Coronavirus neutralizing humanized antibodies and uses thereof
12024551 · 2024-07-02
Assignee
Inventors
- Brooke Nicole Harmon (Livermore, CA)
- Maxwell Stefan (Pleasanton, CA, US)
- Yooli Kim Light (Pleasanton, CA)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
International classification
A61K47/68
HUMAN NECESSITIES
Abstract
The present disclosure relates to an isolated or purified antibody, or a fragment thereof, having a binding domain that binds to a coronavirus (e.g., SARS-COV-2) or a portion thereof. In other embodiments, the antibody includes a binding domain that competes with binding to angiotensin converting enzyme 2 (ACE2) or a portion thereof. Methods of using such antibodies are also described herein, such as methods of treating or delaying the progression of a disease associated with a coronavirus.
Claims
1. An isolated or purified single-domain antibody comprising a variable domain, wherein the variable domain comprises: (i) a first complementarity determining region comprising a polypeptide sequence of SEQ ID NO:40, a second complementarity determining region comprising a polypeptide sequence of SEQ ID NO:74, and a third complementarity determining region comprising a polypeptide sequence of SEQ ID NO:108; or (ii) a first complementarity determining region comprising a polypeptide sequence of SEQ ID NO:41, a second complementarity determining region comprising a polypeptide sequence of SEQ ID NO:75, and a third complementarity determining region comprising a polypeptide sequence of SEQ ID NO:109; or (iii) a first complementarity determining region comprising a polypeptide sequence of SEQ ID NO:42, a second complementarity determining region comprising a polypeptide sequence of SEQ ID NO:76, and a third complementarity determining region comprising a polypeptide sequence of SEQ ID NO:110; or (iv) a first complementarity determining region comprising a polypeptide sequence of SEQ ID NO:43, a second complementarity determining region comprising a polypeptide sequence of SEQ ID NO:77, and a third complementarity determining region comprising a polypeptide sequence of SEQ ID NO:111, wherein the variable domain binds to a spike protein of a coronavirus or a receptor-binding domain of a coronavirus, and wherein the coronavirus is severe acute respiratory syndrome coronavirus 2.
2. The single-domain antibody of claim 1, wherein the variable domain further comprises: a first framework region attached to an N-terminus of the first complementarity determining region; a second framework region disposed between the first and second complementarity determining regions; a third framework region disposed between the second and third complementarity determining regions; and a fourth framework region attached to a C-terminus of the third complementarity determining region.
3. The single-domain antibody of claim 2, wherein the variable domain comprises a polypeptide sequence of any one of SEQ ID NOS:1-4.
4. The single-domain antibody of claim 2, wherein the first framework region comprises a polypeptide sequence of SEQ ID NO:191, wherein the second framework region comprises a polypeptide sequence of SEQ ID NO:221, wherein the third framework region comprises a polypeptide sequence of SEQ ID NO:252, and wherein the fourth framework region comprises a polypeptide sequence of SEQ ID NO:290.
5. The single-domain antibody of claim 2, wherein the first framework region comprises a polypeptide sequence of SEQ ID NO:190, wherein the second framework region comprises a polypeptide sequence of SEQ ID NO:220, wherein the third framework region comprises a polypeptide sequence of SEQ ID NO:251, and wherein the fourth framework region comprises a polypeptide sequence of SEQ ID NO:290.
6. The single-domain antibody of claim 1, wherein the spike protein comprises a polypeptide sequence of any one of SEQ ID NOS:320-326; or wherein the receptor-binding domain comprises a polypeptide sequence of any one of SEQ ID NOS:328-334.
7. The single-domain antibody of claim 1, further comprising a therapeutic agent or a diagnostic agent attached directly or indirectly to the variable domain.
8. A method of treating or prophylactically treating a viral infection, the method comprising: administering a single-domain antibody of claim 1 to a subject in need thereof, wherein the viral infection comprises an infection from a coronavirus, and wherein the coronavirus is severe acute respiratory syndrome coronavirus 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(13) The present invention relates to an isolated or purified antibody or fragment thereof, that binds to SARS-COV-2 or a portion thereof (e.g., the spike protein or receptor-binding domain (RBD) of the spike protein). We have identified CDRs that provide enhanced efficacy, as determined by reducing infectivity of a coronavirus and/or exhibiting binding to the coronavirus. Such CDRs can be provided in an antibody having any useful format, such as a nanobody or other forms described herein.
(14) CDRs can be identified by using a phage library (
(15) The selections from the phage library can be screened. Such screening can include testing by various assays, including a competition enzyme-linked immunosorbent assay (ELISA), a binding assay with the coronavirus or a portion thereof (e.g., SARS-COV-2, the spike protein of SARS-COV-2, or the RBD of SARS-COV-2), a competition assay including the coronavirus (or a portion thereof) and a target of the coronavirus (e.g., human ACE2 or another receptor that binds to SARS-COV-2), and/or a neutralization assay including coronavirus-expressing cells.
(16) The antibody can have any useful form, such as one or more of the following domains: a single variable domain (VHH) or nanobody, a variable heavy domain (VH), a variable light domain (VL), a single-chain variable-fragment (scFv) antibody, a monoclonal antibody (mAb), an antigen-binding fragment (Fab), a fragment crystallizable region (Fc region), a heavy-chain only antibody (HcAb), an Immunoglobulin G (IgG) antibody, as well as bivalent, trivalent, tetravalent, multivalent, biparatropic, bispecific, multispecific chimeric, and humanized forms thereof. Any of the forms can include a linker between a first amino acid sequence (any domain herein, e.g., such as VH) and a second amino acid sequence (any domain herein, e.g. such as VL).
(17) The constructs herein can be employed as a nanobody, preferably human or humanized. In some embodiments, the nanobody has a molecular weight of about 12-15 kDa. In some embodiments, the nanobody can include a single VHH domain. In other embodiments, the nanobody can include a plurality of VHH domains, in which each VHH domain can be the same or different. Furthermore, each VHH domain can bind to the same target, the same portion of the same target, different targets, or different portions of the same target. Non-limiting targets are described herein.
(18) One or more VHH domains can be fused to any useful amino acid sequence, such as a Fc domain or a humanized Fc domain. The VHH domains can be fused to other humanized forms of constant domains, such as Fc, CH1, CH2, CH3, and CL domains. Such VHH domains can be sued to other forms, such as IgG1, IgG3, IgA, or IgM.
(19) Yet other forms can include an Fc region and a Fab; or an Fc region and a VHH. The Fc region can include heavy chains present in any useful isotype (A, E, G, or M), such as Immunoglobulin G (e.g., IgG1, IgG2a, or IgG3). The Fab region can include domains from the heavy and light chains, including the variable heavy (VH) and variable light (VL) domains. Within the VH and VL domains, CDRs can be configured to bind to a target of interest. The VHH domain can include a single variable domain having CDRs configured to bind to a target of interest.
(20) The Fc region of the constructs herein can include a native immunoglobulin, i.e., as formed by the dimeric association of the respective Fc domains (or Fc moieties) of its two heavy chains, in which a native Fc region is homodimeric and comprises two polypeptide chains; or a genetically-fused Fc region or a single-chain Fc region (scFc region), in which a synthetic dimeric Fc region comprised of Fc domains (or Fc moieties) are genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence) as described. In one embodiment, the construct includes a complete Fc region, whether present as one polypeptide chain (an scFc molecule) or in the wild-type form as two polypeptide chains.
(21) The Fc region can include a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc moiety comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, a Fc moiety comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In another embodiment, an Fc moiety comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In another embodiment, an Fc moiety consists of a CH3 domain or portion thereof. In another embodiment, an Fc moiety consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In another embodiment, a Fc moiety consists of a CH2 domain (or portion thereof) and a CH3 domain. In another embodiment, a Fc moiety consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In one embodiment, an Fc moiety lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
(22) The Fc domains or moieties of a polypeptide may be from any isotype (A, E, G, or M) and may be derived from different immunoglobulin molecules. For example, an Fc domain or moiety of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, an Fc domain or moiety can comprise a chimeric hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, an Fc domain or moiety can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
(23) The constructs herein can be modified antibodies, which includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); engineered antibodies having synthetic linkers, such as any described herein; and multispecific forms of antibodies (e.g., bispecific, trispecific, etc., forms of any antibody, such as a nanobody) altered to bind to two or more different antigens e.g., to the RBD of a coronavirus and another therapeutically relevant target binding site.
(24) Modified antibodies can include other types of modifications, such as chemical modification (e.g., pegylation, glycosylation, lipidation, etc.), attachment to a particle or liposome, or bonding to a protein (e.g., a serum protein, a cytokine) or a cell (e.g., a CAR-T cell).
(25) The constructs herein can be chimeric or fusion proteins. Such proteins comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created using methods well known in the art, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
(26) Such forms or fusions can include a linker disposed between any number of domains, in which non-limiting linkers are described herein. Any useful linker can be employed, such as a peptide linker that can be cleavable or non-cleavable. Linkers can include or consist of a sequence according to the formula [(Gly).sub.m(Ser)].sub.n(Gly).sub.p, where each of m, n, and p is, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m=1, 2, 3, 4, 5, or 6; n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p=0, 1, 2, 3, or 4. Alternatively, the linker sequence includes or consists of a sequence according to the formula (Gly).sub.p[(Ser)(Gly).sub.m].sub.n, where each of m, n, and p is, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m=1, 2, 3, 4, 5, or 6; n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p=0, 1, 2, 3, or 4. In another embodiment, the linker sequence includes or consists of a sequence according to the formula [(Gly).sub.m(Ser)(Gly).sub.p].sub.n, where each of m, n, and p is, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m=1, 2, 3, 4, 5, or 6; n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p=0, 1, 2, 3, or 4. Further non-limiting linkers include any described herein, such as in SEQ ID NOs:310-319 (Table 1).
(27) TABLE-US-00001 TABLE1 Non-limitinglinkers Name Sequence SEQIDNO: G.sub.3 GGG 310 G.sub.3S GGGS 311 G.sub.4 GGGG 312 G.sub.4S GGGGS 313 G.sub.2SG GGSG 314 (G.sub.4S).sub.2 GGGGSGGGGS 315 (G.sub.4S).sub.3 GGGGSGGGGSGGGGS 316 (G.sub.4S).sub.4 GGGGSGGGGSGGGGSGGGGS 317 (G.sub.2SG).sub.2 GGSGGGSG 318 (G.sub.2SG).sub.3 GGSGGGSGGGSG 319
(28) The constructs can include other variations. Such variations can include one or more amino acids that facilitate humanization of an initial sequence. Humanization can include use of one or more amino acids present in a human form of the constant or variable regions (e.g., frameworks regions or CDRs). In other embodiments, the variation can include a sequence that lacks Cys and Met residues. In yet other embodiments, the CDR can have an altered length, such as a length from about 4-9 amino acids, 9-12 amino acids, or 12-15 amino acids.
