SARS-cov-2 inhibitors

Abstract

To provide polypeptide inhibitors of SARS-CoV-2, and their use for treating and limiting development of SARS-CoV-2 infection.SOLUTION: Polypeptide inhibitors of SARS-CoV-2 are provided comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence selected from the group consisting of specific amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 051,474, filed July 14, 2020, and U.S. Provisional Application No. 63 / 067,593, filed August 19, 2020, the entire disclosures of which are incorporated herein by reference.

[0002] Explanation of research funding from the U.S. federal government This invention was made with government support under Grant Number FA8750-17-C-0219 awarded by the Defense Advanced Research Projects Agency, and Grant Numbers HHSN272201700059C and R01GM120553 awarded by the National Institutes of Health. The federal government has certain rights in this invention.

[0003] Explanation of the sequence listing The Sequence Listing accompanying this application is submitted electronically in computer-readable form and is incorporated herein by reference in its entirety. This Sequence Listing is contained in a file created on May 25, 2021, with the file name "20-1074-WO_SeqList_ST25" and a file size of 1,112 kb. [Background technology]

[0004] SARS-CoV-2 infection is thought to often begin in the nose, where the virus replicates for several days before spreading to the wider respiratory system. Delivery of high concentrations of virus inhibitors to the nasal cavity and respiratory system in general could therefore potentially provide preventative protection and therapeutic benefit early in the course of infection, and may be particularly useful for healthcare workers and others who frequently come into contact with infected individuals. Summary of the Invention

[0005] In a first aspect, the present disclosure provides a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-101, wherein the polypeptide binds to the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD). In one embodiment, amino acid substitutions relative to the amino acid sequence of the reference polypeptide are selected from the exemplary amino acid substitutions set forth in Table 1. In another embodiment, interface residues are identical to those in the reference polypeptide or are conservatively substituted for interface residues in the reference polypeptide. In a further embodiment, the polypeptide comprises two or more copies of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-101. In one embodiment, the polypeptide comprises the formula Z1-Z2-Z3: Z1 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164; Z2 comprises an optional amino acid linker; and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164: Here, Z1 and Z3 may be the same or different.

[0006] In another embodiment, the polypeptide comprises the formula B1-B2-Z1-Z2-Z3-B3-B4; Z1, Z2, and Z3 are as defined; B2 and B3 comprise an optional amino acid linker; and One or both of B1 and B4 independently comprise an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164, wherein one of B1 and B4 may be absent.

[0007] In one embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-60, 193-355, and 454-588, and a species selected from those listed in the right-hand column of Table 8, wherein species sites X1, X2, X3, and X4 may be present or absent, and, if present, may be any sequence of one or more amino acids.

[0008] In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-453 and 595-692 and a species selected from those listed in the middle column of Table 9, wherein species sites X1, X2, X3, and X4 may be present or absent and, if present, may be any sequence of one or more amino acids.

[0009] In a further embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-96, wherein in embodiments in which the secretory signal (MARAWIFFLLCLAGRALA; SEQ ID NO: 63) is present, it can be replaced with any other secretory signal.

[0010] In other aspects, the present disclosure provides nucleic acids encoding polypeptides of the present disclosure, expression vectors comprising the nucleic acid operably linked to a promoter, host cells comprising the polypeptides, nucleic acids, and / or expression vectors of the present disclosure, oligomers of polypeptides of the present disclosure, compositions comprising two, three, four, or more copies of a polypeptide of any embodiment of the present disclosure attached to a support (including, but not limited to, a polypeptide particulate support), and pharmaceutical compositions comprising the polypeptides, nucleic acids, expression vectors, host cells, oligomers, and / or compositions of the present disclosure and a pharmaceutically acceptable carrier.

[0011] In another aspect, the present disclosure provides methods for treating or suppressing the onset of a severe acute respiratory syndrome (SARS) coronavirus infection (including SARS-Co-V and SARS-CoV-2), comprising administering to a subject in need thereof an amount of a polypeptide, nucleic acid, expression vector, host cell, oligomer, composition, and / or pharmaceutical composition of the present disclosure effective to treat or suppress the onset of the infection. [Brief explanation of the drawings]

[0012] [Figure 1]Designed minibinder proteins for the SARS-CoV-2 spike receptor-binding domain. The Approach 1 and Approach 2 designs were encoded by long oligonucleotides and screened for binding to fluorescently tagged RBD on the yeast cell surface. Deep sequencing identified three Ace2 helix scaffold designs (Approach 1) and 150 de novo interface designs (Approach 2) that were significantly enriched after FACS sorting for RBD binding. The designs were expressed and purified in E. coli; many were soluble expressed and found to bind the RBD in biolayer interference experiments; and were able to effectively compete with ACE-2 for binding to the RBD (examples shown in Figure 2). Based on BLI data (e.g., see Figure 2), the RBD-binding affinities of the minibinders are: LCB1 < 1 nM, LCB3 < 1 nM. The affinities of LCB2, LCB4, LCB5, LCB6, LCB7, and LCB8 range from 1 to 20 nM, and the relative strengths of the different binders are LCB4>LCB2>LCB9=LCB5>LCB6>LCB7. [Figure 2] High-affinity binding of de novo designed minibinders to the SARS-CoV-2 spike RBD. Biotinylated spike RBD protein was loaded onto a streptavidin biolayer interferometry (BLI) sensor tip (ForteBio Octet®). After washing, the tip was immersed in different concentrations of purified Combo 1 anti-RBD minibinder. After loading, the tip was placed in a buffer-only solution. (Left and center) Response curves show an affinity with a Kd of approximately 300 pM. (Right) ACE-2 was loaded onto the RBD tip, followed by the addition of Combo 1; the minibinder rapidly displaced ACE-2 from the BLI tip. [Figure 3] De novo designed minibinders against the SARS-CoV-2 spike RBD are heat stable. Purified Combo 1 minibinders were measured using a circular dichroism spectrophotometer at 25°C, 95°C, and 25°C after heating to 95°C. All CD spectra were very similar in shape, indicating that the protein remained folded under all conditions. [Figure 4]De novo designed minibinders against the SARS-CoV-2 spike RBD are potent in virus neutralization assays. SARS-CoV-2 strain 2019 n-CoV / USA_WA1 / 2020 was obtained from the US Centers for Disease Control and Prevention (a gift from Natalie Thornburg). Virus stocks were produced in Vero CCL81 cells (ATCC) and titered by focus-forming assay in Vero E6 cells. Serial dilutions of mAb or minibinder were incubated with 102 focus-forming units (FFU) of SARS-CoV-2 for 1 hour at 37°C. RBD minibinder (or mAb) / virus complexes were added to a Vero E6 cell monolayer in a 96-well plate and incubated for 1 hour at 37°C. The cells were then overlaid with 1% (w / v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested after 3 minutes by removing the overlay and fixed with 4% PFA in PBS for 20 minutes at room temperature. Plates were washed and sequentially incubated with 1 μg / mL of CR3022 ([1]) anti-S antibody and HRP-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA. SARS-CoV-2-infected cell foci were visualized using TrueBlue® peroxidase substrate (KPL) and quantified using an ImmunoSpot® analyzer (Cellular Technologies). Data were processed using Prism software (GraphPad Prism® 8.0). [Figure 5]LCB1-Fc prophylactic drug prevents SARS-CoV-2 infection. (A) Molecular surface diagram of three LCB1v1.3 miniproteins bound to individual promoters of the SARS-CoV-2 spike protein trimer (left: side view; right: top view). (B) Binding curves of purified LCB1v1.3 and LCB1-Fc to the SARS-CoV-2 RBD monitored by biolayer interferometry (one experiment performed in duplicate). (C) Neutralization curves of LCB1v1.3, LCB1-Fc, or control binders against the SARS-CoV-2 WA1 / 2020 isolate (EC50 values: 14.4 pM, 71.8 pM, and >10,000 nM, respectively; average of two experiments, each performed in duplicate). (D-J) Seven- to eight-week-old female and male K18-hACE2 transgenic mice received 250 μg of LCB1-Fc or a control binder via intraperitoneal injection 1 day before intranasal inoculation with 103 PFU of SARS-CoV-2. Tissues were collected 4 and 7 days after infection. (D) Body weight change after LCB1-Fc administration (mean ± standard error; n = 8, duplicate experiments: 2-way ANOVA and Sidak's post-hoc test: ***P < 0.001, ****P < 0.0001). (E) Infectious virus in the lungs measured by plaque assay 4 and 7 days after infection (n = 8, duplicate experiments: Mann-Whitney test; ***P < 0.001). (F–J) Viral RNA levels 4 and 7 days after infection in lung, heart, spleen, brain, or nasal washes ( n = 8, duplicate experiments: Mann–Whitney test: ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). [Figure 6]LCB1-Fc prophylactic drug prevents SARS-CoV-2-mediated lung disease. (A) Respiratory mechanics parameters: deep inspiratory volume, inspiratory resistance, elastance, tissue damping, quasi-static compliance, and pressure-volume loops measured 7 days after infection (n = 3–6, duplicate experiments; 2-way ANOVA with Tukey's post-hoc test: ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 between indicated groups). (B) Hematoxylin and eosin staining of lung sections taken 7 days after SARS-CoV-2 infection from mice treated with D-1. Images show low (left) and high (right; corresponding to the boxed area from the left panel) magnification. Scale bar for all images is 100 μm. Representative images from n = 3 mice / group. (C) Heatmap of cytokine mRNA levels from lung tissue of SARS-CoV-2-infected mice 4 days after infection. For each cytokine, fold change was calculated relative to age-matched naive control animals after normalization to Gapdh, and Log2(fold change) was plotted (n=8 mice / group relative to n=3 naive controls). [Figure 7]Post-exposure delivery of anti-RBD binders reduces SARS-CoV-2 burden. (A-G) Seven- to eight-week-old female and male K18-hACE2 transgenic mice received 250 μg of LCB1-Fc or a control binder via intraperitoneal injection 1 day after intranasal inoculation with 103 PFU of SARS-CoV-2. Tissues were collected 4 and 7 days after infection. (A) Body weight change after LCB1-Fc administration (mean ± standard error; n = 6, duplicate experiments: 2-way ANOVA and Sidak's post-hoc test: **P < 0.01, ****P < 0.0001). (B) Infectious virus in the lungs measured by plaque assay 4 and 7 days after infection (n = 6, duplicate experiments: **P < 0.01). (C–G) Viral RNA levels 4 and 7 days after infection in lung, heart, spleen, brain, or nasal washes (n = 6, duplicate experiments; Mann-Whitney test: ns, not significant; *P < 0.05, **P < 0.01). (H) Hematoxylin and eosin staining of lung sections taken 7 days after SARS-CoV-2 infection from mice treated on Day 1. Images show low (left) and high (right; corresponding to the boxed area from the left panel) magnification. Scale bars for all images are 100 μm. Representative images from n = 3 mice / group. (I–J) Male K18-hACE2 transgenic mice, 7–8 weeks old, received a single 50 μg intranasal dose of LCB1v1.3 or a control binder 1 or 2 days after inoculation with 103 PFU of SARS-CoV-2. Viral RNA levels in lungs (I) or nasal washes (J) 7 days after infection (n = 6, 2 experiments: 1-way ANOVA: ns, not significant; * P < 0.05, **** P < 0.0001). [Figure 8]Intranasal administration of LCB1v1.3 reduces viral infection even when administered 5 days before exposure to SARS-CoV-2. (A-D) Seven- to eight-week-old female K18-hACE2 transgenic mice received a single 50 μg intranasal dose of LCB1v1.3 or a control binder at the indicated time points before intranasal inoculation with 103 PFU of SARS-CoV-2. Tissues were harvested 4 and 7 days after infection, and viral RNA levels were determined (n = 5-6 animals / group, two experiments: 2-way ANOVA with Sidak's post-hoc test: ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). (E-J) Seven- to eight-week-old female K18-hACE2 transgenic mice received the indicated intranasal doses of LCB1v1.3 or control binder the day before intranasal inoculation with 103 PFU of SARS-CoV-2. (E) Body weight change after administration of LCB1v1.3 or control binder (mean ± standard error; n = 6, duplicate experiments; two-way ANOVA and Sidak post-hoc test compared with the control binder-treated group: **P < 0.01, ****P < 0.0001). (F-J) Viral RNA levels in lung, heart, spleen, brain, or nasal washes 7 days after infection (n = 6, duplicate experiments; Kruskal-Wallis ANOVA and Dunn's post-hoc test: *P < 0.05, **P < 0.01, ***P < 0.001). (K) Hematoxylin and eosin staining of lung sections taken 7 days after SARS-CoV-2 infection from mice administered a single 50 μg dose of LCB1v1.3 or control binder intranasally on D-1. Images show low (left) and high (right; corresponding to the boxed area from the left panel) magnification. Scale bars for all images are 100 μm. Representative images from n=3 mice / group. [Figure 9]Immunogenicity of LCB1v1.3 and protection from infection. (A) Scheme of experimental details. K18-hACE2 transgenic mice (n = 10–12 per group) were treated intranasally with 50 μg of LCB1v1.3 or a control binder every 3 days. 18 days after treatment, animals were bled and anti-LCB1v1.3 antibodies were measured. The next day, animals were inoculated with 103 PFU of SARS-CoV-2, and tissues were collected 7 days post-infection. (B) Serum antibody binding to LCB1v1.3 was measured by ELISA (three experiments). The dashed line indicates the detection limit of the assay. (C) Body weight change after administration of LCB1v1.3 or a control binder (mean ± standard error; two experiments: two-way ANOVA and Sidak's post-hoc test: ****P < 0.0001). (D–H) Viral RNA levels 7 days after infection in lung, heart, spleen, brain, or nasal washes (duplicate experiments: Mann-Whitney test: * P < 0.05, ** P < 0.01, **** P < 0.0001). [Figure 10]LCB1v1.3 protects mice against the B.1.1.7 variant and the WA1 / 2020 E484K / N501Y / D614G strain. (A) Neutralization of LCB1v1.3 against B.1.1.7 or WA1 / 2020 E484K / N501Y / D614G SARS-CoV-2 (EC50 values: 802 pM and 667 pM, respectively; average of two experiments, each performed in duplicate). (B-G) Seven- to eight-week-old female K18-hACE2 transgenic mice were treated with a single 50 μg intranasal dose of LCB1v1.3 or a control binder the day before intranasal inoculation with 103 PFU of B.1.1.7. (B) Body weight change after administration of LCB1v1.3 or a control binder (mean ± standard error; n = 6, duplicate experiments: 2-way ANOVA and Sidak's post-hoc test: ***P < 0.001, ****P < 0.0001). (C-G) Viral RNA levels in the lung, heart, spleen, nasal wash, or brain 6 days after infection (n = 6, duplicate experiments: Mann-Whitney test: *P < 0.05, **P < 0.01). (H-M) Eight-week-old male K18-hACE2 transgenic mice were treated with a single 50 μg intranasal dose of LCB1v1.3 or a control binder the day before intranasal inoculation with 103 PFU of WA1 / 2020 E484K / N501Y / D614G. (H) Body weight change after administration of LCB1v1.3 or control binder (mean ± SEM; n = 6, duplicate experiments: 2-way ANOVA with Sidak's post-hoc test: *P < 0.05, ****P < 0.0001). (I-M) Viral RNA levels in lung, heart, spleen, nasal wash, or brain 6 days after infection (n = 6, duplicate experiments: Mann-Whitney test: *P < 0.05, **P < 0.01). [Figure 11]Induction of cytokines and chemokines after SARS-CoV-2 infection. Individual plots of cytokine and chemokine RNA levels in the lungs of SARS-CoV-2-infected mice 4 days post-infection after treatment with control or LCB1-Fc binders (n = 8 mice / group, 2 experiments; Mann-Whitney test: ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001). These data were used to generate the heatmap in Figure 6C. [Figure 12] Intranasal delivery of LCB1v1.3 the day after or two days after SARS-CoV-2 infection reduces viral load, as shown in Figure 7. (A-C) Male K18-hACE2 transgenic mice aged 7 to 8 weeks received a single 50 μg intranasal dose of LCB1v1.3 or a control binder the day after or two days after intranasal inoculation with 10 PFU of SARS-CoV-2. Viral RNA levels 7 days after infection in the heart (A), spleen (B), or brain (C) (n = 6, duplicate experiments: 1-way ANOVA: *P < 0.05, **P < 0.01). [Figure 13] Related to Figure 8, intranasal prophylaxis with LCB1v1.3 reduces weight loss. Seven- to eight-week-old female K18-hACE2 transgenic mice received a single 50 μg intranasal dose of LCB1v1.3 or a control binder at the indicated time points prior to intranasal inoculation with 10 PFU of SARS-CoV-2. Body weight changes were recorded daily (mean ± standard error; n = 6, duplicate experiments: 2-way ANOVA with Sidak's post-hoc test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). [Figure 14] Multivalent minibinders simultaneously engage multiple epitopes on the pre-fusion SARS-CoV-2 spike protein, resulting in extremely slow dissociation rates. (A, B) Dissociation of minibinder constructs complexed with the receptor binding domain (RBD) (A) or hexapro spike protein (S6P) (B) was followed by competition with a 100-fold molar excess of tag-free M1 using AlphaLISA® (mean ± standard error, n=3). [Figure 15] Cryo-EM structure of a multivalent minibinder in complex with the SARS-CoV-2 S glycoprotein. (A) Ribbon diagram representation of all three minibinders bound to the RBDs. (B) Cryo-EM map of F31-G10 in a complex containing two RBDs. (C) Cryo-EM map of F31-G10 of F231-P24 in a complex containing three RBDs. (D) Design model of H2-1 bound to the S glycoprotein. (E) Cryo-EM maps of H2-1 in complex with the S glycoprotein in two orthogonal orientations. (F) Cryo-EM map showing interacting residues at the interface between H2-1 and the S glycoprotein. [Figure 16] Multivalency enhances both the breadth and potency of neutralization of SARS-CoV-2 mutants by minibinders. (A) Dissociation of minibinder constructs from S6P mutants after 24 hours was measured by competition with tag-free H2-0 using AlphaLISA® (mean, n=3). Cells containing X show insufficient signal in the absence of competitor to quantify the proportion of bound protein. (B) Competition between ACE2 and minibinder constructs for S6P was measured by ELISA (mean, n=2). (C) Neutralization curves of minibinder constructs against SARS-CoV-2 pseudovirus mutants (mean, n=2). (D) Table summarizing the neutralization potency of multivalent minibinder constructs against SARS-CoV-2 pseudovirus mutants. N / A indicates IC50 values ​​higher than the concentration range tested and an IC50 above 50,000 pM. (E) Neutralization curves of minibinder constructs against natural SARS-CoV-2 isolates (mean, n=2). (F) Table summarizing the neutralization potency of multivalent minibinder constructs against natural SARS-CoV-2 isolates. [Figure 17]Top candidate multivalent minibinders are escape-resistant and protect mice from SARS-CoV-2 infection following intranasal administration prior to challenge. (A) Plaque assays were performed to isolate VSV-SARS-CoV-2 chimeric virus escape mutants against a control neutralizing antibody (2B04) and the F231-P12 and H2-1 multivalent minibinders. Images are representative of 35 duplicate wells per multivalent minibinder. Large plaques, highlighted by black arrows, indicate escape. (B, C) K18-hACE2 transgenic mice (n = 6 / time point) received 50 μg of H2-0 intranasally 24 hours (T-24h) before infection with 10 plaque-forming units of SARS-CoV-2 variants B.1.1.7, B1.351, or B.1.1.24 intranasally on day 0 (intranasally, 2 x 25 μl doses / nostril, 100 μl total). (B) Daily body weight change after infection (mean ± standard error; n = 6, 2-way ANOVA with Sidak's post-hoc test: *P < 0.05, ***P < 0.001, ****P < 0.0001). (C) Several days after infection (day 6 post-infection), animals were sacrificed (n = 6 / time point) and analyzed by quantitative real-time RT-PCR for the presence of SARS-CoV-2 viral RNA in lung, heart, spleen, brain, or nasal washes (n = 6; Mann-Whitney test: ns, not significant; * P < 0.05; ** P < 0.01). DETAILED DESCRIPTION OF THE INVENTION

