Protein therapeutics

Abstract

Provided herein are protein therapeutics, such as antibodies and fusion proteins, for preventing, reducing, and / or treating SARS-CoV-2 infection in a subject. The methods provided herein include administering to a subject a therapeutically effective amount of an antigen-binding fragment (Fab fragment) or antibody that binds to a SARS-CoV-2 spike protein, an ACE2 decoy peptide that binds to a SARS-CoV-2 spike protein, and / or a DNA construct encoding an anti-spike Fab fragment or an ACE2 decoy peptide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit under 35 USC §120 of U.S. application Ser. No. 16 / 880,804, filed May 21, 2020. This patent application also claims the benefit of the following U.S. Provisional Application Nos. 62 / 988,328 filed on March 11, 2020, 62 / 991,504 filed on March 18, 2020, 63 / 016,048 filed on April 27, 2020, 63 / 016,241 filed on April 27, 2020, 63 / 022,146 filed on May 8, 2020, 63 / 036,445 filed on June 9, 2020, 63 / 080,887 filed on September 21, 2020, 63 / 082,145 filed on September 23, 2020, and 63 / 121,102 filed on December 3, 2020 under 35 U.S.C. The entire contents of US16 / 880,804, US62 / 988,328, US62 / 991,504, US63 / 016,048, US63 / 016,241, US63 / 022,146, US63 / 036,445, US63 / 080,887, US63 / 082,145, and US63 / 121,102 are hereby incorporated by reference.

[0003] Statement regarding sequence listing

[0004] This disclosure contains references to amino acid sequences and nucleic acid sequences that have been filed concurrently with this disclosure as a sequence listing text file entitled "62803442_1.TXT," with a file size of 788 kilobytes (KB), created on March 10, 2021. The foregoing sequence listing is hereby incorporated by reference in its entirety pursuant to 37 CFR §1.52(e)(5). Technical Field

[0005] The present disclosure relates to proteins that can be administered to a subject to prevent and treat COVID-19 and other such virus-induced diseases. Background Art

[0006] Angiotensin-converting enzyme 2 (ACE2) is a regulatory carboxypeptidase of the renin-angiotensin hormone system and acts as a regulator of cardiovascular homeostasis. ACE2 is expressed in many tissues, including alveolar epithelial cells, small intestinal enterocytes, testicular Leydig cells and Sertoli cells, renal proximal tubules, and testes. ACE2 is also a receptor for human coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The widespread expression of ACE2 in various human tissues enables efficient large-scale infection of human coronaviruses.

[0007] Currently, there are no known effective treatment options that can reduce or even completely avoid the pathogenic effects of coronavirus infection and its sequelae. Although several drugs (e.g., remdesivir, hydroxychloroquine, azithromycin, immunosuppressive steroids, etc.) have been proposed for the treatment of COVID-19, their efficacy is currently unknown. In addition, most of the known proposed drugs do not directly affect the virus or prevent it from entering host cells, but rather generally reduce the overstimulated immune response to the virus. Similarly, there are no known effective vaccine compositions that can help treat or avoid coronavirus disease.

[0008] Batlle et al. (2020) Clin. Sci. 134:543-45 hypothesized that a soluble angiotensin-converting enzyme 2 (ACE2) extracellular domain conjugated to an immunoglobulin G (IgG1) crystallizable fragment (Fc) domain could serve as a therapeutic against COVID-19 infection.

[0009] Batlle and Wysocki described chimeric ACE2-Fc constructs in International Patent Application Publication No. WO 18 / 140456.

[0010] Esvelt (2020, “Designing virus-specific sACE2 mimics for competitive inhibition of SARS-CoV-2,” available at www.covid19-hpc-consortium.org / projects / 5e90e0ca836090007f1ddb39) hypothesized that the ACE2 construct could be mutated to achieve various variants that have higher binding affinity for the receptor binding domain (spike-RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Hypothetically, such mutants would outcompete native ACE2 for the spike-RBD, thereby blocking the ability of the spike-RBD to bind to ACE2 on human cells.

[0011] Jiang et al. (2020) Trends Immunol. 41(5):355-359 reported neutralizing monoclonal antibodies (mAbs) against SARS-CoV-2. Summary of the invention

[0012] Disclosed herein are various protein therapeutics that can be used to treat and prevent COVID-19 infection. In certain embodiments, ACE2 decoy peptides and ACE2 decoy peptide fragments are disclosed, optionally conjugated to an Fc domain, which have a higher affinity for SARS CoV-2 spike RBD than human wild-type ACE2. These ACE2 decoy peptides and fragments thereof can be used to prevent or inhibit viral entry into individuals inactively infected with SARS CoV-2. These decoy peptides can also be used in individuals already infected with SARS-CoV-2 to prevent the spread of the virus from infected cells to uninfected cells, thereby slowing and controlling the disease. Also disclosed herein are nucleotides and nucleotide vectors (including viral vectors) suitable for inducing the subject's own cells to produce these therapeutically effective ACE2 decoy peptides, fragments and / or chimeras.

[0013] Additionally or alternatively, various mAbs that specifically bind to SARS CoV-2 spikes, as well as Fab and scFv fragments of these mAbs, are also disclosed herein. These mAbs and fragments can be used to prevent the virus from entering individuals who are inactively infected with SARS CoV-2. These decoy peptides can also be used in individuals who have been infected with SARS-CoV-2 to prevent the virus from spreading from infected cells to uninfected cells, thereby slowing down and controlling the disease. Also disclosed herein are nucleotides and nucleotide vectors (including viral vectors) suitable for inducing the production of these therapeutically effective mAbs and / or fragments by the subject's own cells.

[0014] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The crystal structure of the SARS-CoV-2 spike protein trimer is depicted. The S1 and S2 domains of the trimer are indicated. The dashed line marks the Figure 2 The trimer part is enlarged in the figure.

[0016] Figure 2 The crystal structure of the receptor binding domain (RBD) of the S1 domain of the spike trimer is depicted.

[0017] Figure 3 depicts SPR results obtained with the N-612-004 mAb. Graph 3A shows affinity results for the cleaved spike peptide, while 3B shows results for the RBD-S1 peptide.

[0018] Figure 4 depicts SPR results obtained with the N-612-017 mAb. Graph 4A shows affinity results for the cleaved spike peptide, while 4B shows results for the RBD-S1 peptide.

[0019] Figure 5 Biofilm interferometry illustrating the ability of selected mAbs to block the SARS-CoV-2 spike to ACE2 is depicted. The line labeled "buffer" shows unimpeded binding of ACE2 to RBD. The lines for N-612-002 and N-612-004 overlap substantially throughout the time series. The N-612-017 line is closest to the horizontal line.

[0020] Figure 6 The K of N-612-004 for RBD peptide by SPR is depicted. D Determination.

[0021] Figure 7 The K of N-612-017 for RBD peptide by SPR is depicted. D Determination.

[0022] Figure 8 Depicted is the K of N-612-002 against the SARS-CoV-2 spike protein construct Sc-S-PP-T4F by SPR. D Determination.

[0023] Fig. 9 Depicted is the K of N-612-004 against the SARS-CoV-2 spike protein construct Sc-S-PP-T4F by SPR. D Determination.

[0024] Fig.10 Depicted is the K of N-612-007 against the SARS-CoV-2 spike protein construct Sc-S-PP-T4F by SPR. D Determination.