(29) The construct can be characterized in any useful manner. In one instance, the construct can bind a target (e.g., any described herein), in which such binding can be characterized by a dissociation constant (K.sub.d) of about 0.5 nM to 1 ?M and/or a half maximal inhibitory concentration (IC.sub.50) of about 0.001-100 nM. In other embodiments, efficacy of binding can be characterized by a half maximal effective concentration (EC.sub.50) of about 0.001-100 nM.
(30) Binding Domains
(31) The binding domain can have any useful sequence. In one embodiment, the binding domain includes or is a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-34 (
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(33) Within the variable domain of the antibody, three CDRs can be present. Non-limiting CDRs can include a first CDR, a second CDR, and a third CDR. Any of these CDRs can include or be a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs:40-141 (
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(35) In some embodiments, each X in SEQ ID NO: 150 can be an amino acid in any one of SEQ ID NOs:40-73 when any one of the sequences in SEQ ID NOs:40-73 is used as a reference sequence to be optimally aligned with SEQ ID NO:150. In other embodiments, each X in SEQ ID NO:150 can be an amino acid in any one of SEQ ID NOs:40-58 when any one of the sequences in SEQ ID NOs:40-58 is used as a reference sequence to be optimally aligned with SEQ ID NO:150.
(36) In some embodiments, the first CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs: 151-154: X.sub.1TFGX.sub.5YR (SEQ ID NO:151), X.sub.1TSGX.sub.5YR (SEQ ID NO:152), X.sub.1AFGX.sub.5YR (SEQ ID NO:153), or X.sub.1FFGX.sub.5YR (SEQ ID NO:154), wherein: X.sub.1 is G, R, H, K, N, Q, S, T, F, Y, or absent (e.g., G, R, H, Q, S, T, F, or absent); and X.sub.5 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., L, R, H, S, T, Y, or W).
(37) In some embodiments, each X in SEQ ID NOs: 151-154 can be an amino acid in any one of SEQ ID NOs:40-73 when any one of the sequences in SEQ ID NOs:40-73 is used as a reference sequence to be optimally aligned with SEQ ID NOs:151-154. In other embodiments, each X in SEQ ID NOs: 151-154 can be an amino acid in any one of SEQ ID NOs:40-58 when any one of the sequences in SEQ ID NOs:40-58 is used as a reference sequence to be optimally aligned with SEQ ID NOs: 151-154.
(38) In some embodiments, the first CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 155: X.sub.1X.sub.2YGX.sub.5YR wherein: X.sub.1 is G, R, H, K, N, Q, S, T, F, Y, or absent (e.g., G, R, H, Q, S, T, F, or absent); X.sub.2 is A, V, I, L, R, H, K, S, T, F, Y, or P (e.g., A, R, S, T, F, or P); and X.sub.5 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., L, R, H, S, T, Y, or W).
(39) In some embodiments, each X in SEQ ID NO: 155 can be an amino acid in any one of SEQ ID NOs:40-73 when any one of the sequences in SEQ ID NOs:40-73 is used as a reference sequence to be optimally aligned with SEQ ID NO:155. In other embodiments, each X in SEQ ID NO:155 can be an amino acid in any one of SEQ ID NOs:40-58 when any one of the sequences in SEQ ID NOs:40-58 is used as a reference sequence to be optimally aligned with SEQ ID NO: 155.
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(41) In some embodiments, each X in SEQ ID NO: 160 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs:74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO:160. In other embodiments, each X in SEQ ID NO:160 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO:160.
(42) In other embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:161: X.sub.5GX.sub.7X.sub.8GHTR, wherein: X.sub.5 is G, D, E, S, T, or absent (e.g., G, D, S, or absent); X.sub.7 is G, A, V, I, L, D, E, R, H, K, S, T, or absent (e.g., G, A, E, H, S, or absent); and X.sub.8 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, F, or Y (e.g., G, A, D, R, Q, T, or F).
(43) In some embodiments, each X in SEQ ID NO:161 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO: 161. In other embodiments, each X in SEQ ID NO:161 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO: 161.
(44) In yet other embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 162: X.sub.1AX.sub.3AGYAQ, wherein: X.sub.1 is G, D, E, R, H, K, or absent (e.g., D, R, or absent); and X.sub.3 is G, D, E, S, T, F, Y, W, or absent (e.g., G, D, S, T, W, or absent).
(45) In some embodiments, each X in SEQ ID NO: 162 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO:162. In other embodiments, each X in SEQ ID NO:162 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs:74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO:162.
(46) In some embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 163: GX.sub.3X.sub.4DX.sub.6STX.sub.9, wherein: X.sub.3 is G, D, E, S, T, F, Y, W, or absent (e.g., G, D, S, T, W, or absent); X.sub.4 is G, A, V, I, L, S, or T (e.g., G, A, or S); X.sub.6 is G, A, V, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, D, K, Q, S, T, Y, or absent); and X.sub.9 is G, A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, R, H, K, Q, T, F, Y, W, or absent).
(47) In some embodiments, each X in SEQ ID NO:163 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO:163. In other embodiments, each X in SEQ ID NO: 163 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO: 163.
(48) In other embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 164: X.sub.2WX.sub.4GX.sub.6SAX.sub.9, wherein: X.sub.2 is G, A, V, I, L, D, E, S, T, or absent (e.g., G, A, D, S, T, or absent); X.sub.4 is G, A, V, I, L, S, or T (e.g., G, A, or S); X.sub.6 is G, A, V, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, D, K, Q, S, T, Y, or absent); and X.sub.9 is G, A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, R, H, K, Q, T, F, Y, W, or absent).
(49) In some embodiments, each X in SEQ ID NO:164 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO:164. In other embodiments, each X in SEQ ID NO:164 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO:164.
(50) In yet other embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 165: X.sub.2WX.sub.4GX.sub.6SX.sub.8X.sub.9, wherein: X.sub.2 is G, A, V, I, L, D, E, S, T, or absent (e.g., G, A, D, S, T, or absent); X.sub.4 is G, A, V, I, L, S, or T (e.g., G, A, or S); X.sub.6 is G, A, V, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, D, K, Q, S, T, Y, or absent); X.sub.8 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., G, A, D, R, Q, T, or F); and X.sub.9 is G, A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, R, H, K, Q, T, F, Y, W, or absent).
(51) In some embodiments, each X in SEQ ID NO: 165 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO:165. In other embodiments, each X in SEQ ID NO:165 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO: 165.
(52) In other embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:166:
(53) TX.sub.3X.sub.4GX.sub.6X.sub.7X.sub.8R, wherein: X.sub.3 is G, D, E, S, T, F, Y, W, or absent (e.g., G, D, S, T, W, or absent); X.sub.4 is G, A, V, I, L, S, or T (e.g., G, A, or S); X.sub.6 is G, A, V, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, D, K, Q, S, T, Y, or absent); X.sub.7 is G, A, V, I, L, D, E, R, H, K, S, T, or absent (e.g., G, A, E, H, S, or absent); and X.sub.8 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., G, A, D, R, Q, T, or F).
(54) In some embodiments, each X in SEQ ID NO:166 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO:166. In other embodiments, each X in SEQ ID NO:166 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO:166.
(55) In yet other embodiments, the second CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:167:
(56) TX.sub.3X.sub.4G-X.sub.7X.sub.8X.sub.9X.sub.10, wherein: X.sub.3 is G, D, E, S, T, F, Y, W, or absent (e.g., G, D, S, T, W, or absent); X.sub.4 is G, A, V, I, L, S, or T (e.g., G, A, or S); X.sub.7 is G, A, V, I, L, D, E, R, H, K, S, T, or absent (e.g., G, A, E, H, S, or absent); X.sub.8 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., G, A, D, R, Q, T, or F); X.sub.9 is G, A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., G, A, R, H, K, Q, T, F, Y, W, or absent); and X.sub.10 is G, S, T, R, H, K, or absent (e.g., S, T, R, H, K, or absent).
(57) In some embodiments, each X in SEQ ID NO: 167 can be an amino acid in any one of SEQ ID NOs: 74-107 when any one of the sequences in SEQ ID NOs: 74-107 is used as a reference sequence to be optimally aligned with SEQ ID NO: 167. In other embodiments, each X in SEQ ID NO:167 can be an amino acid in any one of SEQ ID NOs: 74-92 when any one of the sequences in SEQ ID NOs: 74-92 is used as a reference sequence to be optimally aligned with SEQ ID NO: 167.
(58)
(59) In some embodiments, each X in SEQ ID NO: 170 can be an amino acid in any one of SEQ ID NOs: 108-141 when any one of the sequences in SEQ ID NOs: 108-141 is used as a reference sequence to be optimally aligned with SEQ ID NO:170. In other embodiments, each X in SEQ ID NO:170 can be an amino acid in any one of SEQ ID NOs: 108-126 when any one of the sequences in SEQ ID NOs: 108-126 is used as a reference sequence to be optimally aligned with SEQ ID NO:170.
(60) In some embodiments, the third CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:171: NAQLGPX.sub.22X.sub.23DKIG, wherein: X.sub.22 is G, A, V, I, L, D, E, R, H, K, N, Q, or absent (e.g., G, A, V, E, H, K, Q, or absent); and X.sub.23 is P, F, Y, W, or absent (e.g., F, Y, W, or absent).
(61) In some embodiments, each X in SEQ ID NO: 171 can be an amino acid in any one of SEQ ID NOs: 108-141 when any one of the sequences in SEQ ID NOs: 108-141 is used as a reference sequence to be optimally aligned with SEQ ID NO:171. In other embodiments, each X in SEQ ID NO:171 can be an amino acid in any one of SEQ ID NOs: 108-126 when any one of the sequences in SEQ ID NOs: 108-126 is used as a reference sequence to be optimally aligned with SEQ ID NO: 171.
(62) In other embodiments, the third CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:172: X.sub.6WDX.sub.9X.sub.10GX.sub.12X.sub.13X.sub.14X.sub.15-------FX.sub.24, wherein: X.sub.6 is G, A, V, I, L, D, E, R, H, K, S, T, P, F, Y, or absent (e.g., A, L, D, R, S, Y, or absent); X.sub.9 is G, A, V, I, L, R, H, K, N, Q, P, F, Y, or absent (e.g., A, I, R, Q, F, Y, or absent); X.sub.10 is G, A, V, I, L, D, E, N, Q, S, T, P, F, Y, W, or absent (e.g., V, A, E, N, Q, S, T, P, F, Y, or absent); X.sub.12 is G, A, V, I, L, P, F, Y, W, or absent (e.g., V, I, L, P, W, or absent); X.sub.13 is G, A, V, I, L, N, Q, R, H, K, P, F, Y, W, or absent (e.g., I, V, N, H, P, F, or absent); X.sub.14 is D, E, R, H, K, S, T, P, F, Y, W, or absent (e.g., D, R, H, S, T, P, or absent); X.sub.15 is A, V, I, L, D, E, R, H, K, N, Q, P, F, Y, W, or absent (e.g., A, E, R, Q, F, or absent); and X.sub.24 is D, E, R, H, K, P, F, Y, W, or absent (e.g., D, R, K, P, or absent).