[0013] All cited references are incorporated herein by reference in their entirety. Unless otherwise specifically mentioned within this application, the techniques utilized may be found in any of several well-known references, including "Molecular Cloning: A Laboratory Manual" (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press); "Gene Expression Technology" (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, CA); "Guide to Protein Purification" (Methods in Enzymology, edited by M. P. Deutschner, 1990, Academic Press, Inc.); "PCR Protocols: A Guide to Methods and Applications" (Innis et al., 1990, Academic Press, San Diego, CA); "Animal Cell Culture: A Manual of Basic Technique, 2nd Ed.," RI Freshney, 1987, Liss, Inc., New York, NY); Gene Transfer and Expression Protocols, pp. 109-128, EJ Murray (ed.), The Humana Press Inc., Clifton, NJ); and Ambion 1998 Catalog (Ambion, Austin, TX).

[0014] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0015] As used herein, amino acid residues are represented by the following abbreviations: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0016] In all embodiments of the polypeptides disclosed herein, the N-terminal methionine residue is optional (ie: may or may not be present).

[0017] All embodiments of any aspect of this disclosure may be used in combination unless the context clearly dictates otherwise.

[0018] Unless the context clearly requires otherwise, throughout this specification and the claims, the words "comprise," "comprising," and similar words are to be understood in the inclusive sense, as opposed to the exclusive or exhaustive sense; that is, to mean "including, but not limited to." Terms using the singular or plural number also include the plural and singular number, respectively. Furthermore, the terms "herein," "above," and "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0019] In a first aspect, the present disclosure provides a polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-101, wherein the polypeptide binds to the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD). >LCB1-1 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER (SEQ ID NO: 1) >LCB1-2 DKEEILNKIYEIMRLLDELGNAEASMRVSDLILEFMKKGDERLLEEAERLLEEVER (SEQ ID NO: 2) >LCB1-3 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 3) >LCB1-4 DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 4) >LCB1-5 DKENILQKIYEIMKTLDQLGHAEASMNVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 5) LCB1_v 1.1_Cys DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVERC (SEQ ID NO: 6) >LCB1_v 1.2 DKENILQKIYEIMKTLDQLGHAEASMYVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 7) >LCB1_v 1.3 DKENILQKIYEIMKTLEQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 8) >LCB1_v 1.4 DKENILQKIYEIMKTLEQLGHAEASMQVSDLIYEFMKQGDENLLEEAEQLLQEVER (SEQ ID NO: 9) >LCB1_v 1.5 (LCB1_v 1.3, with N-linked glycosylation) DKENILQKIYEIMKTLEQLGHAEASMNVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 10) >LCB2-1 SDDEDSVRYLLYMAELRYEQGNPEKAKKILEMAEFIAKRNNNEELERLVREVKKRL (SEQ ID NO: 11) >LCB2-2 SDDEDAVRYLLYMAELLYKQGNPEEAKKLLELAEFIAKRNNNEELERLVREVKKRL (SEQ ID NO: 12) >LCB3-1 NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLS (SEQ ID NO: 13) >LCB3-2 NDDELLMLVTDLVAEALLFAKDEEIKKRVFTLFELADKAYKNNDRDTLSKVVSELKELLERLQ (SEQ ID NO: 14) > LCB3_v 1.2 NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID NO: 15) >LCB3-4 NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFENATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID NO: 16) >LCB3_v 1.1 NDDELHMQMTDLVYEALHFAKDEEFQKHVFQLFEKATKAYKNNDRQKLEKVVEELKELLERLLS (SEQ ID NO: 17) >LCB3_v 1.3 NDDELHMQMTDLVYEALHFAKDEEFQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID NO: 19) >LCB3_v 1.4 NDDELHMQMTDLVYEALHKAKDEEFQKHVFQLFEKATKARKNKDRQKLEKVVEELKELLERLLS (SEQ ID NO: 20) >LCB3_v 1.5 NDDELHMQMTDLVYEALHKAKDEEMQKRVFQLFEQADKAYKTKDRQKLEKVVEELKELLERLLS (SEQ ID NO: 21) >LCB4-1 QREKRLKQLEMLLEYAIERNDPYLMFDVAVEMLRLAEENNDERIIERAKRILEEYE (SEQ ID NO: 23) >LCB4-2 DREERLKYLEMLLELAVERNDPYLIFDVAIELLRLAEENNDERIYERAKRILEEVE (SEQ ID NO: 24) >LCB5-1 SLEELKEQVKELKKELSPEMRRLIEEALRFLEEGNPAMAMMVLSDLVYQLGDPRVIDLYMLVTKT (SEQ ID NO: 25) >LCB5-2 SLEEVKEILKELKKELSPEDRRLIEEALRLLEEGNPAMASMVLSDLVFLLGDPRVIELLMLVTKT (SEQ ID NO: 26) >LCB6-1 DREQRLVRFLVRLASKFNLSPEQILQLFEVLEELLERGVSEEEIRKQLEEVAKELG (SEQ ID NO: 27) >LCB6-2 DREQRLVRFLVRLASKFNLSMEQILILFDVLEELLERGVSEEEIRKILEEVAKEL (SEQ ID NO: 28) >LCB7-1 DDDIRYLIYMAKLRLEQGNPEEAEKVLEMARFLAERLGMEELLKEVRELLRKIEELR (SEQ ID NO: 29) >LCB7-2 DDDVRYLIYMAKLLLEQGNPEEAEKVLESARFAAELLGNEELLKEVRELLRKIEELR (SEQ ID NO: 30) >LCB8-1 PIIELLREAKEKNDEFAISDALYLVNELLQRTGDPRLEEVLYLIWRALKEKDPRLLDRAIELFER (SEQ ID NO: 31) >LCB8-2 PVTELLREAKEKNDPMAISDALFLVFELAQRTGDPRLEEVLFLIWRALKEKDPRLLDRAIELFER (SEQ ID NO: 32) >AHB1-1 DEDLEELERLYRKAEEVAKEAKDASRRGDDERAKEQMERAMRLFDQVFELAQELQEKQTDGNRQKATHLDKAVKEAADELYQRVR (SEQ ID NO: 33) >AHB1-2 DEDLEELERLYRKAEEVAKEAEEASRRGDKERAKELLERALHLFDQVFELAQELQEKLTDEKRQKATHLDKAVHEAADELYQRVR (SEQ ID NO: 34) >AHB2-1 ELEERVMHLLDQVSELAHELLHKLTGEELQRATHFDKWANEAILELIKSDDEREIREIEEEARRILEHLEELARK (SEQ ID NO: 100) >AHB2-2_ ELEEQVMHVLDQVSELAHELLHKLTGEELERAAYFNWWATEMMLELIKSDDEREIREIEEEARRILEHLEELARK (SEQ ID NO: 101)

[0020] As described in detail in the Examples below, the polypeptide binds with high affinity to the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD).