[0025] Fig.11 Depicted is the K of N-612-014 against the SARS-CoV-2 spike protein construct Sc-S-PP-T4F by SPR. D Determination.

[0026] Fig.12 Depicted is the K of N-612-017 against the SARS-CoV-2 spike protein construct Sc-S-PP-T4F by SPR. D Determination.

[0027] Fig.13 The K of N-612-017scFv to RBD peptide by SPR is depicted. D Determination.

[0028] Fig.14 The K of N-612-056scFv to RBD peptide by SPR is depicted. D Determination.

[0029] Fig.15 The K of N-612-017scFv against SARS-CoV-2 spike trimer by SPR is depicted. D Determination.

[0030] Fig.16 Depicted is the K of N-612-056scFv against SARS-CoV-2 spike trimer by SPR. D Determination.

[0031] Fig.17 The conformational structure and crystal structure of ACE2-IgG1Fc are depicted.

[0032] FIG. 18 shows the purification of ACE2-IgG1Fc mutant decoy peptide. Fig.18A HPLC purification is depicted. Fig.18B The purity of the recovered ACE2 IgG1 Fc mutant decoy peptides under reducing and non-reducing conditions is shown.

[0033] Fig.19 The binding affinity of wild-type ACE2 decoy to RBD-SD1 is depicted.

[0034] Fig. 20The binding affinity of the T27Y / H34A mutant ACE2 decoy to RBD-SD1 is depicted.

[0035] Fig.21 Depicted is the KD determination of ACE2 wild-type and ACE2 mutant decoy peptides fused to IgG1 Fc by SPR.

[0036] Fig. 22 Live SARS-CoV-2 neutralization data are presented.

[0037] Fig.23 The stability of wild-type and mutant ACE2 decoy peptides is depicted.

[0038] Fig.24 The size of the T27Y / H34A / R273Q triple mutant ACE2 decoy peptide was depicted by size exclusion chromatography.

[0039] Fig.25 The size of the T27Y / H34A / R273K triple mutant ACE2 decoy peptide was depicted by size exclusion chromatography.

[0040] Fig.26 The size of the T27Y / H34A / R273L triple mutant ACE2 decoy peptide was depicted by size exclusion chromatography.

[0041] Fig. 27 The size of the T27Y / H34A / H345A triple mutant ACE2 decoy peptide was depicted by size exclusion chromatography.

[0042] Fig.28 The size of the T27Y / H34A / H505L triple mutant ACE2 decoy peptide was characterized by size exclusion chromatography.

[0043] Fig.29 The size of the T27Y / H34A / H374N triple mutant ACE2 decoy peptide was depicted by size exclusion chromatography.

[0044] Fig.30 The size of the T27Y / H34A / H378N triple mutant ACE2 decoy peptide was depicted by size exclusion chromatography.

[0045] Fig.31 The ACE2 activities of the triple mutant ACE2 decoy peptides are depicted.

[0046] Fig.32 The stability of single-, double-, and triple-mutant ACE2 decoy peptides is depicted.

[0047] Fig.33 The stability of double- and triple-mutant ACE2 decoy peptides is depicted.

[0048] Fig.34 The stability of various triple-mutant ACE2 decoy peptides was profiled.

[0049] Fig.35 The stability of avidin-labeled and unlabeled wild-type ACE2-IgGFc is depicted.

[0050] Fig.36 Depicted are the K values ​​of N-612-017 and N-612-056 against SARS-CoV-2 spike protein and its variants by SPR. D Determination.

[0051] Fig.37 The K values ​​of N-612-004, N-612-017, N-612-017, and N-612-056 against SARS-CoV-2 spike protein and its variants by SPR are depicted. D Determination.

[0052] Fig.38 Figure 3 K of wild-type ACE2 and T27Y / H34A / H374N ACE2 decoy peptides against SARS-CoV-2 spike protein and its variants by SPR. D Determination. DETAILED DESCRIPTION

[0053] I. Definitions

[0054] The following definitions refer to various terms used above and throughout this disclosure.

[0055] "Concomitant or concomitantly" includes administering an agent (e.g., an IL-15 agonist) in the presence of another agent (e.g., an ACE2 mutant). Concomitant administration in a therapeutic treatment method includes a method of co-administering a first, second, third, or additional agent. Concomitant administration also includes a method in which a first or additional agent is administered in the presence of a second or additional agent, wherein the second or additional agent can, for example, be administered in advance. Concomitant therapeutic treatment methods can be performed stepwise by different participants. For example, one participant can administer a first agent (e.g., an ACE2 mutant) to a subject, while a second participant can administer a second agent (e.g., an IL-15 agonist) to a subject, and the administration step can be performed at the same time or at approximately the same time. The participant and the subject can be the same entity (e.g., a person). Therefore, the term includes simultaneous administration and substantially simultaneous administration, i.e., administration at approximately the same time.

[0056] As used herein, mutation positions in ACE2 mutants are specified relative to corresponding positions in SEQ ID NO:135.

[0057] "Encodes" - when used to describe a polynucleotide - means that when transcription is initiated from the polynucleotide in a wild-type human cell, the resulting transcript will be translated into a given protein. That is, when the codon triplets of wild-type human tRNA produce a polypeptide from the polynucleotide according to the normal working principles of transcription and translation in a wild-type human cell, the polynucleotide "encodes" the polypeptide.

[0058] "Effective amount" or "therapeutically effective amount" refers to the amount and / or dosage and / or dosage regimen of one or more agents necessary to bring about the desired result, for example, an amount sufficient to prevent an infection in a subject, an amount sufficient to reduce the occurrence of an infection in a subject, and / or an amount sufficient to treat an infection in a subject.

[0059] "IL-15 agonist" refers to a compound or molecule that binds to and activates the IL-15 receptor ("IL-15Rα"). The type of compound or molecule of the IL-15 agonist is not particularly limited, as long as it binds to and activates IL-15Rα. The IL-15 agonist can be a peptide, a protein, a small molecule (e.g., a drug), or an oligonucleotide. A peptide or protein can be a single amino acid sequence or two or more sequences bound via covalent attachment (e.g., a disulfide bond) or non-covalent attachment (e.g., hydrophilic or hydrophobic interactions, hydrogen bonds). In a specific embodiment, the IL-15 agonist is an antibody, a modified antibody, a chimeric antibody, or a derivative thereof. In a further embodiment, the IL-15 agonist is a super agonist complex, such as an IL-15 derivative bound to an IL-15Rα / IgG1 Fc fusion protein, also known as nogapendekin alfa-imbakicept (NAI). NAI is also referred to in the literature as N-803, ALT-803, or IL-15 N72D :IL-15RαSu / IgG1. US 9,328,159 describing NAI is incorporated herein by reference in its entirety. Clinical trials involving N-803 are described in NCT04385849, which is incorporated herein by reference in its entirety. In some embodiments, the IL-15 agonist is administered alone, and / or as the sole therapeutic agent for the treatment of a disease, disorder, condition, or infection as described herein. In another embodiment, the IL-15 agonist is administered in combination with another compound or molecule, for example, in combination with: an antibody or Fab fragment that binds to a spike protein or a modified variant thereof; an ACE decoy peptide or a variant thereof; and / or an agent that modulates ACE or ACE2 signaling (e.g., an ACE inhibitor or an angiotensin receptor antagonist).