(63) In some embodiments, each X in SEQ ID NO: 172 can be an amino acid in any one of SEQ ID NOs: 108-141 when any one of the sequences in SEQ ID NOs: 108-141 is used as a reference sequence to be optimally aligned with SEQ ID NO:172. In other embodiments, each X in SEQ ID NO:172 can be an amino acid in any one of SEQ ID NOs: 108-126 when any one of the sequences in SEQ ID NOs: 108-126 is used as a reference sequence to be optimally aligned with SEQ ID NO:172.
(64) In other embodiments, the third CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:173: YWX.sub.8X.sub.9NGX.sub.12X.sub.13PF-----X.sub.23X.sub.24, wherein: X.sub.8 is G, A, V, I, L, D, E, R, H, K, P, F, Y, or absent (e.g., A, D, K, F, Y, or absent); X.sub.9 is G, A, V, I, L, R, H, K, N, Q, P, F, Y, or absent (e.g., A, I, R, Q, F, Y, or absent); X.sub.12 is G, A, V, I, L, P, F, Y, W, or absent (e.g., V, I, L, P, W, or absent); X.sub.13 is G, A, V, I, L, N, Q, R, H, K, P, F, Y, W, or absent (e.g., I, V, N, H, P, F, or absent); X.sub.23 is P, F, Y, W, or absent (e.g., F, Y, W, or absent); and X.sub.24 is D, E, R, H, K, P, F, Y, W, or absent (e.g., D, R, K, P, or absent).
(65) In some embodiments, each X in SEQ ID NO:173 can be an amino acid in any one of SEQ ID NOs: 108-141 when any one of the sequences in SEQ ID NOs: 108-141 is used as a reference sequence to be optimally aligned with SEQ ID NO:173. In other embodiments, each X in SEQ ID NO:173 can be an amino acid in any one of SEQ ID NOs: 108-126 when any one of the sequences in SEQ ID NOs:108-126 is used as a reference sequence to be optimally aligned with SEQ ID NO:173.
(66) In other embodiments, the third CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:174: FPX.sub.12X.sub.13D--X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21X.sub.22W, wherein: X.sub.12 is G, A, V, I, L, P, F, Y, W, or absent (e.g., V, I, L, P, W, or absent); X.sub.13 is G, A, V, I, L, N, Q, R, H, K, P, F, Y, W, or absent (e.g., I, V, N, H, P, F, or absent); X.sub.17 is G, A, V, I, L, R, H, K, N, Q, S, T, P, F, Y, W, or absent (e.g., G, A, V, R, K, Q, S, F, or absent); X.sub.18 is G, A, V, I, L, R, H, K, N, Q, or absent (e.g., G, V, L, K, N, Q, or absent); X.sub.19 is A, V, I, L, D, E, R, H, K, S, T, P, F, Y, W, or absent (e.g., A, L, E, R, S, T, P, Y, or absent); X.sub.20 is G, D, E, N, Q, P, F, Y, W, or absent (e.g., G, D, E, N, P, or absent); X.sub.21 is A, V, I, L, R, H, K, D, E, S, T, N, Q, P, F, Y, W, or absent (e.g., L, R, E, T, N, P, Y, or absent); and X.sub.22 is G, A, V, I, L, D, E, R, H, K, N, Q, or absent (e.g., G, A, V, E, H, K, Q, or absent).
(67) In some embodiments, each X in SEQ ID NO:174 can be an amino acid in any one of SEQ ID NOs: 108-141 when any one of the sequences in SEQ ID NOs: 108-141 is used as a reference sequence to be optimally aligned with SEQ ID NO:174. In other embodiments, each X in SEQ ID NO:174 can be an amino acid in any one of SEQ ID NOs: 108-126 when any one of the sequences in SEQ ID NOs: 108-126 is used as a reference sequence to be optimally aligned with SEQ ID NO: 174.
(68) In yet other embodiments, the third CDR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:175: FPLX.sub.13D--X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21GW, wherein: X.sub.13 is G, A, V, I, L, N, Q, R, H, K, P, F, Y, W, or absent (e.g., I, V, N, H, P, F, or absent); X.sub.17 is G, A, V, I, L, R, H, K, N, Q, S, T, P, F, Y, W, or absent (e.g., G, A, V, R, K, Q, S, F, or absent); X.sub.18 is G, A, V, I, L, R, H, K, N, Q, or absent (e.g., G, V, L, K, N, Q, or absent); X.sub.19 is A, V, I, L, D, E, R, H, K, S, T, P, F, Y, W, or absent (e.g., A, L, E, R, S, T, P, Y, or absent); X.sub.20 is G, D, E, N, Q, P, F, Y, W, or absent (e.g., G, D, E, N, P, or absent); and X.sub.21 is A, V, I, L, R, H, K, D, E, S, T, N, Q, P, F, Y, W, or absent (e.g., L, R, E, T, N, P, Y, or absent).
(69) In some embodiments, each X in SEQ ID NO: 175 can be an amino acid in any one of SEQ ID NOs: 108-141 when any one of the sequences in SEQ ID NOs: 108-141 is used as a reference sequence to be optimally aligned with SEQ ID NO:175. In other embodiments, each X in SEQ ID NO: 175 can be an amino acid in any one of SEQ ID NOs: 108-126 when any one of the sequences in SEQ ID NOs: 108-126 is used as a reference sequence to be optimally aligned with SEQ ID NO: 175.
(70) The construct can include one or more binding domains. The binding domain can also be characterized by its binding affinity to a binding sequence. The terms binding sequence, binding domain, or binding site, as used herein, refer to the portion, region, or site of polypeptide that mediates specific binding with a target molecule (e.g., a SARS-COV-2 as the target, including the spike protein or RBD thereof). Exemplary binding domains include an antigen binding site (e.g., a VHH, VH, and/or VL domain) or molecules comprising such a binding site (e.g., an antibody or a single domain antibody). In one instance, a plurality of CDRs can be taken together to form a binding domain for the construct.
(71) Non-limiting constructs have CDRs and framework regions (FRs). As can be seen, each CDR can be disposed between two FRs. As seen in
(72)
(73) In some embodiments, the construct includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:180: X.sub.1VQLX.sub.5X.sub.6SGX.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17LX.sub.19LX.sub.21CX.sub.23X.sub.24SGX.sub.27X.sub.28X.sub.29X.sub.30-CDR1-X.sub.31X.sub.32WX.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39X.sub.40X.sub.41X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49-CDR2-X.sub.50X.sub.51X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59X.sub.60X.sub.61X.sub.62X.sub.63DX.sub.65X.sub.66X.sub.67X.sub.68X.sub.69X.sub.70X.sub.71X.sub.72X.sub.73X.sub.74X.sub.75 X.sub.76X.sub.77X.sub.78X.sub.79X.sub.80DX.sub.82X.sub.83X.sub.84YX.sub.86X.sub.87X.sub.88X.sub.89-CDR3-X.sub.90X.sub.91GX.sub.93GX.sub.95X.sub.96X.sub.97X.sub.98 VSX.sub.101, wherein: X.sub.1 is A, V, I, L, D, E, R, H, K, N, or Q (e.g., A, D, E, H, or Q); X.sub.5 is A, V, I, L, N, or Q (e.g., V, L, or Q); X.sub.6 is A, V, I, L, D, E, N, or Q (e.g., A, E, or Q); X.sub.9 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, or P); X.sub.10 is G, A, V, I, L, D, E, N, Q, S, or T (e.g., G, A, E, N, or S); X.sub.11 is A, V, I, L, S, T, F, Y, or W (e.g., V, L, S, or F); X.sub.12 is A, V, I, L, R, H, K, M, S, or T (e.g., V, K, or M); X.sub.13 is R, H, K, N, or Q (e.g., R, K, or Q); X.sub.14 is A, V, I, L, F, Y, or P (e.g., A or P); X.sub.15 is G, S, or T (e.g., G or S); X.sub.16 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, E, R, Q, or S); X.sub.17 is S, T, N, or Q (e.g., S or T); X.sub.19 is R, H, K, S, or T (e.g., R, K, S, or T); X.sub.21 is S, T, N, or Q (e.g., S or T); X.sub.23 is G, A, V, I, L, R, H, K, S, or T (e.g., A, V, K, or T); X.sub.24 is A, V, I, L, F, Y, or W (e.g., A, V, I, or F); X.sub.27 is G, F, Y, W, or absent (e.g., G, F, or absent); X.sub.28 is R, H, K, S, T, P, F, Y, W, or absent (e.g., K, S, T, P, or absent); X.sub.29 is A, V, I, L, D, E, R, H, K, F, Y, W, or absent (e.g., V, I, E, R, F, or absent); X.sub.30 is G, D, E, N, Q, S, T, F, Y, W, or absent (e.g., G, D, E, N, T, Y, or absent); X.sub.31 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., I, V, M, W, or absent); X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent); X.sub.34 is A, V, I, L, P, F, Y or W (e.g., I, V, F, or Y); X.sub.35 is R, H, or K (e.g., R or K); X.sub.36 is R, H, K, N, or Q (e.g., R or Q); X.sub.37 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., A, V, R, or P); X.sub.38 is R, H, K, S, T, P, F, Y, or W (e.g., H, S, T, or P); X.sub.39 is G, D, or E (e.g., G or E); X.sub.40 is G, R, H, K, N, or Q (e.g., G, R, K, N, or Q); X.sub.41 is G, A, V, I, L, D, E, R, H, K, N, or Q (e.g., G, A, E, R, or Q); X.sub.42 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., L, R, P, or F); X.sub.43 is D, E, S or T (e.g., D or E); X.sub.44 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, L, F, or W); X.sub.45 is A, V, I, L, M, S, or T (e.g., V, I, L, or M); X.sub.46 is G, A, V, I, L, S, or T (e.g., G, A, V, or S); X.sub.47 is A, V, I, L, R, H, K, M, S, T, or absent (e.g., A, V, I, R, S, T, or absent); X.sub.48 is A, V, I, L, M, S, T, or absent (e.g., V, I, L, M, or absent); X.sub.49 is A, V, I, L, D, E, R, H, K, M, S, T, or absent (e.g., A, D, R, S, or absent); X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent); X.sub.51 is R, H, K, S, T, F, Y, W, or absent (e.g., H, S, F, Y, or absent); X.sub.52 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, or absent (e.g., G, A, V, L, D, E, R, N, T, or absent); X.sub.53 is G, D, E, N, Q, S, T, P, F, Y, W, or absent (e.g., G, D, E, Q, S, P, or absent); X.sub.54 is A, V, I, L, R, H, K, S, T, F, Y, W, or absent (e.g., A, K, S, F, or absent); X.sub.55 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., A, V, L, M, F, or absent); X.sub.56 