[0021] In any of the embodiments described herein, the percent identity requirement does not include any additional functional domains that may be incorporated into the polypeptide. In one embodiment, one, two, or three amino acids may be deleted from the N-terminus and / or C-terminus.

[0022] The polypeptide has been subjected to comprehensive mutational analysis, as described in the Examples below, which allows for the determination of permissible substitutions at each residue position within the polypeptide. Exemplary substitutions are shown in Table 1 (where the numbers represent the residue number and the letters represent the single-letter amino acids that can be present at that residue position). That is, in one embodiment, amino acid substitutions for a reference polypeptide amino acid sequence (i.e., one of SEQ ID NOS: 1-17, 19-21, 23-34, and 100-101) are selected from the exemplary amino acid substitutions shown in Table 1.

[0023] [Table 1] TIFF2025170319000002.tif242162TIFF2025170319000003.tif242162TIFF2025170319000004.tif242162TIFF2025170319000005.tif242162TIFF2025170319000006.tif242162TIFF2025170319000007.tif242162TIFF2025170319000008.tif242162TIFF2025170319000009.tif242162TIFF2025170319000010.tif242162TIFF2025170319000011.tif242161TIFF2025170319000012.tif242162TIFF2025170319000013.tif242162TIFF2025170319000014.tif242161TIFF2025170319000015.tif242162TIFF2025170319000016.tif242162TIFF2025170319000017.tif242162TIFF2025170319000018.tif242162TIFF2025170319000019.tif242162TIFF2025170319000020.tif242162TIFF2025170319000021.tif242162TIFF2025170319000022.tif23162

[0024] The residue numbers of interface residues within 8A relative to the RBD target are listed in Table 2 for various design types. In another embodiment, the amino acid residues at the interface residues listed in Table 2 are either identical to the reference sequence at that residue position or may be substituted by conservative amino acid substitutions. Such conservative amino acid substitutions include replacing a residue with a residue having similar physicochemical properties, for example, substituting one aliphatic residue for another (such as substituting Ile, Val, Leu, or Ala for each other), or substituting one polar residue for another (such as between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions are known, for example, substituting entire regions with similar hydrophobic properties. Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another.Particular conservative substitutions include, for example; Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0025] [Table 2]

[0026] In one embodiment, the amino acid residue at the interface residue listed in Table 2 is identical to that residue position in the reference sequence.

[0027] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 13-17, 19-21, 33-34, and 100-101.

[0028] In one embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136 (see Table 3).

[0029] [Table 3] TIFF2025170319000025.tif126162

[0030] The polypeptides may contain a substantial number of mutations while retaining binding activity, as described in detail in the Examples below. In one embodiment, the polypeptides contain amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 residues selected from the group consisting of 2, 4, 5, 14, 15, 17, 18, 27, 28, 32, 37, 38, 39, 41, 42, 49, 52, and 55. In another embodiment, the substitutions are selected from the substitutions listed in Table 4, either individually (i.e., any single mutation listed in the table) or in combination in a given row.

[0031] [Table 4] TIFF2025170319000027.tif124162

[0032] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163 (see Table 5).

[0033] [Table 5] TIFF2025170319000029.tif45162

[0034] The polypeptides may contain a substantial number of mutations while retaining binding activity, as described in detail in the Examples below. In one embodiment, the polypeptides contain amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 13 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 residues selected from the group consisting of 2, 6, 8, 9, 13, 14, 19, 22, 25, 26, 28, 29, 34, 35, 37, 40, 43, 45, 49, and 62. In another embodiment, the substitutions are selected from the substitutions listed in Table 6, either individually or in combination in a given row.

[0035] [Table 6] TIFF2025170319000031.tif161162

[0036] In a further embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164 (see Table 7).

[0037] [Table 7]

[0038] In one embodiment, the polypeptide comprises an amino acid substitution relative to the amino acid sequence of SEQ ID NO: 101 at either or both residues selected from the group consisting of 63 and 75. In a further embodiment, the substitution comprises R63A and / or K75T.

[0039] In all embodiments disclosed herein, the polypeptide may include one or more additional functional groups or residues as deemed appropriate for the intended use. In one embodiment, the polypeptide may further include one or more additional cysteine ​​residues at the N-terminus and / or C-terminus. In another embodiment, the polypeptide may further include an N-linked glycosylation site (i.e., N-X(S / T), where X is any amino acid).

[0040] In another embodiment, the polypeptide may comprise two or more copies (i.e., two, three, four, five, or more copies) of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-101. In this embodiment, two or more binding agents are linked. In one embodiment, the two or more copies of the polypeptide are all identical; in another embodiment, the two or more copies of the polypeptide are not all identical. In any of these embodiments, the two or more copies of the polypeptide may be separated by an amino acid linker sequence, even though such an amino acid linker sequence is not required. The amino acid linkers may be of any length and any amino acid composition appropriate for the intended purpose, hi one embodiment, the amino acid linkers are independently in the range of 2 to 100 amino acids in length, or in the range of 3 to 100 amino acids in length.

[0041] In another embodiment, the amino acid linker sequence comprises a Gly-Ser rich (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Gly-Ser residues) amino acid linker. In a further embodiment, the Gly-Ser rich linker comprises an amino acid sequence selected from the group consisting of GG and SEQ ID NOs: 35-46 and 165-171. GSGS (SEQ ID NO: 35) GGSGGS (SEQ ID NO: 36) SGGSGGSGGSG (SEQ ID NO: 37) GGSGGSGSGGSG (SEQ ID NO: 38) GGSGSSGGSGSGSG (SEQ ID NO: 39) GGSGSGGSGSGSGGS (SEQ ID NO: 40) SGGSGSGSGGSGSGS (SEQ ID NO: 41) GGGSGGGSSGGSGGSSGGGSGGGS (SEQ ID NO: 42) GGGSGGGGSGGGGGSGGGGGSGGGGSGGGGSG (SEQ ID NO: 43) GGGSGGGSGGSGGSGGGSGGGSGSGGSGGGGSGGGS (SEQ ID NO: 44) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 45) SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46) GGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 165) GGSGGGGSGGGGSGG (SEQ ID NO: 166) GGSGGGGSGG (SEQ ID NO: 167) GGGGSGGGG (SEQ ID NO: 168) GGGSGGG (SEQ ID NO: 169) GGSGG (SEQ ID NO: 170) GGSGSSG (SEQ ID NO: 171)

[0042] In another embodiment, the amino acid linker sequence may comprise a Pro-rich (at least 15%, 20%, 25% or more Pro residues) amino acid linker. A non-limiting exemplary embodiment may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 97-98 and 172-176. AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSASG (SEQ ID NO: 97) GSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSGGSGNSSGSGGSPVPSTPPTPSPSTPPTPSPSAS (SEQ ID NO: 98) GGASPAAPAPASPAAPAPSAPAGG (SEQ ID NO: 172) GGASPAAPAPASPAGG (SEQ ID NO: 173) GGASPAAPAPGG (SEQ ID NO: 174) GGASPAAPAGG (SEQ ID NO: 175) GGSSGPSTPPTPSPSTPPTPSPSPGGSSG (SEQ ID NO: 176)

[0043] In a further non-limiting embodiment, the amino acid linker can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 99 and 177-178.

[0044] [Table 8]

[0045] In one embodiment, the polypeptide comprises the formula Z1-Z2-Z3, wherein: Z1 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164; Z2 comprises an optional amino acid linker; and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164; Here, Z1 and Z3 may be the same or different.

[0046] In one embodiment, Z1 and Z3 are the same; in another embodiment, Z1 and Z3 are different. In embodiments in which Z1 and Z3 are different, each may be a variant of a given starting monomer (e.g., Z1 comprises the amino acid sequence of SEQ ID NO:1 (LCB1) and Z3 comprises the amino acid sequence of SEQ ID NOs:102-136). Any such combination of monomers of the present disclosure may be used. It will further be understood that a polypeptide may comprise two, three, four, five, or more monomers of any of the embodiments disclosed herein. In embodiments in which three or more monomers are present, all three monomers may be identical; two monomers may be identical and one different; or all three monomers may be different.

[0047] In one embodiment using LCB1 and variants thereof, Z1 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136; and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136.

[0048] In another embodiment using LCB3 and variants thereof: Z1 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163; and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163.

[0049] In another embodiment using AHB and its variants: Z1 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164; and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164.

[0050] In one embodiment, one of Z1 and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136; and The other of Z1 and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163.

[0051] In another embodiment, one of Z1 and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136, and The other of Z1 and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-100, and 164.

[0052] In a further embodiment, one of Z1 and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163; and The other of Z1 and Z3 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-100, and 164.

[0053] In another embodiment of any of the other embodiments disclosed herein, the polypeptide comprises at least three monomers (i.e., three, four, five, or more). In one such embodiment, the polypeptide comprises the formula B1-B2-Z1-Z2-Z3-B3-B4; wherein Z1, Z2, and Z3 are as defined above; B2 and B3 comprise an optional amino acid linker; and One or both of B1 and B4 independently comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164, wherein one of B1 and B4 may be absent. In one embodiment, one of B1 and B4 is absent. In another embodiment, both B1 and B4 are present. In one embodiment, B1 and B4 independently comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34, and 100-164. In this embodiment, B1 and B4 may be the same or different. In one embodiment, B1, if present, and B4, if present, are identical to one or both of Z1 and Z3. In another embodiment, B1, if present, and B4, if present, are not identical to either Z1 or Z3.

[0054] In one embodiment, B1, if present, and B4, if present, independently comprise an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 13-17, 19-21, 33-34, 100-101, and 102-164.

[0055] In another embodiment, B1, if present, and B4, if present, independently comprise an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136.

[0056] In a further embodiment, B1, if present, and B4, if present, independently comprise an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163.

[0057] In yet a further embodiment, B1, if present, and B4, if present, independently comprise an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164.

[0058] In various embodiments where both B1 and B4 are present, one of B1 and B4 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136, and the other comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163; one of B1 and B4 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136, and the other comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164, or one of B1 and B4 comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21, and 137-163, and the other comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164.

[0059] In various non-limiting embodiments, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-60, 193-355, and 454-588, and comprises a species selected from those listed in the right-hand column of Table 8, wherein species sites X1, X2, X3, and X4 may be present or absent and, if present, may be any sequence of one or more amino acids. In all embodiments, an optional N-terminal methionine residue may or may not be present in the polypeptide. In one embodiment, an optional N-terminal methionine residue is absent in the polypeptide. >LCB1-6GS-LCB1 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER GGSGGS DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER (SEQ ID NO: 47) >LCB1-12GS-LCB1 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER GGSGGSGSGGSGD KEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER (SEQ ID NO: 48) >LCB1-24GS-LCB1 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER GGGSGGGSSGGSGGSSGGGSGGGS DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER (SEQ ID NO: 49) >LCB1-36GS-LCB1 DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER GGGSGGGSGGSGGSGGGSGGGSGSGGSGGGGSGGGS DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAEERLLEEVER (SEQ ID NO: 50) >LCB1_v 1.1-GSLCB1_v 1.1(1GS1) DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 51) >LCB1_v 1.1 - PRO - LCB1_v 1.1(1PRO1) DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSASG DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID: 52) >LCB3_v 1.2 - GS3 - LCB3_v 1.2(3GS3) NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLL SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID: 53) >LCB3_v 1.2 - PRO - LCB3_v 1.2(3PRO3) NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSASG NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID: 54) >LCB1_v 1.1 - GS - LCB3_v 1.2(1GS3) DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID: 55) >LCB3_v 1.2 - GS - LCB1_v 1.1(3GS1) NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLL SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID: 56) >LCB3_v 1.2 - 10GS - LCB1_v 1.1(LCB3 - GS10 - LCB1) NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLSGGSGGSGGSG DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 57) >LCB1_v 1.1-PRO-LCB3_v 1.2(1PRO3) DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSASG NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLLS (SEQ ID NO: 58) >LCB3_v 1.2-PRO-LCB1_v 1.1(3PRO1) NDDELHMQMTDLVYEALHFAKDEEIQKHVFQLFEKATKAYKNKDRQKLEKVVEELKELLERLL SAGSGGGSGGSGGSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSPVPSTPPTPSPSTPPTPSPSASG DKENILQKIYEIMKTLDQLGHAEASMQVSDLIYEFMKQGDERLLEEAERLLEEVER (SEQ ID NO: 59) >36175(5_LCB1_Linker14) DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER GGSGSSGGSGSGSG DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER GGSGSSGGSGSGSG DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER GGSGSSGGSGSGSG DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER GGSGSSGGSGSGSG DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER (SEQ ID NO: 60)

[0060] [Table 9] TIFF2025170319000035.tif242162TIFF2025170319000036.tif242162TIFF2025170319000037.tif242162TIFF2025170319000038.tif242162TIFF2025170319000039.tif242162TIFF2025170319000040.tif242161TIFF2025170319000041.tif242162TIFF2025170319000042.tif242162TIFF2025170319000043.tif242162TIFF2025170319000044.tif242161TIFF2025170319000045.tif242161TIFF2025170319000046.tif242162TIFF2025170319000047.tif242162TIFF2025170319000048.tif242161TIFF2025170319000049.tif242161TIFF2025170319000050.tif242161TIFF2025170319000051.tif242161TIFF2025170319000052.tif242162TIFF2025170319000053.tif242161TIFF2025170319000054.tif242161TIFF2025170319000055.tif242161TIFF2025170319000056.tif242161TIFF2025170319000057.tif242161TIFF2025170319000058.tif242161TIFF2025170319000059.tif242162TIFF2025170319000060.tif242161TIFF2025170319000061.tif242162TIFF2025170319000062.tif242162TIFF2025170319000063.tif157162

[0061]

Table 10

[0062] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a species selected from those listed in the middle column of Table 8. In these embodiments, X1, X2, X3 (if indicated in the species), and X4 (if indicated in the species) can be present or absent, and, if present, can be any sequence of one or more amino acids. For example, the species in the middle column of the sequence in the first column of Table 8 is X1-(SEQ ID NO:4)-X2-(SEQ ID NO:4). In this embodiment, X2 can be present or absent, and, if present, can comprise an amino acid linker of any suitable length and amino acid composition as deemed appropriate (for example). X1 may be present or absent and, if present, may comprise any amino acid residue or residues as deemed appropriate, including but not limited to a leader sequence, a detectable tag, a purification tag, etc.