[0060] The binding of IL-15 agonists to IL-15Rα induces signals from downstream elements to activate the IL-15 signaling pathway and activate cells. Cells expressing IL-15Rα include, but are not limited to, T cells, NK cells, monocytes, macrophages, dendritic cells, keratinocytes, fibroblasts, muscle cells, and nerve cells. Guo et al. Cytokine Growth Factor Rev. [Cytokine and Growth Factor Review], 2017. The binding of IL-15 agonists to IL-15Rα propagates signals (e.g., via conformational changes) through IL-15Rα, which initiates the IL-15 signaling pathway to activate an immune response, such as an antiviral response.

[0061] "Subject", "individual" and "patient" refer interchangeably to mammals, preferably humans or non-human primates, but also to domesticated mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cattle, pigs, sheep). In certain embodiments, the subject may be a person (e.g., adult males, adult females, adolescent males, adolescent females, boys, girls) under the care of a physician or other health worker. In certain embodiments, the subject may not be under the care of a physician or other health worker.

[0062] "SARS-CoV-2" refers to severe acute respiratory syndrome coronavirus 2. It is a highly contagious positive-sense, single-stranded RNA virus that causes a respiratory disease called COVID-19. The sequence of the wild-type virus can be found at www.ncbi.nlm.nih.gov / nuccore / NC_045512.2 (RefSeq NC_045512.2). As used herein, SARS-CoV-2 also includes mutations and variants of typical viruses, including but not limited to SARS-CoV-2 mutants and variants that can induce COVID-19 symptoms in the global population. In some embodiments, SARS-CoV-2 mutants and variants induce any two or more COVID-19 symptoms in at least 10% of the global population.

[0063] COVID-19 is a human respiratory disease caused by infection of a subject with SARS-CoV-2 (including wild-type viruses and their mutants and variants). Symptoms of COVID-19 include, but are not limited to, headache, confusion, loss of smell and taste, nasal congestion and runny nose, cough, muscle aches and / or pain, sore throat, fever, chills, diarrhea, shortness of breath, pneumonia, vomiting, dyspnea, hypoxia, respiratory failure, shock, multiple organ dysfunction, and combinations thereof. In some subjects, symptoms may occur in clusters, for example, cough, sputum, shortness of breath, and fever; or muscle pain, joint pain, headache, and fatigue.

[0064] "An agent that modulates at least one of ACE and ACE2 signaling" refers to a compound or molecule that modulates (e.g., increases or decreases) the expression and / or activity of angiotensin converting enzyme (ACE) or, in particular, ACE2. In some embodiments, the agent that modulates at least one of ACE or ACE2 signaling may be an ACE inhibitor or a compound that increases ACE2 expression. In some embodiments, the compound may be an ACE inhibitor selected from, but not limited to, the group consisting of: Benazepril, Captopril, Fosinopril, Lisinopril, Enalapril, Perindopril, Moexipril, Quinapril, Ramipril, and Trandolapril. In some embodiments, the compound is an angiotensin receptor antagonist selected from, but not limited to, Candesartan, Olmesartan, Valsartan, and Losartan. Additionally or alternatively, the additional agent may be an inhibitor of BDKRB1 / 2, AGTR1, MAS1, or renin.

[0065] "Treat and treatment" each refers to a method of reducing, inhibiting or otherwise improving an infection by administering a therapeutic agent to a subject in need of treatment. In some embodiments, a subject in need of treatment may include a subject suffering from, diagnosed with, or suspected of having an infection. In a specific embodiment, treatment (treat or treatment) includes administering a therapeutic agent to a subject suffering from, diagnosed with, or suspected of having COVID-19. Additionally or alternatively, the subject may be infected with SARS-CoV-2, diagnosed with infection with SARS-CoV-2, or suspected of being infected with SARS-CoV-2. In some embodiments, the subject may be asymptomatic. Treatment includes administering separately any of an ACE2 decoy peptide or variant thereof optionally fused to an Fc domain, a Fab fragment or antibody bound to a spike protein, an IL-15 agonist, or an agent that regulates at least one of ACE and ACE2 signaling, or any combination thereof.

[0066] II. ACE2 decoy peptides and variants thereof

[0067] The natural cell surface receptor of SARS CoV-2 is the extracellular domain of ACE2. By mutating the part of ACE2 that interacts with the SARS-CoV-2 spike-RBD, an ACE2 decoy peptide (or a peptide fragment thereof) that binds to the RBD can be made, wherein the binding affinity is higher than the affinity of the wild-type ACE2 extracellular domain. The modified form of the ACE2 decoy peptide can be changed to retain at least three domains known to interact with the SARS-CoV spike glycoprotein. Similarly, the ACE2 portion preferably includes a leader peptide to allow secretion from recombinant cells that produce ACE2-Fc constructs (discussed herein). Therefore, in the case of truncation at the C-terminus, the truncation can remove the sequence motifs required for ADAM17 cleavage and / or the sequence motifs required for TMPRSS11D and / or TMPRSS2 cleavage. These high-affinity ACE2-Fc constructs can effectively "chelate" the virus away from the wild-type ACE2 on the surface of the subject's cells.

[0068] The ACE2 protein is 805 amino acids long, but in certain preferred embodiments, the ACE2 decoy will not include the entire 805 amino acid ACE2 sequence, but will be composed of truncations, for example, the ACE2 decoy peptide may lack a transmembrane domain. In certain embodiments, the therapeutic protein will contain no more than 700 amino acids, for example no more than 600 amino acids, no more than 500 amino acids, no more than 400 amino acids, no more than 300 amino acids, no more than 200 amino acids, no more than 100 amino acids, or even 50 or less amino acids. For example, certain preferred embodiments will consist of amino acid residues 1-740, or 1-615, or 10-400, or 20-100, or 21-614, or 20-50, or 320-360. Wild-type ACE2 is shown in SEQ ID NO: 135.