is D, E, R, H, K, N, Q, or absent (e.g., E, R, K, Q, or absent); X.sub.57 is G, D, E, S, T, or absent (e.g., G, D, S, or absent); X.sub.58 is R, H, or K (e.g., R or K); X.sub.59 is A, V, I, L, F, Y, or W (e.g., A, V, L, or F); X.sub.60 is A, V, I, L, C, M, S, or T (e.g., A, C, S, or T); X.sub.61 is A, V, I, L, C, M, S, T, F, Y, or W (e.g., A, I, L, M, S, or F); X.sub.62 is C, M, S, or T (e.g., S or T); X.sub.63 is A, V, I, L, R, H, K, N, or Q (e.g., A, V, R, K, or Q); X.sub.65 is A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., I, D, R, K, N, S, or T); X.sub.66 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, R, K, N, or S); X.sub.67 is A, V, I, L, R, H, K, S, or T (e.g., A, R, K, S, or T); X.sub.68 is R, H, K, N, Q, S, or T (e.g., K, N, S, or T); X.sub.69 is A, V, I, L, C, M, N, Q, S, or T (e.g., A, L, M, Q, S, or T); X.sub.70 is A, V, I, L, C, M, F, Y, or W (e.g., A, V, L, M, or F); X.sub.71 is S, T, F, Y, or W (e.g., S, T, F, or Y); X.sub.72 is A, V, I, L, C, or M (e.g., L or M); X.sub.73 is D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., E, R, K, Q, S, or F); X.sub.74 is A, V, I, L, C, or M (e.g., L or M); X.sub.75 is D, E, N, Q, S, or T (e.g., D, N, or S); X.sub.76 is R, H, K, N, Q, S, or T (e.g., R, N, or S); X.sub.77 is A, V, I, or L (e.g., V or L); X.sub.78 is R, H, K, N, Q, S, or T (e.g., R, K, Q, or T); X.sub.79 is A, V, I, L, R, H, K, S, T, P, F, Y, or W (e.g., A, V, L, R, S, T, P, or Y); X.sub.80 is A, V, I, L, D, or E (e.g., A, D, or E); X.sub.82 is C, S, or T (e.g., S or T); X.sub.83 is G, A, V, I, or L (e.g., G or A); X.sub.84 is A, V, I, L, C, M, D, E, S or T (e.g., V, I, M, D, E, or T); X.sub.86 is S, T, F, Y, or W (e.g., T, F, or Y); X.sub.87 is A, V, I, L, C, or M (e.g., V or C); X.sub.88 is A, V, I, L, N, Q, S, T, F, Y, W, or absent (e.g., A, V, N, Q, T, Y, or absent); X.sub.89 is G, A, V, I, L, R, H, K, F, Y, W, or absent (e.g., G, A, V, R, K, Y, or absent); X.sub.90 is any amino acid or absent (e.g., V, H, Q, S, F, Y, or absent); X.sub.91 is R, H, K, F, Y, or W (e.g., R or W); X.sub.93 is R, H, K, N, Q, P, F, Y, or W (e.g., R, K, Q, or P); X.sub.95 is A, V, I, L, S, or T (e.g., L or T); X.sub.96 is A, V, I, L, C, M, N, Q, S, or T (e.g., L, M, Q, S, or T); X.sub.97 is A, V, I, or L (e.g., V or L); X.sub.98 is A, V, I, L, S, or T (e.g., V or T); X.sub.101 is S, T, or absent (e.g., S or absent); CDR1 is any CDR described herein (e.g., SEQ ID NOs:40-73 and 150-155); CDR2 is any CDR described herein (e.g., SEQ ID NOs:74-107 and 160-167); and CDR3 is any CDR described herein (e.g., SEQ ID NOs: 108-141 and 170-175).
(74) In other embodiments, the construct includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:181: X.sub.1X.sub.2X.sub.3LX.sub.5X.sub.6X.sub.7GX.sub.9X.sub.10X.sub.11X.sub.12X.sub.13PX.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21CX.sub.23X.sub.24X.sub.25X.sub.26-CDR1-X.sub.31X.sub.32WX.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39X.sub.40X.sub.41X.sub.42EX.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49-CDR2-X.sub.50X.sub.51X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59X.sub.60X.sub.61X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69X.sub.70X.sub.71X.sub.72X.sub.73 X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78X.sub.79X.sub.80DX.sub.82X.sub.83X.sub.84YX.sub.86X.sub.87X.sub.88X.sub.89-CDR3-X.sub.90WGX.sub.93X.sub.94X.sub.95X.sub.96 X.sub.97X.sub.98VSX.sub.101, wherein: X.sub.1 is A, V, I, L, D, E, R, H, K, N, or Q (e.g., A, D, E, H, or Q); X.sub.2 is A, V, I, or L (e.g., V or L); X.sub.3 is N, Q, S, or T (Q or T); X.sub.5 is A, V, I, L, N, or Q (e.g., V, L, or Q); X.sub.6 is A, V, I, L, D, E, N, or Q (e.g., A, E, or Q); X.sub.7 is S, T, P, F, Y, or W (e.g., S, T, or P); X.sub.9 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, or P); X.sub.10 is G, A, V, I, L, D, E, N, Q, S, or T (e.g., G, A, E, N, or S); X.sub.11 is A, V, I, L, S, T, F, Y, or W (e.g., V, L, S, or F); X.sub.12 is A, V, I, L, R, H, K, M, S, or T (e.g., V, K, or M); X.sub.13 is R, H, K, N, or Q (e.g., R, K, or Q); X.sub.15 is G, S, or T (e.g., G or S); X.sub.16 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, E, R, Q, or S); X.sub.17 is S, T, N, or Q (e.g., S or T); X.sub.18 is A, V, I, or L (e.g., V, I, or L); X.sub.19 is R, H, K, S, or T (e.g., R, K, S, or T); X.sub.20 is A, V, I, or L (e.g., V, I, or L); X.sub.21 is S, T, N, or Q (e.g., S or T); X.sub.23 is G, A, V, I, L, R, H, K, S, or T (e.g., A, V, K, or T); X.sub.24 is A, V, I, L, F, Y, or W (e.g., A, V, I, or F); X.sub.25 is S or T; X.sub.26 is G, A, V, I, L, D, E, R, H, K, or absent (e.g., G, A, E, R, or absent); X.sub.31 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., I, V, M, W, or absent); X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent); X.sub.34 is A, V, I, L, P, F, Y or W (e.g., I, V, F, or Y); X.sub.35 is R, H, or K (e.g., R or K); X.sub.36 is R, H, K, N, or Q (e.g., R or Q); X.sub.37 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., A, V, R, or P); X.sub.38 is R, H, K, S, T, P, F, Y, or W (e.g., H, S, T, or P); X.sub.39 is G, D, or E (e.g., G or E); X.sub.40 is G, R, H, K, N, or Q (e.g., G, R, K, N, or Q); X.sub.41 is G, A, V, I, L, D, E, R, H, K, N, or Q (e.g., G, A, E, R, or Q); X.sub.42 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., L, R, P, or F); X.sub.43 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, L, F, or W); X.sub.45 is A, V, I, L, M, S, or T (e.g., V, I, L, or M); X.sub.46 is G, A, V, I, L, S, or T (e.g., G, A, V, or S); X.sub.47 is A, V, I, L, R, H, K, M, S, T, or absent (e.g., A, V, I, R, S, T, or absent); X.sub.48 is A, V, I, L, M, S, T, or absent (e.g., V, I, L, M, or absent); X.sub.49 is A, V, I, L, D, E, R, H, K, M, S, T, or absent (e.g., A, D, R, S, or absent); X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent); X.sub.51 is R, H, K, S, T, F, Y, W, or absent (e.g., H, S, F, Y, or absent); X.sub.52 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, or absent (e.g., G, A, V, L, D, E, R, N, T, or absent); X.sub.53 is G, D, E, N, Q, S, T, P, F, Y, W, or absent (e.g., G, D, E, Q, S, P, or absent); X.sub.54 is A, V, I, L, R, H, K, S, T, F, Y, W, or absent (e.g., A, K, S, F, or absent); X.sub.55 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., A, V, L, M, F, or absent); X.sub.56 is D, E, R, H, K, N, Q, or absent (e.g., E, R, K, Q, or absent); X.sub.57 is G, D, E, S, T, or absent (e.g., G, D, S, or absent); X.sub.58 is R, H, or K (e.g., R or K); X.sub.59 is A, V, I, L, F, Y, or W (e.g., A, V, L, or F); X.sub.60 is A, V, I, L, C, M, S, or T (e.g., A, C, S, or T); X.sub.61 is A, V, I, L, C, M, S, T, F, Y, or W (e.g., A, I, L, M, S, or F); X.sub.62 is C, M, S, or T (e.g., S or T); X.sub.63 is A, V, I, L, R, H, K, N, or Q (e.g., A, V, R, K, or Q); X.sub.64 is D or E; X.sub.65 is A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., I, D, R, K, N, S, or T); X.sub.66 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, R, K, N, or S); X.sub.67 is A, V, I, L, R, H, K, S, or T (e.g., A, R, K, S, or T); X.sub.68 is R, H, K, N, Q, S, or T (e.g., K, N, S, or T); X.sub.69 is A, V, I, L, C, M, N, Q, S, or T (e.g., A, L, M, Q, S, or T); X.sub.70 is A, V, I, L, C, M, F, Y, or W (e.g., A, V, L, M, or F); X.sub.71 is S, T, F, Y, or W (e.g., S, T, F, or Y); X.sub.72 is A, V, I, L, C, or M (e.g., L or M); X.sub.73 is D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., E, R, K, Q, S, or F); X.sub.74 is A, V, I, L, C, or M (e.g., L or M); X.sub.75 is D, E, N, Q, S, or T (e.g., D, N, or S); X.sub.76 is R, H, K, N, Q, S, or T (e.g., R, N, or S); X.sub.77 is A, V, I, or L (e.g., V or L); X.sub.78 is R, H, K, N, Q, S, or T (e.g., R, K, Q, or T); X.sub.79 is A, V, I, L, R, H, K, S, T, P, F, Y, or W (e.g., A, V, L, R, S, T, P, or Y); X.sub.80 is A, V, I, L, D, or E (e.g., A, D, or E); X.sub.82 is C, S, or T (e.g., S or T); X.sub.83 is G, A, V, I, or L (e.g., G or A); X.sub.84 is A, V, I, L, C, M, D, E, S or T (e.g., V, I, M, D, E, or T); X.sub.86 is S, T, F, Y, or W (e.g., T, F, or Y); X.sub.87 is A, V, I, L, C, or M (e.g., V or C); X.sub.88 is A, V, I, L, N, Q, S, T, F, Y, W, or absent (e.g., A, V, N, Q, T, Y, or absent); X.sub.89 is G, A, V, I, L, R, H, K, F, Y, W, or absent (e.g., G, A, V, R, K, Y, or absent); X.sub.90 is any amino acid or absent (e.g., V, H, Q, S, F, Y, or absent); X.sub.93 is R, H, K, N, Q, P, F, Y, or W (e.g., R, K, Q, or P); X.sub.94 is G, A, V, I, or L (e.g., G); X.sub.95 is A, V, I, L, S, or T (e.g., L or T); X.sub.96 is A, V, I, L, C, M, N, Q, S, or T (e.g., L, M, Q, S, or T); X.sub.97 is A, V, I, or L (e.g., V or L); X.sub.98 is A, V, I, L, S, or T (e.g., V or T); X.sub.101 is S, T, or absent (e.g., S or absent); CDR1 is any CDR described herein (e.g., SEQ ID NOs:40-73 and 150-155); CDR2 is any CDR described herein (e.g., SEQ ID NOs:74-107 and 160-167); and CDR3 is any CDR described herein (e.g., SEQ ID NOs: 108-141 and 170-175).