[0063] In another example, the species shown in the middle column of the sequence in the last row of Table 8 is X1-(SEQ ID NO:155)-X2-(SEQ ID NO:164)-X3-(SEQ ID NO:135)-X4. In this embodiment, X2 and X3 may be present or absent and, if present, may comprise (for example) an amino acid linker of any suitable length and amino acid composition as deemed appropriate. X1 and X4 may be present or absent and, if present, may comprise any amino acid residue or residues as deemed appropriate, including, but not limited to, a leader sequence, a detectable tag, a purification tag, a secretion signal, etc.

[0064] In some embodiments, optional domains present between monomer domains may be present and include an amino acid linker. In this embodiment, (a) in the first example above, X2 is present and includes an amino acid linker of any suitable length and amino acid composition, and X1 may be present or absent; and (b) in the second example above, one or both of X2 and X3 are present and include an amino acid linker of any suitable length and amino acid composition, and X1 and X4 may independently be present or absent.

[0065] In any one embodiment or combination of embodiments of the polypeptides disclosed herein, the polypeptide may further comprise one or more additional functional peptide domains. Any such additional functional peptide domains may be used as appropriate for the intended purpose. In various non-limiting embodiments, the additional functional peptide domain may comprise, for example, a targeting domain, a detectable domain, a scaffolding domain, a secretion signal, an Fc domain, or an additional therapeutic peptide domain. In one embodiment, the additional functional domain comprises an Fc domain, including, but not limited to, an Fc domain comprising an amino acid sequence comprising the amino acid sequence of SEQ ID NO:64. Fc domain: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 64)

[0066] In another embodiment, the added functional domain may comprise an oligomerization domain. Any oligomerization domain may be used as suitable for generating oligomers suitable for the intended purpose. In a non-limiting embodiment, the oligomerization domain may comprise a homotrimerization domain. Exemplary oligomerization domains may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 179-189 and 589-594.

[0067] [Table 11]

[0068] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-453 and 595-692, and a species selected from those listed in the right-hand column of Table 9, wherein species sites X1, X2, X3, and X4 may be present or absent and, if present, may be any sequence of one or more amino acids. In all embodiments, an optional N-terminal methionine residue may be present or absent in the polypeptide. In one embodiment, an optional N-terminal methionine residue is absent in the polypeptide.

[0069] [Table 12] TIFF2025170319000083.tif242162TIFF2025170319000084.tif242162TIFF2025170319000085.tif242162TIFF2025170319000086.tif242161TIFF2025170319000087.tif242162TIFF2025170319000088.tif242161TIFF2025170319000089.tif242161TIFF2025170319000090.tif242161TIFF2025170319000091.tif242161TIFF2025170319000092.tif242162TIFF2025170319000093.tif242162TIFF2025170319000094.tif242162TIFF2025170319000095.tif242161TIFF2025170319000096.tif242162TIFF2025170319000097.tif242161TIFF2025170319000098.tif242162TIFF2025170319000099.tif242161TIFF2025170319000100.tif242161TIFF2025170319000101.tif242162TIFF2025170319000102.tif95162

[0070]

Table 13

[0071] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a species selected from those listed in the middle column of Table 9. In these embodiments, X1, X2, X3 (if indicated in the species), and X4 (if indicated in the species) can be present or absent, and, if present, can be any sequence of one or more amino acids, as described above for the embodiments listed in Table 8. In some embodiments, an optional domain present between monomer domains can be present and can include an amino acid linker, as described above for the embodiments listed in Table 8.

[0072] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:693-701, wherein the optional N-terminal methionine residue may be present or absent, and wherein the residue in parentheses may be present or absent (preferably absent) and is not considered in determining percent identity. In one embodiment, the N-terminal methionine residue is absent, and the optional residue is also absent.

[0073] [Table 14] TIFF2025170319000113.tif27162

[0074] The polypeptide of any one embodiment or combination of embodiments described herein may be further linked to a stabilization domain that promotes extended residence time upon administration to a subject. Any suitable stabilization domain may be used for the intended purpose. Examples of stabilization domains include, but are not limited to, polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and / or Ser ("PAS-modified"), and / or mucin-diffusible polypeptides composed of the amino acids Lys and Ala, with or without Glu.

[0075] Non-limiting embodiments of such mucin-diffusible polypeptides include, but are not limited to: Mucin domain: AKAKAKAKAKAKAKAKAKGG (SEQ ID NO: 61); GGAKAKAKAKAKAKAKAKAKAKAK (SEQ ID NO: 62).

[0076] Exemplary polypeptides of these embodiments may comprise, for example, an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:65-96, wherein in embodiments where a secretion signal is present (MARAWIFFLLCLAGRALA; SEQ ID NO:63), it can be replaced with any other secretion signal. [ka] TIFF2025170319000115.tif242162TIFF2025170319000116.tif242162TIFF20251703190 00117.tif242162TIFF2025170319000118.tif242162TIFF2025170319000119.tif124162

[0077] [Table 15] TIFF2025170319000121.tif141162

[0078] The present disclosure further provides oligomers of the polypeptides of any one embodiment or combination of embodiments described herein. In one embodiment, the oligomer is an oligomer of a polypeptide disclosed herein that includes an oligomerization domain. In one embodiment, the oligomer comprises a trimer, including, but not limited to, a homotrimer.

[0079] In another embodiment, the present disclosure provides a composition comprising two, three, four, or more copies of a polypeptide of any one embodiment or combination of embodiments described herein attached to a support, including but not limited to, a polypeptide particle support, such as a nanoparticle or virus-like particle.

[0080] As disclosed herein, the polypeptides bind to the SARS-CoV-2 spike glycoprotein and are therefore useful (for example) as therapeutic agents for treating SARS-CoV-2 infection. In one embodiment, the polypeptides bind to the SARS-CoV-2 spike glycoprotein with an affinity of at least 10 nM, as measured as described in the accompanying Examples.

[0081] In another aspect, the present disclosure provides nucleic acids encoding the polypeptides of the present disclosure. The nucleic acid sequences may comprise RNA (such as mRNA) or DNA. Such nucleic acid sequences may contain additional sequences useful for promoting expression of the encoded protein and / or facilitating purification of the protein, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the contents of this specification, it will be clear to those skilled in the art which nucleic acid sequences encode the proteins of the present invention.

[0082] In another aspect, the present disclosure provides an expression vector comprising a nucleic acid of any one embodiment or combination of embodiments of the present disclosure operably linked to a suitable control sequence. An "expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of effecting expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present disclosure is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. Control sequences need not be contiguous with the nucleic acid sequence, so long as they function to direct its expression. Thus, for example, a non-translated but transcribed intervening sequence may be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors may be of the type known in the art, including, but not limited to, plasmid- and viral-based expression vectors. The control sequences used to drive expression of the nucleic acid sequences of the present disclosure in mammalian systems can be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters, including but not limited to, tetracycline, ecdysone, steroid responsive ones).

[0083] In one aspect, the present disclosure provides a cell comprising a polypeptide, composition, nucleic acid, and / or expression vector of any one embodiment or combination of embodiments described herein, wherein the cell can be either a prokaryotic cell or a eukaryotic cell (such as a mammalian cell). In one embodiment, the cell may be transiently or stably transformed with a nucleic acid or expression vector of the present disclosure. Such introduction of expression vectors into prokaryotic and eukaryotic cells can be accomplished by any technique known in the art. A method for producing a polypeptide of the present invention is a further part of the present invention. The method comprises: (a) culturing a host according to this aspect of the invention under conditions conducive to expression of the polypeptide; and (b) optionally recovering the expressed polypeptide. In other embodiments, the polypeptide may be produced by any other suitable technique, including, but not limited to, using cell-free protein synthesis (or in vitro transcription and translation).

[0084] In another aspect, the present disclosure provides: (a) a polypeptide, nucleic acid, expression vector, and / or host cell of any one embodiment or combination of embodiments described herein; and (b) a pharmaceutically acceptable carrier; The present invention provides a pharmaceutical composition / vaccine comprising: The composition may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity agent; (e) a stabilizer; (f) a preservative, and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The composition may also comprise a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the composition comprises a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition comprises a bulking agent, such as glycine. In yet another embodiment, the composition includes a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The composition may also include a tonicity agent, such as a compound that renders the formulation substantially isotonic or isosmotic with human blood. Examples of tonicity agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the composition further comprises a stabilizer, e.g., a molecule that substantially prevents or reduces chemical and / or physical instability of the nanostructures in lyophilized or liquid form. Examples of stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0085] The polypeptide, nucleic acid, expression vector, and / or host cell may be the only active agent in the composition, or the composition may further comprise one or more other agents suitable for the intended use.

[0086] In a further aspect, the present disclosure provides a method for treating a severe acute respiratory syndrome (SARS) coronavirus infection (including SARS-CoV and SARS-CoV-2), comprising administering to a subject in need thereof an amount of a polypeptide, nucleic acid, expression vector, host cell, oligomer, composition, and / or pharmaceutical composition of any of the preceding items effective to treat the infection. In one embodiment, the SARS coronavirus includes SARS-CoV-2.

[0087] In another aspect, the disclosure provides a method for limiting the development of severe acute respiratory syndrome (SARS) coronavirus infection (including SARS-CoV and SARS-CoV-2), comprising administering to a subject in need thereof an amount of a polypeptide, nucleic acid, expression vector, host cell, oligomer, composition, and / or pharmaceutical composition of any of the preceding items effective to treat the infection. In one embodiment, the SARS coronavirus includes SARS-CoV-2.

[0088] The polypeptides, nucleic acids, expression vectors, host cells, oligomers, compositions, and / or pharmaceutical compositions may be administered by any suitable route of administration, as deemed appropriate by the attending medical practitioner. In one embodiment, the polypeptides, nucleic acids, expression vectors, host cells, oligomers, compositions, and / or pharmaceutical compositions are administered intranasally. In another embodiment, the polypeptides, nucleic acids, expression vectors, host cells, oligomers, compositions, and / or pharmaceutical compositions are administered systemically.

[0089] Where the method includes treating a SARS-coronavirus infection, one or more polypeptides, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions are administered to a subject already diagnosed with a SARS-coronavirus infection. As used herein, "treat" or "treating" means achieving one or more of the following: (a) reducing the severity of infectious symptoms in a subject; (b) inhibiting the progression of infectious symptoms in a subject; (c) increasing survival; (d) shortening the duration of symptoms; (e) limiting or preventing the onset of symptoms; and (f) reducing the need for and / or length of hospitalization to treat the infection.

[0090] Where the method includes limiting the onset of SARS-coronavirus infection, one or more polypeptides, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions are administered prophylactically to a subject who may or may not be infected with SARS-coronavirus but who may be at risk for such infection. As used herein, "limiting" refers to limiting the onset of SARS-coronavirus infection in a subject (which may be any subject) at risk for SARS-coronavirus infection.

[0091] The subject can be any subject, such as a human subject.

[0092] Examples of SARS-CoV-2 infection symptoms include, but are not limited to, fever, fatigue, cough, shortness of breath, chest tightness and / or pain, loss or reduction of smell, loss or reduction of taste, and breathing problems (including, but not limited to, pneumonia, bronchitis, severe acute respiratory syndrome (SARS), and upper and lower respiratory tract infections).