[0069] Suitable mutations that increase the binding affinity of ACE2 to spike-RBD include glutamine mutation at position 24 of ACE2 (SEQ ID NO: 136), threonine mutation at position 27 (SEQ ID NO: 137), aspartic acid mutation at position 30 (SEQ ID NO: 138), histidine mutation at position 34 (SEQ ID NO: 139), glutamate mutation at position 35 (SEQ ID NO: 140), aspartic acid mutation at position 38 (SEQ ID NO: 141), tyrosine mutation at position 41 (SEQ ID NO: 142), arginine mutation at position 273 (SEQ ID NO: 201), histidine mutation at position 345 (SEQ ID NO: 202), aspartic acid mutation at position 355 (SEQ ID NO: 143), histidine mutation at position 374 (SEQ ID NO: 203), histidine mutation at position 378 (SEQ ID NO: 204), and histidine mutation at position 505 (SEQ ID NO: 205). Various combinations of these single point mutations (e.g., position 24 & 27, 24 & 30, 24 & 34, 24 & 35, 24 & 38, 24 & 41, 24 & 355, 27 & 30, 27 & 34, 37 & 35, 27 & 38, 27 & 41, 27 & 355, 30 & 34, 30 & 35, 30 & 38, 30 & 41, 30 & 355, 34 & 35, 34 & 38, 34 & 41, 34 &355, 35&38, 35&41, 35&355, 38&41, 38&355, 41&355, 24&27&30, 24&27&34, 24&27&35, 24&27&38, 24&27&41, 24&27&355, 24&30&34, 24&30&35, 24&30&38, 24&30&41, 24&30&355, 24&34&3 5. 24&34&38, 24&34&41, 24&34&355, 24&35&38, 24&35&41, 24&35&355, 24&38&41, 24&38&355, 24&41&355, 27&30&34, 27&30&35, 27&30&38, 27&30&41, 27&30&355, 27&34&35, 27&34&38, 2 7&34&41、27&34&273、27&34&345、27&34&355、27&34&374、27&34&505、27&35&38、27&35&41、27&35&355、27&38&41、27&38&355、27&41&355、30&34&35、30&34&38、30&34&41、30&34&355、30&35&38、30&35&41、30&35&355、30&38&41、30&38&355、30&41&355、34&35&38、34&35&41、34&35&355、34&38&41、34&38&355、34&41&355、35&38&41、35&38&355、35&41&355、38&41&355、24&27&30&35、24&27&30&38、24&27&30&41、24&27&30&355、24&30&35&38、24&30&35&41、24&30&35&355、24&35&38&41、24&35&38&355、24&38&41&355、27&30&35&38、27&30&35&41、27&30&35&355、27&35&38&41、27&35&38&355、27&38&41&355、30&35&38&41、30&35&38&355、30&38&41&355、35&38&41&355、24&27&30&35&38、24&27&30&35&41、24&27&30&35&355、27&30&34&35&41、27&30&34&35&355、30&34&35&38&41、30&34&35&38&355、30&34&38&41&355、30&35&38&41&355、34&35&38&41&355、24&27&30&34&35&41、24&27&30&34&35&355、24&27&34&35&41&355、24&27&30&34&41&355、24&27&30&34&35&355、24&30&34&35&38&41、24&30&34&35&38&355、27&30&34&35&38&41、27&30&34&35&38&355、27&30&34&35&41&355、30&34&35&38&41&355、24&27&30&34&35&38&41、24&27&30&35&38&355、24&27&30&34&35&41&355、24&27&30&34&38&41&355、24&27&30&35&38&41&355、24&27&34&35&38&41&355、24&30&34&35&38&41&355、27&30&34&35&38&41&355、and 24&27&30&34&35&38&41&355) can also be used to improve affinity for RBD, and all such mutations are shown in SEQ ID NO: 144.

[0070] SEQ ID NOs: 136-144, 147-159, and 201-212 are provided only as examples of suitable ACE2 decoy peptides, but many variations of these sequences may also be used for anti-COVID19 therapeutic purposes. For example, a polypeptide having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to any one of SEQ ID NOs: 136-144, 147-159, and 201-212 may also be used for therapeutic purposes, provided that the molecule broadly retains the overall binding site structure and orientation of a single SEQ ID NO: 136-144, 147-159, and 201-212 molecule. SEQ ID NO: 136-144 discloses ACE2 decoy peptide mutants with potential single mutations within the protein. SEQ ID NO: 147-159 and 201-205 disclose ACE2 decoy peptide mutants with specific single mutations within the protein. SEQ ID NO: 206-212 describes ACE2 decoy peptide mutants with three point mutations (e.g., triple mutants).

[0071] In one embodiment, double mutants of ACE2 decoy peptides with mutations T27Y and H34A are contemplated. An important mechanism of SARS-CoV-1 and SARS-CoV-2 infection begins with the binding of the viral spike protein to the human receptor protein ACE2. Double-mutated ACE2 decoy peptides, or peptides of binding domains, can bind to the viral spike protein with very high binding affinity, thereby exceeding, reducing, or even completely avoiding viral docking. In one embodiment, the α-helix of the ACE2 decoy peptide has mutations T27Y and H34A. This mutant ACE2 decoy peptide can be generated by introducing T27Y and H34A mutations into the α-helix of ACE2, where the α-helix is ​​close to the RBD when the RBD and ACE2 form a binding complex. It is demonstrated that the mutant α-helix has a higher binding affinity to the RBD than the wild-type α-helix of ACE2. This double-mutated ACE2 decoy peptide can be used to treat or prevent COVID-19.

[0072] Additionally or alternatively, the ACE2 decoy peptide portion can be fused to an Fc domain to produce a therapeutic protein chimera (also known as a hybrid polypeptide, a hybrid construct, a fusion polypeptide, and / or a fusion construct). Although any Fc domain can be used (e.g., an Fc domain from IgA, IgD, IgE, IgG, IgM, etc.), in a preferred embodiment, the chimera will comprise an Fc domain from IgA or IgG. In a further embodiment, IgGFc is selected from the group consisting of IgG1Fc, IgG2Fc, IgG3Fc, and IgG4Fc. For example, the Fc domain may have at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity) with SEQ ID NO: 145 or 146. In some embodiments, ACE2 decoy peptide mutants comprising multiple mutations, such as three mutations, such as T27Y and H34A and additional mutations (e.g., R273K, R273L, R273Q, H345A, H374N, H378N, and H505L) can be transferred to the IgA scaffold. In a preferred embodiment, the three mutations are T27Y, H34A, and H374N mutations. Fusion of the ACE2 portion with the IgFc domain can be achieved by incorporating a nucleotide sequence encoding an ACE2 mutant and an IgFc scaffold (e.g., an IgAFc scaffold) into a vector (e.g., an adenovirus vector). The vector is then formulated in a pharmaceutical composition suitable for administration (e.g., by subcutaneous, intramuscular, or oral delivery). Alternatively, the ACE2 decoy peptide and Fc can be coupled via a flexible peptide linker. Typically, such a linker may have 5 to 25 amino acids, and all known flexible linkers are considered to be applicable herein. Particularly suitable linkers include a series of glycines interspersed with serines (e.g., GGGS). Additionally or alternatively, the ACE2-Fc hybrid construct can be fixed to a carrier. All modifications that allow fixation are suitable, such as biotinylation, cellulose binding domains, etc. Detectable labels can also be added to the ACE2-Fc hybrid construct to achieve in situ detection and / or quantification in quantitative assays. Suitable labels include luminescent labels, radioisotope labels, enzyme labels, etc.

[0073] III. Antigen-binding fragments and antibodies

[0074] Antigen binding fragments (Fab fragments) and antibodies that bind to the SARS-CoV-2 spike protein, particularly antibodies that bind to the spike RBD, can interfere with the entry of SARS-CoV-2 into host cells. Antibodies and Fab fragments disclosed herein can bind to the SARS-CoV-2 spike, including several that can bind to the spike RBD.

[0075] For purposes of illustrating the anti-spike antibodies and Fab fragments disclosed herein, a H CDR1-B-V H CDR2-C-V H The heavy chain variable region (V H ). These exemplary anti-spike Fab fragments also have structures as shown in SEQ ID NO: 131-V L CDR3-E light chain variable region (V L ). V of various Fab fragments of this type H and V L The amino acid sequences of are listed in Table 1 below. The binding affinity of some of these Fab fragments has been confirmed by surface plasmon resonance (SPR).

[0076]

[0077]

[0078] Certain anti-Spike Fab fragments specifically bind to the Spike RBD. The V H and V L The amino acid sequences are listed in Table 2 below. The binding affinity of some of these Fab fragments to the spike protein trimer and / or RBD peptide has been confirmed by surface plasmon resonance (SPR).