(75) In yet other embodiments, the construct includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:182: X.sub.1VX.sub.3LX.sub.5X.sub.6SGGGX.sub.11X.sub.12X.sub.13X.sub.14GX.sub.16SLX.sub.19LSCAASG-CDR1-X.sub.31X.sub.32WX.sub.34RX.sub.36X.sub.37PX.sub.39X.sub.40X.sub.41X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49-CDR2-X.sub.50YX.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57RFX.sub.60X.sub.61SX.sub.63DX.sub.65X.sub.66X.sub.67X.sub.68X.sub.69X.sub.70X.sub.71LX.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 X.sub.79X.sub.80DTX.sub.83X.sub.84YX.sub.86CAX.sub.89-CDR3-X.sub.90X.sub.91GX.sub.93GTX.sub.96VTVSX.sub.101, wherein: X.sub.1 is A, V, I, L, D, E, R, H, K, N, or Q (e.g., A, D, E, H, or Q); X.sub.3 is N, Q, S, or T (Q or T); X.sub.5 is A, V, I, L, N, or Q (e.g., V, L, or Q); X.sub.6 is A, V, I, L, D, E, N, or Q (e.g., A, E, or Q); X.sub.11 is A, V, I, L, S, T, F, Y, or W (e.g., V, L, S, or F); X.sub.12 is A, V, I, L, R, H, K, M, S, or T (e.g., V, K, or M); X.sub.13 is R, H, K, N, or Q (e.g., R, K, or Q); X.sub.14 is A, V, I, L, F, Y, or P (e.g., A or P); X.sub.16 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, E, R, Q, or S); X.sub.19 is R, H, K, S, or T (e.g., R, K, S, or T); X.sub.31 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., I, V, M, W, or absent); X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent); X.sub.34 is A, V, I, L, P, F, Y or W (e.g., I, V, F, or Y); X.sub.36 is R, H, K, N, or Q (e.g., R or Q); X.sub.37 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., A, V, R, or P); X.sub.39 is G, D, or E (e.g., G or E); X.sub.40 is G, R, H, K, N, or Q (e.g., G, R, K, N, or Q); X.sub.41 is G, A, V, I, L, D, E, R, H, K, N, or Q (e.g., G, A, E, R, or Q); X.sub.42 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., L, R, P, or F); X.sub.43 is D or E; X.sub.44 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, L, F, or W); X.sub.45 is A, V, I, L, M, S, or T (e.g., V, I, L, or M); X.sub.46 is G, A, V, I, L, S, or T (e.g., G, A, V, or S); X.sub.47 is A, V, I, L, R, H, K, M, S, T, or absent (e.g., A, V, I, R, S, T, or absent); X.sub.48 is A, V, I, L, M, S, T, or absent (e.g., V, I, L, M, or absent); X.sub.49 is A, V, I, L, D, E, R, H, K, M, S, T, or absent (e.g., A, D, R, S, or absent); X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent); X.sub.52 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, or absent (e.g., G, A, V, L, D, E, R, N, T, or absent); X.sub.53 is G, D, E, N, Q, S, T, P, F, Y, W, or absent (e.g., G, D, E, Q, S, P, or absent); X.sub.54 is A, V, I, L, R, H, K, S, T, F, Y, W, or absent (e.g., A, K, S, F, or absent); X.sub.55 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., A, V, L, M, F, or absent); X.sub.56 is D, E, R, H, K, N, Q, or absent (e.g., E, R, K, Q, or absent); X.sub.57 is G, D, E, S, T, or absent (e.g., G, D, S, or absent); X.sub.60 is A, V, I, L, C, M, S, or T (e.g., A, C, S, or T); X.sub.61 is A, V, I, L, C, M, S, T, F, Y, or W (e.g., A, I, L, M, S, or F); X.sub.63 is A, V, I, L, R, H, K, N, or Q (e.g., A, V, R, K, or Q); X.sub.65 is A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., I, D, R, K, N, S, or T); X.sub.66 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, R, K, N, or S); X.sub.67 is A, V, I, L, R, H, K, S, or T (e.g., A, R, K, S, or T); X.sub.68 is R, H, K, N, Q, S, or T (e.g., K, N, S, or T); X.sub.69 is A, V, I, L, C, M, N, Q, S, or T (e.g., A, L, M, Q, S, or T); X.sub.70 is A, V, I, L, C, M, F, Y, or W (e.g., A, V, L, M, or F); X.sub.71 is S, T, F, Y, or W (e.g., S, T, F, or Y); X.sub.73 is D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., E, R, K, Q, S, or F); X.sub.74 is A, V, I, L, C, or M (e.g., L or M); X.sub.75 is D, E, N, Q, S, or T (e.g., D, N, or S); X.sub.76 is R, H, K, N, Q, S, or T (e.g., R, N, or S); X.sub.77 is A, V, I, or L (e.g., V or L); X.sub.78 is R, H, K, N, Q, S, or T (e.g., R, K, Q, or T); X.sub.79 is A, V, I, L, R, H, K, S, T, P, F, Y, or W (e.g., A, V, L, R, S, T, P, or Y); X.sub.80 is A, V, I, L, D, or E (e.g., A, D, or E); X.sub.83 is G, A, V, I, or L (e.g., G or A); X.sub.84 is A, V, I, L, C, M, D, E, S or T (e.g., V, I, M, D, E, or T); X.sub.86 is S, T, F, Y, or W (e.g., T, F, or Y); X.sub.89 is G, A, V, I, L, R, H, K, F, Y, W, or absent (e.g., G, A, V, R, K, Y, or absent); X.sub.90 is any amino acid or absent (e.g., V, H, Q, S, F, Y, or absent); X.sub.91 is F, Y, or W (e.g., W); X.sub.93 is R, H, K, N, Q, P, F, Y, or W (e.g., R, K, Q, or P); X.sub.96 is A, V, I, L, C, M, N, Q, S, or T (e.g., L, M, Q, S, or T); X.sub.101 is S, T, or absent (e.g., S or absent); CDR1 is any CDR described herein (e.g., SEQ ID NOs:40-73 and 150-155); CDR2 is any CDR described herein (e.g., SEQ ID NOs:74-107 and 160-167); and CDR3 is any CDR described herein (e.g., SEQ ID NOs: 108-141 and 170-175).
(76) In some embodiments, the construct includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs: 183-186. In particular embodiments, CDR1 is any CDR described herein (e.g., SEQ ID NOs:40-73 and 150-155); CDR2 is any CDR described herein (e.g., SEQ ID NOs: 74-107 and 160-167); and CDR3 is any CDR described herein (e.g., SEQ ID NOs:108-141 and 170-175).
(77) Any of the constructs herein can include any of the FRs described herein.
(78) In some embodiments, each X in SEQ ID NO:213 can be an amino acid in any one of SEQ ID NOs: 190-212 when any one of the sequences in SEQ ID NOs: 190-212 is used as a reference sequence to be optimally aligned with SEQ ID NO:213.
(79) In other embodiments, the first FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:214: X.sub.1X.sub.2X.sub.3LX.sub.5X.sub.6X.sub.7GX.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21CX.sub.23X.sub.24X.sub.25X.sub.26, wherein: X.sub.1 is A, V, I, L, D, E, R, H, K, N, or Q (e.g., A, D, E, H, or Q); X.sub.2 is A, V, I, or L (e.g., V or L); X.sub.3 is N, Q, S, or T (Q or T); X.sub.5 is A, V, I, L, N, or Q (e.g., V, L, or Q); X.sub.6 is A, V, I, L, D, E, N, or Q (e.g., A, E, or Q); X.sub.7 is S, T, P, F, Y, or W (e.g., S, T, or P); X.sub.9 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, or P); X.sub.10 is G, A, V, I, L, D, E, N, Q, S, or T (e.g., G, A, E, N, or S); X.sub.11 is A, V, I, L, S, T, F, Y, or W (e.g., V, L, S, or F); X.sub.12 is A, V, I, L, R, H, K, M, S, or T (e.g., V, K, or M); X.sub.13 is R, H, K, N, or Q (e.g., R, K, or Q); X.sub.14 is A, V, I, L, F, Y, or P (e.g., A or P); X.sub.15 is G, S, or T (e.g., G or S); X.sub.16 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, E, R, Q, or S); X.sub.17 is S, T, N, or Q (e.g., S or T); X.sub.18 is A, V, I, or L (e.g., V, I, or L); X.sub.19 is R, H, K, S, or T (e.g., R, K, S, or T); X.sub.20 is A, V, I, or L (e.g., V, I, or L); X.sub.21 is S, T, N, or Q (e.g., S or T); X.sub.23 is G, A, V, I, L, R, H, K, S, or T (e.g., A, V, K, or T); X.sub.24 is A, V, I, L, F, Y, or W (e.g., A, V, I, or F); X.sub.25 is S or T; and X.sub.26 is G, A, V, I, L, D, E, R, H, K, or absent (e.g., G, A, E, R, or absent).
(80) In some embodiments, each X in SEQ ID NO:214 can be an amino acid in any one of SEQ ID NOs: 190-212 when any one of the sequences in SEQ ID NOs: 190-212 is used as a reference sequence to be optimally aligned with SEQ ID NO:214.