[0093] As used herein, "effective amount" refers to an amount of a composition that is effective for treating and / or limiting SARS-CoV-2 infection. The polypeptides, compositions, nucleic acids, or compositions of any of the embodiments described herein are typically formulated as pharmaceutical compositions, such as those disclosed above, and may be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, or topically, in a dosage unit formulation containing conventional pharmaceutically acceptable carriers, adjuvants, and carriers. The term "parenteral," as used herein, includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intrathecal, intracranial, intrapleural, infusion techniques, or intraperitoneally. Polypeptide compositions may also be administered via microspheres, liposomes, immune stimulating complexes (ISCOMs), or other particulate delivery systems, or sustained-release formulations introduced into a suitable tissue (such as blood). Dosage regimens may be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). A suitable dose range is, for example, 0.1 μg / kg to 100 mg / kg body weight of the polypeptide or nanoparticle thereof. The composition may be delivered in a single bolus, or may be administered two or more times (e.g., two, three, four, five, or more times) as determined by the attending medical personnel.

[0094] The present disclosure also provides methods for designing polypeptides that bind to the receptor binding site (RBD) of SARS-Cov-2, the methods comprising the steps as described in the Examples below. Such methods may include the steps of polypeptide design (as described in any one embodiment or combination of embodiments of the Examples), cell-free synthesis, and evaluation for SARS-Cov-2 RBD binding using any suitable technique.

[0095] Example overview Effective therapeutic agents against SARS-CoV-2 are needed. We attempted to use computational protein design to create high-affinity binders for the SARS-CoV-2 receptor-binding domain (RBD), which blocks its interaction with the Ace2 receptor, a necessary pathway for cellular entry. Two strategies were used to create small protein scaffolds with complementary morphologies to the Ace2-binding site on the RBD: first, scaffolds were built around the helices of Ace2 responsible for the majority of interactions with the RBD; and second, diverse de novo-designed scaffolds less than 65 residues in length were attached to this region. In both cases, scaffold residues at the RBD interface were then optimized for high-affinity binding, and residues in the remainder of the protein were optimized for folding into the target structure and stability. 50,000 designs predicted to bind most tightly to the virus were encoded on large oligonucleotide arrays and screened for binding to the RBD using yeast surface display by fluorescence-activated cell sorting; deep sequencing of the populations before and after sorting identified hundreds of designs that bound the target. The binding modes of the highest affinity binders (maximally enriched by sorting) were confirmed by high-resolution sequence mapping, and their affinities were further enhanced by combining one to four beneficial substitutions. Eight optimized designs with distinct binding sites near the Ace2 interface on the RBD and completely distinct sequences were expressed at high levels in E. coli and found to bind the RBD with Kds ranging from 100 pM to 10 nM. These designs inhibited live virus infection of Vero-6 cells with IC50s ranging from 10 nM to 20 pM. These polypeptides are thus useful, for example, in both intranasal and systemic SARS-CoV-2 therapeutics, and more generally, our results demonstrate the power of computational protein design to rapidly generate potential therapeutic candidates against pandemic threats.

[0096] SARS-CoV-2 infection is thought to often begin in the nose, where the virus is thought to replicate for several days before spreading to the wider respiratory system. Delivery of high-concentration virus inhibitors to the nasal cavity and respiratory system in general could potentially provide preventative protection and therapeutic efficacy early in infection and may be particularly useful for healthcare workers and others who frequently come into contact with infected individuals. While numerous monoclonal antibodies are under development as systemic SARS-CoV-2 therapeutics, these compounds are not ideal for intranasal delivery because antibodies are often large, not very stable molecules, and have a low density of binding sites (two per 150 Kd antibody); Fc domains offer little additional benefit. More desirable would be inhibitory proteins with very high affinity for a single clone of the virus, but with greater stability and a smaller size to maximize inhibitory domain density and enable direct delivery into the respiratory system via aerosolization.

[0097] We set out to de novo design high-affinity binders to RBD that would compete with Ace2 binding. We tried two strategies: first, we attempted to scaffold the alpha helices in Ace2, which constitute the majority of interactions with the RBD, into small designed proteins that create additional interactions with the RBD to further enhance affinity; and second, we attempted to design binders entirely from scratch, without incorporating any known binding interactions with the RBD. The advantage of the second approach is that the range of design possibilities is greater, and potentially higher affinity binding modes can be identified. For the first approach, we used Rosetta® blueprint builder to create mini-proteins containing the Ace2 helices, and for the second approach, we used RIF docking and design using a large library of mini-proteins. The designs interact with different regions of the RBD surface around the Ace2 binding site (Figure 1). Designs from Approach 1 and Approach 2 were screened for binding to fluorescently tagged RBDs encoded by long oligonucleotides on the yeast cell surface. Deep sequencing identified three designs with an Ace2 helical scaffold (Approach 1) and 150 de novo interface designs (Approach 2) that were significantly enriched after FACS sorting for RBD binding. The designs were expressed and purified in E. coli; many were soluble expressed and found to bind RBD in biolayer interference experiments and could effectively compete with ACE-2 for binding to the RBD (examples shown in Figure 2). Based on BLI data (e.g., see Figure 2), the RBD-binding affinities of the minibinders are: LCB1 < 1 nM, LCB3 < 1 nM. The affinities of LCB2, LCB4, LCB5, LCB6, LCB7, and LCB8 range from 1 to 20 nM, and the relative strengths of the different binders are LCB4 > LCB2 > LCB9 = LCB5 > LCB6 > LCB7.

[0098] To determine whether the designs bind RBD through the engineered interface, site-saturation libraries were constructed in which all residues in each design were substituted, one residue at a time, with each of 20 amino acids and subjected to FACS sorting for RBD binding. Deep sequencing showed that binding interface residues and protein core residues were conserved in many of the designs for which such site-saturation libraries (SSMs) were constructed (SSMs were used to define permissible positions for amino acid changes in Table 1). For the majority of designs, a few substitutions were enriched in FACS sorting, suggesting that they enhanced binding affinity to RBD. For the Approach 1 designs and the eight highest-affinity Approach 2 designs, combinatorial libraries incorporating these substitutions were constructed and rescreened for binding by FACS; due to the very high binding affinity, the concentration used in sorting was as low as 20 pM. Each library converged to a small number of closely related sequences, and for each design, one optimized variant was expressed in E. coli and purified.

[0099] The binding of eight optimized designs with different binding modes to the RBD was examined by biolayer interferometry (Figure 1). For many designs, the Kd ranged from 1 to 20 nM; for the remainder, the Kd was lower than 1 nM, too strong to be reliably measured by this technique (see Figure 2). Circular dichroism spectra of the designs were consistent with the design model, and the designs retained full binding activity after numerous days at room temperature (Figure 3).

[0100] We investigated the ability of our designs to block live virus infection of human cells by infecting 100 FFU of SARS-CoV-2 with 2.5–3 × 10 antibodies in the presence of various amounts of our designed binders. 4 The antibodies were added to Vero cells. Details are described in the legend to Figure 4. Potent inhibition of infection was observed for all of the designs, with IC50 values ​​ranging from 1 nM to 0.02 nM.

[0101] Details regarding specific designs are provided in Table 10.

[0102] [Table 16] TIFF2025170319000123.tif242162TIFF2025170319000124.tif242162TIFF2025170319000125.tif242162TIFF20251703190 00126.tif242162TIFF2025170319000127.tif242162TIFF2025170319000128.tif242162TIFF2025170319000129.tif163162

[0103] The designed binding agents have multiple advantages over antibodies as potential therapeutics. Overall, they span a variety of binding modes, and when combined, viral escape is unlikely. Retention of activity after extended periods at elevated temperatures suggests they do not require cold chain distribution. The designs are 20 times smaller than full-body antibody molecules and therefore possess 20 times more potent neutralizing sites for the same mass, enhancing the potential efficacy of topically administered drugs. The cost of goods and the ability to scale to large-scale production should be lower for simpler miniproteins, which, unlike antibodies, do not require expression in mammalian cells for proper folding. Their small size and high stability should make them suitable for direct delivery to the respiratory system via nebulization. While immunogenicity is a potential issue with any foreign molecule, previously characterized de novo designed miniproteins have elicited little or no immune responses; likely because their small size, combined with their high solubility and stability, reduces the likelihood of presentation on dendritic cells. [Table 17]

[0104] Ultrapotent miniproteins targeting receptor-binding domains prevent SARS-CoV-2 infection and disease Despite public health responses and the introduction of spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and associated deaths continue to increase. Herein, we investigated the ability of a modified form of LCB1, a key binder, to prevent SARS-CoV-2-mediated lung disease in transgenic mice expressing human ACE2. Systemic administration of LCB1-Fc reduced viral load, attenuated immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lungs, even when administered 5 days before or 2 days after viral inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1 / 2020), an emerging B.1.1.7 strain, and strains encoding the critical E484K and N501Y spike protein substitutions. These data support the use of LCB1v1.3 for the prevention or treatment of SARS-CoV-2 infection.

[0105] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease 2019 (COVID-19) pandemic, has resulted in global illness, suffering, and economic hardship. Despite public health responses, SARS-CoV-2 transmission continues primarily through person-to-person transmission (Day, 2020; Li et al., 2020; Standl et al., 2020). SARS-CoV-2-induced clinical symptoms range from asymptomatic infection to severe pneumonia, multiple organ failure, and death. While the underlying mechanisms determining disease severity are poorly understood, immunocompromised individuals, the elderly, and individuals with certain comorbidities (e.g., a history of cardiovascular disease, diabetes, or obesity) are at increased risk for poor outcomes (Zhou et al., 2020).

[0106] Herein, we evaluated the in vivo efficacy of an exemplary miniprotein binder, LCB1, using a rigorous model of SARS-CoV-2 disease pathogenesis in human ACE2 (hACE2)-expressing transgenic mice (Golden et al., 2020; Winkler et al., 2020a). In our in vivo experiments, we evaluated two forms of LCB1: (a) LCB1-hIgG-Fc9 (LCB1-Fc), an Fc-modified bivalent form that may extend in vivo half-life and engage effector components of the immune system; and (b) LCB1v1.3, a further optimized, monomeric form of LCB1 lacking the Fc domain. Intraperitoneal administration of LCB1-Fc the day before or the day after exposure to SARS-CoV-2 conferred substantial protection, including no weight loss, near-detectable reductions in viral load, and inhibition of lung inflammation and pathological changes. Intranasal delivery of LCB1v1.3 conferred protection even 5 days before or 2 days after SARS-CoV-2 inoculation. Dosing experiments revealed that LCB1v1.3 maintained efficacy at pharmacologically achievable concentrations and was weakly immunogenic. Most importantly, LCB1v1.3 protected animals against SARS-CoV-2 strains encoding key spike substitutions E484K and N501Y present in both the current emerging B.1.1.7 UK variant and the South African (B.1.351) and Brazilian (B.1.1.248) variants of concern. Overall, these studies establish LCB1-Fc and LCB1v1.3 as potential therapeutics for preventing or mitigating SARS-CoV-2 disease.

[0107] result LCB1v1.3 prophylactic drug limits viral load and clinical disease LCB1 was modified to generate two variants for in vivo testing: (a) polar mutations were introduced into LCB1 to increase expression and solubility without altering RBD binding (LCB1v1.3), and (b) LCB1 was modified by fusing it to the human IgG1 Fc domain (LCB1-Fc) to enhance bioavailability. LCB1v1.3 and LCB1-Fc bind strongly to the single RBD within the S trimer (Figure 5A), with dissociation constants (K) of <625 pM and <156 pM, respectively. D ) (Figure 5B). LCB1v1.3 and LCB1-Fc also potently neutralized a natural SARS-CoV-2 isolate (2019n-CoV / USA_WA1 / 2020 [WA1 / 2020]) (EC 50 = 14.4 pM and 71.8 pM; Figure 5C).

[0108] To determine the protective potential of these miniproteins against SARS-CoV-2, we used K18 human hACE2-expressing transgenic mice, which develop severe pulmonary infection and disease after intranasal inoculation with SARS-CoV-2 (Golden et al., 2020; Winkler et al., 2020a). 3A single dose of 250 μg (10 mg / kg) of LCB1-Fc administered by intraperitoneal injection (ip) the day before intranasal inoculation with PFU of SARS-CoV-2 WA1 / 2020 prevented weight loss compared to animals receiving a control protein (influenza A virus hemagglutinin minibinder) designed using a similar computational method (Figure 5D). After prophylaxis with LCB1-Fc, infectious virus was not detected in the lungs at 4 and 7 days post-infection, although high levels were observed in animals receiving the control protein (Figure 5E, top and bottom). Similarly, viral RNA levels in the lungs, heart, spleen, and brain of LCB1-Fc-treated animals were at or near the detection limit of the assay at 4 and 7 days post-infection (Figure 5F-I). LCB1-Fc treatment did not affect viral RNA levels in nasal wash samples obtained 4 days after infection (Figure 5J); these results are similar to a recent study of neutralizing human antibodies in hamsters (Zhou et al., 2021). However, viral RNA levels were reduced 7 days after infection, suggesting that LCB1-Fc treatment accelerated viral clearance or prevented spread in the upper respiratory tract.