[0079]

[0080] Although specific examples are listed in Tables 1 and 2, the skilled artisan understands that the affinity of an antibody or Fab fragment is determined by the complementarity determining region (CDR), and that a large number of changes and adjustments can be made to the non-CDR portions of the variable domains without substantially changing the usefulness of these antibodies or Fab fragments in neutralizing SARS-CoV-2 infectivity. Therefore, many sequences beyond those exemplified above are equally useful for treating COVID-19 disease. Particularly suitable sequences will retain at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 127, at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 128, at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 129, at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 130, NO: 130, and at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 132. Similarly, particularly suitable sequences will retain at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 131, but such sequences will retain 100% identity to the light chain CDR1 and CDR2 within the SEQ ID NO: 131 sequence.

[0081] In some embodiments, the Fab fragments and antibodies that bind to the spike protein (e.g., spike RBD) may additionally or alternatively bind to modified and / or mutated spike proteins. Modified and / or mutated spike proteins may be found on Sars-Cov-2 variants. The modified spike protein may be different from the wild-type or naturally occurring spike protein by containing point mutations, insertions, deletions, and / or inversions. In an embodiment, one or more point mutations are selected from the group consisting of: S13I, L18F, T20N, P26S, D80A, D138Y, W152C, R190S, D215G, L242H, K417N, K417T, E484K, L452R, N501Y, A570D, D614G, H655Y, P681H, T716I, S982A, D1118H, T1027I, and combinations thereof. Specific multiple mutations include, but are not limited to, those listed in Table 3 below:

[0082]

[0083]

[0084] IV. Polynucleotides and Vectors

[0085] Contemporary molecular biologists know how to make nucleotides that express proteins described herein, and how to express such nucleotides in cells to obtain related proteins. Further embodiments provided herein include polynucleotides encoding polypeptides comprising ACE2 decoy peptides, wherein the ACE2 decoy peptide is a polypeptide of SEQ ID NO: 135 or a variant thereof. For example, a polynucleotide encoding wild-type human ACE2 is presented herein as SEQ ID NO: 168. Ordinary molecular biologists know how to change the sequence of SEQ ID NO: 168 to encode SEQ ID NO: 136-144, 147-159, and 201-212 and appropriate variants thereof (e.g., variants having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with any one of SEQ ID NO: 136-144). Non-limiting examples of polynucleotides encoding SEQ ID NO: 206-212 are provided herein as SEQ ID NO: 213-219, respectively. Non-limiting examples of polynucleotides encoding suitable Fc domains are provided herein as SEQ ID NOs: 169 and 170. In particular embodiments, the polynucleotide encodes an ACE2 decoy polypeptide in which the threonine at position 27 is replaced by tyrosine (T27Y), the histidine at position 34 is replaced by alanine (H34A), and the histidine at position 374 is replaced by asparagine (H374N). In particular embodiments, the polynucleotide is characterized by SEQ ID NO: 216. In further embodiments, the ACE2 decoy peptide or a variant thereof is fused or otherwise connected to IgFc to form a fusion polypeptide. In particular embodiments, IgFc is selected from the group consisting of: IgAFc, IgDFc, IgEFc, IgGFc, and IgMFc. In the case where IgFc is IgGFc, Fc can be IgG1Fc, IgG2Fc, IgG3Fc, or IgG4Fc.

[0086] Similarly, examples of suitable polynucleotides encoding Fab heavy and light chains are presented herein as SEQ ID NOs: 171 and 172. An ordinary molecular biologist would know how to change the sequences of SEQ ID NOs: 171 and 172 to encode Fab fragments having the relevant CDRs of the anti-spike and anti-RBD antibodies disclosed herein. Similarly, if the user wishes to express these Fab fragments in the context of a complete immunoglobulin, an ordinary molecular biologist would know how to further change SEQ ID NOs: 171 and 172 to encode an Fc domain or other such extended structures. In an embodiment, the polynucleotide encodes a heavy variable (V) having at least 85% sequence identity to each of SEQ ID NOs: 127-130. H) chain and a light variable (V) having at least 85% identity to each of SEQ ID NOs: 131 and 132 L ) chain Fab fragment, wherein V H Chain and V L Each of the chains has three complementarity determining regions (CDRs).In a further embodiment, the polynucleotide encodes a Fab fragment and a fragment crystallizable (Fc) domain.

[0087] In certain embodiments, the above-mentioned polynucleotides can be expressed in a supporting cell line. Mammalian cell lines such as Chinese hamster ovary (CHO) cells or 293T cells are particularly suitable for these purposes. The above-mentioned proteins are usually soluble and therefore can be discharged from the production cells unless they are modified to be retained in the cell. The protein produced in this way can be purified from the culture medium. If necessary, the protein can be labeled with (for example) polyhistidine tags or other such commercially common tags to facilitate purification. The protein produced and purified in this way can then be applied to a subject in need as described below.

[0088] Additionally or alternatively, the above polynucleotides can be incorporated into a vector (e.g., a transfection vector or a viral transduction vector). Such vectors can then be transfected or transduced into the subject's own cells. In this way, the subject's own cells (e.g., the subject's respiratory mucosa) will produce ACE2 chimeras and / or anti-spike antibodies to protect these tissues from SARS-CoV-2 infection. Non-limiting examples of vectors comprising the above polynucleotides are provided herein as SEQ ID NO: 220-233.

[0089] V. Treatment Methods

[0090] The above proteins, polynucleotides and vectors can be used to treat and / or prevent COVID-19 disease and / or SARS-CoV-2 or its variant infection. In order to treat and / or prevent such infection and disease, the above proteins, polynucleotides and vectors must be administered to a subject in need thereof in a therapeutically effective amount. The subject can be symptomatic or asymptomatic.

[0091] In the case of transfection protein, any suitable route of administration can be used, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection and inhalation (e.g., aerosol inhalation). In a specific embodiment, the Fab fragment or polypeptide comprising the ACE2 decoy peptide is applied to the respiratory mucosa of the subject by inhalation (e.g., aerosol inhalation). In some embodiments, the Fab fragment or polypeptide comprising the ACE2 decoy peptide is provided to the respiratory mucosa of the subject. The therapeutically effective amount of these proteins includes but is not limited to 1 μg protein / kg subject body weight, 5 μg / kg, 10 μg / kg, 50 μg / kg, 100 μg / kg, 500 μg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, 500 mg / kg, and 1 mg / kg or higher.

[0092] Where polynucleotides are to be transfected, any suitable amount may be administered, including but not limited to 10 ng, 50 ng, 100 ng, 500 ng, 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, and 500 mg or more. In order to transfect a subject's cells with a polynucleotide as described herein, it would be useful to extract the cells from the subject, transfect them according to known techniques, and then return the transfected cells to the subject. Particularly suitable cells circulate throughout the body, such as circulating lymphocytes.

[0093] In the case of transducing polynucleotides, the viral vector can be directly administered to the subject, or cells can be extracted for transduction and re-infusion. The viral vector can be administered to the subject by any suitable route of administration, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection and inhalation (e.g., aerosol inhalation). In a specific embodiment, the viral vector is administered by inhalation (e.g., aerosol inhalation). In another embodiment, the viral vector is provided to the respiratory mucosa of the subject.