(81) In yet other embodiments, the first FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:215: X.sub.1VQLX.sub.5X.sub.6SGX.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17LX.sub.19LX.sub.21CX.sub.23X.sub.24SG, wherein: X.sub.1 is A, V, I, L, D, E, R, H, K, N, or Q (e.g., A, D, E, H, or Q); X.sub.5 is A, V, I, L, N, or Q (e.g., V, L, or Q); X.sub.6 is A, V, I, L, D, E, N, or Q (e.g., A, E, or Q); X.sub.9 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, or P); X.sub.10 is G, A, V, I, L, D, E, N, Q, S, or T (e.g., G, A, E, N, or S); X.sub.11 is A, V, I, L, S, T, F, Y, or W (e.g., V, L, S, or F); X.sub.12 is A, V, I, L, R, H, K, M, S, or T (e.g., V, K, or M); X.sub.13 is R, H, K, N, or Q (e.g., R, K, or Q); X.sub.14 is A, V, I, L, F, Y, or P (e.g., A or P); X.sub.15 is G, S, or T (e.g., G or S); X.sub.16 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, E, R, Q, or S); X.sub.17 is S, T, N, or Q (e.g., S or T); X.sub.19 is R, H, K, S, or T (e.g., R, K, S, or T); X.sub.21 is S, T, N, or Q (e.g., S or T); X.sub.23 is G, A, V, I, L, R, H, K, S, or T (e.g., A, V, K, or T); and X.sub.24 is A, V, I, L, F, Y, or W (e.g., A, V, I, or F).
(82) In some embodiments, each X in SEQ ID NO:215 can be an amino acid in any one of SEQ ID NOs: 190-212 when any one of the sequences in SEQ ID NOs: 190-212 is used as a reference sequence to be optimally aligned with SEQ ID NO:215.
(83) In some embodiments, the first FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:216: QVQLVESGGGL VQX.sub.14GGSLRLSCAASGX.sub.27X.sub.28X.sub.29X.sub.30, wherein: X.sub.14 is A, V, I, L, F, Y, or P (e.g., A or P); X.sub.27 is G, F, Y, W, or absent (e.g., G, F, or absent); X.sub.28 is R, H, K, S, T, P, F, Y, W, or absent (e.g., K, S, T, P, or absent); X.sub.29 is A, V, I, L, D, E, R, H, K, F, Y, W, or absent (e.g., V, I, E, R, F, or absent); and X.sub.30 is G, D, E, N, Q, S, T, F, Y, W, or absent (e.g., G, D, E, N, T, Y, or absent).
(84) In other embodiments, each X in SEQ ID NO:216 can be an amino acid in any one of SEQ ID NOs: 190-212 when any one of the sequences in SEQ ID NOs: 190-212 is used as a reference sequence to be optimally aligned with SEQ ID NO:216.
(85) In yet other embodiments, the first FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:217: QVQLVESGGGLVQX.sub.14GGSLRLSCAASG, wherein: X.sub.14 is A, V, I, L, F, Y, or P (e.g., A or P).
(86)
(87) In yet other embodiments, each X in SEQ ID NO:244 can be an amino acid in any one of SEQ ID NOs:220-243 when any one of the sequences in SEQ ID NOs:220-243 is used as a reference sequence to be optimally aligned with SEQ ID NO:244.
(88) In some embodiments, the second FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:245: X.sub.31X.sub.32WX.sub.34RQAX.sub.38GX.sub.40X.sub.41X.sub.42EX.sub.4X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49, wherein: X.sub.31 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., I, V, M, W, or absent); X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent); X.sub.34 is A, V, I, L, P, F, Y or W (e.g., I, V, F, or Y); X.sub.38 is R, H, K, S, T, P, F, Y, or W (e.g., H, S, T, or P); X.sub.40 is G, R, H, K, N, or Q (e.g., G, R, K, N, or Q); X.sub.41 is G, A, V, I, L, D, E, R, H, K, N, or Q (e.g., G, A, E, R, or Q); X.sub.42 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., L, R, P, or F); X.sub.44 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, L, F, or W); X.sub.45 is A, V, I, L, M, S, or T (e.g., V, I, L, or M); X.sub.46 is G, A, V, I, L, S, or T (e.g., G, A, V, or S); X.sub.47 is A, V, I, L, R, H, K, M, S, T, or absent (e.g., A, V, I, R, S, T, or absent); X.sub.48 is A, V, I, L, M, S, T, or absent (e.g., V, I, L, M, or absent); and X.sub.49 is A, V, I, L, D, E, R, H, K, M, S, T, or absent (e.g., A, D, R, S, or absent).
(89) In other embodiments, each X in SEQ ID NO:245 can be an amino acid in any one of SEQ ID NOs:220-243 when any one of the sequences in SEQ ID NOs:220-243 is used as a reference sequence to be optimally aligned with SEQ ID NO:245.
(90) In some embodiments, the second FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:246: X.sub.31X.sub.32WX.sub.34RQAX.sub.38GX.sub.40X.sub.41X.sub.42EX.sub.44X.sub.45X.sub.46, wherein: X.sub.31 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., I, V, M, W, or absent); X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent); X.sub.34 is A, V, I, L, P, F, Y or W (e.g., I, V, F, or Y); X.sub.38 is R, H, K, S, T, P, F, Y, or W (e.g., H, S, T, or P); X.sub.40 is G, R, H, K, N, or Q (e.g., G, R, K, N, or Q); X.sub.41 is G, A, V, I, L, D, E, R, H, K, N, or Q (e.g., G, A, E, R, or Q); X.sub.42 is A, V, I, L, R, H, K, P, F, Y, or W (e.g., L, R, P, or F); X.sub.43 is G, A, V, I, L, P, F, Y, or W (e.g., G, A, L, F, or W); X.sub.45 is A, V, I, L, M, S, or T (e.g., V, I, L, or M); and X.sub.46 is G, A, V, I, L, S, or T (e.g., G, A, V, or S).
(91) In other embodiments, each X in SEQ ID NO:246 can be an amino acid in any one of SEQ ID NOs:220-243 when any one of the sequences in SEQ ID NOs:220-243 is used as a reference sequence to be optimally aligned with SEQ ID NO:246.
(92) In some embodiments, the second FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:247: MX.sub.32WFRQAPGKEREWVAX.sub.47X.sub.48X.sub.49, wherein: X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent); X.sub.47 is A, V, I, L, R, H, K, M, S, T, or absent (e.g., A, V, I, R, S, T, or absent); X.sub.48 is A, V, I, L, M, S, T, or absent (e.g., V, I, L, M, or absent); and X.sub.49 is A, V, I, L, D, E, R, H, K, M, S, T, or absent (e.g., A, D, R, S, or absent).
(93) In other embodiments, each X in SEQ ID NO:247 can be an amino acid in any one of SEQ ID NOs:220-243 when any one of the sequences in SEQ ID NOs:220-243 is used as a reference sequence to be optimally aligned with SEQ ID NO:247.
(94) In yet other embodiments, the second FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:248: MX.sub.32WFRQAPGKEREWVA, wherein: X.sub.32 is G, A, V, I, L, N, Q, R, H, K, or absent (e.g., G, A, N, H, or absent).
(95)
(96) In other embodiments, each X in SEQ ID NO:285 can be an amino acid in any one of SEQ ID NOs:250-284 when any one of the sequences in SEQ ID NOs:250-284 is used as a reference sequence to be optimally aligned with SEQ ID NO:285.
(97) In some embodiments, the third FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:286: X.sub.50X.sub.51X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57RX.sub.59X.sub.60IX.sub.62RDX.sub.65X.sub.66X.sub.67X.sub.68X.sub.69VX.sub.71LX.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 X.sub.79EDTAX.sub.84YYX.sub.87X.sub.88X.sub.89, wherein: X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent); X.sub.51 is R, H, K, S, T, F, Y, W, or absent (e.g., H, S, F, Y, or absent); X.sub.52 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, or absent (e.g., G, A, V, L, D, E, R, N, T, or absent); X.sub.53 is G, D, E, N, Q, S, T, P, F, Y, W, or absent (e.g., G, D, E, Q, S, P, or absent); X.sub.54 is A, V, I, L, R, H, K, S, T, F, Y, W, or absent (e.g., A, K, S, F, or absent); X.sub.55 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., A, V, L, M, F, or absent); X.sub.56 is D, E, R, H, K, N, Q, or absent (e.g., E, R, K, Q, or absent); X.sub.57 is G, D, E, S, T, or absent (e.g., G, D, S, or absent); X.sub.59 is A, V, I, L, F, Y, or W (e.g., A, V, L, or F); X.sub.60 is A, V, I, L, C, M, S, or T (e.g., A, C, S, or T); X.sub.62 is C, M, S, or T (e.g., S or T); X.sub.65 is A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., I, D, R, K, N, S, or T); X.sub.66 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, R, K, N, or S); X.sub.67 is A, V, I, L, R, H, K, S, or T (e.g., A, R, K, S, or T); X.sub.68 is R, H, K, N, Q, S, or T (e.g., K, N, S, or T); X.sub.69 is A, V, I, L, C, M, N, Q, S, or T (e.g., A, L, M, Q, S, or T); X.sub.71 is S, T, F, Y, or W (e.g., S, T, F, or Y); X.sub.73 is D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., E, R, K, Q, S, or F); X.sub.74 is A, V, I, L, C, or M (e.g., L or M); X.sub.75 is D, E, N, Q, S, or T (e.g., D, N, or S); X.sub.76 is R, H, K, N, Q, S, or T (e.g., R, N, or S); X.sub.77 is A, V, I, or L (e.g., V or L); X.sub.78 is R, H, K, N, Q, S, or T (e.g., R, K, Q, or T); X.sub.79 is A, V, I, L, R, H, K, S, T, P, F, Y, or W (e.g., A, V, L, R, S, T, P, or Y); X.sub.84 is A, V, I, L, C, M, D, E, S or T (e.g., V, I, M, D, E, or T); X.sub.87 is F, Y, or W (e.g., Y); X.sub.88 is A, V, I, L, N, Q, S, T, F, Y, W, or absent (e.g., A, V, N, Q, T, Y, or absent); and X.sub.89 is G, A, V, I, L, R, H, K, F, Y, W, or absent (e.g., G, A, V, R, K, Y, or absent).
(98) In other embodiments, each X in SEQ ID NO:286 can be an amino acid in any one of SEQ ID NOs:250-284 when any one of the sequences in SEQ ID NOs:250-284 is used as a reference sequence to be optimally aligned with SEQ ID NO:286.