[0109] Extensive alveolar damage, inflammation, and pneumonia are symptoms of COVID-19 lung disease, leading to respiratory failure and the need for mechanical ventilation (Johnson et al., 2020; Kordzadeh-Kermani et al., 2020). We evaluated the ability of LCB1-Fc to prevent the pulmonary dysfunction seen after SARS-CoV-2 infection in K18-hACE2 mice (Winkler et al., 2020a). At 7 days postinfection, mechanical ventilation testing of pulmonary biomechanics in LCB1-Fc-treated animals showed no differences from naive animals (Figure 6A), whereas mice receiving a control binder protein exhibited reduced inspiratory capacity and lung compliance, as well as increased lung resistance, elastance, and tissue damping, all consistent with pulmonary dysfunction. These biophysical properties resulted in distinct pressure-volume loops between control binder and LCB1-Fc-treated or naive animals. We also evaluated the effect of LCB1-Fc treatment on SARS-CoV-2-induced lung pathology. Lung sections taken 7 days after SARS-CoV-2 infection showed widespread inflammation characterized by cellular infiltration and dense infiltrates in LCB1-Fc-treated animals, but not in control protein-treated or naive mice (Figure 6B). At 4 days postinfection, RNA signatures of inflammatory cytokines and chemokines were observed in the lungs of LCB1-Fc-treated animals but not in control binder-treated animals, suggesting that LCB1-Fc treatment prevents viral infection and inflammation in the lungs (Figures 6C and 11).

[0110] Post-exposure treatment with an anti-RBD binder reduces viral load To evaluate the efficacy of LCB1-Fc in a post-exposure setting, LCB1-Fc was administered by intraperitoneal injection 1 day post-infection. Treatment with LCB1-Fc prevented weight loss (Fig. 7A) and reduced viral load in all examined tissues 4 and 7 days post-infection (Fig. 7B–G). No infectious virus was recovered from the lungs of LCB1-Fc-treated animals harvested at either time point. Lung sections confirmed that treatment with LCB1-Fc improved the pathological outcome (Fig. 7H). At 7 days post-infection, immune cell infiltration was absent in lung sections from LCB1-Fc-treated animals but was observed in control binder-treated animals.

[0111] We next tested the efficacy of LCB1v1.3 as an intranasally delivered post-exposure treatment. Intranasal delivery would enable self-administration of anti-SARS-CoV-2 biologic agents. Indeed, miniprotein inhibitors against influenza virus have shown efficacy as intranasal sprays (Chevalier et al., 2017). For these studies, LCB1v1.3 was used because it can bind a larger number of RBD molecules per given mass, resulting in increased neutralizing activity (Figure 5C). High levels of SARS-CoV-2 RNA were detected in the lungs and other peripheral tissues of animals treated with a control binder at day 7 post-infection, whereas infection was reduced in animals receiving LCB1v1.3 intranasally on days D+1 or D+2 post-SARS-CoV-2 inoculation (Figures 7I and 12). Viral RNA levels were reduced in nasal washes from animals receiving LCB1v1.3 on post-treatment D+1, but not on post-treatment D+2, compared to animals treated with the control binder (Figure 7J).

[0112] Intranasal delivery of LCB1v1.3 confers protection against SARS-CoV-2 when administered up to 5 days before infection The persistence of intranasally administered LCB1v1.3 prophylaxis was then assessed. 3K18-hACE2 transgenic mice received a single 50 μg intranasal dose of LCB1v1.3 or a control binder 5 days, 3 days, 1 day, or 6 hours before inoculation with PFU of SARS-CoV-2. Viral load in tissues was determined by RT-qPCR on days 4 and 7 postinfection. As expected, protection by LCB1v1.3 was greater when administered closer to the time of SARS-CoV-2 challenge, as reflected by significantly reduced viral load and weight loss (Figures 8A-D and S3). However, even mice administered LCB1v1.3 5 days prior to inoculation and harvested 7 days postinfection showed reduced viral RNA levels in the lungs compared to animals treated with the control binder. Regardless of the time of collection, lung viral RNA levels were reduced in animals that received LCB1v1.3 3 days prior to SARS-CoV-2 inoculation.

[0113] A range of intranasal doses of LCB1v1.3 were tested for efficacy (Figure 8E-J). Treatment with as little as 2 μg (0.1 mg / kg) of LCB1v1.3 prevented SARS-CoV-2-induced weight loss. Doses of 2–10 μg (0.1–0.5 mg / kg) of LCB1v1.3 reduced viral RNA levels in the lung, heart, and spleen at 7 days postinfection compared with animals treated with a control binder. Furthermore, animals receiving a 50 μg dose of LCB1v1.3 showed minimal, if any, lung inflammation (Figure 8K). Together, these results demonstrate that even low doses of LCB1v1.3, when administered intranasally prior to challenge, can limit SARS-CoV-2 infection and disease in a rigorous K18-hACE transgenic mouse model of disease pathogenesis.

[0114] LCB1v1.3 is weakly immunogenic and retains protective activity after repeated administration K18-hACE2 transgenic mice were treated with 50 μg of control binder or LCB1v1.3 every 3 days for a total of 18 days (Figure 9A). At this time point, serum was collected and assessed for the presence of anti-LCB1v1.3 antibodies. Only 1 in 10 mice produced IgG antibodies to LCB1v1.3 (Figure 9B). To determine whether repeated administration affected LCB1v1.3-mediated protection, 10 3 Cohorts were inoculated with PFU of SARS-CoV-2. Again, substantial protection against weight loss (Figure 9C) and viral infection in the lungs and other organs (Figure 9D-H) was observed in all animals receiving LCB1v1.3.

[0115] LCB1v1.3 protects against emerging SARS-CoV-2 variants The activity of LCB1v1.3 was evaluated against the B.1.1.7 isolate, which contains deletions at positions 69-70 and 144-145 and substitutions at N501Y, A570D, D614G, and P681H, and against a recombinant WA1 / 2020 strain containing the major substitutions present in the B.1.351 and B.1.248 mutants at residues E484K, N501Y, and D614G (Xie et al., 2021a). The neutralizing activity of LCB1v1.3 against the B.1.1.7 and E484K / N501Y / D614G strains was approximately 45-50-fold lower than that for the WA1 / 2020 strain, but the EC50 values ​​were still approximately 800 pM and 667 pM, respectively (Figure 10A). To determine whether LCB1v1.3 protects against SARS-CoV-2 strains harboring spike protein substitutions of interest in vivo, 3K18-hACE2 transgenic mice were treated with a single intranasal 50 μg dose of LCB1v1.3 or a control binder 1 day before inoculation with full-fledged ...

[0116] Consideration Here, using the rigorous K18-hACE2 mouse model of SARS-CoV-2 pathogenesis, we show that LCB1-Fc prevented SARS-CoV-2 infection and disease when administered 1 day before or 1 day after viral inoculation. Lung biomechanics in LCB1-Fc-treated mice mirrored those of naive animals in all parameters tested.

[0117] We also evaluated the efficacy of LCB1v1.3, an optimized monomeric form of LCB1 lacking the Fc domain. A single intranasal dose of LCB1v1.3 reduced viral load even when administered 5 days before or S2 days after SARS-CoV-2 infection. Our intranasal delivery approach is unique. Intranasal therapy of SARS-CoV-2 has only been reported with type I interferon therapy in a diseased hamster model (Hoagland et al., 2021), with limited efficacy. The K18-hACE2 mouse model recapitulates multiple aspects of severe COVID-19, including pulmonary inflammation and impaired lung function (Golden et al., 2020; Winkler et al., 2020a). Because K18-hACE2 mice are highly susceptible to infection, the therapeutic window of treatment is limited (Winkler et al., 2020b), and our miniprotein may only have suppressed viral infection. Importantly, our data demonstrate that treatment with the LCB1v1.3 binder before or after infection suppressed immune cell infiltration and lung inflammation, which prevented tissue damage and respiratory dysfunction. As part of our proof-of-principle studies for intranasal prophylaxis, little immunogenicity was observed with LCB1v1.3, suggesting that repeated administration is possible.

[0118] Although multiple antibody-based therapeutics have shown promise against SARS-CoV-2, and some have already been approved for emergency use, viral evolution may threaten these interventions, as evidenced by emerging variants in the UK (B.1.1.7), South Africa (B.1.351), Brazil (B.1.248), and elsewhere. Indeed, we and other researchers have observed that many monoclonal and polyclonal antibodies exhibited reduced neutralizing activity against several of these variants (Chen et al., 2021; Wang et al., 2021a; Wang et al., 2021b; Wibmer et al., 2021; Xie et al., 2021b). In comparison, LCB1v1.3 demonstrated efficacy against historical (WA1 / 2020) and emerging (B.1.1.7 and E484K / N501Y / D614G) SARS-CoV-2 strains. Based on the cryo-EM structure of the parent LCB1 binder in complex with the SARS-CoV-2 RBD (Cao et al., 2020), only the N501Y mutation is expected to affect binding. Although we observed a decrease in the neutralizing activity of LCB1v1.3 against the emerging variants, the EC50 value was still below 800 pM, suggesting substantial potency was retained.

[0119] Compared to other potential SARS-CoV-2 antibody-based therapeutics, miniproteins have several advantages: (a) due to their smaller size, they can combine each promoter of a single trimeric spike, resulting in greater efficacy for a given dose; (b) they can be produced cost-effectively; and (c) they can be mixed using linker proteins to create multimerized constructs that limit resistance.

[0120] Experimental model and subject details Cells and viruses Vero E6 (CRL-1586, American Type Culture Collection (ATCC), Vero CCL81 (ATCC), Vero-Furin (Mukherjee et al., 2016), and Vero-hACE2-TMPRSS2 (gifts from A. Creanga and B. Graham, NIH) were cultured in 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 1× Cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with non-essential amino acids and 100 U / ml penicillin-streptomycin. Additionally, Vero-hACE2-TMPRSS2 cells were cultured in the presence of 5 mg / mL puromycin. The SARS-CoV-2 WA1 / 202 (2019n-CoV / USA_WA1 / 2020) isolate was obtained from the US Centers for Disease Control and Prevention (CDC). The B.1.1.7 and WA1 / 2020 E484K / N501Y / D614G viruses have been previously described (Chen et al., 2021; Xie et al., 2021a). Infectious stocks were obtained from Vero The cells were propagated by inoculation into CCL81 or Vero-hACE2-TMPRSS2 cells. Supernatants were collected, aliquoted, and stored at -80°C. All experiments with infectious SARS-CoV-2 were performed in BSL3 and A-BSL3 facilities approved by the Washington University School of Medicine Institutional Biosafety Committee using positive pressure respirators and protective equipment.

[0121] Mouse experiments Animal experiments were performed in accordance with the recommendations in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Ethics Committee of the Washington University School of Medicine (approval number: A3381-01). Virus inoculation was performed under anesthesia induced and maintained with ketamine hydrochloride and xylazine, and every effort was made to minimize animal suffering.

[0122] Heterozygous K18-hACE c57BL / 6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn / J) were obtained from Jackson Laboratory. Animals were group-housed and fed a standard oral diet. Mice of different ages and both sexes were administered 10 mg of ACE2-hACE2-1000-mg ... 3 PFU of SARS-CoV-2 was administered.

[0123] Learn more about how Miniprotein production LCB1-Fc was synthesized and cloned into the pCMVR plasmid by kanamycin resistance using GenScript. The plasmid was transformed into E. coli strain NEB5-alpha (New England Biolabs) to recover DNA for transient transfection into Expi293F mammalian cells. Expi293F cells were grown in suspension using Expi293F expression medium (Life Technologies) at 33°C, 70% humidity, and 8% CO with shaking at 150 rpm. Cultures were grown at 3x10 6 Cells grown to a density of 1000 cells / mL were transformed with PEI-MAX (Polyscience) and cultured for 3 days. The supernatant was clarified by centrifugation (4000 x g for 5 minutes, with PDADMAC solution (Sigma-Aldrich, #409014) added to a final concentration of 0.0375%) and a second centrifugation (4000 x g for 5 minutes). The clarified supernatant was purified using a MabSelect PrismA® 2.6 x 5 cm column (Cytiva) on an AKTA Avant150 FPLC (Cytiva). Bound proteins were washed with 5 column volumes of 20 mM NaPO4 and 150 mM NaCl (pH 7.2), followed by 5 column volumes of 20 mM NaPO4 and 1 M NaCl (pH 7.4), and eluted with 3 column volumes of 100 mM glycine (pH 3.0). The eluate was neutralized with 2 M Tris base to a final concentration of 50 mM. Protein purity was assessed using SDS-PAGE, and the protein was passed through a 0.22 μm filter and stored at 4°C until use.

[0124] LCB1v1.3, which contains polar mutations (4N, 14K, 15T, 17E, 18Q, 27Q, 38Q) relative to the original LCB1, was cloned into the pet29b vector. LCB1v1.3 was expressed in Lemo21(DE3) (NEB) in TB medium and grown in 2L baffled shake flasks. Bacteria were grown at 37°C to an OD of approximately 0.8 and induced with 1 mM IPTG. The expression temperature was lowered to 18°C, and the cells were shaken for approximately 16 hours. Cells were harvested, lysed using heat treatment, and incubated at 80°C for 10 minutes with agitation. The lysate was clarified by centrifugation at 24,000 × g for 30 minutes and loaded onto a 2.6 × 10 cm Ni Sepharose® 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein was eluted with a linear gradient of 30 to 500 mM imidazole in a buffer of 50 mM Tris (pH 8.0) and 500 mM NaCl. Peak fractions were pooled, concentrated with a 10 kDa MWCO centrifugal filter (Millipore), sterile filtered (0.22 μm), and loaded onto either a Superdex® 200 Increase 10 / 300 or a HiLoad S200 pg GL SEC column (Cytiva) using a buffer of 50 mM phosphate (pH 7.4), 150 mM NaCl. After size exclusion chromatography, bacterial components were tested to confirm low levels of endotoxin.