[0094] The effective amount of virus for treatment includes but is not limited to 1×10 7 Virus particles (VP), 5×10 7 VP, 1×10 8 VP, 5×10 8 VP, 1×10 9 VP, 5×10 9 VP, 1×10 10 VP, or more than 1×10 10VP. Adenoviral vectors are particularly suitable for this purpose due to their large cargo capacity. Suitable adenoviral vectors include those disclosed in WO 98 / 17783, WO 02 / 27007, WO 09 / 6479, and WO 14 / 31178, each of which is incorporated herein by reference in its entirety. Suitable methods of administering these adenoviral vectors are disclosed in WO 16 / 112188, which is incorporated herein by reference in its entirety.

[0095] Alternatively and / or additionally, administration of a protein or polypeptide (e.g., an ACE2 decoy peptide or Fab fragment that binds to the SARS-CoV-2 spike protein) can be combined with a protein or polynucleotide encoding a protein that stimulates an immune response to a viral infection (e.g., a SARS-CoV-2 infection). Where appropriate, such treatments include administration of an "IL-15 agonist" (e.g., NAI), an immunostimulatory cytokine or an analog thereof, a checkpoint inhibitor, an OX40 ligand, and / or a cell lysate to provide a source of DAMPS and PAMPS. The polynucleotide can be a viral vector, e.g., from a non-pathogenic virus. In some embodiments, the NAI is administered at a dose of about 5 μg / kg / day to about 15 μg / kg / day. In certain embodiments, the NAI is administered at a dose of about 5 μg / kg / day, about 6 μg / kg / day, about 7 μg / kg / day, about 8 μg / kg / day, about 9 μg / kg / day, about 10 μg / kg / day, about 11 μg / kg / day, about 12 μg / kg / day, about 13 μg / kg / day, about 14 μg / kg / day, or about 15 μg / kg / day. In a specific embodiment, the NAI is administered at about 10 μg / kg / day.

[0096] In still further embodiments, another agent that regulates at least one of ACE and ACE2 signal transduction is administered. The agent that regulates at least one of ACE or ACE2 signal transduction can be an ACE inhibitor or a compound that increases ACE2 expression. In some embodiments, the compound can be an ACE inhibitor selected from, but not limited to, the group consisting of: Benazepril, Captopril, Fosinopril, Lisinopril, Enalapril, Perindopril, Moexipril, Quinapril, Ramipril and Trandolapril. In some embodiments, the compound is an angiotensin receptor antagonist selected from, but not limited to, Candesartan, Olmesartan, Valsartan and Losartan. Additionally or alternatively, the additional agent may be an inhibitor of BDKRB1 / 2, AGTR1, MAS1 or renin.

[0097] In another embodiment, treatment of a subject suffering from, diagnosed with, or suspected of having SARS-CoV-2 comprises administering an IL-15 agonist as the sole therapeutic agent. Alternatively, the IL-15 agonist is administered in combination with another active agent, such as an agent that regulates at least one of ACE or ACE2 signaling. In another embodiment, treatment of a subject infected, diagnosed with, or suspected of having COVID-19 comprises an IL-15 agonist as the sole therapeutic agent or in combination with another active agent, such as an agent that regulates at least one of ACE or ACE2 signaling. In a further embodiment, the IL-15 agonist used to treat SARS-CoV-2 and / or COVID-19 infection is a NAI. In some embodiments, the NAI is administered at a dose of about 5 μg / kg / day to about 15 μg / kg / day. In certain embodiments, the NAI is administered at a dose of about 5 μg / kg / day, about 6 μg / kg / day, about 7 μg / kg / day, about 8 μg / kg / day, about 9 μg / kg / day, about 10 μg / kg / day, about 11 μg / kg / day, about 12 μg / kg / day, about 13 μg / kg / day, about 14 μg / kg / day, and about 15 μg / kg / day. In a specific embodiment, the NAI is administered at about 10 μg / kg / day.

[0098] With regard to non-pathogenic viruses, it should be understood that all viruses suitable for gene therapy are considered suitable for use herein. However, particularly preferred viruses are those that have been established in therapy, including adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, and the like. Among other suitable choices, adenoviruses are particularly preferred. It should be particularly understood that these viruses are suitable for the delivery of two payloads, namely nucleic acid sequences of host proteins that are critical for viral entry / replication and nucleic acid sequences of antigenic viral proteins.

[0099] In addition, it is generally further preferred that the virus is a replication-deficient and non-immunogenic virus. For example, suitable viruses include genetically modified alphaviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like. However, adenoviruses are particularly preferred. For example, genetically modified replication-deficient adenoviruses are preferred, which are not only suitable for multiple vaccinations, but also suitable for vaccination in individuals with pre-existing immunity to adenoviruses (see, for example, WO 2009 / 006479 and WO 2014 / 031178, which are incorporated by reference in their entirety). In some embodiments, the replication-deficient adenoviral vector includes a replication-deficient adenoviral 5 vector. In some embodiments, the replication-deficient adenoviral vector comprises a deletion in the E2b region. In some embodiments, the replication-deficient adenoviral vector further comprises a deletion in the E1 region. In this regard, it should be noted that the E2b gene and other late proteins are deleted in the genetically modified replication-deficient adenovirus to reduce immunogenicity. Moreover, due to these specific deletions, such genetically modified viruses are replication-deficient and allow for use in relatively large recombinant cargoes.

[0100] Examples

[0101] The following examples are provided to further illustrate the invention disclosed herein, but of course should not be construed as limiting the scope thereof in any way.

[0102] Example 1: Selected exemplary constructs presented herein were expressed in CHO-S using Maxcyte electroporation. 3.2×10 8The cells were electroporated in Maxcyte electroporation buffer and cultured in CD Opti CHO medium with CD CHO efficient feed at 32°C and 3% CO2 for 14 days. The ACE2-Fc hybrid constructs were isolated according to known isolation procedures. The ACE2-Fc IgG hybrid protein has the amino acid sequence of SEQ ID NO: 234, while the ACE2-Fc IgG R273Q hybrid protein (lacking ACE2 activity) has the amino acid sequence of SEQ ID NO: 235. The ACE2-Fc IgA hybrid protein has the amino acid sequence of SEQ ID NO: 236, while the ACE2-Fc IgA R273Q hybrid protein (lacking ACE2 activity) has the amino acid sequence of SEQ ID NO: 237.

[0103] The yields and relative purity of small scale Maxcyte production of IgG-Fc hybrid constructs were within the ideal range. Table 4 provides the numerical results of size exclusion chromatography.

[0104]

[0105] ACE2-IgAFc expression was as efficient as ACE2-IgG1Fc expression, with no significant difference in the mutant form (R273Q) relative to the non-mutated form. Conventional IgG purification processes are generally ineffective for IgA, so ion exchange chromatography and affinity media selective for IgA were used to separate the hybrid construct. The purified ACE2-Fc hybrid construct was tested for binding and affinity to the 2019-n-CoV spike protein. Table 5 lists exemplary test results.

[0106]

[0107]

[0108] In the case where the modified ACE2-Fc hybrid construct is used for diagnostic testing, a variety of test formats can be selected. An exemplary system uses a biotin-labeled ACE2-Fc hybrid construct fixed on an MSD streptavidin plate and another modified ACE2-Fc hybrid construct for detection (sulfo tag MSD label). As shown, the ACE2-Fc hybrid construct has high sensitivity and specificity.