(99) In some embodiments, the third FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:287: X.sub.50X.sub.51X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57RX.sub.59X.sub.60IX.sub.62RDX.sub.65X.sub.66X.sub.67X.sub.68X.sub.69VX.sub.71LX.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 X.sub.79EDTAX.sub.84YYX.sub.87X.sub.88, wherein: X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent); X.sub.51 is R, H, K, S, T, F, Y, W, or absent (e.g., H, S, F, Y, or absent); X.sub.52 is G, A, V, I, L, D, E, R, H, K, N, Q, S, T, or absent (e.g., G, A, V, L, D, E, R, N, T, or absent); X.sub.53 is G, D, E, N, Q, S, T, P, F, Y, W, or absent (e.g., G, D, E, Q, S, P, or absent); X.sub.54 is A, V, I, L, R, H, K, S, T, F, Y, W, or absent (e.g., A, K, S, F, or absent); X.sub.55 is A, V, I, L, M, S, T, F, Y, W, or absent (e.g., A, V, L, M, F, or absent); X.sub.56 is D, E, R, H, K, N, Q, or absent (e.g., E, R, K, Q, or absent); X.sub.57 is G, D, E, S, T, or absent (e.g., G, D, S, or absent); X.sub.59 is A, V, I, L, F, Y, or W (e.g., A, V, L, or F); X.sub.60 is A, V, I, L, C, M, S, or T (e.g., A, C, S, or T); X.sub.62 is C, M, S, or T (e.g., S or T); X.sub.65 is A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., I, D, R, K, N, S, or T); X.sub.66 is G, A, V, I, L, D, E, R, H, K, N, Q, S, or T (e.g., G, A, D, R, K, N, or S); X.sub.67 is A, V, I, L, R, H, K, S, or T (e.g., A, R, K, S, or T); X.sub.68 is R, H, K, N, Q, S, or T (e.g., K, N, S, or T); X.sub.69 is A, V, I, L, C, M, N, Q, S, or T (e.g., A, L, M, Q, S, or T); X.sub.71 is S, T, F, Y, or W (e.g., S, T, F, or Y); X.sub.73 is D, E, R, H, K, N, Q, S, T, F, Y, or W (e.g., E, R, K, Q, S, or F); X.sub.74 is A, V, I, L, C, or M (e.g., L or M); X.sub.75 is D, E, N, Q, S, or T (e.g., D, N, or S); X.sub.76 is R, H, K, N, Q, S, or T (e.g., R, N, or S); X.sub.77 is A, V, I, or L (e.g., V or L); X.sub.78 is R, H, K, N, Q, S, or T (e.g., R, K, Q, or T); X.sub.79 is A, V, I, L, R, H, K, S, T, P, F, Y, or W (e.g., A, V, L, R, S, T, P, or Y); X.sub.84 is A, V, I, L, C, M, D, E, S or T (e.g., V, I, M, D, E, or T); X.sub.87 is F, Y, or W (e.g., Y); and X.sub.88 is A, V, I, L, N, Q, S, T, F, Y, W, or absent (e.g., A, V, N, Q, T, Y, or absent).
(100) In other embodiments, each X in SEQ ID NO:287 can be an amino acid in any one of SEQ ID NOs:250-284 when any one of the sequences in SEQ ID NOs:250-284 is used as a reference sequence to be optimally aligned with SEQ ID NO:287.
(101) In some embodiments, the third FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:288: X.sub.50YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAX.sub.89, wherein: X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent); and X.sub.89 is G, A, V, I, L, R, H, K, F, Y, W, or absent (e.g., G, A, V, R, K, Y, or absent).
(102) In other embodiments, the third FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:289: X.sub.50YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA, wherein: X.sub.50 is A, V, I, L, D, E, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., L, D, R, N, T, Y, or absent).
(103)
(104) In other embodiments, each X in SEQ ID NO:302 can be an amino acid in any one of SEQ ID NOs:290-301 when any one of the sequences in SEQ ID NOs:290-301 is used as a reference sequence to be optimally aligned with SEQ ID NO:302.
(105) In some embodiments, the fourth FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:303: X.sub.90WGX.sub.93GX.sub.95X.sub.96X.sub.97TVS, wherein: X.sub.90 is A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., V, H, Q, S, F, Y, or absent); X.sub.93 is R, H, K, N, Q, P, F, Y, or W (e.g., R, K, Q, or P); X.sub.95 is A, V, I, L, S, or T (e.g., L or T); X.sub.96 is A, V, I, L, C, M, N, Q, S, or T (e.g., L, M, Q, S, or T); and X.sub.97 is A, V, I, or L (e.g., V or L).
(106) In other embodiments, each X in SEQ ID NO:303 can be an amino acid in any one of SEQ ID NOs:290-301 when any one of the sequences in SEQ ID NOs:290-301 is used as a reference sequence to be optimally aligned with SEQ ID NO:303.
(107) In some embodiments, the fourth FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:304: WGX.sub.93GX.sub.95X.sub.96X.sub.97TVSX.sub.101, wherein: X.sub.93 is R, H, K, N, Q, P, F, Y, or W (e.g., R, K, Q, or P); X.sub.95 is A, V, I, L, S, or T (e.g., L or T); X.sub.96 is A, V, I, L, C, M, N, Q, S, or T (e.g., L, M, Q, S, or T); X.sub.97 is A, V, I, or L (e.g., V or L); and X.sub.101 is S, T, or absent (e.g., S or absent).
(108) In other embodiments, each X in SEQ ID NO:304 can be an amino acid in any one of SEQ ID NOs:290-301 when any one of the sequences in SEQ ID NOs:290-301 is used as a reference sequence to be optimally aligned with SEQ ID NO:304.
(109) In some embodiments, the fourth FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:305: X.sub.90X.sub.91GX.sub.93GTX.sub.96X.sub.97X.sub.98VSX.sub.101, wherein: X.sub.90 is A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., V, H, Q, S, F, Y, or absent); X.sub.91 is P, F, Y, or W (e.g., W); X.sub.93 is R, H, K, N, Q, P, F, Y, or W (e.g., R, K, Q, or P); X.sub.96 is A, V, I, L, C, M, N, Q, S, or T (e.g., L, M, Q, S, or T); X.sub.97 is A, V, I, or L (e.g., V or L); X.sub.98 is S or T; and X.sub.101 is S, T, or absent (e.g., S or absent).
(110) In other embodiments, each X in SEQ ID NO:305 can be an amino acid in any one of SEQ ID NOs:290-301 when any one of the sequences in SEQ ID NOs:290-301 is used as a reference sequence to be optimally aligned with SEQ ID NO:305.
(111) In some embodiments, the fourth FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:306: X.sub.90WGQGTQVTVSS, wherein: X.sub.90 is A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., V, H, Q, S, F, Y, or absent).
(112) In other embodiments, the fourth FR includes or is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:307: X.sub.90WGQGTQVTVS, wherein: X.sub.90 is A, V, I, L, R, H, K, N, Q, S, T, F, Y, W, or absent (e.g., V, H, Q, S, F, Y, or absent).
Targets
(113) A target can be an antigen that can be bound by any construct described herein. Non-limiting targets include a coronavirus or a portion thereof. Non-limiting portions of a coronavirus includes a spike protein (e.g., a S-glycoprotein) or a receptor-binding domain (RBD). Non-limiting sequences for such spike proteins and RBDs include one or more of the following: UniProtKB No. P0DTC2 (amino acids 13-1273 for the spike glycoprotein, amino acids 13-685 for the spike protein 1, amino acids 319-541 for the RBD, or amino acids 437-508 for a receptor-binding motif that binds to human ACE2); UniProtKB No. A0A6B9WHD3 (amino acids 31-1228 for the spike glycoprotein, amino acids 31-592 for the spike protein 1, or amino acids 349-526 for the RBD); UniProtKB No. P59594 (amino acids 14-1255 for the spike glycoprotein, amino acids 14-667 for the spike protein S1, amino acids 306-527 for the RBD, or amino acids 424-494 for a receptor-binding motif that binds to human ACE2); UniProtKB No. Q5GDB5 (amino acids 14-667 for spike protein S1, amino acids 306-527 for the RBD, or amino acids 335-512 for the RBD); UniProtKB No. Q3LZX1 (amino acids 14-1242 for the spike glycoprotein, amino acids 14-654 for the spike protein S1, or amino acids 310-514 for the RBD); UniProtKB No. Q315J5 (amino acids 14-1241 for the spike glycoprotein, amino acids 14-653 for the spike protein S1, or amino acids 310-513 for the RBD); and UniProtKB No. QOQ475 (amino acids 14-1241 for the spike glycoprotein, amino acids 14-653 for the spike protein S1, or amino acids 310-513 for the RBD).
(114) Targets can also include a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs:320-327 (
(115) In yet other embodiments, the target can include the following one or more mutations: LSF, V341I, K417N, K417T, A435S, N439K, L452R, K458R, 1472V, E484K, N501Y, D614G, H655Y, R682Q, D936Y, S939F, and/or S943T, using the numbering provided for SEQ ID NO:320 or for another sequence that is optimally aligned with SEQ ID NO:320.
