhibiting the ability of the SARS-CoV-2 spike protein variant from binding to a receptor. For example, the one or more antigen-binding proteins can inhibit the SARS-CoV-2 spike protein variant from binding to receptor ACE2. For instance, the one or more antigen-binding proteins can form one or more salt bridges with the RBD of the SARS-CoV-2 spike protein variant to facilitate the inhibiting at 904. In another instance, one or more hydrophobic residues of the one or more antigen-binding proteins can interact with one or more carbon chains of K484 in the RBD of the SARS-CoV-2 spike protein variant to facilitate the inhibiting at 904. In a further instance, the one or more antigen-binding proteins can form one or more cation-π interactions with the RBD of the SARS-CoV-2 spike protein variant to facilitate the inhibiting at 904.

FIG.10illustrates a flow diagram of an example, non-limiting method 1000 that can facilitate treating a CoV infection with one or more neutralizing antigen-binding proteins (e.g., nanobodies, antibodies and/or Fabs thereof) in accordance with one or more embodiments described herein. Repetitive description of like elements employed in other embodiments described herein is omitted for the sake of brevity. In various embodiments, method 1000 can be employed to facilitate treatment of a CoV infection comprising SARS-CoV-2 carrying the L452R,Q mutation.

At 1002, the method 1000 can comprise contacting one or more antigen-binding proteins with the RBD of a SARS-CoV-2 spike protein variant carrying the L452R,Q mutation. For example, the RBD of the SARS-CoV-2 spike protein variant can have the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6. In various embodiments, the one or more antigen-binding proteins can be a variant of Bamlanivimab's VHand/or VLregions (e.g., be a variant of Bamlanivimab), and can comprise one or more amino acid substitutions of the R50, 154, L55, and/or R96 amino acids in accordance with various embodiments described herein. For instance, the one or more antigen-binding proteins can comprise variant of Bamlanivimab's VHand/or VLregions (e.g., be a variant of Bamlanivimab) comprising one or more amino acid substitutions of the 154, L55, and/or R96 amino acids with: one or more negatively charged amino acids (e.g., D and/or E), one or more hydrophobic residues (e.g., L, V, or I), one or more phosphorylated residues (e.g., serine, threonine, or tyrosine), and/or one or more molecules (e.g., amino acids W, F, and/or Y) that can establish cation-π interactions with the RBD.

In various embodiments, the contacting at 1002 can be facilitated by administering an effective concentration (e.g., 35 milligrams per milliliter (mg/mL)) of the one or more antigen-binding proteins to a patient inflicted with a CoV infection. For example, the one or more antigen-binding proteins can be included in one or more therapeutic compositions (e.g., comprising a pharmaceutically acceptable carrier) that can be injected into the patient via a needle.

At 1004, the method 1000 can comprise inhibiting the ability of the SARS-CoV-2 spike protein variant from binding to a receptor. For example, the one or more antigen-binding proteins can inhibit the SARS-CoV-2 spike protein variant from binding to receptor ACE2. For instance, the one or more antigen-binding proteins can form one or more salt bridges with the RBD of the SARS-CoV-2 spike protein variant to facilitate the inhibiting at 1004. In another instance, one or more hydrophobic residues of the one or more antigen-binding proteins can interact with one or more carbon chains of the RBD of the SARS-CoV-2 spike protein variant to facilitate the inhibiting at 1004. In a further instance, the one or more antigen-binding proteins can form one or more cation-π interactions with the RBD of the SARS-CoV-2 spike protein variant to facilitate the inhibiting at 1004.