[0125] Biolayer Interferometry Biolayer interference data were collected using an Octet® RED96 (ForteBio) and processed using the instrument's built-in software. Briefly, biotinylated RBD (Acro Biosystems) was loaded onto a streptavidin-coated biosensor (SA ForteBio) at 20 nM in binding buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, and 0.5% nonfat dry milk) for 360 s. The assay protein (LCB1v1.3 or LCB1-Fc) was diluted from a concentrated stock solution in binding buffer. After a baseline measurement in binding buffer alone, the binding kinetics was monitored by immersing the biosensor in a well containing the target protein at the indicated concentration (association step) for 3,600 s and then returning the sensor to baseline / buffer for 7,200 s (dissociation step).

[0126] Plaque assay Vero-Furin cells ( Mukherjee et al., 2016 ) were cultured at 2.5 × 10 5 Cells were seeded into 12-well flat-bottom tissue culture plates at a density of 1000 cells / well. The next day, the medium was removed and replaced with 200 μL of 10-fold serial dilutions of the material to be titrated in DMEM + 2% FBS, and the plates were incubated at 37°C with shaking at regular intervals. After 1 hour, 1 mL of methylcellulose was overlaid. The plates were incubated at 37°C for 72 hours and then fixed with 4% paraformaldehyde (final concentration) in PBS for 20 minutes. The fixed monolayer cells were stained with 0.05% (w / v) crystal violet in 20% methanol and washed twice with distilled, deionized water.

[0127] Measurement of viral load Tissues were weighed and homogenized with zirconia beads in a MagNA Lyse® device (Roche Life Science) in 1,000 μL of DMEM medium supplemented with heat-inactivated 2% FBS. The homogenized tissue was clarified by centrifugation at 10,000 rpm for 5 minutes and stored at -80°C. RNA was extracted using the MagMax mirVana® Total RNA Isolation Kit (Thermo Scientific) on a Kingfisher Flex extraction robot (Thermo Scientific). RNA was reverse transcribed and amplified using the TaqMan® RNA-to-CT 1-Step Kit (ThermoFisher). Reverse transcription was performed at 48°C for 15 minutes, followed by heating at 95°C for 2 minutes. Amplification was performed for 50 cycles as follows: 95°C for 15 seconds and 60°C for 1 minute. The copy number of SARS-CoV-2 N gene RNA in the samples was determined using a previously reported assay (Case et al., 2020; Hassan et al., 2020). Briefly, a TaqMan® assay was designed to target a highly conserved region of the N gene (forward primer: ATGCTGCAATCGTGCTACAA (SEQ ID NO: 190); reverse primer: GACTGCCGCCTCTGCTC (SEQ ID NO: 191); probe: / 56-FAM / TCAAGGAAC / ZEN / AACATTGCCAA / 3IABkFQ / ) (SEQ ID NO: 192). This region was included in the RNA standard to allow copy number determination up to 10 copies per reaction. The reaction mixture contained final primer and probe concentrations of 500 nM and 100 nM, respectively.

[0128] Cytokine and chemokine mRNA measurements RNA was isolated from lung homogenates as described above, and cDNA was synthesized from DNAse-treated RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific) with the addition of RNase inhibitors according to the manufacturer's protocol. Cytokine and chemokine expression was determined using TaqMan® Fast Universal PCR Master Mix (Thermo Scientific) with commercially available primer / probe sets specific for the following: IFN-g (IDT: Mm.PT.5 8.41769240), IL-6 (Mm.PT.5 8.10005566), IL-1b (Mm.PT.5 8.41616450), Tnfa (Mm.PT.5 8.12575861), CXCL10 (Mm.PT.5 8.43575827), CCL2 (Mm.PT.5 8.42151692), CCL5 (Mm.PT.5 8.43548565), CXCL11 (Mm.PT.5 Results were normalized to GAPDH (Mm.PT.39a.1) levels. Fold changes were calculated comparing treated mice to naive controls. -ΔΔCt was determined using the method.

[0129] Lung lesions Animals were euthanized before tissue collection and fixation. The left lung was first ligated at the level of the left main bronchus and collected for viral RNA analysis. The right lung was inflated with approximately 1.2 mL of 10% neutral buffered formalin using a 3 mL syringe and a catheter inserted into the trachea. Tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin. Slides were scanned using a Hamamatsu NanoZoomer® slide scanning system, and images were viewed using NDP viewing software (ver. 1.2.46).

[0130] respiratory mechanism Mice were anesthetized with ketamine / xylazine (100 mg / kg and 10 mg / kg, respectively, intraperitoneally). The trachea was isolated by dissection of the cervical region and cannulated using an 18-gauge blunt-tip metal cannula (typical resistance of 0.18 cmH2O.s / mL), which was secured in place with nylon sutures. The mouse was then connected to a flexiVent® computer-controlled piston-type ventilator (SCIREQ Inc.) via the cannula attached to an FX adapter Y-tube. Mechanical ventilation was initiated, and the mouse received an additional 100 mg / kg of ketamine and 0.1 mg / mouse of the paralytic pancuronium bromide via the intraperitoneal route to prevent breathing against the ventilator and during measurements. Mice were ventilated using the default settings for mice; these consisted of a positive end-expiratory pressure of 3 cm H2O, a tidal volume (Vt) of 10 mL / kg, a respiratory rate of 150 breaths per minute (bpm), and an FiO2 of 0.21 (i.e., room air). Respiratory mechanics were assessed using the forced oscillation method, as previously reported (McGovern et al., 2013), utilizing the latest version of the flexiVent® operating software (flexiWare v8.1.3). Pressure-volume loop and inspiratory volume measurements were also performed.

[0131] Neutralization assay Serial dilutions of binder proteins were incubated at 37°C for 1 hour at 10°C. 2The binder / virus complexes were incubated with focus-forming units (FFUs) of SARS-CoV-2. The binder / virus complexes were added to monolayers of Vero E6 (WA1 / 2020) or Vero-hACE2-TMPRSS2 (B.1.1.7 and WA1 / 2020 E484K / N501Y / D614G) cells in 96-well plates and incubated for 1 hour at 37°C. The cells were then overlaid with 1% (w / v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 24–30 hours later by removing the overlay and fixing with 4% PFA in PBS for 20 minutes at room temperature. Plates were washed and sequentially incubated with the following oligoclone pools in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin: SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 anti-spike protein antibodies (Zhou et al., 2021), and HRP-conjugated goat anti-mouse IgG. SARS-CoV-2-infected cell foci were visualized using TrueBlue® peroxidase substrate (KPL) and quantified using an ImmunoSpot® analyzer (Cellular Technologies). Data were processed using Prism® software (GraphPad Prism® 8.0).

[0132] ELISA C-terminally biotinylated LCB1.1v3 was immobilized on streptavidin-coated plates (RayBiotech #7C-SCP-1) at 2.5 μg / mL in a total volume of 100 μL per well and incubated overnight at 4°C. The plates were washed with wash buffer (TBS + 0.1% (w / v) BSA + 0.05% (v / v) Tween 20) and blocked with 200 μL / well of blocking buffer (TBS + 2% (w / v) BSA + 0.05% (v / v) Tween 20) for 1 hour at room temperature. The plates were rinsed with wash buffer using 200 μL / well, and 100 μL of a 1:100 diluted serum sample in blocking buffer was added to each well. For the positive control, Fc-RBD was serially diluted 1:5 in 100 μL of blocking buffer, starting at 240 ng / mL. All samples were incubated at room temperature for 1 hour. The plate was washed with 200 μL / well of wash buffer. For serum samples, HRP-conjugated horse anti-mouse IgG antibody (Vector Laboratories #PI-2000-1) was diluted 1:200 in blocking buffer, and 100 μL was incubated in each well for 30 minutes at room temperature. For the positive control, HRP-conjugated mouse anti-human IgG antibody (Invitrogen #05-4220) was diluted 1:500 in blocking buffer, and 100 μL was incubated in each well for 30 minutes at room temperature. The plate was rinsed with wash buffer, and 100 μL of TMB (SeraCare) was added to each well for 2 minutes. The reaction was stopped by adding 100 μL of 1N HCl. Optical density was measured at 450 nm using a Synergy Neo2® plate reader (BioTek Instruments).

[0133] Quantitative and statistical analysis Statistical significance was assigned when P values ​​were <0.05 using Prism® version 8 (GraphPad). The test, animal number, median, and statistical comparison group are described in each figure legend. Analysis of body weight change was determined by two-way analysis of variance. Changes in functional or immune parameters were compared to control binder-treated animals and analyzed by one-way analysis of variance and multiple comparison tests. Statistical analysis of viral load between two groups was determined by the Mann-Whitney test. [Table 18] TIFF2025170319000132.tif242161TIFF2025170319000133.tif242162TIFF2025170319000134.tif242162TIFF2025170319000135.tif73161

[0134] Multivalent Design SARS-CoV-2 escape mutants pose a significant threat for prolonging the COVID-19 pandemic. Here, we developed multivalent minibinders as potential prophylactic and therapeutic agents to address this issue. We designed multivalent minibinders containing three copies of the minibinder (self-assembled homotrimers) or three linked minibinders (multi-domain fusions) targeting different sites, geometrically adapted to the spike trimer and optimized for their composition using a rapid cell-free expression and evaluation workflow. The optimized designs have very slow dissociation rates from the SARS-CoV-2 S-glycoprotein, with complex half-lives exceeding two weeks. Cryo-EM structures reveal that both the homotrimer and fusion minibinder constructs can engage all three RBDs on a single spike protein. The top trimeric and fusion candidates neutralized wild-type SARS-CoV-2 virus in addition to the B.1.1.7, B.1.351, and B.1.1.28 mutants with IC50s in the low pM range. Furthermore, the top homotrimeric candidate provided prophylactic protection against the same mutant strains in transgenic mice expressing human ACE2. Our approach highlights the utility of computational protein design coupled with experimental rapid prototyping to engineer potent multivalent inhibitors capable of broadly neutralizing circulating variants of concern.

[0135] We attempted to develop multivalent versions of our computationally designed miniproteins to block SARS-CoV-2 receptor-binding domain (RBD) interactions with its host receptor, ACE2. In principle, the small size of the designed minibinders would allow simultaneous engagement of multiple RBDs within a single spike protein trimer. We hypothesized that this multivalent binding would result in ultra-high affinity inhibitors that are more resistant to escape mutations than their monomeric counterparts. The avidity gained from these multivalent interactions could improve the effectiveness of mutations that escape individual domains. Furthermore, a single protein containing domains targeting multiple different epitopes or containing different combinations of contacts with target epitopes could further enhance the stability of the design against mutational escape. Starting from the LCB1, AHB2, and LCB3 minibinders (hereafter referred to as M1, M2, and M3, respectively; Table 11) and their known binding modes, we pursued two parallel strategies to design multivalent inhibitors: self-assembled homotrimers and multi-domain fusions.

[0136] [Table 19]

[0137] To enable rapid prototyping of designed proteins, we developed a cell-free DNA assembly and protein expression workflow that allows for a significantly shortened design / build / test cycle better suited to the urgency of the pandemic. This workflow combines a cell-free DNA assembly step using Gibson assembly followed by PCR to generate linear expression templates for directing cell-free protein synthesis (CFPS). The developed workflow allows for translation of synthetic DNA into purified protein in as little as six hours, is easily scaled up to high-throughput formats (e.g., 96- or 384-well plates), and is amenable to automated liquid handling. Furthermore, we coupled this cell-free workflow to an AlphaLISA® protein / protein interaction (PPI) competition assay to enable comparison of the dissociation rates of designed proteins toward either the monomeric RBD or the trimeric hexaproARS-CoV-2 S-glycoprotein (S6P). Because multivalency largely affects only the dissociation rate constant of an interaction, we reasoned that in-solution dissociation rate screening would enable us to distinguish monovalent from multivalent binding. The resulting workflow can evaluate hundreds of candidate multivalent proteins per week.

[0138] Design and validation of multivalent binders In the first strategy, we designed self-assembling trimeric forms of the M1, M2, and M3 miniproteins that geometrically matched the three RBDs in the spike trimer (hereafter referred to as H[binding domain #]-[homotrimer #]; e.g., H1-1 represents the homotrimer of M1 with homotrimerization domain 1; Table 11). Using our cell-free expression and multivalency screening workflow, we designed, expressed, and evaluated over 100 different proteins containing various homotrimerization domains and linker lengths. We identified each homotrimeric form that exhibited slow dissociation rates, potentially suggesting multivalent binding (Figure 14).

[0139] In the second strategy, we created two- and three-domain fusions of the M1, M2, and M3 binding domains separated by a flexible linker (hereafter referred to as F[binding domain #s]-[linker]; e.g., F231-P12 represents an M2 to M3 to M1 fusion, all separated by a PAS12 linker; Table 11). A range of linker lengths, selected to span the distance between the ends of the domains when bound in the "open" and "closed" states of the RBD, was evaluated. To optimize multivalency, over 100 different designs, varying in binding domain connectivity and linker length, were reexpressed and evaluated. Several identified two- and three-domain fusions exhibited slow dissociation rates comparable to the homotrimeric constructs described above (Figure 14).