(116) Targets can also include a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 328-336 (
(117) In some embodiments, the target is or includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:335:
(118) RVX.sub.321PX.sub.323X.sub.324X.sub.325X.sub.326X.sub.327RFPNITNX.sub.335CPFX.sub.339X.sub.340X.sub.341FNAX.sub.345X.sub.346FX.sub.348 X.sub.349VYAWX.sub.354RX.sub.356X.sub.357ISX.sub.360CVADYX.sub.366VLYNSX.sub.372X.sub.373FSTFKCYGVSX.sub.384 X.sub.385KLX.sub.388DLCFX.sub.393X.sub.394VYADX.sub.399FX.sub.401X.sub.402X.sub.403X.sub.404X.sub.405X.sub.406VRQX.sub.410APGX.sub.414TGX.sub.417IADYNYKLPDDFX.sub.430GCVX.sub.434X.sub.435WNX.sub.438X.sub.439X.sub.440X.sub.441DX.sub.443X.sub.444X.sub.445X.sub.446G X.sub.448X.sub.449X.sub.450YX.sub.452YRX.sub.455X.sub.456RX.sub.458X.sub.459X.sub.460LX.sub.462PFERDX.sub.468SX.sub.470X.sub.471X.sub.472X.sub.473 X.sub.474X.sub.475X.sub.476X.sub.477X.sub.478X.sub.479X.sub.480X.sub.481X.sub.482X.sub.483X.sub.484X.sub.485X.sub.486NX.sub.488X.sub.489X.sub.490X.sub.491LX.sub.493X.sub.494YX.sub.496FX.sub.498X.sub.499X.sub.500X.sub.501X.sub.502X.sub.503X.sub.504X.sub.505QX.sub.507X.sub.508RVVVLSFELLX.sub.519APATVCGPKX.sub.529S TX.sub.532LX.sub.534KNX.sub.537CVNF wherein: X.sub.321 is A, V, I, L, N, Q, S, or T (e.g., V, Q, S, or T); X.sub.323, X.sub.345, X.sub.366, X.sub.385, X.sub.393, X.sub.399, X.sub.404, and X.sub.438 is, independently, G, S, or T (e.g., S or T); X.sub.324, X.sub.339, X.sub.354, X.sub.360, X.sub.414, and X.sub.532 is, independently, G, D, E, N or Q (e.g., G, D, E, or Q; E or N; D or N; or E or Q); X.sub.325 and X.sub.405 is, independently, D, E, S, or T (e.g., D, E, or S); X.sub.326, X.sub.327, X.sub.341, X.sub.401, X.sub.402, X.sub.410, X.sub.434, X.sub.468, X.sub.503, and X.sub.534 is, independently, A, V, I, or L (e.g., V or I; or V or L); X.sub.335 and X.sub.529 is, independently, A, V, I, L, R, H, or K (e.g., L or R; or V or K); X.sub.340 is D, E, R, H, or K (e.g., E or K); X.sub.346, X.sub.356, and X.sub.403 is, independently, R, H, K, S, or T (e.g., R, K, or T); X.sub.348, X.sub.384, X.sub.489, X.sub.502, and X.sub.507 is, independently, G, A, V, I, L, P, F, Y, or W (e.g., A or P; V or W; G or P; G or W); X.sub.349, X.sub.394, X.sub.470, and X.sub.500 is, independently, N, Q, S, or T (e.g., N or S); X.sub.357, X.sub.458, and X.sub.462 is, independently, R, H, or K (e.g., R or K); X.sub.372 and X.sub.435 is, independently, A, V, I, L, S, or T (e.g., A or T; or A or S); X.sub.373, X.sub.491, and X.sub.499 is, independently, S, T, P, F, Y, or W (e.g., S or F; or T or P); X.sub.388 is A, V, I, L, N, or Q (e.g., I or N); X.sub.406, X.sub.496, and X.sub.504 is, independently, G, A, V, I, L, D, or E (e.g., D or E; or G, A, or E); X.sub.417, X.sub.439, X.sub.441, and X.sub.443 is, independently, A, V, I, L, R, H, K, N, Q, S, or T (e.g., V, K, N, or T; A, R, K, or N; A, Q, S, or T; or L, I, H, or Q); X.sub.430 is L, C, M, S, or T (e.g., M or T); X.sub.440, X.sub.450, X.sub.460, X.sub.519, and X.sub.537 is, independently, R, H, K, N or Q (e.g., N, K, or Q); X.sub.444, X.sub.446, and X.sub.478 is, independently, G, R, H, K, S, T, or absent (e.g., K, T, or absent; or G, T, or absent; or K, T, or absent); X.sub.445, X.sub.459, X.sub.471, X.sub.476, X.sub.477, and X.sub.485 is, independently, G, A, V, I, L, D, E, S, T, or absent (e.g., V, E, S, or absent; G, A, E, S, or T; V, D, or E; G, D, E, or absent; G, S, or absent; E, T, or absent; or G, A, or absent); X.sub.448 is N, Q, or absent (e.g., N or absent); X.sub.449, X.sub.473, X.sub.479, and X.sub.482 is, independently, G, P, F, Y, W, or absent (e.g., F, Y, or absent; P or absent; or G, P, or absent); X.sub.452, X.sub.456, X.sub.490, and X.sub.505 is, independently, A, V, I, L, R, H, K, F, Y, or W (e.g., L, R, K or Y; or L, H, F, or Y; or R, F, Y, or W; or H or Y); X.sub.455 and X.sub.508 is, independently, A, V, I, L, S, T, F, Y, or W (e.g., L, S, or Y; or T or Y); X.sub.472, X.sub.475, and X.sub.483 is, independently, A, V, I, L, D, E, N, Q, P, or absent (e.g., V, I, D, P, or absent; A, P, or absent; or V, Q, P, or absent); X.sub.474 and X.sub.481 is, independently, N, Q, S, T, or absent (e.g., Q, S, or absent; N, T, or absent); X.sub.480 and X.sub.488 is, independently, G, C, M, or absent (e.g., C or absent); X.sub.486 is G, A, V, I, L, D, E, P, F, Y, or W (e.g., G, L, E, P, or F); X.sub.493 and X.sub.498 is, independently, N, Q, S, T, F, Y, or W (e.g., N, Q, S, or Y; or N, Q, or Y); X.sub.484 and X.sub.494 is, independently, G, D, E, R, H, K, S, T, or absent (e.g., G, D, R, S, or T; or E, K, T, or absent); and X.sub.501 is any amino acid, such as A, V, I, L, D, E, N, Q, S, T, F, Y, or W (e.g., V, I, D, N, S, T, or Y).
(119) In yet other embodiments, the target can include the following one or more mutations: V341I, K417N, K417T, A435S, N439K, L452R, K458R, 1472V, E484K, and/or N501Y using the numbering provided for SEQ ID NOs:335-336 or for another sequence that is optimally aligned with one of SEQ ID NOs:335-336.
(120) Therapeutic or Diagnostic Agents
(121) The present disclosure also encompasses a construct that can be directly or indirectly attached to one or more therapeutic or diagnostic agents. Such agents can include a therapeutic antibody, a complementarity determining region (CDR), a small molecule, a chemotherapeutic agent, an antiviral agent, an antibacterial agent, an anti-inflammatory agent, a scavenging agent, an imaging agent, a marker, a dye, a detectable moiety, or a label.
(122) Any of the constructs herein (e.g., isolated or purified antibodies or fragments thereof) can be employed to bind to a target. Binding can be accomplished, e.g., by using CDRs specific for that target. In one embodiment, the therapeutic or diagnostic agent includes one or more CDRs for viral targets. Exemplary targets include a virus, such as Coronaviridae (e.g., severe acute respiratory syndrome-related coronavirus (SARS-COV), severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), or variants thereof); or a portion of a virus, such as a spike protein or a receptor-binding domain (RBD) of a coronavirus.
(123) Other non-limiting therapeutic or diagnostic agents include a nucleic acid (e.g., oligonucleotides, polynucleotides, nucleotides, nucleosides, molecules of DNA, or molecules of RNA, including a chromosome, a plasmid, a viral genome, a primer, or a gene); a protein (e.g., a glycoprotein, a metalloprotein, an enzyme, a prion, or an immunoglobulin); a metabolite; a sugar; a lipid; or a lipopolysaccharide.
(124) Non-limiting detectable moieties may be a radioisotope (e.g., 32P), a fluorescent or chemiluminescent compound such as rhodamine or luciferin, or an enzyme, such as alkaline phosphatase or horseradish peroxidase. Non-limiting labels include a radiolabel, an isotope, a visible or near-infrared fluorescent label, a reporter molecule, biotin, or the like.
(125) The therapeutic or diagnostic agent can be a peptide, an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, glucose-6-phosphatase or beta-galactosidase), a nucleic acid, a virus, a fluorophore (e.g., green fluorescent protein (GFP), blue fluorescent dyes excited at wavelengths in the ultraviolet (UV) part of the spectrum (e.g., AMCA (7-amino-4-methylcoumarin-3-acetic acid); Alexa Fluor 350), green fluorescent dyes excited by blue light (e.g., FITC, Cy2, Alexa Fluor 488), red fluorescent dyes excited by green light (e.g., rhodamines, Texas Red, Cy3, Alexa Fluor dyes 546, 564 and 594), or dyes excited with far-red light (e.g., Cy5) to be visualized with electronic detectors (CCD cameras, photomultipliers)), a heavy metal (including chelates thereof, such as those including europium, lanthanum or yttrium), a chemical entity, or a radioisotope (e.g., [.sup.18F]fluorodeoxy glucose, .sup.11C-, .sup.125I-, .sup.131I-, .sup.3H-, .sup.14C-, .sup.35S-, or .sup.99Tc-labelled compounds).
(126) The therapeutic or diagnostic agent can include a drug, an antigen binding fragment of an antibody molecule or portion thereof (e.g., F(ab), scFv, a VH domain, or a VL domain) (e.g., to impart, induce or block a biological response), a ligand binding portion of a receptor or a receptor binding portion of a ligand, an enzyme, therapeutically useful amino acids, peptides, proteins, nucleic acids, including but not limited to polynucleotides, oligonucleotides, carbohydrates and lipids. Yet other exemplary agents include cytokines, neurotrophic factors, growth factors, enzymes, antibodies, neurotransmitters, neuromodulators, antibiotics, antiviral agents, antifungal agents, imaging or detectable agents, isotopes, and chemotherapeutic agents, and the like. The therapeutic or diagnostic agents can also include drugs, prodrugs, and precursors that can be activated when the therapeutic agent is delivered to the target tissue.
(127) Methods
(128) The present disclosure also encompasses methods that employ any construct described herein. In particular embodiments, the methods include method of treatment or prophylaxis of one or more diseases or conditions. Non-limiting diseases and conditions include a viral infection.
(129) Methods can also include use of the construct as a therapeutic or diagnostic agent, which can be administered to a subject (a mammal or a human) by injection, preferably by intravenous, intraperitoneal, intramuscular or subcutaneous injection. The constructs herein (e.g., with a therapeutic or diagnostic agent) can be used in imaging or in diagnosing viral spread.
(130) Methods can also include providing a construct or a pharmaceutical composition thereof (e.g., as described herein) for use in the treatment of viral infections or any disorder or condition herein. A pharmaceutical composition can include any construct, described herein either with a therapeutic or diagnostic agent, and a pharmaceutically acceptable carrier.
(131) As used herein, pharmaceutically acceptable carrier is intended to include any and all solvents, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes, cationic lipids and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with a therapeutic agent as defined hereabove, use thereof in the composition of the present invention is contemplated.
EXAMPLES
Example 1: Highly Effective SARS-COV-2 Neutralizing Humanized Nanobodies
(132) A high-diversity synthetic nanobody phage library was used to identify 34 humanized nanobodies that show nanomolar to low picomolar efficacy in preventing cell infection by replicating VSV-SARSCOV-2 virus. In particular, we developed a high diversity humanized nanobody library (more than 3?10.sup.10 nanobody variants), which was designed to have three different CDR3 lengths and incorporated the natural prevalence of amino acids at specific CDR positions for CDR1 and CDR2 derived from numerous effective nanobodies. For CDR3, all amino acids were used with the exception of cysteine and methionine.
(133)
(134) From this biopanning campaign, we identified numerous potential candidate nanobodies. Reconformation screening using nanobody-human Fc fusion proteins elucidated 34 nanobodies, which specifically prevented infection of green monkey kidney cells (vero) with VSV-SARS-COV-2 virus with enhanced efficacy. These candidates also were able to compete with human ACE2 binding on full length SARS-COV-2 spike protein in an in vitro competition enzyme-linked immunosorbent assay (ELISA). Results for the competition assays and neutralization assays are seen in
OTHER EMBODIMENTS
(135) All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
(136) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
(137) Other embodiments are within the claims.