[0109] Example 2: To confirm the affinity of various anti-spike antibodies disclosed herein for SARS-CoV-2 spikes, KD was measured by surface plasmon resonance (SPR). All SPR experiments were performed on Pioneer FE (Sartorius Corporation) and the data were analyzed by Qdat analysis software. Antibodies were captured on an SPR sensor coated with anti-human IgG Fc. One-step kinetic assays were performed using 50nM RBD-SD1 or 20nM sc-S-PP-T4F samples. mAbs with the ability to bind to spike trimers and / or RBD peptides are shown in Tables 1 and 2 above.

[0110] Example 3: To study the epitope of the antibody, sc-S-PP-T4F, RBD-SD1, and RBD were used as analytes. These different constructs are shown in Figure 1 and 2 All epitope analyses were performed on Octet Red96e (Sartorius). Antibodies were captured on anti-human capture (AHC) biosensors. A 2× dilution series starting with 20 nM sc-S-PP-T4F and another series starting with 100 nM RBS-SD1 and RBD were used for kinetic assay cycles. Table 3 below summarizes these epitope assay experiments using several selected mAbs. Figures 3 and 4 show SPR plots for some of these experiments. N-612-004 and N-612-017 each bound to the cleaved spike peptide in a 1:1 binding mode, which was observed when bound to RDB-SD1, indicating that the cleaved spike contains only the S1 domain. The calculated k off As follows: N-612-004 has a cutting spike of 9.8×10 -4 , N-612-004 to RBD-S1 is 3.4×10 -4 , N-612-017 has a cutting spike of 3.9×10 -3 , and N-612-017 to RBD-S1 is 1.9×10 -3 .

[0111] Example 4: Ni-NTA biosensor was coated with His-tagged RBD-SD1 and quenched with anti-RBD-SD1 antibody. Immediately after quenching, the sensor was incubated with ACE2-IgG1Fc solution to determine which antibody blocked the binding of ACE2-IgGFc to RBD-SD1 on the sensor. Figure 5In the , N-612-004 binds both S and S-RBD-SD1, while N-612-002 binds only S. In other words, N-612-002 binds to either the RBD or the spike, but does not interfere with ACE2 binding. Steric hindrance may explain the low N-612-002 binding. The overlapping N-612-002 and N-612-004 lines extend discernibly below the buffer line, suggesting that ACE2 / RBD binding is somehow hindered. Notably, mAb N-612-017 was able to almost completely block the binding of the spike to ACE2 under these in vitro conditions.

[0112]

[0113] Example 5: The binding affinity between IgG1 and recombinant spike trimer extracellular domain or RBD-SD1 protein was measured using the Pioneer FE instrument. Figure 6 and 7 The SPR affinity curves of two anti-spike IgG1 to RBD-SD1 peptide show K D Determination. Figure 8-12 The SPR affinity curves of a selected series of anti-spike IgG1 for Sc-S-PP-T4F show K D The spike binding data are avidity enhancing data, resulting in a flat dissociation curve.

[0114] Example 6: Binding affinity between scFv and recombinant spike trimer extracellular domain or RBD-SD1 protein was measured on a Pioneer FE instrument. In brief, biotinylated anti-FLAG M2 antibodies were immobilized on a PCH biosensor (Molecular Devices / ForteBio) coated with neutral avidin. Triple FLAG-tagged scFvs were captured with anti-FLAG M2 on the sensor, and binding affinity was measured using a one-step injection of recombinant analyte protein. The avidity effect of the spike trimer resulted in an unfitted flat dissociation curve. Fig.13 and 14 The SPR affinity curves of the two anti-spike scFvs for the RBD-SD1 peptide show K D Determination. Fig.15 and 16 The SPR affinity curve of the anti-spike scFv in SARS-CoV-2 spike trimer shows K D Determination.

[0115] Example 7: The RBD structure can be likened to a human foot, with a "heel", "arch", and "toe" region. A careful analysis of the binding interface revealed a pocket structure between the "arch" portion and the ACE2 α-helix. Water can be displaced from this pocket structure to increase the binding strength between the RBD and the ACE2 α-helix. Mutants of this α-helix can be used as competitive inhibitors of ACE2 / RBD binding or as molecular traps to isolate RBD from cell surface ACE2. Fig.17 Table 7 shows the ACE2-IgG1 Fc mutation study, confirming that the "heel" is the most important element for binding affinity to RBD. Although the H34A mutant ACE2 α-helix was shown to bind to RBD with a Kd of 9.4 nM, and the T27Y mutant ACE2 α-helix was shown to bind to RBD with a Kd of 3.8 nM, the presence of the double mutations H34A and T27Y produced a synergistic effect and increased the binding affinity to about 1 nM.

[0116]

[0117]

[0118] Fig.18A and 18B It was demonstrated that the ACE2-IgG1Fc mutant was successfully expressed and purified.

[0119] The purification summary in Table 8 shows the purification of the single point mutations H34A and T27Y and the double mutant (H34A and T27Y).

[0120]

[0121] Example 8: The binding affinity of wild-type ACE2-IgG1 Fc antibody, T27Y and H34A single mutants, and T27Y / H34A double mutant ACE2-IgG1 Fc antibody was measured. Antibodies were captured on AHC biosensors as in Example 3. A two-fold dilution series starting from 200 μM RBD-SP1 was used for kinetic assay cycles. Fig.19 and 20 SPR plots for some of these experiments are shown. Table 9 below summarizes the binding data for the two antibodies.

[0122]

[0123] Fig.21 It was demonstrated that the ACE2-IgG1Fc double mutation (T27Y / H34A) had superior binding affinity to RBD compared to wild-type ACE2-IgG1Fc and ACE2-IgG1Fc single mutant.

[0124] Example 9: Fig. 22 Live SARS-CoV-2 neutralization data are presented. Neutralization of SARS-CoV-2 was tested against wild-type and mutant ACE2-IgG1Fc. Wild-type ACE2-IgG1Fc and its enzyme-inactivating mutation (R273Q) were sufficient to neutralize SARS-CoV-2. ACE2-IgG1Fc with H34A and T27Y mutations (ACE2 dbl) was much better than the wild-type variant in neutralizing SARS-CoV-2. The ACE2 double mutant with H34A and T27Y mutations was as good as antibody 017 in neutralizing SARS-CoV-2. Table 10 reports the IC50 values ​​of various neutralizing antibodies and ACE2 decoy peptides calculated from the neutralization experiments.

[0125]

[0126] The neutralizing ability of the combination of ACE2-IgG1Fc or ACE2 IgA with H34A and T27Y, which was expressed as an adenovirus vaccine with various antibodies, was also tested. It was found that combining the antibody N-612-017 with either or both of N-612-007 (binding to S2) or N-612-004 (binding to SD1) improved neutralization, although neither N-612-004 or N-612-007 alone had much neutralizing activity. Finally, it was found that combining N-612-017 with N-612-014 and / or N-612-056 significantly improved neutralization.

[0127] Example 10: Stability of wild-type, R273Q mutant, and T27Y / H34A double mutant ACE2 decoy peptides was measured by differential scanning fluorimetry (DSF). 20 μL of 1 mg / mL antibody was mixed with 10 μL of 20X SYPRO Orange. The plate was scanned from 20°C to 70°C in 0.5°C increments in a CFX96 Real-Time System (BioRad). The DSF results for wild-type, R273Q mutant, and T27Y / H34A double mutant ACE2 decoy peptides are shown in Fig.23 middle.