[0140] The best candidates from each strategy showed little to no dissociation after 14 days with the competitor, and further measurements were limited by the stability of S6P. From these data, it can be seen that the complexes were 1x10 -7 s -1 To our knowledge, these are the slowest measured dissociation rate constants for an artificial protein / protein interaction reported to date.

[0141] Next, we used single-particle cryo-electron microscopy (cryo-EM) to characterize the complexes formed between S6P and the top candidate minibinder constructs (Figure 15). The cryo-EM structures of the H2-1, F31-G10, and F231-P24 constructs were determined at resolutions of 2.6 Å, 4.5 Å, and 3.9 Å, respectively. H2-1 was found to simultaneously engage all three RBDs, forcing all three RBDs to adopt an open state. The designed model closely matches the observed structure. F31-G10 binds two RBDs, both of which appear to adopt an open conformation upon binding. The structure indicates that this linker length allowed simultaneous binding of the two RBDs in their native state. The third free RBD adopted either an open or closed conformation in the structure. F231-P24 bound to three RBDs, with M1 bound to the RBD in the closed conformation and M2 and M3 bound to the RBD in the open conformation. This suggests that the linker length is long enough to allow all three binding domains to simultaneously engage all three RBDs without significant distortion of the native state. Although the flexible linker does not provide density in the EM maps to confirm domain connectivity, the maps for both F31-G10 and F231-P24 strongly suggest multivalent binding.

[0142] Multivalent minibinders neutralize widely circulating SARS-CoV-2 variants Next, we sought to determine the ability of multivalent constructs to neutralize SARS-CoV-2 mutants. We screened the dissociation rates of the best multivalent minibinders against a panel of mutant spike proteins (Figure 16). The homotrimers were most mutation-resistant, with the H2 homotrimer showing little dissociation after 24 hours against any of the mutant spikes. The two-domain fusions showed little increased dissociation against the point mutants tested. The three-domain fusions showed significantly more consistent binding to the point mutants tested, although some still had affected binding.

[0143] We further evaluated the potency of these proteins in neutralization assays against both SARS-CoV-2 HIV pseudoviruses, in addition to natural SARS-CoV-2 isolates (Figure 16). H2-0 and H2-1 homotrimers were consistently the best among all tested constructs, with IC values ​​in the low pM range. 50 The three-domain fusions were also successful, with IC in the sub-nM range for all tested variants. 50 The greater neutralization breadth of the H2 homotrimer likely reflects closer mimicry of the ACE2 binding site by the M2 monomer, an inherent advantage made possible by protein design.

[0144] Multivalent minibinders counter viral escape In addition to assessing the ability of the best candidates to neutralize currently circulating SARS-CoV-2 variants, we also tested the inhibitors' ability to counter viral escape (Figure 17). To do this, plaque assays were performed, replicating VSV-SARS-CoV-2 chimeric viruses in Vero E6 cells. To select for mutants that were resistant to the inhibitors, the inhibitors were included in the overlay to block replication of non-resistant viruses. For the positive control neutralizing antibody (2B04), multiple escape mutants were selected per plate. For both F231-P12 and H2-1, no escape mutants were isolated in 35 replicate wells of each inhibitor.

[0145] H2-0 provides preventative protection in transgenic mice expressing human ACE2 To determine the ability of our multivalent minibinders to protect in an in vivo model, we evaluated them as pre-exposure prophylaxis in human ACE2-expressing transgenic mice (Figure 17). A single 50 μg dose of H2-0 was administered to 100,000 SARS-CoV-2 mutants B.1.1.7, B1.351, and B.1.1.24. 3The mice were administered intranasally (intranasally) the day before inoculation with focus-forming units (FFU). In both cases, intranasal administration of H2-0 protected mice from SARS-CoV-2-induced weight loss. Six days after infection, viral loads were determined in various tissues by RT-qPCR. Notably, lung viral loads were reduced in all cases. These results demonstrate that intranasally administered H2-0 provides prophylactic protection against SARS-CoV-2 infection in a valid mouse model.

[0146] conclusion The cell-free protein expression and evaluation workflow is expected to be practical in many different applications where evaluation of individual protein variants is a rate-limiting processing step. Furthermore, the multivalent screen we have developed will accelerate researchers' ability to develop multivalent protein therapeutics.

[0147] Engineered protein constructs may have multiple advantages over monoclonal antibodies for preventing and treating COVID-19 infection: (1) direct administration into the respiratory system, (2) low cost of goods and amenability to very large-scale production, (3) high stability and no need for cold chain, and (4) very broad-spectrum resistance to escape mutants in a single compound. More generally, engineered high-affinity multivalent minibinders may provide a powerful platform for fighting viral pandemics.

Claims

1. A polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101, wherein the polypeptide binds to the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD).

2. The polypeptide of claim 1, wherein the amino acid substitutions relative to the amino acid sequence of the reference polypeptide are selected from the exemplary amino acid substitutions provided in Table 1.

3. 3. The polypeptide of claim 1 or 2, wherein the interface residues are identical to or conservatively substituted for the interface residues in the reference polypeptide.

4. The polypeptide of any one of claims 1 to 3, comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101.

5. The polypeptide of any one of claims 1 to 4, comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101.

6. 6. The polypeptide of any one of claims 1 to 5, comprising an amino acid substitution at one or both residues selected from the group consisting of 63 and 75 relative to the amino acid sequence of SEQ ID NO:

101.

7. The polypeptide of claim 6, wherein the substitutions include R63A and / or K75T.

8. The polypeptide of any one of claims 1 to 7, further comprising one or more additional cysteine ​​residues at the N-terminus and / or C-terminus.

9. The polypeptide of any one of claims 1 to 8, comprising an N-linked glycosylation site (i.e.: NX(S / T), where X is any amino acid).

10. 2. The polypeptide of claim 1, comprising two or more copies of an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101.

11. 11. The polypeptide of claim 10, wherein the two or more copies of the polypeptide are all identical.

12. 11. The polypeptide of claim 10, wherein the two or more copies of the polypeptide are not all identical.

13. The polypeptide of any one of claims 10 to 12, wherein the two or more copies of the polypeptide are separated by an amino acid linker.

14. 14. The polypeptide of claim 13, wherein the amino acid linker comprises at least 90% Gly-Ser residues.

15. 15. The polypeptide of claim 14, wherein the amino acid linker comprises an amino acid sequence selected from the group consisting of GG and SEQ ID NOs: 35-46 and 165-171.

16. The polypeptide of claim 14, wherein the amino acid linker comprises at least 15%.

17. 17. The polypeptide of claim 16, wherein the amino acid linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 97-98 and 172-176.

18. 14. The polypeptide of claim 13, wherein the amino acid linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99 and 177-178.

19. 19. The polypeptide of any one of claims 13 to 18, wherein the amino acid linkers are independently 2 to 100 amino acids in length.

20. comprising the formula Z1-Z2-Z3, Z1 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101; Z2 comprises an optional amino acid linker; and Z3 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34 and 100-164; Z1 and Z3 may be the same or different; A polypeptide according to any one of claims 10 to 19.

21. comprising the formula Z1-Z2-Z3, Z1 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34 and 100-164; Z2 comprises an optional amino acid linker; and Z3 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101; Z1 and Z3 may be the same or different; A polypeptide according to any one of claims 10 to 19.

22. 22. The polypeptide of claim 20 or 21, wherein Z1 and Z3 are identical.

23. 22. The polypeptide of claim 20 or 21, wherein Z1 and Z3 are different.

24. 21. The polypeptide of claim 20, wherein Z3 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-10, 13-17, 19-21, 33-34, and 102-163.

25. 22. The polypeptide of claim 21, wherein Z1 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 13-17, 19-21, 33-34, and 102-163.

26. 22. The polypeptide of claim 21, wherein Z comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100-101, and 164.

27. Comprising the formula B1-B2-Z1-Z2-Z3-B3-B4; Z1, Z2 and Z3 are as defined in any one of claims 20 to 26; B2 and B3 comprise an optional amino acid linker; and one or both of B1 and B4 independently comprise an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34 and 100-164, and one of B1 and B4 may be absent; A polypeptide according to any one of claims 20 to 26.

28. 28. The polypeptide of claim 27, wherein one of B1 and B4 is absent.

29. 28. The polypeptide of claim 27, wherein B1 and B4 independently comprise an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, 19-21, 23-34 and 100-164.

30. 30. The polypeptide of claim 29, wherein B1 and B4 are identical.

31. 30. The polypeptide of claim 29, wherein B1 and B4 are not identical.

32. 32. The polypeptide of any one of claims 27 to 31, wherein B1, when present, and B4, when present, are identical to one or both of Z1 and Z3, or B1, when present, and B4, when present, are not identical to either Z1 or Z3.

33. 33. The polypeptide of any one of claims 27-32, wherein B1, when present, and B4, when present, independently comprise an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 13-17, 19-21, 33-34, and 100-164.

34. 34. The polypeptide of any one of claims 27 to 33, wherein B1, when present, and B4, when present, independently comprise an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164.

35. Both B1 and B4 are present, and one of B1 and B4 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10 and 102-136, and the other comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101, and 164; or one of B1 and B4 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17, 19-21 and 137-163, and the other comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34, 100-101 and 164; A polypeptide according to any one of claims 27 to 33.

36. 36. The polypeptide of any one of claims 1-35, comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 267, 269-270, 343-365, 377-380, 383-384, 387-388, 390-396, 400-403, 452-453, 506, 508-509, 576-582, 584-588, 595-604, 616-619, 622-623, 626-627, 629-635, 639-682, 691-695, and 697-699, wherein the optional N-terminal methionine residue may be present or absent in the polypeptide, and any residue may be present or absent and is not considered in determining percent identity.

37. A polypeptide according to any one of claims 1 to 35, comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 693 to 701, and wherein any N-terminal methionine residue is optional and may be present or deleted.

38. A polypeptide according to any one of claims 1 to 35, comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 693, wherein the N-terminal methionine residue is optional and may be present or deleted.

39. 39. The polypeptide of any one of claims 1 to 38, further comprising an additional functional peptide domain.

40. 40. The polypeptide of claim 39, wherein the additional functional peptide domain comprises a targeting domain, a detectable domain, a scaffolding domain, a secretion signal, an Fc domain, or an additional therapeutic peptide domain.

41. 41. The polypeptide of claim 40, wherein the additional functional domain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO:

64.

42. The polypeptide of any one of claims 39 to 41, wherein the added functional domain comprises an oligomerization domain.

43. 43. The polypeptide of claim 42, wherein the oligomerization domain comprises a homotrimerization domain.

44. 42. The polypeptide of claim 40 or 41, wherein the oligomerization domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 179-189 and 589-594.

45. 43. The polypeptide of any one of claims 40-42, comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 356-365, 377-380, 383-384, 387-388, 390-396, 400-403, 452-453, and 595-604, 616-619, 622-623, 626-627, 629-635, 639-682, 691-692, wherein the optional N-terminal methionine residue may or may not be present in the polypeptide.

46. 46. ​​The polypeptide of any one of claims 1 to 45, wherein the polypeptide is linked to a stabilization domain selected from the group consisting of polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), a conformationally disordered polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser ("PASylated"), and / or a mucin diffusible polypeptide composed of the amino acids Lys and Ala, with or without Glu.

47. 47. The polypeptide of any one of claims 1 to 46, which binds to the SARS-CoV-2 spike glycoprotein with an affinity of at least 10 nM, measured as described in the accompanying examples.

48. A nucleic acid encoding the polypeptide of any one of claims 1 to 45.

49. 49. An expression vector comprising the nucleic acid of claim 48 operably linked to a promoter.

50. A host cell comprising the polypeptide, nucleic acid, and / or expression vector of any of claims 1 to 49.

51. An oligomer of the polypeptide of any one of claims 1 to 47.

52. 52. The oligomer of claim 51, comprising a trimer.

53. A composition comprising two, three, four or more copies of the polypeptide of any one of claims 1 to 47 attached to a support.

54. 54. A pharmaceutical composition comprising the polypeptide, nucleic acid, expression vector, host cell, oligomer, and / or composition of any of claims 1 to 53 and a pharmaceutically acceptable carrier.

55. 55. The pharmaceutical composition of claim 54 for use in treating Severe Acute Respiratory Syndrome (SARS) coronavirus infection.

56. 56. The pharmaceutical composition of claim 55, wherein the SARS coronavirus comprises SARS-CoV-2.

57. 55. The pharmaceutical composition of claim 54 for use in limiting Severe Acute Respiratory Syndrome (SARS) coronavirus infection.

58. 58. The pharmaceutical composition of claim 57, wherein the SARS coronavirus comprises SARS-CoV-2.

59. 59. The pharmaceutical composition of any one of claims 55 to 58, wherein the polypeptide, nucleic acid, expression vector, host cell, oligomer, composition and / or pharmaceutical composition is administered intranasally.

60. 59. The pharmaceutical composition of any one of claims 55 to 58, wherein the polypeptide, nucleic acid, expression vector, host cell, oligomer, composition, and / or pharmaceutical composition is administered systemically.

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