[0128] Example 11: Seven triple mutant ACE2 decoy peptides were prepared. The size of each triple mutant ACE2 decoy peptide was measured by size exclusion chromatography. Briefly, the purified triple mutant ACE2 decoy peptide was run on a Zenix-CSEC-300 (Sepax) (3 μm, 7.8 x 300 mm) in a running buffer containing 50 mM sodium phosphate, 250 mM sodium chloride, and pH 6.8. Figure 24-30The size of each triple mutant ACE2 bait peptide is shown. The ACE2 activity of wild-type ACE2 bait peptide, R273Q mutant ACE2 bait peptide, T27Y / H34A double mutant ACE2 bait peptide and each triple mutant ACE2 bait peptide was also determined. In 75mM HEPES, 1M sodium chloride and pH 7.4, 1μg / mL of each sample was mixed with 20μM substrate (MCA-Tyr-Val-Ala-Asp-Pro-Lys(DNP)-OH (R&D systems)). The sample was shaken (700rpm) and incubated for 30 minutes at room temperature. FRET signals (top readings) were measured at excitation and emission wavelengths of 320nm and 405nm, respectively. Fig.31 The ACE2 activity of wild-type and mutant ACE2 decoy peptides is shown. Table 11 summarizes the binding data of triple mutant ACE2 decoy peptides. The stability of various triple mutant ACE2 decoy peptides was measured and compared with single and double ACE2 mutant decoy peptides. DSF was performed as described in Example 7 above. Fig.32 The stability of R273Q, T27Y / H34A, and T27Y / H34A / R273Q mutant ACE2 decoy peptides is shown. Fig.33 The stability of three triple mutant ACE2 decoy peptides as compared to the double mutant ACE2 decoy peptide is shown. Fig.34 The stability of five triple-mutant ACE2 decoy peptides is shown.

[0129]

[0130] Example 12: Fusion of wild-type ACE2 decoy peptide to avidin at the C-terminus of the antibody As described in Example 7, the stability of avidin-labeled and unlabeled wild-type ACE2 decoy peptides was measured by DSF. Fig.35 The stability of avidin-labeled and unlabeled wild-type ACE2 decoy peptides is shown.

[0131] Example 13: Expose anti-spike antibodies to wild-type and mutant spike proteins and measure the binding affinity of the antibodies to the spike proteins. Unless otherwise indicated, all experiments used a running buffer consisting of 10mM HEPES (pH 7.4), 150mM NaCl, 0.02% tween 20, 0.1% BSA. For the 1:1 binding affinity determination of nAbs to SARS-CoV-2 RBD wild type and mutants and S1 variants, nAbs were immobilized on AHC sensors (Sartorius) and RBD (wild type or mutant) and S1 concentration series of 200, 100, 50, 25, 12.5, 6.25, 3.125nM were tested to determine K D. Fig.36 Binding of N-612-017 or N-612-056 to wild-type or mutant spike proteins is shown. Fig.37 The binding of N-612-004, N-612-014, N-612-017, and N-612-056 to wild-type spike or UK variant spike proteins is shown. Tables 12 and 13 present the K values ​​of various anti-spike antibodies to wild-type and mutant spike proteins. D .

[0132]

[0133]

[0134] Example 14: Expose ACE2 decoy peptides to wild-type and mutant spike proteins and measure the binding affinity of ACE2 decoy peptides to spike proteins. Unless otherwise indicated, all experiments used a running buffer consisting of 10mM HEPES (pH 7.4), 150mMNaCl, 0.02% tween 20, 0.1% BSA. For the 1:1 binding affinity determination of ACE2 decoy peptides to SARS-CoV-2RBD wild-type and mutants, the ACE2 decoy peptides were immobilized on an AHC sensor (Sartorius) and a concentration series of 200, 100, 50, 25, 12.5, 6.25, 3.125nM RBD (wild-type or mutant) was used to determine K D The binding of wild-type ACE2-IgG1Fc and T27Y / H34A / H374N triple mutant ACE2 decoy-IgG1Fc to wild-type or mutant spike proteins is shown in Fig.38 Table 14 presents the K values ​​of ACE2 decoy peptides against wild-type and mutant spike proteins. on , K off , and K D .

[0135]

[0136] All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0137] Unless otherwise indicated herein or clearly contradicted by context, the use of the terms "a and an" and "the" and "at least one" and similar referents in the context of describing the present invention (especially in the context of the appended claims) should be interpreted as including both the singular and the plural. Unless otherwise indicated herein or clearly contradicted by context, the use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be understood to refer to one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B). Unless otherwise stated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the statement of numerical ranges herein is intended only to be used as a shorthand method of referring individually to each independent value within the range, and each independent value is incorporated into the specification as if the value were stated individually herein. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (such as "for example") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention otherwise claimed. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the present invention.

[0138] Specific embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, variations of those specific embodiments will become apparent to those of ordinary skill in the art. The inventors expect that skilled technicians will appropriately adopt such variations, and the inventors intend to practice the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter stated in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

Claims

1. A Fab fragment that binds to the SARS-CoV-2 spike protein, comprising a heavy chain variable region (V H ) and light chain variable region (V L ), where V H having three complementarity determining regions (CDRs), wherein the CDRs are selected from the group consisting of: V as shown in SEQ ID NO: 67 H CDR1, V as shown in SEQ ID NO:68 H CDR2, and V as shown in SEQ ID NO:69 H CDR3, and wherein V L Having SEQ ID NO:131, V as shown in SEQ ID NO:70 connected in sequence L CDR3 and SEQ ID NO:

132.

2. The Fab fragment of claim 1, wherein the V H Having SEQ ID NO: 127, V H CDR1, SEQ ID NO: 128, V H CDR2, SEQ ID NO: 129, V H CDR3 and SEQ ID NO:

130.

3. The Fab fragment of claim 1 or 2, wherein the Fab fragment further comprises an Fc domain.

4. A polynucleotide encoding the Fab fragment according to any one of claims 1 to 3.

5. The Fab fragment of claim 1 or 2, wherein the spike protein has one or more point mutations selected from the group consisting of N501Y, K417N, K417T, E484K, L452R, and combinations thereof. A viral vector comprising the polynucleotide according to claim 4 .

7. The viral vector of claim 6, wherein the viral vector is an adenoviral vector comprising a deletion of the E1 gene region and a deletion of the E2b gene region.

8. The viral vector of claim 7, further comprising a deletion of the E3 gene region.

9. Use of the Fab fragment as described in any one of claims 1-3 and 5 in the preparation of a medicament for treating COVID-19 in a subject in need thereof, comprising administering the Fab fragment as described in any one of claims 1-3 and 5 to the respiratory mucosa of the subject.

10. Use of a viral vector as described in any one of claims 6-8 in the preparation of a medicament for treating COVID-19 in a subject in need thereof, comprising administering a viral vector as described in any one of claims 6-8 to the respiratory mucosa of the subject.

11. The use according to claim 10, wherein the viral vector is administered by inhalation.

12. The use according to claim 11, wherein 5×10 8 to 5×10 9 Virus particles.

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